Tyrosine kinase inhibitors and uses thereof

Abstract

The present disclosure relates to the technical field of tyrosine kinase inhibitors and uses thereof. In particular, the present disclosure relates generally to novel tyrosine kinase (TK) multi-target inhibitors that can suppress angiogenesis, metastasis, neogenesis and / or immunomodulatory activity by inhibiting TK and have very potent immunomodulatory activity. The present disclosure also relates to a method of treating cancer using a tyrosine kinase inhibitor, alone or in combination with an HDAC inhibitor, particularly in cancer immunotherapy by modulating the tumor microenvironment, including reducing tumor hypoxia, reducing lactic acid accumulation, activating CTLs, inhibiting the number and activity of immunosuppressive cells, and in particular in cancer immunotherapy. Therefore, excellent anti-cancer benefits are finally obtained and / or lasting immune memory is generated.

Description

Technical Field

[0001] The present disclosure generally relates to novel tyrosine kinase (TK) multi-target inhibitors, which can suppress angiogenesis, metastasis, tumorigenesis and / or immunomodulatory activity by inhibiting TK, and have very effective immunomodulatory activity. The present disclosure also relates to a method for treating cancer using a tyrosine kinase inhibitor alone or in combination with an HDAC inhibitor, particularly in cancer immunotherapy, the method being achieved by: regulating the tumor microenvironment, including reducing tumor hypoxia, reducing lactic acid accumulation, activating CTLs, suppressing the number and activity of immunosuppressive cells, and ultimately obtaining excellent anti-cancer benefits and / or generating lasting immune memory. Background Art

[0002] TK inhibitors have been widely developed for the treatment of cancer and have been classified as targeted therapies. The emergence of cancer immunotherapy and the clinical success of immune checkpoint inhibitors (ICIs) have opened a new turning point in cancer treatment. However, with the continuous expansion of clinical applications, these emerging ICI therapeutic drugs face challenges and their ORR (objective response rate) is quite low. Therefore, a combination drug strategy is needed to improve ORR and enable patients to obtain better treatment benefits.

[0003] Although experts have made every effort to find effective combination regimens of ICIs to enhance the therapeutic benefit, the results have shown very limited potential. There is still a strong need to develop combination therapies of anticancer drugs. Summary of the invention

[0004] The present disclosure relates to a series of novel structures of tyrosine kinase (TK) multi-target inhibitors.

[0005] In one embodiment, the present disclosure provides a compound of formula (I):

[0006]

[0007] in:

[0008] A is –OD or The wavy lines indicate the connection locations;

[0009] D is H, C 1-4 Alkyl or Ar;

[0010] Ar is The dotted lines indicate the connection locations;

[0011] X is N, CH or CR d ; n is an integer ranging from 0 to 3;

[0012] When present, each R dindependently selected from the group consisting of methyl, NH2, OH, SH, methoxy, dimethylamino, trifluoromethoxy, cyanide, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Cl and Br;

[0013] R p Selected from the group consisting of: H, methyl, NH2, OH, SH, methoxy, dimethylamino, trifluoromethoxy, cyanide, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Cl and Br;

[0014] L1, L2 are (i) -NH-C(=O)- or (ii) -(C=O)-NH-; and

[0015] L3, L4 are (i) –NH-C(=O)- or (ii) –(C=O)-NH-;

[0016] or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0017] In one embodiment, the present disclosure provides a compound of formula (II):

[0018]

[0019] wherein D, L1, L2, L3 and L4 have the same meanings as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0020] In one embodiment, the present disclosure provides a compound of formula (III):

[0021]

[0022] wherein D, L3 and L4 have the same meanings as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0023] In one embodiment, the present disclosure provides a compound of formula (IV):

[0024]

[0025] wherein D has the same meaning as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0026] In one embodiment, the present disclosure provides a compound selected from the group consisting of:

[0027]

[0028]

[0029] or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

[0030] In other embodiments, the present disclosure provides a pharmaceutical composition or combination comprising a compound described herein.

[0031] In other embodiments, the present disclosure provides a method for suppressing angiogenesis, metastasis, tumorigenesis and / or immunomodulatory activity in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0032] In other embodiments, the present disclosure provides a method for treating a disease or condition associated with tyrosine kinases and / or tumors or cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chemical structure and detailed NMR data of Cbo-23 are shown.

[0034] Figure 2A-2G Animal test designs and results for cabozantinib, Cbo-02, and drug combinations for evaluating therapeutic efficacy are shown.

[0035] Figures 3A-3G Animal test designs and results for cabozantinib, Cbo-20, and drug combinations for evaluating therapeutic efficacy are shown.

[0036] Figures 4A-4F Animal testing results for cabozantinib, Cbo-22, and drug combinations for evaluating therapeutic efficacy are shown.

[0037] Figure 5A-5G Animal test designs and results for cabozantinib, Cbo-23, and drug combinations for evaluating therapeutic efficacy are shown.

[0038] Figures 6A-6G Animal test designs and results for cabozantinib, Cbo-23, and drug combinations for evaluating therapeutic efficacy are shown.

[0039] Figures 7A-7G Animal test designs and results for zanzalintinib, Cbo-23-k30, and drug combinations for evaluating therapeutic efficacy are shown. DETAILED DESCRIPTION

[0040] definition

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. The terms used in this specification are only used to describe specific embodiments and are not intended to limit the content of the present disclosure.

[0042] Where a range of values ​​is provided, it is understood that each intervening value (to one-tenth of the lower limit, unless the context clearly indicates otherwise) between the upper and lower limits of the range (such as in the case of a group containing a number of carbon atoms, in which case each number of carbon atoms falling within the range is provided) and any other stated value or intervening value within the range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the disclosure, subject to any explicitly excluded limits within the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also encompassed within the disclosure.

[0043] As used herein and in the appended claims, the articles "a" and "an" are intended to refer to one or to more than one (ie, to at least one) of the grammatical object of the article unless the context clearly dictates otherwise. By way of example, "an element" means one element or more than one element.

[0044] As used herein in the specification and in the claims, the term "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements present together in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" elements so combined. In addition to the elements specifically listed by the "and / or" phrase, other elements may optionally be present, whether related or unrelated to those elements specifically identified.

[0045] As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0046] The term "alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon atoms and hydrogen atoms, which is free of unsaturation and has one to more carbon atoms. In some embodiments, the alkyl group contains one to six carbon atoms (e.g., C1-C6 alkyl) or one to five carbon atoms (e.g., C1-C5 alkyl). In some embodiments, the alkyl group contains one to four carbon atoms (e.g., C1-C4 alkyl). In other embodiments, the alkyl group contains one to three carbon atoms (e.g., C1-C3 alkyl). In other embodiments, the alkyl group contains one to two carbon atoms (e.g., C1-C2 alkyl). In other embodiments, the alkyl group contains one carbon atom (e.g., C1 alkyl). In other embodiments, the alkyl group contains two to five carbon atoms (e.g., C2-C5 alkyl). In other embodiments, the alkyl group contains three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl group is connected to the rest of the molecule by a single bond. Unless otherwise specifically stated in the specification, the alkyl group is optionally substituted with one or more substituents.

[0047] The term "alkoxy" refers to a radical bonded through an oxygen atom of the formula -O-alkyl, wherein alkyl is an alkyl chain as defined above.

[0048] As used herein, the term "cycloalkyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic compound. Examples of C3-C8-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl.

[0049] As used herein, "aryl" refers to a monocyclic or polycyclic carbocyclic ring system having one or more fused or non-fused aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.

[0050] As used herein, the term "heteroaryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic or more) fused or non-fused group or ring system having at least one aromatic ring having five to ten ring atoms, one of which is selected from S, O and N; zero, one or two of which are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, phenylthio, furanyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like.

[0051] As used herein, the term "heterocycloalkyl" refers to a non-aromatic 3-, 4-, 5-, 6-, or 7-membered ring or a bicyclic or tricyclic group fused or non-fused system, wherein (i) at least one ring contains between one and three heteroatoms independently selected from oxygen, sulfur, and nitrogen; (ii) each 5-membered ring has 0 to 1 double bonds, and each 6-membered ring has 0 to 2 double bonds; (iii) the nitrogen and sulfur heteroatoms may be optionally oxidized; (iv) the nitrogen heteroatom may be optionally quaternized; and (v) any of the above rings may be fused to a benzene ring. Representative heterocycloalkyls include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuranyl.

[0052] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed by the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present disclosure, which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of the basic compounds of the present disclosure include, but are not limited to, the following: acetate, ascorbate, adipate, alginate, draw salt, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, camphorsulfonate, carbonate, chloride, clavulanate, citrate, cyclopentanepropionate, diethylacetate, digluconate, dihydrochloride, dodecylsulfate, ethylenediaminetetraacetate, ethylenediaminetetraacetate, ester salt, sulfonate, ethanesulfonate, formate, fumarate, gluconate, gluconate, glutamate, glycerophosphate, glycol dimethylarsonic acid salt, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrobromide, hydrochloride , 2-hydroxyethanesulfonate, hydroxynaphthoate, hydroiodide, iodide, isonicotinate, isonicotinate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methyl nitrate, methyl sulfate, methanesulfonate, acetalate, 2-naphthalenesulfonate, naphthenate, nicotinate, nitrate, oleate, oxalate, pamoate (spicate), palmitate, pantothenate, pectinate, persulfate, phosphate / diphosphate, pimelate, phenylpropionate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theocyanate, toluenesulfonate, trithiolate, trifluoroacetate, undecylenate, valerate, etc. In addition, when the compounds of the present disclosure carry an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganate, potassium, sodium, zinc, etc. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, cyclic amines, dicyclohexylamine and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.Also included are basic nitrogen-containing groups which may be quaternized with such agents as lower alkyl halides (e.g., methyl, ethyl, propyl and butyl chlorides, bromides and iodides); dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and diamyl sulfates); long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides); aralkyl halides (e.g., benzyl and phenethyl bromides, etc.).

[0053] The term "subject" includes living organisms, such as humans, monkeys, cows, sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, cultured cells, and transgenic species thereof. In a preferred embodiment, the subject is a human.

[0054] The term "administering" includes routes of administration that allow the active ingredients of the present disclosure to perform their intended functions.

[0055] The terms "treat" or "treatment" refer to methods of lessening the effects of a disease or condition. Treatment may also refer to methods of reducing the underlying cause of the disease or condition itself, rather than just the symptoms. Treatment may be any reduction from natural levels, and may be, but is not limited to, complete ablation of the disease, condition, or symptoms of the disease or condition.

[0056] The terms "prevent," "prevention," or "preventing" refer to inhibiting or avoiding symptoms associated with a target disease.

[0057] The term "therapeutically effective amount" refers to an amount of a compound, material, or composition comprising a compound of the present disclosure effective to produce the desired therapeutic effect, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0058] Tyrosine kinase (TK) inhibitor compounds

[0059] In terms of clinical application, tyrosine kinase inhibitors are considered to be a successful class of anticancer drugs. Potential TK inhibition targets include c-MER, AXL, ROS1, BTK, TYRO3 (RSE), NTRK2 (TRKB), LCK, LYNB, FLT4 (VEGFR3), EPHA2, HCK, FER, FLT3, FGR, FRK (PTK5), RET, KDR (VEGFR2), SRC, c-MET, PDGFRA, CSFIR (FMS), MST1R (RON), LYNA, INSRR (IRR), and TEK (Tie-2). The US FDA has approved many TK inhibitors, including for the treatment of tumor indications and non-tumor indications. Some TK inhibitors are also believed to have immunomodulatory activity in the tumor microenvironment, but it is difficult to confirm whether their inhibition of these TKs is closely related to immunomodulation. Many TK inhibitors that have been marketed (such as regorafenib, lenvatinib, cabozantinib) are used in combination with ICI (such as anti-PD-1 / anti-PD-L1Ab) in clinical trials, hoping to enhance the anti-tumor therapeutic benefits of ICI through the immunomodulatory ability of TK inhibitors. However, the vast majority of combination treatments have not met expectations, nor have they demonstrated efficacy in the primary endpoints of clinical trials. This also shows that in order to significantly improve the anti-cancer efficacy of cancer immunotherapy, it is necessary to overcome the rigidity of ICI and create new drugs with different new cancer immunotherapy mechanisms, used alone or in combination.

