Camptothecin compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to camptothecin compounds and a preparation method thereof. The compound shows good tumor inhibition activity and can be used as a cancer therapeutic agent.

Description

Camptothecin compounds and their preparation methods and uses

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on December 16, 2022, with application number CN202211624940.9, and invention name “Camptothecin compounds, preparation methods and uses thereof”, and the Chinese patent application filed with the Patent Office of China on June 28, 2023, with application number CN202310778837.8, and invention name “Camptothecin compounds, preparation methods and uses thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to a class of camptothecin compounds and a preparation method thereof. The compounds exhibit good tumor-suppressing activity and can be used as cancer therapeutic agents. Background Art

[0003] Traditional chemotherapy is one of the main methods of cancer treatment. Although chemotherapy drugs mainly act on tumor cells with disordered proliferation, they will inevitably damage normal cells at the same time. The strategy of traditional chemotherapy is to combine drugs with different mechanisms of action to achieve synergistic anti-tumor effects while avoiding overlapping toxicities. The non-selectivity and high systemic toxicity of traditional chemotherapy drugs are the main reasons that limit their application. In order to reduce the occurrence of adverse events, targeted drugs have become the research direction of anti-tumor drugs. By detecting gene targets specifically expressed by tumor cells and further developing corresponding targeted drugs, the specificity of drugs for tumor cells can be improved, the killing of normal cells can be reduced, and thus adverse reactions can be alleviated.

[0004] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via a stable chemical linker. These drugs leverage the specificity of antibodies for binding to surface antigens on both normal and tumor cells and the high efficacy of cytotoxic agents, while avoiding the drawbacks of the former, such as the lower efficacy and the more toxic side effects of the latter. Compared to traditional chemotherapy drugs, ADCs can more precisely bind to tumor cells while minimizing their effects on normal cells. Cytotoxic molecules are attracting increasing attention in the pursuit of more effective ADCs, with researchers striving for greater improvements in efficacy.

[0005] Summary of the Invention

[0006] The first aspect of the present invention provides a compound represented by Formula I, or a pharmaceutically acceptable salt thereof:

[0007] Wherein, R1 is selected from H, -CN, -CH2CN, C1-4 Alkyl, C 1-4 Haloalkyl; preferably H, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, trichloromethyl; more preferably H;

[0008] R2 is selected from -L1-Rx;

[0009] L1 is selected from chemical bonds, C 1-3 Alkylene, carbonyl, -C(=NH)-, -CH(CN)-, -S(=O)2-; the C 1-3 Alkylene is selected from methylene, ethylene, n-propylene, preferably methylene;

[0010] Rx is selected from C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl-carbonyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl, -N(OH)-Ry, -C(Rz)=N(OH), -CH(Rb)2, -N(Rb)2, The 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl, Ry, Rz may be optionally substituted with 1, 2, 3, or 4 Ra; the C 1-4 The alkyl group is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, and tert-butyl; the 3-6 membered cycloalkyl group is independently selected from cyclobutane, cyclopentane, and cyclohexane; the 3-6 membered heterocycloalkyl group is preferably azetidine, azopentane, and azohexane; the 5-7 membered aryl group is preferably phenyl; the 5-7 membered heteroaryl group is preferably pyridyl; the Ry group is independently selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl, 5-7 membered aryl, benzyloxy-C 1-4 Alkyl, preferably H, methyl, ethyl, cyclopropyl, cyclobutyl, phenyl; said Rz is independently selected from C 1-4 Alkyl, C 1-4 Alkyl-carbonyl, benzyloxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl, benzoyl, 3-6 membered cycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl, preferably methyl, ethyl, acetyl, benzyloxymethyl, aminomethyl, benzoyl, cyclopropyl, phenyl, pyridyl; wherein Ra is independently selected from halogen, hydroxy, oxo (=O), amino, hydroxy-C 1-4 Alkyl, benzyloxy, benzyloxy-C 1-4 Alkyl, C 1-4 Alkyl, C3-5 cycloalkyl, C 1-4 Halogenated alkyl, C 1-4Alkoxy, preferably, Ra is selected from Cl, F, hydroxy, oxo (=O), amino, hydroxymethyl, 1-hydroxyethylbenzyloxy, benzyloxymethyl, methyl, ethyl, cyclopropyl, methoxy, trifluoromethyl; said Rb is independently selected from H, hydroxy, hydroxyamino, C 1-4 Alkyl, C 3-5 Cycloalkyl, hydroxyamino-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, N-methyl-N-hydroxyamino, NH2-C 1-4 Alkyl-carbonyl, preferably H, methyl, ethyl, cyclopropyl, hydroxy, hydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, N-methyl-N-hydroxyamino, hydroxymethyl, hydroxyethyl, aminoacetyl, 3-aminopropionyl; Can be substituted by 1-3 Rc; said Rc is selected from C 1-4 Alkyl, amino-C 1-4 Alkyl, preferably methyl, aminomethyl, 1-aminoethyl;

[0011] Or R1, R2 and the N atom to which they are commonly connected form a 4-membered heterocycloalkyl, a 5-membered heterocycloalkyl or a 6-membered heterocycloalkyl; the 4-membered heterocycloalkyl is preferably azetidine, the 5-membered heterocycloalkyl is preferably azacyclopentane, and the 6-membered heterocycloalkyl is preferably piperidine; and the heterocycloalkyl formed by R1, R2 and the N atom to which they are commonly connected may be optionally substituted by 1, 2, 3, or 4 Rb; the Rb is independently selected from C 1-4 Alkyl, C 1- 4-haloalkyl, hydroxy, hydroxy-C 1-4 Alkyl groups are preferably methyl, ethyl and hydroxyl groups.

[0012] In one technical solution of formula I of the present invention, L1 is selected from a chemical bond; Rx is selected from a 3-6 membered cycloalkyl group; the 3-6 membered cycloalkyl group is independently selected from cyclobutane, cyclopentane, and cyclohexane, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, and is preferably hydroxyl, benzyloxy, hydroxymethyl, benzyloxymethyl, or methoxy.

[0013] In one technical solution of formula I of the present invention, L1 is selected from C 1-3 Alkylene, preferably methylene or ethylene; Rx is selected from 5-7 membered aryl, 5-7 membered heteroaryl; the 5-7 membered aryl is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, and is preferably hydroxyl, hydroxymethyl, 1-hydroxyethyl.

[0014] In a technical solution of formula I of the present invention, L1 is selected from a chemical bond; Rx is selected from C 1-4 Alkyl; the C 1-4The alkyl group is preferably ethyl or n-propyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in formula I, and is preferably hydroxy, hydroxymethyl, trifluoromethyl, or cyclopropyl.

[0015] In a technical solution of formula I of the present invention, L1 is selected from carbonyl; Rx is selected from C 1-4 Alkyl, 5-7 membered aryl; said C 1-4 The alkyl group is preferably ethyl or n-propyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the 5-7 membered aryl group is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I; preferably, Ra is selected from hydroxyl, hydroxymethyl, and amino.

[0016] In one technical solution of formula I of the present invention, L1 is selected from carbonyl; Rx is selected from -N(OH)-Ry; Ry has the same meaning as in formula I, and is preferably H, methyl, cyclopropyl, or phenyl.

[0017] In one technical solution of formula I of the present invention, L1 is selected from a chemical bond; Rx is selected from -C(Rz)=N(OH); Rz has the same meaning as in formula I, preferably methyl, ethyl, acetyl, benzyloxymethyl, aminomethyl, benzoyl, cyclopropyl, phenyl, pyridyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, preferably Cl, F, methyl, trifluoromethyl, hydroxyl, hydroxymethyl.

[0018] In one technical solution of formula I of the present invention, L1 is selected from carbonyl; Rx is selected from -C(Rz)=N(OH); Rz has the same meaning as in formula I, preferably methyl, trifluoromethyl, phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in formula I.

[0019] In a technical solution of formula I of the present invention, L1 is selected from -C(=NH)-; Rx is selected from 5-7 membered aryl, 5-7 membered heteroaryl, N(Rb)2; the 5-7 membered aryl is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, preferably F, hydroxyl; the Rb is independently selected from H, hydroxyl, C1-4 alkyl, preferably independently selected from H, hydroxyl, methyl.

[0020] In one technical solution of formula I of the present invention, L1 is selected from -S(=O)2-; Rx is selected from C 1-4 Alkyl, 5-7 membered aryl, 5-7 membered heteroaryl, preferably methyl, ethyl, n-propyl, phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in formula I, preferably methyl, methoxy, hydroxy, 1-hydroxyethyl.

[0021] In a technical solution of formula I of the present invention, R1 is selected from -CN or -CH2CN; R2 is selected from -L1-Rx; L1 is a chemical bond; Rx has the same meaning as in formula I, preferably H, hydroxy-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl-carbonyl, more preferably H, hydroxyethyl, hydroxyacetyl.

[0022] In a technical solution of formula I of the present invention, R1 is H; R2 is selected from -L1-Rx, L1 is -CH(CN)-, Rx is C 1-4 Alkyl, hydroxy-C 1-4 Alkyl; the C 1-4 The alkyl group is selected from methyl and ethyl; preferably, Rx is selected from methyl and hydroxymethyl.

[0023] In a technical solution of formula I of the present invention, R1 is H; R2 is selected from -L1-Rx, L1 is -S(O)2-, Rx is -N(Rb)2; said Rb is independently selected from H, hydroxyl, C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl-carbonyl, C 3-5 Cycloalkyl is preferably H, methyl, hydroxy, hydroxymethyl, aminoacetyl, 3-aminopropionyl, or cyclopropyl.

[0024] In a technical solution of formula I of the present invention, R1 is H, R2 is selected from -L1-Rx, L1 is carbonyl, Rx is -N(Rb)2; said Rb is independently selected from H, hydroxyl, C 1-4 Alkyl, C 3-5 Cycloalkyl, hydroxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl-carbonyl, preferably H, methyl, ethyl, cyclopropyl, hydroxy, hydroxymethyl, hydroxyethyl. In a more specific technical solution, one Rb in -N(Rb)2 is selected from H, methyl, ethyl, cyclopropyl, and the other Rb is selected from H, hydroxyethyl.

[0025] In a technical solution of formula I of the present invention, R2 is -L1-Rx, L1 is a carbonyl group, and Rx is CH(Rb)2; the Rb has the same meaning as in formula I, preferably H, methyl, cyclopropylhydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, N-methyl-N-hydroxyamino; in a more specific technical solution, one Rb in CH(Rb)2 is selected from H, methyl, cyclopropyl, and the other Rb is selected from hydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, and N-methyl-N-hydroxyamino.

[0026] In a technical solution of formula I of the present invention, R2 is -L1-Rx, L1 is a carbonyl group, and Rx is described may be optionally substituted with 1, 2, 3, or 4 Rc; wherein Rc is selected from C 1-4 Alkyl, amino-C 1-4 Alkyl, preferably methyl, aminomethyl, 1-aminoethyl;

[0027] In one embodiment of Formula I of the present invention, R2 is -L1-Rx, L1 is a carbonyl group, and Rx is a 3-6 membered heterocycloalkyl group; the 3-6 membered heterocycloalkyl group is preferably azetidine, azopentane, or azohexane. The 3-6 membered heterocycloalkyl group may be optionally substituted with 1, 2, 3, or 4 Ra groups; Ra has the same meaning as in Formula I, and is preferably methyl or ethyl.

[0028] In one technical solution of formula I of the present invention, R2 is -L1-Rx, L1 is a carbonyl group, and Rx is a pyridyl group; the pyridyl group may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, preferably Cl, F, oxo (=O), methyl, hydroxyl, hydroxymethyl, or trifluoromethyl.

[0029] In one technical solution of formula I of the present invention, the compound represented by formula I is preferably a compound represented by the following formula I-1, I-2, I-3, I-4, I-5, I-6, I-7, or I-8, I-9, I-10, I-11, or I-12:

[0030] wherein m is selected from 1, 2, or 3; n is selected from 1, 2, 3, or 4; R1, Ra, Rc, Ry, and Rz have the same meanings as in Formula I or any of the above technical solutions.

[0031] In one technical solution of the present invention, the following compound or a pharmaceutically acceptable salt thereof is provided:

[0032] In some embodiments, the cytotoxic drug, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is selected from:

[0033] In some embodiments, the antibody drug conjugate, its stereoisomers, or pharmaceutically acceptable salts thereof is selected from the structure represented by formula (IA),

[0034] in:

[0035] Ab is an antibody or antigen-binding fragment;

[0036] Y is the linker site connected to Ab;

[0037] M is a peptide residue consisting of 2 to 4 amino groups selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), alanine (A), glutamic acid, and aspartic acid;

[0038] X is a structural fragment connecting Dr and M;

[0039] Dr is a cytotoxic drug fragment selected from

[0040] t is a value between 4 and 8.

[0041] In some embodiments, the antibody drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, wherein X is selected from -CH2-NH- or

[0042] In some embodiments, the antibody drug conjugate, its stereoisomers, or pharmaceutically acceptable salts thereof, wherein M is a peptide residue consisting of 2 to 4 amino groups selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), and alanine (A);

[0043] Preferably, M is selected from

[0044] In some embodiments, the antibody drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof, wherein Y is -C(=O)-Y 1 -Y 2 -S-,

[0045] Y 1 Selected from -(CH2) p 、-(CH2) p -NH-SO2-, -CH2-N + (CH3)2-(CH2) p -NH-SO2-, -C 2-4 Alkynylidene-NH-SO2-, -C 2-5 Alkynylidene-, -(CH2) p -OC 2-4 Alkynylidene-, -C 3-5 Cycloalkyl-C 2-4 Alkynylidene-, -(CH2) p -6-membered heterocyclic-SO2- or -C 6-10 Aryl-SO2-NH-;

[0046] p is selected from 0, 1, 2, 3, 4 or 5;

[0047] Y 2 is selected from 5-6 membered heteroaryl or 5-6 membered heterocyclic group.