[0060] More and more studies have found that the ORR of cancer immunotherapy is controlled by the tumor microenvironment. However, the tumor microenvironment is dynamic and complex, and its composition is uncertain as the tumor growth state changes. Therefore, it is indeed very difficult to effectively regulate the tumor microenvironment. The applicant has been focusing on this research field and has made important discoveries. Without being bound by theory, the applicant was surprised to note that the following tasks will be important for achieving the goal of regulating the tumor microenvironment. The primary task of regulating the tumor microenvironment is to normalize tumor blood vessels. Only the normalization of tumor blood vessels can reshape the tumor microenvironment. This will effectively reverse the attraction of a large number of immunosuppressive cells (such as Treg, TAM, MDSC) to the tumor microenvironment, and promote the attraction of CTL and NK to the tumor microenvironment, thereby attacking tumors. The second important task is to alleviate the problem of hypoxia in the tumor microenvironment and reduce the expression of HIF-1, which will help CTL continue to maintain the ability to attack tumors. The third important task is to effectively overcome the anaerobic respiration that regulates the tumor microenvironment, reduce the synthesis and accumulation of lactic acid, and reduce the continuous maintenance of the acidic environment in the tumor microenvironment, which is conducive to the activity and survival of CTL against tumor attacks. The fourth important task is to attract more CTLs that already have the ability to identify tumors and allow them to enter the tumor microenvironment, thereby effectively attacking the tumor and achieving the benefits of tumor resection. Among them, increasing antigen presentation is very important for improving the cancer cell recognition ability of CTLs. The last important task is to induce immune memory that the host can produce, so that tumors with the same surface antigens are eradicated and no longer recur, so that the host can obtain long-term therapeutic benefits.

[0061] Therefore, the present disclosure relates to a class of multikinase inhibitors that can be used in combination with a class of HDAC inhibitors, wherein the combination can meet the needs of the above mechanism, obtain excellent ORR, and produce long-term immune memory for cancer immunotherapy. Surprisingly, the compounds of formula (I) (or compounds of formula (II) to (IV), such as Cbo-23 and its derivatives) described herein have been proven to have the dual properties of multikinase inhibitors and tumor immune activation through repeated verification in animal tests, and when they are combined with certain HDAC inhibitors, they exhibit excellent cancer immunotherapy activity and produce persistent immune memory. In particular, compounds of formula (I), (II), (III) or (IV), such as Cbo-23 and its derivatives, can be further developed into novel drugs for treating tumors and are a component of a new generation of cancer immunotherapy with immunomodulatory activity in the tumor microenvironment.

[0062] Tyrosine kinase (TK) multi-target inhibitors

[0063] As described herein, compounds of Formula (I), (II), (III) and / or (IV), or pharmaceutically acceptable salts, hydrates, stereoisomers, solvates or prodrugs thereof, can act as tyrosine kinase multi-target inhibitors.

[0064] In any of the foregoing embodiments, Ar is X is N, CH or CR o ; R p is selected from the group consisting of: H, NH2 and nitro; each R m is independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl; and each R o is independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl. In one embodiment, Ar is X is N, CH or CR o ; Rp is selected from the group consisting of: H, NH2 and nitro; and each R o Independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl.

[0065] In any of the foregoing embodiments, Ar is selected from the group consisting of:

[0066]

[0067]

[0068] In one embodiment, in any of the compounds of formula (I), (II), (III) and / or (IV), L1, L2 are -NH-C(=O)-, and L3, L4 are -NH-C(=O)-. In one embodiment, in any of the compounds of formula (I), (II), (III) and / or (IV), L1, L2 are -NH-C(=O)-, and L3, L4 are -(C=O)-NH-. In one embodiment, in any of the compounds of formula (I), (II), (III) and / or (IV), L1, L2 are -(C=O)-NH-, and L3, L4 together represent -NH-C(=O)-. In one embodiment, in any of the compounds of formula (I), (II), (III) and / or (IV), L1, L2 are -(C=O)-NH-, and L3, L4 are -(C=O)-NH-.

[0069] In any of the foregoing embodiments, R p Selected from the group consisting of: H, NH2 and nitro (NO2).

[0070] In any of the preceding embodiments, when present, each R d is independently selected from the group consisting of methyl, NH2, nitro (NO2), C1-C3 alkyl, F, Cl and Br. In any of the foregoing embodiments, R p is H, n is 1, and R d In any of the foregoing embodiments, n is 1 or 2, and each R d Located at an ortho position relative to the dotted line.

[0071] The compounds of formula (I) described herein (or formula (II) to (IV), such as Cbo-23 and its derivatives) are effective multi-kinase inhibitors, and the mechanism of action of Cbo-23 and its derivatives is as kinase-specific inhibitors. The compounds of formula (I) described herein (or formula (II) to (IV), such as Cbo-23 and its derivatives) are also multi-kinase inhibitors with very effective immunomodulatory activity. For example, the results of animal studies show that Cbo-23 and its derivatives, alone or in combination with HDAC inhibitors, exhibit very good anti-cancer activity, and the combination particularly has excellent anti-cancer therapeutic benefits. The superior anti-cancer benefit of this combination is related to the anti-cancer activity of cancer immunotherapy. Without being bound by theory, the mechanism of cancer immunotherapy involves regulating the tumor microenvironment, including reducing tumor hypoxia, reducing lactic acid accumulation, activating CTLs, inhibiting the number and activity of immunosuppressive cells, etc., which, through complex interactions, will produce excellent anti-cancer benefits and produce lasting immune memory. The compounds disclosed herein, including but not limited to Cbo-23 and its derivatives, can be important components of cancer immunotherapy drug combinations and have the potential to treat cancer.

[0072] The present disclosure encompasses all stereoisomeric forms of compounds of formula (I) to (IV) or their pharmaceutically acceptable salts, hydrates, stereoisomers, solvates or prodrugs. Asymmetric centers present in compounds of formula (I) to (IV) may have (R) configuration or (S) configuration independently of one another. When the bond to a chiral carbon is depicted as a straight line in the structural formula of the present disclosure, it is understood that (R) and (S) are both configurations of chiral carbon, and therefore enantiomers and mixtures thereof are both included in the formula. When describing a specific configuration, it refers to the enantiomer (at the (R) or (S) of the center). Similarly, when the compound name is set forth in the absence of a chiral name of a chiral carbon, it is understood that (R) and (S) are both configurations of chiral carbon, and therefore individual enantiomers and mixtures thereof are both included in the name.

[0073] The present disclosure includes all possible enantiomers, regioisomers, diastereomers and mixtures of two or more stereoisomers in all ratios, such as mixtures of enantiomers and / or diastereomers. Therefore, enantiomers are a subject of the present disclosure, which are in the form of enantiomer-pure, as both left-handed and right-handed enantiomers, in the form of racemates and in the form of mixtures of two enantiomers in all ratios. In the case of cis / trans isomerism, the present disclosure includes mixtures of cis and trans and these forms in all ratios. If desired, the preparation of a single stereoisomer can be carried out by separating the mixture by conventional methods, such as by chromatography or crystallization, by using a single synthetic starting material of stereochemistry or by stereoselective synthesis. Optionally, derivatization can be carried out before separating stereoisomers. The separation of a mixture of stereoisomers can be carried out in an intermediate step of synthesizing a compound of formula (I) to (IV) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or can be carried out on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of the crystalline product or crystalline intermediate, which is derivatized, if necessary, with a reagent containing a stereocenter of known configuration. When the compounds of the present disclosure are capable of tautomerism, all individual tautomers and mixtures thereof are included within the scope of the present disclosure. The present disclosure includes all such isomers, as well as salts, solvates (including hydrates), and solvated salts of such racemates, enantiomers, diastereomers, and tautomers and mixtures thereof.

[0074] As used herein, the symbols and conventions used in these methods, schemes, and examples, whether or not specific abbreviations are explicitly defined, are consistent with the symbols and conventions used in the contemporary scientific literature (e.g., Journal of the American Chemical Society or Journal of Biological Chemistry). Specifically, but not limited to, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimole); M (micromole); Hz (hertz); MHz (megahertz); mmol (millimole); hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); and HPLC (high pressure liquid chromatography). For all of the following examples, standard processing and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in ° C (degrees Celsius). Unless otherwise stated, all reactions were carried out at room temperature. The synthetic methods described herein are intended to illustrate applicable chemistry through the use of specific examples and are not indicative of the scope of the disclosure.

[0075] The compounds of formula (I) to (IV) of the present disclosure are prepared according to general chemical synthesis procedures. An exemplary synthesis route is shown below:

[0076]

[0077] Step A involves a base and a nucleophilic aromatic substitution reaction.

[0078] Step B is the reduction to produce formula (I).

[0079]

[0080] As needed, those skilled in the art may also make other appropriate modifications to the method, for example, using appropriate protecting / deprotecting agents on groups that are sensitive to certain reaction conditions during the synthesis, isolating and purifying intermediates for subsequent reactions, selecting appropriate solvents, etc. For example, the -OH group of the starting reagent or intermediate in the route depicted above may be protected prior to the reaction (for example, compound (β) having a protecting group "-OBn" may be derived from an unprotected reagent and then used for a subsequent reaction with compound (α)), and the resulting (intermediate) product having a protecting group may be deprotected to obtain a subsequent or final product (for example, compound (γ)), ​​etc.

[0081] Pharmaceutical composition / drug combination

[0082] In another aspect, the present disclosure provides a pharmaceutical composition or pharmaceutical combination comprising a compound of any one of Formulae (I) to (IV) or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

[0083] The pharmaceutical composition or drug combination may include one or more second or additional agents. In one embodiment, the second or additional agent is an HDAC inhibitor or an immune checkpoint inhibitor. In another embodiment, the pharmaceutical composition or drug combination includes a compound described herein and an HDAC inhibitor. In another embodiment, the pharmaceutical composition or drug combination includes a compound described herein and an immune checkpoint inhibitor. In another embodiment, the pharmaceutical composition or drug combination includes a compound described herein and an HDAC inhibitor and an immune checkpoint inhibitor.

[0084] In one embodiment, HDAC inhibitors include but are not limited to chidamide, GNTbm-38 (E) -N- (2-aminophenyl) -4- (4- (pyridin-3-yl) but-3-enylamino) benzamide, entinostat, mocetinostat, vorinostat, romidepsin, belinostat, panobinostat, zabadinostat and OKI-179 (Bocodepsin).

[0085] In one embodiment, immune checkpoint inhibitors can be used in drug combinations described herein to stimulate the immune system against cancer cells and treat cancer. Immune checkpoint inhibitors are anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibodies or agents, anti-programmed cell death protein 1 (PD-1) antibodies or agents, anti-programmed death ligand 1 (PD-L1) antibodies or agents, anti-T cell immunoglobulin and mucin domain-3 (TIM-3) antibodies or agents, anti-B and T lymphocyte attenuation factor (BTLA) antibodies or agents, anti-V domain Ig-containing T cell activation inhibitor (VISTA) antibodies or agents, anti-lymphocyte activation gene-3 (LAG-3) antibodies or agents, KIR (killer cell immunoglobulin-like receptor) inhibitors or antibodies, A2AR (adenosine A2A receptor) inhibitors or antibodies, CD276 inhibitors or antibodies, or VTCN1 inhibitors or antibodies. More preferably, the immune checkpoint inhibitor is pembrolizumab, lambrolizumab, pidilizumab, nivolumab, durvalumab, avelumab or atezolizumab. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as larumumab (anti-PD-1Ab, trade name Keytruda) or pidilizumab (anti-PD-1Ab), Bavencio (anti-PD-L1 Ab, avelumab), Imfinzi (anti-PD-L1Ab, durvalumab) and Tecentriq (anti-PD-L1 Ab, atezolizumab) and fully human antibodies, such as nivolumab (anti-PD-1Ab, trade name Opdivo) and cemiplimab-rwlc (anti-PD-1Ab, trade name Libtayo). Other PD-1 inhibitors may include the presence of soluble PD-1 ligands, including but not limited to PD-L2 Fc fusion proteins, also known as B7-DC-Ig or AMP-244, and other PD-1 inhibitors currently being studied and / or developed for therapy. Additionally, immune checkpoint inhibitors may include, but are not limited to, humanized or fully human antibodies that block PD-L1, such as durvalumab and MIH1 (anti-CD274 (PD-L1, B7-H1) monoclonal antibody) and other PD-L1 inhibitors currently under investigation.

[0086] Other anticancer agents or therapies are any anticancer agents or therapies described herein or known in the art. In one embodiment, other anticancer agents or therapies are chemotherapy, or specifically dual chemotherapy based on platinum. In one embodiment, other anticancer agents are anti-VEGF or anti-VEGFR antibodies or compounds. In other embodiments, anticancer agents are platinum agents (e.g., cisplatin, carboplatin), mitotic inhibitors (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel, taxotere, docecard), fluorinated vinca alkaloids (e.g., vinflunine, javlor), vinorelbine, vinblastine, etoposide or pemetrexed gemcitabine. In one embodiment, other anticancer agents are 5-fluorouracil (5-FU). In certain embodiments, other anticancer agents are any other anticancer agents known in the art.