[0048] In some embodiments, the antibody drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, wherein Y 1 Selected from -(CH2)5-, -CH2-NH-SO2-, -(CH2)2-NH-SO2-, -(CH2)3-NH-SO2-, -CH2-N + (CH3)2-(CH2)2-NH-SO2-, -C≡C-CH2-NH-SO2-, -(CH2)3-C≡C-, -CH2-O-CH2-C≡C-,

[0049] In some embodiments, the antibody drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein Y 2 Selected from 5-6 membered N-containing heteroaryl or 5-6 membered N-containing heterocyclic group;

[0050] Preferably, Y 2 Selected from

[0051] In some embodiments, the antibody drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein Y is selected from

[0052] In some embodiments, the antibody drug conjugate, its stereoisomers or pharmaceutically acceptable salts thereof, wherein the structural unit -XMY- is selected from:

[0053] In some embodiments, the cytotoxic drug-linker, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug-linker has a structure represented by Formula (IB), Dr—X—M—Y′ (IB)

[0054] wherein said Y′ is a structure in which Y is inserted before Ab in formula (IA), and X, M, Y and Dr are as defined in formula (IA);

[0055] In some embodiments, Dr is a cytotoxic drug fragment selected from

[0056] In some embodiments, Y' is selected from:

[0057] In some embodiments, X is selected from -CH2-NH- or

[0058] In some embodiments, M is selected from

[0059] In some embodiments, the cytotoxic drug-linker, its stereoisomers or pharmaceutically acceptable salts thereof are selected from:

[0060] The Ab is an antibody;

[0061] t is a value between 2 and 8.

[0062] In certain embodiments, t is a value between 4 and 8.

[0063] The present invention also provides a pharmaceutical composition comprising the compound of formula (I) of the present invention, the antibody-drug conjugate of formula (IA), the cytotoxic drug-linker of formula (IB), and a pharmaceutically acceptable carrier.

[0064] The present invention also provides use of the compound of formula (I), the antibody-drug conjugate of formula (IA), and the cytotoxic drug-linker of formula (IB) in the preparation of drugs for treating cancer.

[0065] The present invention also provides a method for treating cancer, characterized in that an effective dose of the compound of formula (I), the antibody-drug conjugate of formula (IA), the cytotoxic drug-linker of formula (IB) and a pharmaceutical composition thereof is administered to a patient in need.

[0066] In the present invention, the cancer is preferably a solid tumor.

[0067] Description and Definition

[0068] In the present invention, the halogen or halogen atom refers to fluorine, chlorine, bromine, and iodine; the "halogenated" refers to a group formed after one or more hydrogen atoms in a substituted group are replaced by a halogen atom.

[0069] The term "alkyl" refers to a straight or branched hydrocarbon group in which carbon atoms and carbon atoms are connected to hydrogen atoms by single bonds. 1- 4 or C 1-6 Alkyl; C 1-4 Alkyl refers to an alkyl group having 1 to 4 carbon atoms. 1-6 Alkyl refers to an alkyl group having 1 to 6 carbon atoms. Examples of alkyl groups include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl. In the present invention, the C 1-4 The alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl, preferably methyl, ethyl, n-propyl and isopropyl, more preferably methyl and ethyl.

[0070] The term "alkylene" refers to a divalent substituent formed by an alkyl group losing one hydrogen atom. Examples of alkylene groups include, but are not limited to, methylene, ethylene, and n-propylene.

[0071] The term "hydroxy-C 1-4 "Alkyl" refers to C 1-4 A group formed by replacing one H atom on the terminal carbon atom of an alkyl group with a hydroxyl group, specific examples of which include but are not limited to hydroxymethyl, hydroxyethyl, and hydroxy-n-propyl.

[0072] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen atoms. 1-6 or C 1-4 Examples of haloalkyl include, but are not limited to, monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, and tribromomethyl.

[0073] The term "alkoxy" refers to a group in which the hydrogen atom of a hydroxy group is replaced by an alkyl group. 1-4 Alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy.

[0074] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by halogen atoms. 1-4 Alkoxy refers to C 1-4 The substituents obtained by replacing the hydrogen atom of the hydroxy group with an alkyl group include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, and 2,2,2-trichloroethoxy.

[0075] The term "cycloalkyl" refers to a saturated hydrocarbon group in which carbon atoms are linked by single bonds to form a ring. 3-7 Cycloalkyl refers to a cycloalkyl group having a carbon ring skeleton containing 3 to 7 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, wait.

[0076] The term "heterocyclyl" or "heterocycloalkyl" refers to a group in which one or more carbon atoms in the cyclic skeleton structure of a cycloalkyl group are replaced by a heteroatom. The heteroatom is generally selected from N, O, and S, and the heterocyclyl group may be further oxo-substituted. Heterocyclyl groups are preferably 3-6-membered, 3-7-membered, 4-6-membered, 4-7-membered, 5-6-membered, or 5-7-membered heterocyclyl groups. Examples of heterocyclyl groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrothienyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydropyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, wait.

[0077] The "alkynylene" mentioned in the present invention refers to a group derived from a straight-chain or branched alkyne (containing at least one triple bond) by removing two hydrogen atoms, including "C 2-5 "Alkynylidene", specific examples include but are not limited to: -C≡C-, -C≡C-CH2-, -CH2-C≡C-, -CH2CH2CH2-C≡C-, -C≡C-CH2-, -C≡CC≡C-, etc.

[0078] The term "aryl" refers to an unsaturated, typically aromatic hydrocarbon group which may be a single ring or multiple rings fused together. Examples of aryl groups include, but are not limited to, phenyl and naphthyl.

[0079] The term "heteroaryl" refers to a stable monocyclic or polycyclic aromatic hydrocarbon containing at least one heteroatom ring member selected from N, O, and S. When the heteroaryl contains a nitrogen atom, it also includes nitrogen oxides thereof. Heteroaryl groups include 5-6 membered heteroaryl groups and 8-14 membered fused heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, pyrazinyl, pyridazinyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, indazolyl, indolyl, isoquinolyl, quinoxalinyl, benzoxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, benzimidazolyl, quinolyl, quinazolinyl, and 2-oxopyridyl.

[0080] The term "optionally" means that one or more hydrogen atoms on the specified group may not be further substituted or may be further substituted with the specified substituent.

[0081] The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0082] The term "pharmaceutically acceptable carrier" refers to media and excipients generally accepted in the art for delivering biologically active agents to animals, especially mammals, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants.

[0083] The term "effective amount" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, which is a sufficient amount of the compound to treat the disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention.