[0087] In order to prepare the pharmaceutical composition or drug combination of the present disclosure, one or more compounds of the present disclosure as active ingredients can be fully mixed with a pharmaceutical carrier according to conventional pharmaceutical mixing techniques, and the carrier can take a variety of forms, depending on the desired formulation form of administration, such as oral or parenteral administration, such as intramuscular administration. When preparing a composition in an oral dosage form, any conventional pharmaceutical medium can be used. Therefore, for liquid oral preparations (such as, for example, suspensions, elixirs and solutions), suitable carriers and additives include water, ethylene glycol, oil, alcohol, flavoring agents, preservatives, colorants, etc.; for solid oral preparations (such as, for example, powders, capsules, caplets, gel capsules and tablets); Suitable carriers and additives include starch, sugar, diluents, granulators, lubricants, adhesives, disintegrants, etc. Tablets and capsules represent the most advantageous oral unit dosage forms due to their ease of application, and solid pharmaceutical carriers are obviously used in the case. If desired, tablets can be sugar-coated or enteric-coated by standard techniques. For parenteral administration, the carrier generally comprises sterile water, although other ingredients may also be included, for example, to aid solubility or preservation purposes. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, etc. may be employed. The pharmaceutical compositions herein will contain a certain amount of active ingredient necessary to deliver an effective dose as described above in each dosage unit (e.g., tablet, capsule, powder, injection, teaspoon, etc.).

[0088] Liquid forms of the novel compositions of the present disclosure that can be incorporated for oral administration or injection include aqueous solutions, appropriately flavored syrups, aqueous or oily suspensions, and flavored emulsions containing edible oils (such as cottonseed oil, sesame oil, coconut oil or peanut oil) and elixirs and similar pharmaceutical vehicles. Dispersants or suspending agents suitable for aqueous suspensions include: synthetic and natural gums, such as tragacanth, acacia, alginates, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

[0089] Tablets and capsules for oral administration are usually presented in unit dosage form and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g., polyvinyl pyrrolidone and starch derivatives such as sodium glycolate starch), colorants, flavoring agents, and wetting agents (e.g., sodium lauryl sulfate).

[0090] Oral solid compositions can be prepared by conventional methods of blending, filling or tableting. The blending operation can be repeated to distribute the active ingredient in a composition containing a large amount of filler. Such operations are conventional.

[0091] For parenteral administration, a liquid unit dose containing a compound and a sterile vehicle can be prepared. The compound can be suspended or dissolved, depending on the vehicle and concentration. Parenteral solutions are usually prepared by dissolving the compound in a vehicle, sterilizing by filtration, filling a suitable vial and sealing. Advantageously, adjuvants such as local anesthetics, preservatives and buffers can also be dissolved in the vehicle. In order to increase stability, the composition can be frozen after filling the vial and removing water under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound can be suspended in a vehicle instead of dissolving, and sterilized by exposure to ethylene oxide before being suspended in a sterile vehicle. Advantageously, a surfactant or wetting agent can be included in the composition to promote the uniform distribution of the compound used.

[0092] Pharmaceutical formulations for administration by inhalation may be delivered from pressurized packs from an insufflator or nebulizer.

[0093] Therapeutic applications

[0094] In another aspect, the present disclosure provides a method for suppressing angiogenesis in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0095] In another aspect, the present disclosure provides a method for suppressing metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0096] In another aspect, the present disclosure provides a method for suppressing tumorigenesis in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0097] In another aspect, the present disclosure provides a method for suppressing immunomodulatory activity in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0098] On the other hand, the present disclosure provides a compound or pharmaceutical composition or drug combination described herein, which is used in a method for inhibiting angiogenesis, metastasis, tumorigenesis and / or immunomodulatory activity in an individual in need, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or drug combination described herein. On the other hand, the present disclosure provides a use of a compound or pharmaceutical composition or drug combination described herein, which is used to inhibit angiogenesis, metastasis, tumorigenesis and / or immunomodulatory activity in an individual in need. On the other hand, the present disclosure provides a use of a compound or pharmaceutical composition or drug combination described herein, which is used to prepare a medicament for inhibiting angiogenesis, metastasis, tumorigenesis and / or immunomodulatory activity in an individual in need.

[0099] In another aspect, the present disclosure provides a method for treating a disease or condition associated with tyrosine kinase in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0100] In another aspect, the present disclosure provides a method for treating a tumor or cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or pharmaceutical combination described herein.

[0101] In another aspect, the present disclosure provides a compound, pharmaceutical composition, or drug combination as described herein, for use in a method for treating a disease or condition associated with tyrosine kinases or treating a tumor or cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound, pharmaceutical composition, or drug combination as described herein. In another aspect, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, for treating a disease or condition associated with tyrosine kinases, or treating a tumor or cancer in an individual in need thereof. In another aspect, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, for preparing a medicament for treating a disease or condition associated with tyrosine kinases, or treating a tumor or cancer in an individual in need thereof.

[0102] On the other hand, the present disclosure provides a cancer immunotherapy for treating a tumor or cancer in an individual in need thereof, comprising administering to the individual an effective amount of a compound, pharmaceutical composition or drug combination described herein, for example, by modulating the tumor microenvironment, including reducing tumor hypoxia, reducing lactate accumulation, activating CTLs, inhibiting the number and activity of immunosuppressive cells, to ultimately achieve excellent anti-cancer benefits and / or generate persistent immune memory.

[0103] In another aspect, the present disclosure provides a compound, pharmaceutical composition, or drug combination as described herein, which is used in a cancer immunotherapy for treating a tumor or cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of a compound, pharmaceutical composition, or drug combination as described herein. In another aspect, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, which is used in a cancer immunotherapy for treating a tumor or cancer in an individual in need thereof. In another aspect, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, which is used in the preparation of a medicament for treating a tumor or cancer in an individual in need thereof.

[0104] In another aspect, the present disclosure provides a method for treating a tumor or cancer in an individual in need thereof, the method having an enhanced objective response rate (ORR) compared to tumor or cancer treatment based on a pharmaceutical composition or drug combination comprising a conventional TK inhibitor, the method comprising administering to the individual an effective amount of a compound or pharmaceutical composition or drug combination described herein.

[0105] On the other hand, the present disclosure provides a compound, pharmaceutical composition, or drug combination as described herein, which is used in a method for treating a tumor or cancer in an individual in need thereof, and has an enhanced objective response rate (ORR) compared to a tumor or cancer treatment based on a pharmaceutical composition or drug combination comprising a conventional TK inhibitor, the method comprising administering to the individual an effective amount of a compound, pharmaceutical composition, or drug combination as described herein. On the other hand, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, which is used to treat a tumor or cancer in an individual in need thereof, and has an enhanced objective response rate (ORR) compared to a tumor or cancer treatment based on a pharmaceutical composition or drug combination comprising a conventional TK inhibitor. On the other hand, the present disclosure provides a use of a compound, pharmaceutical composition, or drug combination as described herein, which is used to prepare a drug for treating a tumor or cancer in an individual in need thereof, and has an enhanced objective response rate (ORR) compared to a tumor or cancer treatment based on a pharmaceutical composition or drug combination comprising a conventional TK inhibitor.

[0106] Examples of cancers that may be treated in accordance with the present disclosure include, but are not limited to, invasive breast cancer, adenocarcinoma, lung cancer (non-small cell, squamous cell carcinoma, adenocarcinoma, and large cell lung cancer), liver cancer, colorectal cancer, brain cancer, head and neck cancer (e.g., neuro / glioblastoma), breast cancer, ovarian cancer, bladder transitional cell carcinoma, prostate cancer, oral squamous cell carcinoma, osteosarcoma, adrenocortical carcinoma, gastrointestinal tumors (including colorectal cancer), biliary tract cancer such as gallbladder cancer (GBC), bladder cancer, esophageal cancer, gastric cancer, cervical cancer, salivary gland cancer, diarrheal benign tumors, ductal carcinoma in situ, paronychia, bile duct cancer, kidney cancer, pancreatic cancer, medulloblastoma, glioblastoma, luminal tumor, HER2 positive and triple negative breast tumors, hematological malignancies and leukemias (acute myeloid leukemia (AML), B-precursor acute lymphoblastic leukemia (ALL), a portion of T-cell ALL, and chronic myeloid leukemia (CML)).

[0107] The compound or its pharmaceutically acceptable salt is administered orally, nasally, transdermally, pulmonary, inhaled, buccal, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally or parenterally. In one embodiment, the compound is administered orally. Those skilled in the art will recognize the advantages of certain administration routes.

[0108] The dosage regimen using the compound is selected based on a variety of factors, including the type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the patient's renal or liver function; and the specific compound or salt thereof employed. A generally skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progression of the patient.

[0109] Having now described the disclosure by written description, those skilled in the art will recognize that the disclosure can be practiced in various embodiments and that the foregoing description and following examples are for illustrative purposes only and are not limiting of the claims that follow.

[0110] Examples

[0111] Materials and methods for preparing exemplary compounds of the present disclosure are described below.

[0112] Unless otherwise stated, all commercial chemicals and solvents were of reagent grade and used without further purification. 254 All reactions were monitored for completion by thin layer chromatography on silica gel glass backing (20 x 20 cm). The resulting chromatograms were observed under UV radiation (254 nm). The chromatograms were recorded on a Bruker AVANCE III HD 600 MHz spectrometer. 1H NMR spectra, and chemical shifts are recorded in parts per million (ppm, δ). Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), quin (quintet), sex (sextet), and m (multiplet). Coupling constants (J) are expressed in Hertz. Electrospray mass spectrometry (ESMS) is recorded as m / z values ​​using a Waters mass spectrometer. The purity of the final compound is determined using a Waters ACQUITY Arc system using a C18 column (Waters XSelect HSS T3 5μm, 4.6mm×250mm) operated at 40°C. Water containing 0.1% trifluoroacetic acid is used as mobile phase A and methanol as mobile phase B for elution. Elution conditions: at 0 minutes, phase A 90% + phase B 10%; at 6 minutes, phase A 70% + phase B 30%; at 12 minutes, phase A 50% + phase B 50%; at 18 minutes, phase A 10% + phase B 90%; at 23 minutes, phase A 90% + phase B 10%. The flow rate of the mobile phase was 1 ml / min, the injection volume of the sample was 20 μL, and the run time was 30 minutes. Peaks were detected at 210-400 nm. The purity of the final compound was found to be >95%.

[0113] Preparation Example

[0114] Example 1Cbo-02

[0115] Scheme 1. Synthesis scheme for preparing Cbo-02

[0116]

[0117] 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluoroaniline (1)

[0118] A solution of 4-chloro-6,7-dimethoxyquinoline (1 eq.), 4-amino-2-fluorophenol (1.2-2 eq.) and NaH (1.2-2.2 eq.) in DMF was stirred at 100 °C for 10-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 1 as a beige solid (yield 38%).

[0119] Tert-butyl (1-((4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)carbamoyl)cyclopropyl)carbamate (2)

[0120] To a solution of compound 1 (1 eq.), 1-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-4 eq.). The mixture was stirred at room temperature for 1-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 2 (yield 97%) as a yellow solid.

[0121] 1-Amino-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)cyclopropane-1-carboxamide (3)

[0122] To a solution of compound 2 (1 eq.) in DCM was added TFA (1 eq.). The mixture was stirred at room temperature for 2-8 h. The mixture was diluted with DCM and washed with saturated NaHCO 3(水溶液) Neutralized to pH = 7. The organic layer was washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness to give Compound 3 as a yellow solid (yield 86%).

[0123] N-(1-((4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)carbamoyl)cyclopropyl)-4-fluorobenzamide (Cbo-02)

[0124] To a solution of compound 3 (1 eq.), 4-fluorobenzoic acid (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (1-3 eq.). The mixture was stirred at room temperature for 2-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give Cbo-02 as a white solid (yield 78%).

[0125] 1H NMR(600MHz,DMSO-d6)δ9.92(s,1H),9.09(s,1H),8.46(d,J=5.2Hz,1H),8.0 4(dd,J=8.6,5.6Hz,2H),7.87(dd,J=13.3,2.3Hz,1H),7.56(d,J=7.5Hz,1H) ,7.52(s,1H),7.41(t,J=8.0Hz,2H),7.33(t,J=8.8Hz,2H),6.44(d,J=5.2Hz ,1H),3.95(s,3H),3.94(s,3H),1.51(q,J=3.9Hz,2H),1.15(q,J=4.0Hz,2H). LCMS (ESI) m / z 520.4 [M+H] + . HPLC purity: 98.88%.

[0126] Example 2Cbo-03

[0127] Scheme 2. Synthesis scheme for preparing Cbo-03

[0128]

[0129] 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorobenzoic acid (4)

[0130] A solution of 4-chloro-6,7-dimethoxyquinoline (1 equivalent), methyl 3-fluoro-4-hydroxybenzoate (1-1.5 equivalents) and Cs2CO3 (2-3 equivalents) in DMF was stirred at 130-160°C for 8-20 hours. The mixture was diluted with EA and neutralized with 2N HCl to a pH of 6. The mixture was washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain compound 4 (yield 13%) as a beige solid.

[0131] 4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluoro-N-(1-((4-fluorophenyl)carbamoyl)cyclopropyl)benzamide (Cbo-03)

[0132] To a solution of compound 4 (1 eq.), 1-amino-N-(4-fluorophenyl)cyclopropane-1-carboxamide (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 1-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give Cbo-03 as a white solid (yield 44%).

[0133] 1 H NMR (600MHz, DMSO-d6) δ9.71(s,1H),9.19(s,1H),8.52(d,J=5.2Hz,1H),8.08(dd,J=11.6,1.8Hz,1H),7.93(dd,J=8.4,1.5Hz,1H),7.63-7.55(m,2 H),7.52(s,1H),7.44(s,1H),7.14(t,J=8.9Hz,2H),6.49(d,J=5.1Hz,1H ), 3.96 (s, 3H), 3.95 (s, 3H), 1.49 (q, J = 3.9Hz, 2H), 1.13 (q, J = 3.9Hz, 2H). LCMS(ESI)m / z 520.4[M+H] + . HPLC purity: 98.98%.

[0134] Scheme 3. Synthetic scheme for preparing starting material 5

[0135]

[0136] 2-Methoxy-4-nitrophenyl acetate

[0137] To a solution of 2-methoxy-4-nitrophenol (1 eq.) in DCM was added pyridine (1-1.5 eq.) and AcO (1-1.5 eq.). The mixture was stirred at room temperature for 2-5 hours. The mixture was washed with water, brine, and dried over NaSO. The mixture was filtered and concentrated to dryness to give 2-methoxy-4-nitrophenyl acetate (12.3 g, 98%) as a yellow solid.

[0138] 4-Amino-2-methoxyphenyl acetate

[0139] To a solution of 2-methoxy-4-nitrophenyl acetate (1 eq.) in MeOH was added Pd / C (3-5% w / w), and the mixture was stirred under a hydrogen atmosphere at room temperature for 8-20 hours. The mixture was filtered through a pad of celite, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography to give 4-amino-2-methoxyphenyl acetate as a yellow oil (yield 62%).

[0140] 5-(Ethoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione

[0141] A solution of 2,2-dimethyl-1,3-dioxane-4,6-dione in triethyl orthoformate was stirred at 80° C. for 2-5 hours. The mixture was concentrated to dryness to give 5-(ethoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione as a white solid (yield 91%).

[0142] 4-(((2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-ylidene)methyl)amino)-2-methoxyphenyl acetate

[0143] A solution of 4-amino-2-methoxyphenyl acetate (1 eq) and 5-(ethoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (1 eq) in EtOH was stirred at 100° C. for 1-4 h. The resulting solid was collected by filtration to give 4-(((2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-ylidene)methyl)amino)-2-methoxyphenyl acetate as a yellow solid (yield 88%).

[0144] 7-Methoxy-4-oxo-1,4-dihydroquinolin-6-yl acetate

[0145] A solution of 4-(((2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-ylidene)methyl)amino)-2-methoxyphenyl acetate in diphenyl ether was stirred at 160-200° C. for 1-4 hours. The resulting solid was collected by filtration and washed with hexanes to give 7-methoxy-4-oxo-1,4-dihydroquinolin-6-yl acetate as a brown solid (yield 81%).

[0146] 4-Chloro-7-methoxyquinolin-6-yl acetate

[0147] To a solution of 7-methoxy-4-oxo-1,4-dihydroquinolin-6-yl acetate (1 eq.) in CHCl3 was added POCl3 (2-6 eq.) and the mixture was stirred at 60-80°C for 1-2 h. The mixture was concentrated to dryness. The residue was diluted with EA and washed with saturated NaHCO 3(水溶液) Neutralized to pH 7. The organic layer was washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give 4-chloro-7-methoxyquinolin-6-yl acetate as a yellow solid (yield 60%).

[0148] 4-Chloro-7-methoxyquinolin-6-ol

[0149] To a solution of 4-chloro-7-methoxyquinolin-6-yl acetate (1 eq.) in EtOH was added NaOH (1-1.2 eq.), and the mixture was stirred at 0° C. for 6-16 h. And the mixture was acidified with 1N HCl to pH 7. The resulting solid was collected by filtration to give 4-chloro-7-methoxyquinolin-6-ol as a white solid (yield 88%).

[0150] 6-(Benzyloxy)-4-chloro-7-methoxyquinoline (5)

[0151] To a solution of 4-chloro-7-methoxyquinoline-6-ol (1 eq.) in DMF was added KCO (2-3 eq.) and benzyl bromide (1-1.5 eq.). The mixture was stirred at room temperature for 6-16 hours. Water was added to the mixture and the resulting solid was collected by filtration to give compound 5 as a white solid (yield 81%).

[0152] Example 3Cbo-07

[0153] Scheme 4. Synthesis scheme for preparing Cbo-07

[0154]

[0155] 4-((6-(Benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluoroaniline (6)

[0156] A solution of compound 5 (1 eq.), 4-amino-2-fluorophenol (2-4 eq.) and NaH (2-4 eq.) in DMF was stirred at 100-130 °C for 8-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 6 as a beige solid (yield 66%).

[0157] Tert-butyl (1-((4-((6-(Benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)carbamoyl)cyclopropyl)carbamate (7)

[0158] To a solution of compound 6, 1-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid (1 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (1-3 eq.). The mixture was stirred at room temperature for 2-8 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 7 (yield 89%) as a yellow solid.

[0159] 1-Amino-N-(4-((6-(benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)cyclopropane-1-carboxamide (8)

[0160] To a solution of compound 7 (1 eq.) in DCM was added TFA (3-10 eq.). The mixture was stirred at room temperature for 2-8 h. The mixture was diluted with DCM and washed with saturated NaHCO 3(水溶液) Neutralized to pH 7. The organic layer was washed with water, brine, and dried over Na 2 SO 4 . The mixture was filtered and concentrated to dryness to give Compound 8 (yield 99%) as a yellow solid.

[0161] N-(1-((4-((6-(Benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)carbamoyl)cyclopropyl)-4-fluorobenzamide (9)

[0162] To a solution of compound 8 (1 eq.), 4-fluorobenzoic acid (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 2-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 9 (yield 83%) as a white solid.

[0163] 4-Fluoro-N-(1-((3-fluoro-4-((6-hydroxy-7-methoxyquinolin-4-yl)oxy)phenyl)carbamoyl)cyclopropyl)benzamide (Cbo-07)

[0164] A solution of compound 9 (1 eq.), 1,4-cyclohexadiene (5-10 eq.) and Pd / C (20% w / w) in EtOH was stirred at 50-100 °C for 1-4 hours. The mixture was filtered through a pad of celite, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography to afford Cbo-07 as a white solid (yield 95%).

[0165] 1H NMR (600MHz, DMSO-d6) δ10.06(s,1H),9.91(s,1H),9.10(s,1H),8.39(d,J=5.2Hz,1H),8.03(dd,J=8.5,5.7Hz,2H),7.85(dd,J=13.3,2.1Hz, 1H), 7.54 (d, J = 9.4Hz, 1H), 7.47 (s, 1H), 7.39-7.30 (m, 4H), 6.39 (d, J = 5.2Hz, 1H), 3.94 (s, 3H), 1.50 (q, J = 3.8Hz, 2H), 1.14 (d, J = 3.9Hz, 2H). LCMS(ESI)m / z 506.2[M+H] + . HPLC purity: 98.02%.

[0166] Examples 4Cbo-05, 06, 08-10 and 14 and 10a-10f

[0167] Scheme 5. Synthetic scheme for preparing Cbo-05, 06, 08-10 and 14

[0168]

[0169] General procedure for the synthesis of 10a-f

[0170] To a solution of Cbo-07 (1 eq.) and CsCO (2-3 eq.) in DMF, p-fluoronitrobenzene or m-fluoronitrobenzene (1-1.5 eq.) was added. The mixture was stirred at room temperature for 1-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over NaSO. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain the desired product.

[0171]

[0172] General procedure for the synthesis of Cbo-05, 06, 08, 09, 10, and 14

[0173] Nitro adduct (1 eq.), Fe (3-6 eq.) in EtOH / saturated NH4Cl (水溶液) The mixture was stirred at 50-100°C for 1-4 hours. The mixture was filtered through a celite pad. The filtrate was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain the desired product.

[0174]

[0175] Yield: 65 mg, 43%.1 H NMR (600MHz, DMSO-d6) δ9.86 (s, 1H), 9.03 (s, 1H), 8.47 (d, J = 5.2Hz, 1H), 8.03 (d, J=5.8Hz,1H),7.82(d,J=13.1Hz,1H),7.51(d,J=8.6Hz,1H),7.50(s,1H),7.35-7. 28(m,3H),7.24(s,1H),6.86(d,J=8.5Hz,2H),6.62(d,J=8.5Hz,2H),6.40(d,J=5 .2Hz,1H),5.03(s,2H),4.00(s,3H),1.50(q,J=3.7Hz,2H),1.14(q,J=3.7Hz,2H). LCMS (ESI) m / z 597.3 [M+H] + . HPLC purity: 95.69%.

[0176]

[0177] Yield: 138 mg, 73%. 1 H NMR (600MHz, DMSO-d6) δ9.88(s,1H),9.04(s,1H),8.54(d,J=5.2Hz,1H),8.04(d,J=5.8Hz,1H),8.02(d ,J=5.7Hz,1H),7.84(dd,J=13.1,1.7Hz,1H),7.63(s,1H),7.53(d,J=12.4Hz,1H),7.52(s,1H)7.47(d, J=2.7Hz,1H),7.37(t,J=9.1Hz,1H),7.33(t,J=8.8Hz,2H),7.10(dd,J=8.6,2.8Hz,1H),6.85(d,J=8.6 Hz, 1H), 6.45 (d, J = 5.2Hz, 1H), 5.05 (s, 2H), 3.90 (s, 3H), 1.50 (q, J = 3.8Hz, 2H), 1.14 (q, J = 3.8Hz, 2H). LCMS(ESI)m / z 598.2[M+H] + . HPLC purity: 99.16%.

[0178]

[0179] Yield: 86 mg, 53%. 1H NMR(600MHz,DMSO-d6)δ9.91(s,1H),9.09(s,1H),8.53(d,J=5.2Hz,1H),8.03(dd,J=8.7 ,5.6Hz,2H),7.84(dd,J=13.3,2.3Hz,1H),7.56(d,J=3.4Hz,2H),7.53(d,J=8.9Hz,1H), 7.38-7.29(m,3H),7.01(t,J=7.9Hz,1H),6.44(d,J=5.2Hz,1H),6.33(d,J=7.2Hz,1H),6 .21-6.17(m,2H),5.23(s,2H),3.97(s,3H),1.50(q,J=3.9Hz,2H),1.14(q,J=4.0Hz,2H). LCMS(ESI)m / z 597.3[M+H] + . HPLC purity: 97.69%.

[0180]

[0181] Yield: 90 mg, 47%. 1 H NMR(600MHz,DMSO-d6)δ9.87(s,1H),9.06(s,1H),8.46(d,J=5.2Hz,1H),8.03(dd,J=8.9,5 .6Hz,2H),7.81(dd,J=13.3,2.4Hz,1H),7.53-7.49(m,2H),7.35-7.28(m,3H),7.05(s,1H) ,6.78(d,J=8.5Hz,1H),6.52(d,J=2.6Hz,1H),6.46(dd,J=8.5,2.7Hz,1H),6.39(d,J=5.1H z, 1H), 4.97 (s, 2H), 4.02 (s, 3H), 1.97 (s, 3H), 1.49 (q, J = 3.9Hz, 2H), 1.13 (q, J = 4.0Hz, 2H). LCMS(ESI)m / z 611.3[M+H] + . HPLC purity: 97.80%.