[0084] The term "pharmaceutically acceptable salt" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Such salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately. The base can be a pharmaceutically acceptable metal cation hydroxide, an organic primary, secondary, or tertiary amine, or the like.

[0085] The term "cytotoxic drug" or "drug" refers to a small molecule compound that has the potential to disrupt the normal growth of tumor cells. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects.

[0086] The term "antibody-drug conjugate" refers to an antibody linked to a biologically active cytotoxic drug via a stable linker.

[0087] The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains or two identical light chains connected by interchain disulfide bonds;

[0088] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen;

[0089] The term "linker site" refers to the chemical structure segment or bond that connects the Ab;

[0090] The term "linker" refers to a chemical structure fragment connecting a cytotoxic drug and an Ab. In an embodiment of the present invention, the linker is -XMY-, wherein the cytotoxic drug is connected to the X end and the Ab is connected to the Y end.

[0091] It should be noted that for the structural formula "(Dr-XMY-) t -Ab", Y is -C(=O)-Y 1 -Y2 -S-, those skilled in the art will understand that Y 2 It is connected to the sulfhydryl group contained in Ab (such as antibody) after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing the disulfide bond by TCEP to generate sulfhydryl SH), that is, Y 2 The S between Ab is not an additional external sulfur atom.

[0092] For example, In the figure, -S- is not an additional external sulfur atom, but the sulfhydryl group contained in Ab after Ab opens the disulfide bond and binds to Y 2 For example The right end is connected to form -S-.

[0093] For example, In the example, -S- is not an additional external sulfur atom, but the sulfhydryl group contained in Ab after opening the disulfide bond and Y 2 For example The right end is connected to form -S-.

[0094] In the examples of the present invention, Yangshen (positive control compound) SN-38 and Dxd were purchased from MedChemexpress (MCE).

[0095] In the embodiments of the present invention, the composition ratio of the eluent is a volume ratio. For example, "eluent: petroleum ether / ethyl acetate = 10 / 1" means that the eluent is composed of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether:ethyl acetate is 10:1. In the embodiments of the present invention, the "prep-HPLC" refers to preparative high-performance liquid chromatography; the "ACN" refers to acetonitrile.

[0096] The abbreviations used in the examples of the present invention and their corresponding chemical names are as follows: DETAILED DESCRIPTION

[0097] The present invention is further described in detail below through specific examples, but it should be understood that these examples are used to describe the present invention in more detail and should not be understood as limiting the present invention in any way. It will be clear to those skilled in the art that, hereinafter, unless otherwise specified, the materials used are well known in the art and can be purchased from the market or obtained by those skilled in the art according to published literature or conventional methods; the reactions of the present invention are all carried out under continuous magnetic stirring, the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature, which generally refers to 15-35°C, preferably 20-30°C, and more preferably 20-25°C;

[0098] Test equipment:

[0099] The structures of the compounds of the present invention were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using a Bruker Neo 400M or Bruker Ascend 400 NMR instrument, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and / or deuterated chloroform (CDCl3) as the solvent, and tetramethylsilane (TMS) as the internal standard.

[0100] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1260-6125B single quadrupole mass spectrometer or a Waters H-Class SQD2 mass spectrometer (electrospray ionization source). HPLC was performed using a Waters e2695-2998 or Waters ARC and an Agilent 1260 or Agilent Poroshell HPH high performance liquid chromatograph.

[0101] Preparative high performance liquid chromatography was performed using a Waters 2555-2489 (10 μm, ODS 250 cm×5 cm) or a GILSON Trilution LC, and a Welch XB-C18 column (5 μm, 21.2*150 mm) was used as the chromatographic column.

[0102] Example 1:

[0103] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypyrrolidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 1)

[0104] Reaction route:

[0105] Steps:

[0106] Step A: Dissolve compound 1-1 (50.0 mg, 0.10 mmol), 1,4-dibromo-2-butanol (26.0 mg, 0.11 mmol), and NaHCO₃ (16.8 mg, 0.33 mmol) in isopropanol (3 mL). Heat the reaction system to 80°C and stir for 16 hours. LCMS monitoring indicates the disappearance of the starting material. Filter the reaction solution, and the resulting residue is purified by prep-HPLC using 0-100% acetonitrile / water (0.01% formic acid) to afford the title compound (2.2 mg, 4.3% yield).

[0107] LCMS (ESI) m / z: 506.2 [M+H] + .

[0108] 1 H NMR(400MHz,MeOD)δ7.76(d,J=10.5Hz,1H),7.64(s,1H),5.63–5.37(m,4H),5.01(s,1H),4.58(s,1H),3.97–3.65(m,1H),3.60–3 .35(m,2H),3.17(d,J=35.7Hz,2H),2.93–2.59(m,1H),2.47(s,5H),2.03–1.92(m,3H),1.45–1.20(m,1H),1.01(t,J=7.3Hz,3H).

[0109] Example 2:

[0110] Preparation of N-((S)-7-benzyl-1-((4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indol[1,2-b]quinolin-1-yl)amino)cyclohexyl)oxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridec-13-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound 2)

[0111] Reaction route:

[0112] Steps:

[0113] Step A: Dissolve compound 2-1 (300.0 mg, 0.82 mmol) and 4-hydroxycyclohexanone (743.0 mg, 6.50 mmol) in anhydrous dichloromethane (20 mL). Add pyridine p-toluenesulfonate (61.0 mg, 0.20 mmol) and stir the reaction at room temperature for 16 hours. After LCMS monitoring shows the disappearance of the starting material, quench the reaction by adding water (20 mL). The mixture is extracted with dichloromethane (10 mL x 3). The combined organic phases are washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. Purification by column chromatography (eluent: dichloromethane / methanol = 25 / 1) affords compound 2-2 (290 mg, 84.0% yield).

[0114] LCMS (ESI) m / z: 440.2 [M+18]+ .

[0115] Step B: Dissolve compound 2-2 (290.0 mg, 0.68 mmol) and compound 1-1 (290.0 mg, 0.68 mmol) in anhydrous N,N-dimethylformamide (30 mL). Add sodium cyanoborohydride (252 mg, 4 mmol) at room temperature. Stir the reaction at room temperature for 16 hours. After LCMS monitoring shows the disappearance of the starting material, quench the reaction by adding water (20 mL). The mixture is extracted with dichloromethane (10 mL x 3). The combined organic phases are washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. Separate the mixture by column chromatography (eluent: dichloromethane / methanol = 25 / 1) to afford pure compound 2-3 (55.0 mg, 10.0% yield).

[0116] LCMS (ESI) m / z: 842.3 [M+H] + .

[0117] Step C: Dissolve compound 2-3 (18.0 mg, 0.02 mmol) and piperidine (0.02 mL) in anhydrous N,N-dimethylformamide (1.5 mL). Stir the reaction at room temperature for 2 hours. After LCMS monitoring indicated the disappearance of the starting material, the mixture was concentrated under reduced pressure to afford crude compound 2-4 (20.0 mg).

[0118] LCMS (ESI) m / z: 620.3 [M+H] + .

[0119] Step D: Compound 2-4 (20.0 mg, 0.02 mmol) and compound 2-5 (20.0 mg, 0.04 mmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (26.0 mg, 0.01 mmol) was added at 0°C. The reaction was stirred at 0°C for 0.5 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction was quenched by adding water (20 mL). The mixture was extracted with dichloromethane (10 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was purified by column chromatography (eluent: dichloromethane / methanol = 25 / 1) to afford the title compound (2.9 mg, 12.0% yield).

[0120] LCMS (ESI) m / z: 1074.4 [M+H] +

[0121] 1H NMR(400MHz,MeOD)δ8.35(s,1H),7.80(d,J=10.7Hz,1H),7.66(s,1H),7.32–7.18(m,5H),6.78(s ,1H),5.62–5.36(m,3H),5.19(s,1H),4.73(s,2H),4.59–4.44(m,1H),3.94–3.69(m,6H),3.58–3. 42(m,4H),3.21–3.13(m,2H),3.02(dd,J=13.9,9.3Hz,1H),2.48(s,3H),2.40(s,3H),2.20(t,J= 7.5Hz,5H),1.96(s,2H),1.53(d,J=10.4Hz,7H),1.42(s,2H),1.29(s,6H),1.01(t,J=7.3Hz,3H).

[0122] Example 3:

[0123] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-hydroxycyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 3-P1 or Compound 3-P2) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-hydroxycyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 3-P2 or Compound 3-P1)

[0124] Reaction route:

[0125] Steps:

[0126] Step A: Dissolve compound 1-1 (50.0 mg, 0.1 mmol), 4-hydroxycyclohexanone (13.0 mg, 0.11 mmol), and sodium cyanoborohydride (22.0 mg, 0.3 mmol) in dichloromethane (2 mL). Stir the reaction at room temperature for 16 hours. LCMS monitoring indicates the disappearance of the starting material. Filter the reaction mixture, and the resulting residue is purified by Prep-HPLC (acetonitrile / 0.01% formic acid / water) to afford compound 3-P1 (8.9 mg, 16.9% yield) and compound 3-P2 (15.0 mg, 29.0% yield).

[0127] Compound 3-P1:

[0128] LCMS (ESI) m / z: 534.3 [M+H] + .

[0129] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 6.355 min.

[0130] 1 H NMR(400MHz,MeOD)δ7.79(d,J=10.3Hz,1H),7.66(s,1H),5.62-5.58(m,J=16.1Hz,1H),5.52-5.39(m,J =53.6Hz,3H),5.23(s,1H),3.62-3.55(m,J=29.2Hz,2H),3.44-3.38(m,J=22.4Hz,1H),3.19-3.12(m,J= 25.2Hz,1H),2.86(d,J=18.1Hz,1H),2.49(s,3H),2.44-2.31(m,J=48.4Hz,2H),2.29-2.22(m,J=26.4Hz ,1H),2.16–2.07(m,J=36.8Hz,2H),2.01–1.94(m,J=28Hz,2H),1.66(s,2H),1.49(s,2H),1.01(t,J=7.3 Hz,3H).

[0131] Compound 3-P2:

[0132] LCMS (ESI) m / z: 534.3 [M+H] + .

[0133] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 6.514 min.

[0134] 1H NMR (400MHz, MeOD) δ7.77(d,J=10.6Hz,1H),7.64(s,1H),5.60(d,J=15.8Hz,1H),5.44(dd,J= 35.4,10.4Hz,3H),5.27(s,1H),4.05(s,1H),3.66–3.52(m,1H),3.45–3.36(m,1H),3.25–3.1 3(m,1H),2.86(dd,J=17.6,8.7Hz,1H),2.70(s,1H),2.49(s,3H),2.41(d,J=16.7Hz,1H),2.1 7(d,J=8.1Hz,1H),2.03–1.92(m,7H),1.80–1.59(m,3H),1.34(s,1H),1.01(t,J=7.3Hz,3H).

[0135] Example 4:

[0136] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxy-2-oxopyrrolidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 4)

[0137] Reaction route:

[0138] Steps:

[0139] Step A: Dissolve compound 4-1 (1.0 g, 5.74 mmol) in tetrahydrofuran (10 mL) and add borane tetrahydrofuran complex (0.6 mL, 6.31 mmol) dropwise under nitrogen in an ice bath. Stir the reaction mixture under nitrogen at room temperature for 4 hours. TLC analysis revealed the formation of new spots. Add methanol (5 mL) and stir for 0.5 hours. The reaction mixture was then concentrated to yield the crude product of target compound 4-2 (0.8 g), which was used directly in the next step.

[0140] Step B: Dissolve compound 4-2 (0.7 g, 4.37 mmol) in dichloromethane (15 mL). Add pyridinium chlorochromate (1.0 g, 4.81 mmol) under nitrogen in an ice bath. Stir the reaction under nitrogen at room temperature for 2 hours. TLC analysis indicated the disappearance of the starting material spot. The reaction was quenched with water and extracted with dichloromethane. The mixture was dried, concentrated, and purified by column chromatography to afford compound 4-3 (200.0 mg, 29.0% yield).

[0141] Step C: Compounds 1-1 (50.0 mg, 0.10 mmol) and 4-3 (17.0 mg, 0.11 mmol) were dissolved in 1,2-dichloroethane (5 mL). Acetic acid (7.0 mg, 0.12 mmol) was added at room temperature. After stirring for 0.5 hour, sodium triacetoxyborohydride (117.0 mg, 0.55 mmol) was added and the reaction continued at room temperature for 4 hours. TLC analysis revealed the formation of a new spot, and LCMS analysis revealed the formation of the desired product ion peak. The reaction solution was concentrated and purified by preparative HPLC in 0-100% acetonitrile / water (0.1% ammonium bicarbonate) to afford the title compound (1.9 mg, 3.3% yield).

[0142] LCMS (ESI) m / z: 520.0 [M+H] + .

[0143] 1 H NMR (400MHz, Chloroform-d) δ7.65(s,1H),7.63(s,1H),5.80(t,J=7.2Hz,1H),5.70(d,J=16. 4Hz,1H),5.25(d,J=16.3Hz,1H),5.06–4.90(m,2H),4.49(t,J=7.7Hz,1H),3.31(dd,J=19.3,1 2.7Hz,2H),3.18–3.06(m,1H),3.03–2.90(m,1H),2.42(s,3H),2.33(q,J=6.6Hz,2H),2.22(t, J=7.6Hz,1H),2.07–1.99(m,2H),1.92–1.87(m,2H),1.49–1.41(m,1H),1.04(t,J=7.3Hz,3H).