[0182]

[0183] Yield: 135 mg, 64%. 1H NMR (600MHz, DMSO-d6) δ9.90 (s, 1H), 9.09 (s, 1H), 8.49 (d, J = 5.2Hz, 1H), 8.06-7.99 (m,2H),7.82(dd,J=13.3,2.4Hz,1H),7.52(s,1H),7.51(d,J=1.4Hz,1H),7.32(t,J= 8.8Hz,3H),7.17(s,1H),7.01(t,J=9.1Hz,1H),6.52(dd,J=13.2,2.6Hz,1H),6.44- 6.40(m,2H),5.39(s,2H),4.02(s,3H),1.49(q,J=4.0Hz,2H),1.13(q,J=4.0Hz,2H). LCMS(ESI)m / z 615.3[M+H] + . HPLC purity: 98.10%.

[0184]

[0185] Yield: 226 mg, 91%. 1 H NMR(600MHz,DMSO-d6)δ9.90(s,1H),9.10(s,1H),8.51(d,J=5.2Hz,1H),8.05- 8.00(m,2H),7.83(dd,J=13.3,2.4Hz,1H),7.54(s,1H),7.52(dd,J=9.0,1.4Hz, 1H),7.33(q,J=9.0Hz,3H),7.18(s,1H),6.44(d,J=5.0Hz,1H),6.39(d,J=10.6 Hz, 2H), 5.76 (s, 2H), 4.04 (s, 3H), 1.50 (q, J = 4.0Hz, 2H), 1.14 (q, J = 4.0Hz, 2H). LCMS (ESI) m / z 633.3 [M+H] + . HPLC purity: 99.34%.

[0186] Example 5Cbo-20

[0187] Scheme 6. Synthetic scheme for preparing Cbo-20

[0188]

[0189] 1-((4-Fluorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (11)

[0190] To a solution of cyclopropane-1,1-dicarboxylic acid (1 eq.) in THF was added triethylamine (1-1.5 eq.). The solution was stirred at 0°C for 15 min, then SOCl2 (1-1.5 eq.) was added via syringe. After stirring for another 15 min, a solution of 4-fluoroaniline (1-1.5 eq.) in THF was added at 0°C, and the solution was stirred at room temperature for 8-20 h. The reaction mixture was washed with NaOH. (水溶液) Quenched and diluted with EA. The phases were separated and the organic phase was washed with brine NaOH. (水溶液) The combined alkaline extracts were then acidified to pH 2 with HCl and filtered by suction to afford the title compound as a white solid (yield 13%).

[0191] 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluoroaniline (12)

[0192] A solution of 4-chloro-6,7-dimethoxyquinoline (1 eq.), 4-amino-2-fluorophenol (1.5-4 eq.) and NaH (1.5-4 eq.) in DMF was stirred at 70-120 °C for 8-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 12 as a beige solid (yield 41%).

[0193] N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (Cbo-20)

[0194] To a solution of compound 11 (1 eq.), compound 12 (0.9-1.5 eq.) and HATU (0.9-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 8-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain compound Cbo-20 (yield 51%) as a white solid.

[0195] 1H NMR (600MHz, DMSO-d6) δ10.42(s,1H),10.01(s,1H),8.53(d,J=5.2Hz,1H),7.92(d,J=13.0Hz,1H),7.65(dd,J=8.3,5.1Hz,2H),7.56( s,1H),7.54(d,J=8.9Hz,1H),7.44(t,J=8.9Hz,2H),7.15(t,J=8.8Hz,2H),6.50(d,J=4.5Hz,1H),3.96(s,6H),1.48(d,J=6.4Hz,4H). LCMS(ESI)m / z 520.73[M+H]+. HPLC purity: 99.65%.

[0196] Example 6Cbo-22

[0197] Scheme 7. Synthetic scheme for preparing Cbo-22

[0198]

[0199] Tert-butyl (2-(4-chloropyridine-2-carboxamido)propyl)carbamate (13)

[0200] To a solution of 4-chloropyridine-2-carboxylic acid (1-1.5 equiv.), tert-butyl (2-aminopropyl)carbamate (1 equiv.) and HATU (1-1.5 equiv.) in DMF was added DIPEA (2-5 equiv.). The mixture was stirred at room temperature for 2-8 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 13 as a white solid (yield 75%).

[0201] Tert-butyl (2-(4-(4-amino-2-fluorophenoxy)pyridine-2-carboxamido)propyl)carbamate (14)

[0202] A solution of tert-butyl (2-(4-chloropyridine-2-carboxamido)propyl)carbamate (1 eq.), 4-amino-2-fluorophenol (1-2 eq.) and CsCO (2-3 eq.) in DMF was stirred at 70-120° C. for 2-8 h. The mixture was diluted with EA, washed with water, brine, and dried over NaSO. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 14 as a beige solid (yield 65%).

[0203] Tert-butyl (2-(4-(2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxamido)phenoxy)pyridine-2-carboxamido)propyl)carbamate (15)

[0204] To a solution of compound 11 (1-1.5 eq.), compound 3 (1 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (1.5-5 eq.). The mixture was stirred at room temperature for 8-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 15 (yield 76%) as a white solid.

[0205] N-(4-((2-((1-aminopropan-2-yl)carbamoyl)pyridin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (Cbo-22)

[0206] To a solution of compound 4 (1 eq.) in DCM was added TFA (5-10 eq.). The mixture was stirred at room temperature for 2-8 hours. The mixture was diluted with EA and neutralized to pH=8 with saturated NaHCO3 (aq.). The mixture was washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain Cbo-22 (yield 54%).

[0207] 1 H NMR (600MHz, DMSO-d6) δ10.38(brs,1H),10.03(brs,1H),8.53(t,J=6.5Hz,2H),7.90(d,J=13. 1Hz,1H),7.64(dd,J=8.7,5.1Hz,2H),7.50(d,J=8.7Hz,1H),7.38(t,J=9.0Hz,1H),7.35(d,J=2 .3Hz,1H),7.23(dd,J=5.5,2.5Hz,1H),7.15(t,J=8.8Hz,2H),3.90-3.83(m,1H),2.67(dd,J=12 .6,5.9Hz,1H),2.60(dd,J=12.6,5.5Hz,1H),1.60(brs,2H)1.46(s,4H),1.11(d,J=6.6Hz,3H). LCMS(ESI)m / z 510.69[M+H]+. HPLC purity: 99.82%.

[0208] Example 7Cbo-23

[0209] Scheme 8. Synthetic scheme for preparing Cbo-23

[0210]

[0211] 4-((6-(Benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluoroaniline (16)

[0212] To a solution of 6-(benzyloxy)-4-chloro-7-methoxyquinoline (1 eq.), 4-amino-2-fluorophenol (2-5 eq.) in DMF was added NaH (2-5 eq.), and the mixture was stirred at 70-120° C. for 16-30 h. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 16 as a beige solid (yield 27%).

[0213] N-(4-((6-(Benzyloxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (17)

[0214] To a solution of compound 16 (1 eq.), 1-((4-fluorophenyl)carbamoyl)-cyclopropane-1-carboxylic acid (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 10 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 17 (yield 82%) as a white solid.

[0215] N-(3-Fluoro-4-((6-hydroxy-7-methoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (18)

[0216] Pd / C (10-40% w / w) was added to a solution of compound 17 (1 eq.), 1,4-cyclohexadiene (5-10 eq.) in EtOH (30 mL), and the mixture was stirred at 50-100 °C for 1-6 hours. The mixture was filtered through a pad of celite, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography to give 18 as a white solid (yield 99%).

[0217] N-(3-Fluoro-4-((6-(2-fluoro-4-nitrophenoxy)-7-methoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (19)

[0218] 1,2-difluoro-4-nitrobenzene (1-1.5 eq.) was added to a solution of compound 18 (1 eq.) and Cs2CO3 (2-3 eq.) in DMF. The mixture was stirred at room temperature for 1-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain compound 19 (yield 75%) as a pink solid.

[0219] N-(4-((6-(4-amino-2-fluorophenoxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (Cbo-23)

[0220] To a solution of compound 19 (1 eq.), Fe (3-6 eq.) in EtOH was added saturated NH4Cl (水溶液) The mixture was stirred at 50-100 °C for 1-6 hours. The mixture was filtered through a pad of celite. The filtrate was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give Cbo-23 as a pink solid (yield 83%).

[0221] 1 H NMR (600MHz, DMSO-d6) δ10.35(s,1H),10.00(s,1H),8.50(d,J=5.2Hz,1H),7.85(d,J=13.0H z,1H),7.63(dd,J=8.8,5.1Hz,2H),7.53(s,1H),7.47(d,J=8.6Hz,1H),7.35(t,J=9.0Hz,1H) ,7.19(s,1H),7.15(t,J=8.9Hz,2H),7.02(t,J=9.0Hz,1H),6.53(dd,J=13.2,2.3Hz,1H),6.4 3(dd,J=8.7,2.1Hz,1H),6.39(d,J=5.2Hz,1H),5.40(s,2H),4.02(s,3H),1.49-1.44(m,4H). LCMS(ESI)m / z 615.74[M+H] + . HPLC purity: 98.41%. Detailed NMR data of Cbo-23 can be found at Figure 1 and Table 1.

[0222] Example 8Cbo-24

[0223] Scheme 9. Synthetic scheme for preparing Cbo-24

[0224]

[0225] 4-(2-Fluoro-4-nitrophenoxy)-7-methoxyquinoline-6-carboxylic acid methyl ester (20)

[0226] A solution of methyl 4-chloro-7-methoxyquinoline-6-carboxylate (1 equivalent) and 2-fluoro-4-nitrophenol (1.5-3 equivalents) in chlorobenzene was stirred at 120-200° C. for 8-16 hours. The mixture was diluted with EA and washed with 10% NaOH. (水溶液) , water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 20 as a yellow solid (yield 95%).

[0227] 4-(4-Amino-2-fluorophenoxy)-7-methoxyquinoline-6-carboxylic acid methyl ester (21)

[0228] To a solution of compound 20 (1 eq.) in MeOH was added Pd(OH)2 (40% w / w). The mixture was stirred at room temperature under H2 for 8-20 h. The mixture was filtered through a pad of celite and the filtrate was concentrated to dryness to afford compound 21 as a yellow solid (yield 68%).

[0229] 4-(2-Fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxamido)phenoxy)-7-methoxyquinoline-6-carboxylic acid methyl ester (22)

[0230] To a solution of compound 21 (1 eq.), 1-((4-fluorophenyl)carbamoyl)-cyclopropane-1-carboxylic acid (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 8-20 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give compound 22 as a white solid (yield 99%).

[0231] 4-(2-Fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxamido)phenoxy)-7-methoxyquinoline-6-carboxylic acid (23)

[0232] To a solution of 22 (1 eq) in THF was added LiOH (2-5 eq) and the mixture was stirred at room temperature for 1-4 h. The mixture was concentrated to remove THF. The aqueous solution was washed with 1 N HCl (水溶液) Acidified to pH 3. The resulting solid was filtered to give Compound 23 as a white solid (yield 84%).

[0233] Tert-butyl (2-(4-(2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxamido)phenoxy)-7-methoxyquinoline-6-carboxamido)propyl)carbamate (24)

[0234] To a solution of compound 23 (1 eq.), tert-butyl (2-aminopropyl)carbamate (1-1.5 eq.) and HATU (1-1.5 eq.) in DMF was added DIPEA (2-3 eq.). The mixture was stirred at room temperature for 1-4 hours. The mixture was diluted with EA, washed with water, brine, and dried over Na2SO4. The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to obtain compound 24 (yield 98%) as a yellow solid.

[0235] o-Phenyl-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide(Cbo-24)

[0236] To a solution of compound 24 (1 eq.) in DCM was added TFA (5-10 eq.). The mixture was stirred at room temperature for 8-20 h. The mixture was diluted with EA and washed with saturated NaHCO 3(水溶液) Neutralized to pH = 8. The resulting solid was filtered to give Cbo-24 as a yellow solid (yield 68%).