[0144] Example 5:

[0145] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-hydroxypiperidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 5)

[0146] Reaction route:

[0147] Steps:

[0148] Step A: Dissolve compound 1-1 (50.0 mg, 0.1 mmol), 1,5-dichloro-3-pentanone (31.0 mg, 0.2 mmol), and sodium bicarbonate (20.0 mg, 0.2 mmol) in methanol (15 mL). Heat the reaction to reflux and continue stirring for 2 hours. After LCMS monitoring shows the disappearance of the starting material, the reaction solution is concentrated to dryness, cooled to room temperature, and quenched by the addition of water (10 mL). The mixture is extracted with dichloromethane (10 mL x 3). The combined organic phases are washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1) to provide compound 5-2 (35.0 mg, yield 67.0%).

[0149] LCMS (ESI) m / z: 518.2 [M+H] + .

[0150] Step B: Compound 5-2 (25.0 mg, 0.05 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). Sodium borohydride (7.6 mg, 0.20 mmol, dissolved in 0.1 mL of ethanol) was added dropwise at 0°C. The reaction was stirred at 0°C for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction was cooled to room temperature and quenched by the addition of saturated aqueous ammonium chloride (3 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The title compound was purified by preparative HPLC using 0-100% acetonitrile / water (0.1% ammonia) to afford the title compound (3.0 mg, 12.0% yield).

[0151] LCMS (ESI) m / z: 520.3 [M+H] + .

[0152] 1 H NMR(400MHz,MeOD)δ7.63(d,J=13.3Hz,2H),5.68–5.48(m,1H),5.49–5.19(m,3H),5.11-5.18(s,2H),3.64(s,1H),2.97 -3.00(m,2H),2.78-2.70(m,2H),2.41(s,3H),2.30–2.06(m,3H),2.06–1.80(m,5H),1.60(s,2H),1.01(t,J=7.4Hz,3H).

[0153] Example 6:

[0154] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-hydroxy-4-methylpiperidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 6)

[0155] Reaction route:

[0156] Steps:

[0157] Step A: Compound 5-2 (10.0 mg, 0.02 mmol) was dissolved in tetrahydrofuran (3 mL). Methylmagnesium bromide (0.1 mL, 0.03 mmol) was added dropwise at 0°C. The reaction was stirred at 0°C for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of saturated aqueous ammonium chloride (10 mL). The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Preparative HPLC was performed using 0-100% acetonitrile / water (0.1% ammonia) to obtain the title compound (2.6 mg, 24.0% yield).

[0158] LCMS (ESI) m / z: 534.1 [M+H] + .

[0159] 1 H NMR(400MHz,MeOD)δ7.82(d,J=10.5Hz,1H),7.67(s,1H),5.57(dd,J=30.5,17.7Hz,2H),5.46–5.35(m,2H),5.06(s,1H),3.5 7(s,1H),3.18(s,1H),2.53–2.48(m,3H),2.37(s,1H),2.19(m,1H),2.03–1.81(m,6H),1.70–1.48(m,4H),0.99–1.11(m,3H).

[0160] Example 7:

[0161] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((3-hydroxycyclobutyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 7)

[0162] Reaction route:

[0163] Steps:

[0164] Step A: Compound 1-1 (50.0 mg, 0.11 mmol), 3-(benzyloxy)-1-cyclobutanone (60.0 mg, 0.33 mmol), and sodium cyanoborohydride (22.0 mg, 0.33 mmol) were dissolved in N,N-dimethylformamide (1 mL). The reaction system was stirred at room temperature for 16 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of water (10 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to provide compound 7-1 (25.0 mg, yield 36.0%).

[0165] LCMS (ESI) m / z: 596.3 [M+H] + .

[0166] 1 H NMR(400MHz, DMSO-d6)δ7.69(d,J=10.7Hz,1H),7.44–7.18(m,6H),6.50(s,1H),5.42(s,2H),5.30( dd,J=29.7,14.3Hz,2H),4.35(d,J=14.0Hz,2H),4.21(d,J=22.7Hz,1H),3.79–3.65(m,1H),3.54(d, J=44.1Hz,1H),3.18(s,1H),2.93(d,J=33.7Hz,1H),2.58(s,1H),2.34(s,3H),2.13(d,J=17.5Hz,2H ),2.00(d,J=10.4Hz,1H),1.94–1.77(m,2H),1.79–1.63(m,1H),1.23(s,2H),0.86(t,J=6.8Hz,3H).

[0167] Step B: Compound 7-1 (26.0 mg, 0.04 mmol) was dissolved in dichloromethane (5 mL), and trifluoromethanesulfonic acid (1 mL) was added dropwise. The reaction was stirred at room temperature for 4 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was diluted with dichloromethane (30 mL) and quenched with saturated sodium bicarbonate solution (10 mL × 1) with stirring. The mixture was then washed with saturated brine (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by prep-HPLC in 0-100% acetonitrile / water (0.01% formic acid) to afford the title compound (16 mg, 73.0% yield).

[0168] LCMS (ESI) m / z: 506.2 [M+H] + .

[0169] 1 H NMR (400MHz, MeOD) δ8.24(s,1H),7.59(d,J=9.5Hz,2H),5.54(dd,J=34.1,17.7Hz,2H),5.36(t,J=18.2Hz,2H),4.38(t,J=41.1Hz,1H),3.93( dd,J=49.0,40.5Hz,1H),3.10(d,J=6.6Hz,2H),2.81–2.61(m,1H),2.42(m,3H),2.34–2.11(m,4H),2.07–1.73(m,4H),1.01(t,J=7.3Hz,3H).

[0170] Example 8:

[0171] Preparation of (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2,3-dihydroxypropylamine (Compound 8)

[0172] Reaction route:

[0173] Steps:

[0174] Step A: Dissolve compound 1-1 (50.0 mg, 0.10 mmol), compound 8-1 (28.0 mg, 0.2 mmol), and N,N-diisopropylethylamine (0.5 mL) in N,N-dimethylformamide (5 mL). Add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76.0 mg, 0.20 mmol). Stir the reaction system at room temperature for 5 hours. After LCMS monitoring showed the disappearance of the starting material, water (10 mL) was added to quench the reaction mixture. The mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield compound 8-2 (50.0 mg).

[0175] LCMS (ESI) m / z: 564.2 [M+H] + .

[0176] Step B: Compound 8-2 (25.0 mg, 0.05 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL). Sodium borohydride (7.6 mg, 0.20 mmol, dissolved in 0.1 mL of ethanol) was added dropwise at 0°C. The reaction was stirred at 0°C for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction was cooled to room temperature and quenched by the addition of saturated aqueous ammonium chloride (3 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The title compound was purified by preparative HPLC using 0-100% acetonitrile / water (0.1% ammonia) to afford the title compound (10.0 mg, 12.0% yield).

[0177] LCMS (ESI) m / z: 524.2 [M+H] + .

[0178] 1H NMR(400MHz,DMSO-d6)δ8.35(d,J=8.9Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H) ,6.50(s,1H),5.57(td,J=9.4,8.0,5.4Hz,1H),5.42(s,2H),5.33–5.04(m,2H),4. 69(s,1H),4.01(d,J=4.9Hz,1H),3.78–3.54(m,2H),3.20–3.08(m,3H),2.39(d,J= 1.9Hz, 3H), 2.30–2.08 (m, 2H), 1.94–1.76 (m, J = 7.1Hz, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0179] Example 9:

[0180] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)-2-(2-(2-hydroxyethoxy)ethoxy)acetamide (Compound 9)

[0181] Reaction route:

[0182] Steps:

[0183] Step A: Dissolve diethylene glycol (1.0 g, 0.9 mL, 9.43 mmol) in dry N,N-dimethylformamide (18 mL). Then, slowly add 60% sodium hydride (416.0 mg, 10.34 mmol) at 0°C and stir for half an hour. Then, slowly add tert-butyl bromoacetate (2.2 g, 1.8 mL, 11.32 mmol). The reaction is stirred at room temperature overnight. After TLC monitoring shows the disappearance of the starting material, quench the reaction by adding ice water (5 mL). The mixture is diluted with ethyl acetate (200 mL) and washed sequentially with water (100 mL × 3) and saturated sodium chloride solution (100 mL × 1). The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to provide compound 9-1 (500.0 mg, 46.0% yield).

[0184] Step B: Dissolve compound 9-1 (200.0 mg, 0.91 mmol) in dichloromethane (5 mL), then add 1 mL of trifluoroacetic acid dropwise. Stir the reaction system at room temperature for 4 hours. After TLC monitoring shows the disappearance of the starting material, dilute the reaction solution with dichloromethane (30 mL) and quench with the addition of saturated sodium bicarbonate solution (10 mL x 1) with stirring. Then separate the layers, wash the organic phase with saturated brine (10 mL x 1), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude compound 9-2 (200.0 mg). The crude product is used directly in the next step.

[0185] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (2 mL), followed by the addition of N,N-diisopropylethylamine (29.0 mg, 0.28 mmol), compound 9-2 (20.0 mg, 0.14 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (100.0 mg, 0.19 mmol). The reaction was stirred at room temperature for 16 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was diluted with ethyl acetate (30 mL) and washed sequentially with water (10 mL × 3) and saturated brine (10 mL × 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (acetonitrile / 0.01% NH4HCO3 and water) to afford the title compound (1.9 mg, 3.5% yield).

[0186] LCMS (ESI) m / z: 582.2 [M+H] + .

[0187] 1 HNMR(400MHz,DMSO-d6)δ8.42(d,J=8.7Hz,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H),6.51(s,1H),5.61(s,1H),5.42(s,2H),5.20(s,2H),4.04 (s,2H),3.63(s,2H),3.54(s,2H),3.17(s,2H),2.39(s,3H),2.19(s,2 H), 2.03–1.95 (m, 1H), 1.86 (m, 2H), 1.24 (s, 4H), 0.87 (t, J = 7.1Hz, 3H).

[0188] Example 10:

[0189] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-(2-hydroxyethoxy)ethoxy)ethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[en]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 10)

[0190] Reaction route:

[0191] Steps:

[0192] Step A: Compound 1-1 (50.0 mg, 0.09 mmol) and bromotriethylene glycol (24.0 mg, 0.11 mmol) were dissolved in dry tetrahydrofuran (2 mL). Sodium iodide (28.0 mg, 0.19 mmol) was then added and the mixture was stirred in a 60°C oil bath for 16 hours. After LCMS monitoring indicated the disappearance of the starting material, the mixture was diluted with dichloromethane (50 mL) and washed sequentially with water (10 mL x 2) and saturated sodium chloride solution (10 mL x 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC (eluent: ACN / 0.1% NH4HCO3 and water) to afford the title compound (0.7 mg, 1.3% yield).

[0193] LCMS (ESI) m / z: 568.3 [M+H] + .

[0194] 1 H NMR(400MHz,DMSO-d6)δ9.09(s,1H),7.89(d,J=11.0Hz,1H),7.36(s,1H),6 .58(s,1H),5.62–5.36(m,3H),5.11(s,1H),4.57(s,1H),3.73(s,2H),3.63 (s,4H),3.50–3.45(m,6H),3.21–3.16(m,2H),2.77(s,1H),2.42(s,3H),2. 18(s,1H),1.89(dt,J=14.5,7.1Hz,2H),1.24(s,1H),0.89(t,J=7.3Hz,3H).

[0195] Example 11:

[0196] Preparation of (1S,9S)-1-((4-(benzyloxy)phenyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 11)

[0197] Reaction route:

[0198] Steps:

[0199] Step A: Compound 1-1 (50.0 mg, 0.11 mmol) and triethylamine (44.0 mg, 0.44 mmol) were added to dichloromethane (10 mL). The mixture was stirred at room temperature for 10 minutes, followed by the addition of 4-benzyloxyphenylboronic acid (54.0 mg, 0.22 mmol) and copper acetate (44.0 mg, 0.22 mmol). The reaction system was stirred at room temperature under oxygen for 6 hours. After LCMS monitoring indicated no change in the starting material content, the reaction solution was quenched by adding water (10 mL). The mixture was diluted with dichloromethane (30 mL) and washed sequentially with water (10 mL x 3) and saturated sodium chloride solution (10 mL x 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash preparative liquid chromatography to afford the title compound (5.0 mg, 7.0% yield).

[0200] LCMS (ESI): m / z: 618.2 [M+H] + .

[0201] 1 H NMR(400MHz, DMSO-d6)δ8.42(s,1H),7.79(d,J=10.7Hz,1H),7.47–7.30(m,6H),6.83(dd,J=38.9,7.5Hz,4H),6.52(s,1H),5.65(d,J=9.1Hz,1H), 5.40(s,2H),5.33–5.08(m,3H),5.01(s,2H),2.40(s,3H),2.18(d,J=4.9 Hz,2H),1.87(td,J=14.5,7.3Hz,2H),1.24(s,2H),0.87(t,J=6.8Hz,3H).

[0202] Example 12:

[0203] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-(hydroxymethyl)cyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 12)

[0204] Reaction route:

[0205] Steps:

[0206] Step A: Compound 12-1 (200.0 mg, 1.56 mmol) and imidazole (265.0 mg, 3.90 mmol) were dissolved in N,N-dimethylformamide (2 mL). Tert-butyldiphenylsilyl chloride (644.0 mg, 2.34 mmol) was then added with stirring at 0°C. The reaction system was allowed to warm to room temperature and stirred for 24 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was quenched by adding water (10 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to afford compound 12-2 (200.0 mg, 35.0% yield).

[0207] Step B: Compound 1-1 (30.0 mg, 0.06 mmol) and compound 12-2 (124.0 mg, 0.34 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and sodium cyanoborohydride (21.0 mg, 0.34 mmol) was added. The reaction system was purged with argon three times and stirred at room temperature for 16 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was diluted with ethyl acetate (100 mL), washed sequentially with water (30 mL × 3), and then with saturated brine (30 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC (ACN / 0.01% HCl and water) to provide compound 12-3 (50.0 mg, 92.0% yield).