[0237] 1 H NMR (600MHz, DMSO-d6) δ10.42(s,1H),9.99(s,1H),8.67(d,J=5.2Hz,1H),8.60(s,1H),8.32( d,J=8.1Hz,1H),7.92(d,J=12.9Hz,1H),7.64(dd,J=8.9,5.1Hz,2H),7.55-7.52(m,2H),7.44 (t,J=9.0Hz,1H),7.16(t,J=8.9Hz,2H),6.88(brs,2H),6.49(d,J=5.2Hz,1H),4.22(dt,J=13 .9, 6.8Hz, 1H), 4.02 (s, 3H), 2.90 (d, J = 6.5Hz, 2H), 1.50-1.44 (m, 4H), 1.22 (d, J = 6.7Hz, 3H). LCMS(ESI)m / z 590.33[M+H] + . HPLC purity: 99.67%.

[0238] Example 9Cbo-25

[0239] Scheme 10. Synthesis scheme for preparing Cbo-25

[0240]

[0241] N-(4-((6-(2-chloro-4-nitrophenoxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (25)

[0242] To a solution of compound 18 (1 eq.) and Cs2CO3 (2-5 eq.) in DMF was added 2-chloro-1-fluoro-4-nitrobenzene (1-2 eq.). The mixture was stirred at room temperature for 1-4 hours. Water and EA were added to the mixture. The resulting solid was filtered to give compound 25 as a white solid (yield 82%).

[0243] N-(4-((6-(4-amino-2-chlorophenoxy)-7-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (Cbo-25)

[0244] To a solution of compound 25 (1 eq.) and Fe (2-5 eq.) in EtOH was added saturated NH4Cl (水溶液) . The mixture was stirred at 70-100° C. for 1-4 hours. The mixture was filtered through a pad of celite. The filtrate was diluted with EA, washed with water, and then washed with brine, and H 2 O was removed with Na 2 SO 4 . The mixture was filtered and concentrated to dryness. The crude product was purified by column chromatography to give Cbo-25 as a beige solid (yield 88%).

[0245] 1 H NMR (600MHz, DMSO-d6) δ10.35(s,1H),9.99(s,1H),8.50(d,J=5.2Hz,1H),7.85(dd,J=13 .4,1.8Hz,1H),7.65-7.61(m,2H),7.53(s,1H),7.47(d,J=8.9Hz,1H),7.35(t,J=9.0Hz,1 H),7.17-7.12(m,2H),7.10(s,1H),7.01(d,J=8.6Hz,1H),6.76(d,J=2.6Hz,1H),6.59(d d,J=8.6,2.6Hz,1H),6.38(d,J=5.2Hz,1H),5.38(s,2H),4.03(s,3H),1.48-1.43(m,4H). LCMS(ESI)m / z 631.62[M+H] + . HPLC purity: 99.26%.

[0246] Efficacy Test Examples

[0247] Example 10 In vitro cytotoxicity assay

[0248] Six different cell lines were used, including the human breast cancer cell line MDA-MB-231 (6 × 10 3 ) and MDA-MB-453 (2.4×10 4 ), human colorectal cancer cell line SW48 (2.4×10 4 ), human mammary epithelial cell line M10 (6×10 3 ), human lung cancer cell line A549 (1.4×10 3 ) and mouse colorectal cancer CT26 (6×10 3 The cell lines were obtained from the BCRC Bioresource Collection and Research Center. The compound series and cabozantinib and zanzalintinib ( All cell lines were treated with compounds of catalog number HY-13016 and catalog number HY-138696) at doses ranging from 25, 12.5, 6.25, 3.125 and 1.56 μM and then incubated at 37° C. under 5% CO 2 for 72 hours. After 72 hours, the MTT assay (Cayman TM ) to determine cell viability. MDA-MB-231, MDA-MB-453, and SW48 cell lines were maintained in culture medium supplemented with 10% FBS, 0.2% antibiotics (MycoZap TM , Plus-CL). The M10 cell line was maintained in DMEM / F12 supplemented with 10% FBS, 0.2% antibiotics (MycoZap TM , Plus-CL) of MEMα (Gibco TM CT26 cell line was maintained in medium supplemented with 10% FBS, 0.2% antibiotics (MycoZap TM ,Pluse-CL) RPMI 1640(CORNING TM A549 cell line was maintained in medium supplemented with 10% FBS, 0.2% antibiotics (MycoZap TM , Plus-CL) in DMEM (CORNING TM )middle.

[0249] Example 11 Screening method of 60 human cancer cell lines

[0250] The human tumor cell lines of the cancer screening panel are grown in the RPMI1640 culture medium containing 5% fetal bovine serum and 2mM L-glutamine. For typical screening experiments, cells are inoculated into 96-well microtiter plates in 100 μL with an inoculation density ranging from 5,000 to 40,000 cells / well, depending on the doubling time of the individual cell lines. After cell inoculation, before adding the experimental drug, the microtiter plate is incubated for 24 hours at 37°C, 5% CO2, 95% air and 100% relative humidity. After 24 hours, two plates of each cell line are fixed in situ with TCA to represent the measured value (Tz) of the cell colony of each cell line when the drug is added. The experimental drug is dissolved in dimethyl sulfoxide, and the dissolved concentration is 400 times of the expected final maximum test concentration, and is frozen and stored before use. When the drug is added, the aliquot of the frozen concentrate is thawed, and diluted to twice the expected final maximum test concentration with the complete culture medium containing 50 μg / ml gentamicin. Additional four-fold, 10-fold or 1 / 2 logarithmic serial dilutions were performed to provide a total of five drug concentrations plus controls. 100 μl aliquots of these different drug dilutions were added to appropriate microtiter wells already containing 100 μl culture medium to obtain the desired final drug concentration. After drug addition, the plate was incubated for another 48 hours at 37°C, 5% CO2, 95% air and 100% relative humidity. For adherent cells, the assay was terminated by adding cold TCA. Cells were fixed in situ by gentle addition of 50 μl cold 50% (w / v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded and the plate was washed five times with tap water and air-dried. A 0.4% (w / v) sulforhodamine B (SRB) solution (100 μl) in 1% acetic acid was added to each well and the plate was incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid, and the plate was air-dried. The bound colorant was then dissolved with 10 mM trizma base, and the absorbance was read on an automatic plate reader at a wavelength of 515 nm. For suspended cells, the method was the same, except that the settled cells were fixed to the bottom of the wells by gently adding 50 μl 80% TCA (final concentration, 16% TCA) to terminate the assay.

[0251] Example 12 Tyrosine kinase inhibition assay

[0252] Tyrosine kinase inhibition assays were performed at ThermoFisher Life Technologies (Madison, WI, USA) using standard protocols (SelectScreen Biochemical Profiling Services, Thermo Fisher Scientific). The ATP concentration used was the apparent Km. Test compounds were screened in 1% dimethyl sulfoxide (DMSO) (final concentration) in the wells. For a 10-point titration, a 3-fold serial dilution was performed starting from a starting concentration of 10 μM. All peptide / kinase mixtures were diluted to 2X working concentrations in appropriate kinase buffer. All ATP solutions were diluted to 4X working concentrations in kinase buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA). The apparent ATP Km was previously determined using the Z′-LYTE assay. The chromogenic reagent was diluted in chromogenic buffer.

[0253] Example 13 Cell lines and cell culture for animal studies

[0254] LLC1 and CT26 were obtained from the Biological Resources Collection and Research Center. LLC1 was cultured in RPMI1640 (Catalog No. 10-040-CM, ) at 37°C in a humidified atmosphere supplemented with CO2. After the cancer cells were cultured, they were implanted (1×10 6 cells / mouse) into the back of mice for growth.

[0255] Example 14 Experimental tumor models and therapies

[0256] Animal studies were approved and supervised by the Taipei Medical University Animal Care and Use Committee (TMU IACUC, No. LAC-2022-0197 and LAC-2022-0306). Six- to eight-week-old male Balb / c mice were used for all animal experiments. 6 CT26 cells were incubated with Matrigel (354248, ) was injected subcutaneously into the left flank of mice to establish tumors, and growth was determined by measuring two perpendicular diameters. Tumors were allowed to grow for 10-12 days (tumor size approximately 250-300 mm) before randomization and treatment. 3The tumor volume of mice was measured every three or four days (twice a week). When the tumor diameter reached more than 3000 mm 3 Animals were euthanized at 1:10 p.m. . Cabozantinib (HY-13016, MedChemExpress USA), regorafenib (HY-10331, MedChemExpress USA), cedamide-K30, GNTbm-38-K30, GNTbm-38-HCL, Cbo-02, Cbo-20, Cbo-22, Cbo-23, and Cbo-24 were orally administered once daily for 12 or 16 days. Cabozantinib, regorafenib, Cbo-02, Cbo-20, Cbo-22, Cbo-23, and Cbo-24 were dissolved in DMSO and diluted in water (the final concentration of DMSO was 5% v / v) before administration. Cbo-23-K30, cedamide-K30, and GNTbm-38-K30 were dissolved directly in water before administration. Relapse / recurrence is defined as the recurrence of tumor growth and tumor size reaching at least 5 times in mice with CR or PR responses after the second tumor assessment. In this study, complete response (CR, <0.5 times tumor growth in tumor-bearing mice at the time of tumor assessment after the end of treatment) was defined; partial response (PR, tumor size ≥0.5 times tumor growth in tumor-bearing mice at the time of tumor assessment after the end of treatment, but <1 times tumor growth); stable disease (SD, ≥1 times tumor growth in tumor-bearing mice at the time of tumor assessment after the end of treatment, but <5 times tumor growth); and progressive disease (PD, ≥5 times tumor growth in tumor-bearing mice at the time of tumor assessment after the end of treatment) to evaluate the efficacy of treatment.

[0257] Example 15 Tumor re-challenge test

[0258] Mice assessed as CR / PR could enter the rechallenge study. All mice with a PR / CR response underwent rechallenge with CT26 cells on the contralateral side. The first rechallenge was performed after the second assessment, with a tumor dose of 1 × 10 per mouse. 6 cells. After 7 days of growth, CT26 and LLC1 tumor size was used as a baseline. Tumor growth was monitored for 11 days, and on day 11, a tumor size assessment relative to baseline was performed. A response was considered a relapse if the following two criteria were met: first, tumor size was greater than 1.5 times when compared to baseline tumor size; second, tumor volume was greater than 150 mm 3 .

[0259] result

[0260] Tables 1 to 8 referred to herein are provided below:

[0261]

[0262] Table 1. Compound Cbo-23 1 H-NMR spectral data (400 MHz, d-DMSO).

[0263]

[0264]

[0265] Table 2. Growth inhibition of various cancer cell lines treated with various compounds over 72 hours.

[0266]

[0267]

[0268] Table 3. Growth inhibition of NCI-60 human cancer cell line treated with Cbo-02.

[0269]

[0270]

[0271]

[0272] Table 4. Growth inhibition of NCI-60 human cancer cell line treated with Cbo-10.

[0273]

[0274]

[0275]

[0276] Table 5. Growth inhibition of NCI-60 human cancer cell line treated with Cbo-23.

[0277]

[0278]

[0279]

[0280] The mean percentage of cell lethality was subtracted.

[0281] Table 6. Various compounds such as Cbo-02, Cbo-10, Cbo-14 and Cbo-23 were tested for inhibition of the enzymatic activity of tyrosine kinases. Cabozantinib was used as a positive control for the assay.

[0282]

[0283]

[0284]

[0285] The inhibitory activity of the compounds was determined in a biochemical assay using a single concentration (1.0 μM) measurement. The results are provided as percent inhibition values. Compounds showing percent inhibition values ​​greater than 80% were defined as hits. 99 different tyrosine kinases were used.

[0286] Table 7. Selective tyrosine kinase inhibition profiles of cabozantinib, zanzalintinib, and Cbo-23.

[0287]

[0288]

[0289] *:IC 50 Lower than cabozantinib and zanzalintinib.

[0290] Table 8. Re-challenge test for evaluating the induction of immune memory following treatment with Cbo-20 in combination with GNTbm-38-K30.

[0291] CT26 attacks again

[0292]

[0293] CT26 attacks again

[0294]

[0295] LLC1 attacks again

[0296]

[0297] Responses were considered relapses if the following two criteria were met: first, a tumor size greater than 1.5 times when compared to the baseline tumor size; second, a tumor volume greater than 150 mm at day 67. 3 .

[0298] Table 9. Re-challenge test for evaluating immune memory induction following treatment with Cbo-23 in combination with GNTbm-38-K30.

[0299] CT26 attacks again

[0300]

[0301] LLC1 attacks again

[0302]

[0303] LLC1 attacks again

[0304]

[0305] Responses were considered relapses if the following two criteria were met: first, a tumor size increase of more than 1.5 times when compared to the baseline tumor size; second, a tumor volume greater than 150 mm at day 56. 3 .

[0306] Synthesis of kinase inhibitors with novel structures and potent immunomodulatory activities.

[0307] The immunocompetent mouse animal testing platform is used to screen compounds to evaluate their immunomodulatory activity. The anticancer activity of these structurally similar compounds is tested in in vivo tests by monotherapy or combination therapy, wherein the anticancer activity is demonstrated to be achieved by selectively attacking tumors and generating immune memory by the mechanism of cancer immunotherapy. In the present disclosure, the inventors focus on the mechanism of these orally available and strongly immunomodulatory multikinase inhibitors, especially in combination with specific HDAC inhibitors, which can significantly inhibit tumor growth. The inventors have demonstrated that this anticancer mechanism is related to the cancer immunotherapy mechanism. The chemical structures of all novel compounds are confirmed by NMR and mass spectrometry, followed by a series of cell growth and enzyme activity inhibition assays and animal tests. In the present disclosure, three well-known multikinase inhibitors (cabozantinib, zanzalintinib and regorafenib) are particularly used as positive controls. At the same time, two unique HDAC inhibitors (cedabendamine / tucidinostat (Tucidinostat) and GNTbm-38) are also used in combination with novel compounds to confirm unique anticancer immunotherapy activity. Synthetic routes, conditions and spectral data for all new compounds can be found in the Materials and Methods section.

[0308] The newly synthesized compound has been shown to inhibit the growth of multiple cancer cell lines.

[0309] As shown in Table 2, four human cancer cell lines (MDA-MB-453, MDA-MB-231, SW48 and A549) and one human normal cell line (M10) and a mouse colorectal cancer cell line (CT-26) were tested for growth inhibition after treatment with these compounds. When compared to two positive controls (cabozantinib and zanzalintinib), many novel compounds such as Cbo-02, Cbo-05, Cbo-07, Cbo-08, Cbo-10, Cbo-14, Cbo-20 and Cbo-23 showed excellent growth inhibition activity against these cancer cell lines. In addition, Cbo-02, Cbo-10 and Cbo-23 were tested on the NCI-60 human cancer cell line to analyze growth inhibition, as shown in Tables 3, 4 and 5. The GI of these compounds was analyzed.50 Compounds with potent in vitro anticancer activity were further screened for inhibitory activity against tyrosine kinases, and the anticancer activity was analyzed by in vivo animal testing. Cabozantinib was used as a positive control for these analyses. The tyrosine kinase enzyme inhibitory activity of Cbo-02, Cbo-10, Cbo-14, and Cbo-23 was tested to determine whether there were differences in the tyrosine kinase inhibition spectrum, as shown in Table 6.

[0310] In vitro tyrosine kinase inhibition profiles of Cbo-02, Cbo-10, Cbo-14, and Cbo-23.

[0311] To determine the selective inhibitory activity of a given compound against various tyrosine kinases, the SelectScreen kinase profiling service from Thermo Fisher Scientific was used. A variety of potential compounds such as Cbo-02, Cbo-10, Cbo-14, and Cbo-23 were further entered into the tyrosine kinase enzyme platform for inhibitory activity determination. The data was determined using the SelectScreen biochemical kinase profiling service and was performed at Thermo Fisher Life Sciences. The inhibitory activity of the compound was determined in the biochemical assay using a single concentration (1.0 μM) measurement. The results are provided in the form of inhibition percentage values. Compounds showing inhibition percentage values ​​greater than 80% are defined as hits. 99 different tyrosine kinases were used, and the kinase lists of these corresponding groups are provided in Table 6. Among these compounds, the most noteworthy is Cbo-23, which has been shown to have an inhibitory tyrosine kinase spectrum similar to that of cabozantinib, indicating that inhibition may be primarily associated with inhibition of tumor development, inhibition of angiogenesis, inhibition of tumor invasion, and tumor metastasis. As shown in Table 6, the difference in the spectrum may indicate that Cbo-23 has immunomodulatory activity within the tumor microenvironment. Interestingly, Cbo-23 significantly inhibited more than 20 kinase targets, such as AXL, BTK, CSF1R, FGR, FLT3, FLT4, KDR (VEGFR2), c-MER, c-MET, RON, PDGFR-A, RET, ROS-1, SRC, TEK (Tie-2), TYRO3, FER, FRK, HCK, INSRR, LCK, TYN-A / -B, MST1R, NTRK-2, EPHA, EPHB, and YES1. As shown in Table 7, Cbo-23 significantly inhibited the enzyme activity of 16 tyrosine kinases, such as MERTK (c-MER), ROS1, BTK, NTRK2 (TRKB), LYNB, FLT4 (VEGFR3), EPHA2, HCK, FER, FGR, SRC, PDGFRA, MST1R (RON), LYNA, INSRR (IRR), and TEK (Tie-2), which were significantly stronger than those of cabozantinib and zanzalintinib. There are several important tyrosine kinase targets, and Cbo-23, although not significantly superior to cabozantinib and zanzalintinib, also has very strong inhibitory activity against kinases such as AXL, TYRO3 (RSE), LCK, FLT3, FRK (PTK5), RET, KDR (VEGFR2), and MET (c-MET). It is clear from the above results that Cbo-23 is a very promising multi-kinase inhibitor with immunomodulatory activity.

[0312] Therapeutic response of Cbo-02 in combination with chidamide-K30 in CT26 tumor-bearing mice.

[0313] After confirming the multi-kinase inhibitory activity, the in vivo anticancer activity of the compounds was evaluated by using immunocompetent tumor-bearing mice, where the CT-26 cold tumor cancer cell line was selected as a stringent test for evaluating tumor growth inhibition. Figure 2A The timing of compound administration and efficacy and survival assessments were carefully designed as shown. For evaluation, monotherapy and combination therapy were tested for inhibition of tumor growth, respectively. Figure 2B and Figure 2C As shown. It seems that the anticancer activity of the combination of cabozantinib plus cedamide-K30 is significantly superior to the anticancer activity of the combination of Cbo-02 and cedamide-K30, and is superior to the treatment of any single therapy. In addition to the anticancer effects of these monotherapies, the inventors are particularly interested in the regulatory mechanism when multi-kinase inhibitors are combined with special HDAC inhibitors, which show significantly excellent immunomodulatory activity within the tumor microenvironment, which can greatly inhibit tumor growth and obtain high ORR. When combined with Chidamide-K30 (HDAC inhibitor), the results showed that the efficacy of treatment with Cabozantinib (30 mg / kg, as a positive control) + Chidamide-K30 (50 mg / kg) (ORR = 60%) > Cbo-02 (30 mg / kg) + Chidamide-K30 (50 mg / kg) (ORR = 30%) > Chidamide-K30 (50 mg / kg) (ORR = 11%) > Cbo-02 (30 mg / kg) (ORR = 10%) > Cabozantinib (30 mg / kg) (ORR = 0%), as shown in Figure 2. Figure 2E The results clearly demonstrate that the combination of Cbo-02 and Chidamide-K30 has an additive anticancer effect; however, the combination of cabozantinib and Chidamide-K30 appears to have a synergistic anticancer effect. Figure 2F As shown in Figure 2, the survival rate data also demonstrated that the anticancer activity of the combination of cabozantinib and cedamide-K30 was significantly better than that of the combination of Cbo-02 and cedamide-K30. Although the combination of cabozantinib plus cedamide-K30 has excellent anticancer effects, after stopping treatment, it was found that the tumor recurrence rate was higher, such as Figure 2G As shown. Figure 2D As shown, body weight showed little change after cessation of treatment with the compound or drug, and no significant differences occurred.

[0314] Therapeutic response of Cbo-20 in combination with GNTbm-38-K30 in CT26 tumor-bearing mice.

[0315] The design of in vivo tests is as follows Figure 3A All compounds or drugs were orally administered for 12 days, and then tumor growth inhibition was assessed 10 days after cessation of treatment, during which time tumor size measurements were taken twice a week, as shown in Table 1. Figure 3Band 3C As shown. Monotherapy with Cbo-20 was superior to monotherapy with cabozantinib in terms of anticancer activity. However, in terms of combination therapy, the combination of Cbo-20 with GNTbm-38 was as good as the combination of cabozantinib with GNTbm-38, and both showed very excellent anticancer activity, as shown in Figure 2. Figure 3B and 3C As shown. Figure 3E As shown, the individual tumor size change fold and ORR indicated that the GNTbm-38-K30 (40 mg / kg) group achieved 0CR, 0PR, 1SD and 5PD, with an ORR of 0%; the cabozantinib (30 mg / kg) group achieved 0CR, 0PR, 2SD and 5PD, with an ORR of 0%; the Cbo-20 (30 mg / kg) group achieved 2CR, 2PR, 2SD and 2PD, with an ORR of 50%; the cabozantinib (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) group achieved 5CR, 1PR, 1SD and 0PD, with an ORR of 86%; the Cbo-20 (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) group achieved 6CR, 1PR, 1SD and 0PD, with an ORR of 88%. These results show that the Cbo-20 and GNTbm-38 combination group is as good as the cabozantinib and GNTbm-38 combination group, and both show a very superior ORR. The results show that treatment with Cbo-20 (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (ORR = 88%) > cabozantinib (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (ORR = 86%) > Cbo-20 (30 mg / kg) (ORR = 50%) > cabozantinib (30 mg / kg) (ORR = 0%) = GNTbm-38-K30 (40 mg / kg) group (ORR = 0%).

[0316] Survival analysis showed that the combination of Cbo-20 and GNTbm-38 was slightly better than the combination of cabozantinib and GNTbm-38. Figure 3F As shown, it shows that Cbo-20 (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (survival rate = 88%) > cabozantinib (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (survival rate = 71%) > Cbo-20 (30 mg / kg) (survival rate = 25%) > GNTbm-38-K30 (40 mg / kg) group (survival rate = 16%) > cabozantinib (30 mg / kg) group (survival rate = 0%). The recurrence rate was evaluated as follows. Figure 3GAs shown. Only in the Cbo-20 (30 mg / kg) group, tumor recurrence was observed in 3 mice (recurrence rate = 75% (3 / 4)), while the other two combination groups did not show any recurrence on day 60. This also illustrates the superiority of these two combinations in preventing cancer recurrence. Figure 3D As shown, after all treatments alone or in combination, the weight of the mice did not change much, and no significant changes occurred. In order to further evaluate whether immune memory is induced, a re-challenge test was used for evaluation. As shown in Table 8, the combination of Cbo-20 and GNTbm-38 significantly inhibited tumor growth and significantly induced immune memory. In the re-challenge test, two tumors of two different cancer cell lines were implanted in the backs of mice with CR and PR responses after treatment. The two cancer cell lines are CT-26, which is induced to be recognized by the immune system during treatment, and LLC-1 cell lines, which are not induced to be recognized by the immune system during treatment. Therefore, CT-26 tumors did not grow, and LLC-1 tumors grew significantly.

[0317] Therapeutic response of Cbo-22 in combination with GNTbm-38-K30 in CT26 tumor-bearing mice.

[0318] like Figure 4A and 4B As shown, Cbo-22 was tested for tumor suppressive activity in CT-26-bearing mice using either monotherapy or combination therapy with GNTbm-38. The anticancer activity of the combination of cabozantinib and GNTbm-38 was significantly superior to that of the combination of Cbo-22 and GNTbm-38. Figure 4A and 4B As shown, this combination is significantly superior to other treatments. The results show that treatment with cabozantinib (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (ORR = 86%) > Cbo-22 (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) (ORR = 25%) > Cbo-22 (30 mg / kg) (ORR = 0%) = cabozantinib (30 mg / kg), and the synergistic anticancer effect of the combination of cabozantinib and GNTbm-38 is significantly better than the combination of Cbo-22 and GNTbm-38, as shown in Figure 2. Figure 4D The overall survival rate is shown in Figure 4EAs shown, the cabozantinib (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) group (survival rate = 71%) > Cbo-22 (30 mg / kg) + GNTbm-38-K30 (40 mg / kg) group (survival rate = 25%) > GNTbm-38-K30 (40 mg / kg) group (survival rate = 16%) > cabozantinib (30 mg / kg) group (survival rate = 0%) > Cbo-22 (30 mg / kg) group (survival rate = 0%). The recurrence rate was evaluated as shown in the figure. Figure 4F The recurrence rate results showed that there was no recurrence in these two combination groups. Figure 4C As shown, treatment did not induce significant differences in body weight.

[0319] Therapeutic response of Cbo-23 in combination with GNTbm-38-K30 in CT26 tumor-bearing mice.

[0320] Animal testing protocols such as Figure 5A As shown. It is recognized that in addition to cabozantinib, the multikinase inhibitor regorafenib also shows similar immunomodulatory activity as cabozantinib. Therefore, in this animal experiment, regorafenib was specifically used as a positive control. Figure 5B and 5C As shown, the combination of Cbo-23 and GNTbm-38 was superior to the combination of regorafenib and GNTbm-38 in inhibiting tumor growth. After all treatments, either monotherapy or combination therapy, there was no significant change in the body weight of the mice, as shown in Figure 2. Figure 5D As shown. The results showed that treatment with Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) (ORR = 89%) > regorafenib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) (ORR = 56%) > GNTbm-38-K30 (5 mg / kg) (ORR = 25%) > Cbo-23 (30 mg / kg) (ORR = 22%) > regorafenib (30 mg / kg) (ORR = 0%), as shown. Figure 5E As shown. Figure 5EAs shown, the individual tumor size change fold and ORR indicated that the GNTbm-38-K30 (5 mg / kg) group achieved 1CR, 1PR, 0SD and 6PD, with an ORR of 25%; the regorafenib (30 mg / kg) group achieved 0CR, 0PR, 2SD and 7PD, with an ORR of 0%; the Cbo-23 (30 mg / kg) group achieved 2CR, 0PR, 0SD and 7PD, with an ORR of 22%; the regorafenib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group achieved 5CR, 0PR, 3SD and 1PD, with an ORR of 56%; the Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group achieved 8CR, 0PR, 0SD and 1PD, with an ORR of 89%. It is clear from these results that Cbo-23 combined with GNTbm-38 has excellent anticancer benefits and that Cbo-23 is a very promising multikinase inhibitor with immunomodulatory activity. Fig. 5F As shown, Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group (survival rate = 89%) > regorafenib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group (survival rate = 67%) > GNTbm-38-K30 (5 mg / kg) group (survival rate = 13%) > Cbo-23 (30 mg / kg) group (survival rate = 11%) > regorafenib (30 mg / kg) group (survival rate = 0%). The recurrence rate was evaluated as shown in the figure. Figure 5G The relapse rate results showed that on day 60, one mouse in the GNTbm-38-K30 (5 mg / kg) group relapsed (relapse rate = 50% (1 / 2), one mouse in the Cbo-23 (30 mg / kg) group relapsed (relapse rate = 50% (1 / 2), one mouse in the Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group relapsed (relapse rate = 13% (1 / 8), and one mouse in the regorafenib (30 mg / kg) + GNTbm-38-K 30 No mice relapsed. To further confirm whether immune memory was induced, a re-challenge test was performed as shown in Table 9. The inventors found that the combination of Cbo-23 and GNTbm-38 showed induction of immune memory, which is interesting, as evidenced by the inhibition of LLC1 tumor growth as well as CT-26 tumor growth. This indeed proves that the combination of Cbo-23 and GNTbm-38 is an effective regimen that can activate the immune system, selectively attack tumors, and generate immune memory, as shown in Table 9.

[0321] Comparison of therapeutic responses of Cbo-23 and cabozantinib, either monotherapy or in combination, in CT26 tumor-bearing mice.

[0322] It is well known that the multikinase inhibitor cabozantinib has immunomodulatory activity. Therefore, animal experiments were designed to compare the immunomodulatory activity between Cbo-23 and cabozantinib. Fig. 6A As shown. Figure 6B and 6C As shown, the activity of Cbo-23 combined with GNTbm-38 in inhibiting tumor growth was superior to that of cabozantinib combined with GNTbm-38. After all treatments, either monotherapy or combination therapy, there was no significant change in the body weight of the mice, as shown in Figure 2. Fig.6D As shown. The results showed that treatment with Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) (ORR = 90%) > cabozantinib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) (ORR = 70%) > cabozantinib (30 mg / kg) (ORR = 44%) > Cbo-23 (30 mg / kg) (ORR = 30%) > GNTbm-38-K30 (20 mg / kg) (ORR = 25%), as Fig. 6E As shown. Fig. 6E As shown, the individual tumor size change fold and ORR indicated that the GNTbm-38-K30 (20 mg / kg) group achieved 2CR, 0PR, 0SD and 6PD, with an ORR of 25%; the cabozantinib (30 mg / kg) group achieved 3CR, 1PR, 3SD and 2PD, with an ORR of 44%; the Cbo-23 (30 mg / kg) group achieved 3CR, 0PR, 0SD and 7PD, with an ORR of 30%; the cabozantinib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group achieved 6CR, 1PR, 1SD and 2PD, with an ORR of 70%; the Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group achieved 9CR, 0PR, 0SD and 1PD, with an ORR of 90%. It is clear from these results that Cbo-23 combined with GNTbm-38 has excellent anticancer benefits and that Cbo-23 is a very promising multikinase inhibitor with immunomodulatory activity. Fig. 6FAs shown, Cbo-23 (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group (survival rate = 90%) > cabozantinib (30 mg / kg) + GNTbm-38-K30 (20 mg / kg) group (survival rate = 70%) > cabozantinib (30 mg / kg) group (survival rate = 33%) > GNTbm-38-K30 (20 mg / kg) group (survival rate = 25%) > Cbo-23 (30 mg / kg) group (survival rate = 20%). The recurrence rate was evaluated as shown in the figure. Figure 6G shown.

[0323] Therapeutic response of Cbo-23-K30 in combination with GNTbm-38-2HCL in CT26 tumor-bearing mice.

[0324] Zanzalintinib is a new generation multikinase inhibitor also known for its immunomodulatory activity. In order to increase the solubility and bioavailability of Cbo-23, Cbo-23 was formulated into a fumarate salt form and an amorphous solid dispersion (ASD) in PVP K-30 based on the salt form of zanzalintinib (zanzalintinib hemifumarate). Fig. 7A As shown, the initial tumor size before treatment was 263.9 mm 3 Therefore, in this animal experiment, zanzalintinib hemifumarate was specifically used as a positive control. Figure 7B and 7C As shown, on day 28, the tumor inhibition rates of the single agent Zanzalintinib hemifumarate (30 mg / kg) and Cbo-23-K30 (30 mg / kg) groups were 2% and 22%, respectively. The activity of Cbo-23-K30 (30 mg / kg) in combination with GNTbm-38-2HCL (40 mg / kg) in inhibiting tumor growth was superior to that of Zanzalintinib hemifumarate (30 mg / kg) and GNTbm-38-2HCL (40 mg / kg), and the tumor inhibition rates on day 28 were 87% and 76%, respectively. After all treatments, monotherapy or combination therapy, there was no significant change in the body weight of the mice, as shown in Table 1. Fig.7D The results showed that the treatment with Cbo-23-K30 (30 mg / kg) + GNTbm-38--2HCL (40 mg / kg) (ORR = 66.6%) was more effective than that with Zanzalintinib hemifumarate (30 mg / kg) + GNTbm-38-2HCL (40 mg / kg) (ORR = 0%). Fig. 7E As shown. Fig. 7EAs shown, the individual tumor size change fold and ORR indicated that the GNTbm-38--2HCL (40 mg / kg) group achieved 0CR, 0PR, 0SD and 9PD, and ORR 0%; the semi-fumarate zanzalintinib (30 mg / kg) group achieved 0CR, 0PR, 0SD and 9PD, and ORR 0%; the Cbo-23-K30 (30 mg / kg) group achieved 0CR, 0PR, 0SD and 9PD, and ORR 0%; the semi-fumarate zanzalintinib (30 mg / kg) + GNTbm-38-2HCL (40 mg / kg) group achieved 0CR, 0PR, 2SD and 6PD, and ORR 0%; the Cbo-23-K30 (30 mg / kg) + GNTbm-38-2HCL (40 mg / kg) group achieved 4CR, 2PR, 1SD and 2PD, and ORR 66.6%. It is clear from these results that the combination of Cbo-23-K30 and GNTbm-38-2HCL has an excellent anticancer effect through a synergistic mechanism. Cbo-23 is a promising multikinase inhibitor with immunomodulatory activity. Overall survival rate Figure 7F As shown, the Cbo-23-K30 (30 mg / kg) + GNTbm-38-2HCL (40 mg / kg) group (survival rate = 77.7%) > Zanzalintinib hemifumarate (30 mg / kg) + GNTbm-38-2HCL (40 mg / kg) group (survival rate = 0%). In the OS analysis on day 61, it can be observed that there are still 3 CRs in the Cbo-23-K30 combination group, which indicates that it has a significant tumor immune activation ability compared with the Zanzalintinib hemifumarate combination group. The recurrence rate was evaluated as follows Figure 7G shown.

[0325] Those skilled in the art of the present disclosure should understand that the teachings and disclosures of the present disclosure may be changed and modified without departing from the spirit and scope of the present application. Based on the above, the present application is intended to cover any changes and modifications thereof, provided that the changes or modifications fall within the scope defined by the appended claims or their equivalents.

Claims

1. A compound of formula (I): in: A is –OD or The wavy lines indicate the connection locations; D is H, C 1-3 Alkyl or Ar; Ar is The dotted lines indicate the connection locations; X is N, CH or CR d ; n is an integer ranging from 0 to 3; When present, each R d independently selected from the group consisting of methyl, NH2, OH, SH, methoxy, dimethylamino, trifluoromethoxy, cyanide, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Cl and Br; R p Selected from the group consisting of: H, methyl, NH2, OH, SH, methoxy, dimethylamino, trifluoromethoxy, cyanide, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Cl and Br; L1, L2 are (i) -NH-C(=O)- or (ii) -(C=O)-NH-; and L3, L4 are (i) –NH-C(=O)- or (ii) –(C=O)-NH-; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

2. The compound according to claim 1, which has formula (II): wherein D, L1, L2, L3 and L4 have the same meanings as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

3. The compound according to claim 1, which has formula (III): wherein D, L3 and L4 have the same meanings as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

4. The compound according to claim 1, which has formula (IV): wherein D has the same meaning as described in formula (I); or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

5. A compound according to any one of claims 1 to 4, wherein Ar is X is N, CH or CR o ; R p Selected from the group consisting of: H, NH2 and nitro; Each R m independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl; Each R o Independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

6. The compound according to claim 5, wherein Ar is X is N, CH or CR o ; R p Selected from the group consisting of: H, NH2 and nitro; Each R o Independently selected from the group consisting of: H, methyl, NH2, nitro, nitroso, sulfonate, sulfonamide, C1-C6 alkyl, C1-C6 alkoxy, formamide, F, Br and Cl; or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

7. The compound according to any one of claims 1 to 4, wherein Ar is selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

8. The compound according to claim 1, selected from the group consisting of: or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof.

9. A pharmaceutical composition or pharmaceutical combination comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition or pharmaceutical combination according to claim 9, further comprising one or more second agents.

11. The pharmaceutical composition or pharmaceutical combination according to claim 10, wherein the second agent is an immune checkpoint inhibitor, an anticancer agent or a combination thereof.

12. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or pharmaceutical combination according to any one of claims 9 to 11, for the preparation of a medicament for inhibiting angiogenesis in a subject in need thereof.

13. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or pharmaceutical combination according to any one of claims 9 to 11, for the preparation of a medicament for suppressing metastasis in a subject in need thereof.

14. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or pharmaceutical combination according to any one of claims 9 to 11, for the preparation of a medicament for suppressing tumorigenesis in a subject in need thereof.

15. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or drug combination according to any one of claims 9 to 11, for the preparation of a medicament for modulating immunomodulatory activity in a subject in need thereof.

16. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or pharmaceutical combination according to any one of claims 9 to 11, for the preparation of a medicament for treating or preventing a disease associated with tyrosine kinase TK in a subject in need thereof.

17. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or pharmaceutical combination according to any one of claims 9 to 11, for the preparation of a medicament for treating a tumor or cancer in a subject in need thereof.

18. The use according to claim 17, wherein the drug is used in a method involving cancer immunotherapy, which is achieved by: regulating the tumor microenvironment, reducing tumor hypoxia, reducing lactic acid accumulation, activating CTLs, inhibiting the number and activity of immunosuppressive cells, obtaining excellent anti-cancer benefits and / or inducing persistent immune memory.

19. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, hydrate, stereoisomer, solvate or prodrug thereof, or a pharmaceutical composition or drug combination according to any one of claims 9 to 11, for preparing a medicament for treating a tumor or cancer, wherein the medicament has an enhanced objective response rate (ORR) compared to a tumor or cancer treatment based on a pharmaceutical composition or drug combination comprising a conventional tyrosine kinase TK inhibitor.

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