[0208] LCMS (ESI) m / z: 786.3 [M+H] +

[0209] Step C: Dissolve hydrogen fluoride (triethylamine salt) (100.0 mg, 0.64 mmol) in tetrahydrofuran (5 mL), then add triethylamine (64.0 mg, 0.64 mmol) and stir for 30 min. Compound 12-3 (50.0 mg, 0.06 mmol) is then dissolved in a minimal amount of tetrahydrofuran and added to the prepared triethylamine solution with stirring. The reaction system is allowed to react at room temperature for 24 hours. LCMS monitoring indicates the reaction is complete. The reaction solution is diluted with ethyl acetate (100 mL) and washed sequentially with saturated sodium bicarbonate solution (30 mL × 1), water (30 mL × 1), and then saturated brine (30 mL × 1). The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue is purified by Prep-HPLC (ACN / 0.01% HCl and water) to afford the title compound (6.5 mg, 19.0% yield).

[0210] LCMS (ESI) m / z: 548.4 [M+H] +

[0211] 1 H NMR(400MHz,DMSO-d6)δ9.79(s,1H),8.96(d,J=39.2Hz,1H),7.87(d,J=10.6Hz,1H),7.34(s,1H),6 .54(s,1H),6.05–5.85(m,1H),5.48(d,J=25.9Hz,3H),5.20(s,1H),3.24(d,J=5.9Hz,2H),2.84–2.7 0(m,1H),2.40(s,3H),2.36–2.29(m,1H),2.22–1.98(m,2H),1.88(d,J=7.0Hz,5H),1.71(d,J=11.6 Hz,2H),1.49(s,1H),1.35(s,1H),1.21(d,J=14.7Hz,2H),1.11–0.98(m,1H),0.89(t,J=7.0Hz,3H).

[0212] Example 13:

[0213] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((3-(hydroxymethyl)cyclobutyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 13)

[0214] Reaction route:

[0215] Steps:

[0216] Step A: Dissolve compound 1-1 (43.5 mg, 0.1 mmol) and compound 13-1 (11.0 mg, 0.6 mmol) in N,N-dimethylformamide (8 mL). Add sodium cyanoborohydride (37.0 mg, 0.6 mmol), and stir the reaction at room temperature for 3 hours. After LCMS monitoring shows the disappearance of the starting material, quench the reaction by adding water (20 mL). The mixture is extracted with ethyl acetate (10 mL x 3). The combined organic phases are washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Prep-HPLC is performed using 0-100% acetonitrile / water (0.1% formic acid) to afford compound 13-2 (15.0 mg, 25.0% yield).

[0217] LCMS (ESI) m / z: 610.2 [M+H] + .

[0218] 1 H NMR (400MHz, DMSO-d6) δ7.74 (d, J=11.0Hz, 1H), 7.40–7.18 (m, 6H), 6.52 (d, J=5. 6Hz,1H),5.51–5.22(m,4H),4.46(d,J=13.3Hz,2H),4.22(d,J=4.5Hz,1H),3.48- 3.53(m,1H),3.32-3.39(m,2H),3.17-3.24(m,2H),2.99-3.01(m,1H),2.37(d,J= 1.8Hz,4H),2.19(d,J=14.8Hz,2H),1.88(m,4H),1.59–1.43(m,1H),0.88(m,3H).

[0219] Step B: Dissolve compound 13-2 (5 mg, 0.01 mmol) in anhydrous dichloromethane (10 mL) and add trifluoromethanesulfonic acid (0.01 mL) dropwise at room temperature. Stir the reaction at 25°C for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was concentrated under reduced pressure and purified by preparative HPLC in 0-100% acetonitrile / water (0.1% formic acid) to afford the title compound (4.0 mg, 96.0% yield).

[0220] HRMS (ESI) m / z: 520.2 [M+H] + .

[0221] 1H NMR(400MHz, DMSO-d6)δ8.23(s,1H),7.74(d,J=11.2Hz,1H),7.31(s,1H),6.51(s,1H),5.43-5.36(m,4H),3.54-3.48(m ,2H),3.03-2.89(m,4H),2.40-2.29(m,3H),2.27-2.25(m,3H),202-1.83(m,5H),1.83-1.48(m,1H),0.86-0.89(m,3H).

[0222] Example 14:

[0223] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((3R)-(benzyloxy)cyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 14-P1 or Compound 14-P2) and ( Preparation of 1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((3S)-(benzyloxy)cyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 14-P2 or Compound 14-P1)

[0224] Reaction route:

[0225] Steps:

[0226] Step A: Dissolve compound 1-1 (50.0 mg, 0.12 mmol), compound 14-1 (145.0 mg, 0.70 mmol), and sodium cyanoborohydride (45.0 mg, 0.70 mmol) in N,N-dimethylformamide (2 mL); stir the reaction at room temperature for 6 hours. After LCMS monitoring indicated the disappearance of the starting material, cool the reaction to room temperature and quench with water (10 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL x 3) and saturated sodium chloride solution (10 mL x 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1) to afford compound 14-2 (15.0 mg, 30.0% yield).

[0227] LCMS (ESI) m / z: 624.3 [M+H] + .

[0228] 1 H NMR(400MHz, DMSO-d6)δ8.27(s,1H),7.74(d,J=11.2Hz,1H),7.46–7.08(m,5H),6.51(s,1H),5.67–5.14(m,3H),4.60–4.48(m,2H),4 .42(s,1H),3.04(s,2H),2.77(s,1H),2.38(s,3H),2.16(s,3H),2.02(s,2H),1.95–1.67(m,4H),1.13(s,4H),0.88(t,J=7.4Hz,3H).

[0229] Step B: Compound 14-2 (15.0 mg, 0.02 mmol) was dissolved in dichloromethane (2 mL), cooled to 0°C, and trifluoromethanesulfonic acid (0.5 mL) was added dropwise. The mixture was naturally warmed to room temperature and stirred for 4 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was evaporated to dryness and purified by prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid). The preparative solution was lyophilized to provide compounds 14-P1 (5.1 mg, 39.7% yield) and 14-P2 (5.0 mg, 39.0% yield).

[0230] Compound 14-P1:

[0231] LCMS (ESI) m / z: 534.2 [M+H] + .

[0232] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 9.02 min.

[0233] 1 H NMR(400MHz,MeOD)δ7.79(d,J=10.6Hz,1H),7.66(s,1H),5.65–5.36(m,5H),4.33–4.23(m,1H),3.82–3.69(m,1H),3.51–3.37 (m,1H),3.22–3.11(m,1H),2.86(s,1H),2.49(s,3H),2.20(m,3H),1.99–1.92(m,3H),1.80(s,1H),1.57(m,6H),0.88(m,3H).

[0234] Compound 14-P2:

[0235] LCMS (ESI) m / z: 534.2 [M+H] + .

[0236] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 9.47 min.

[0237] 1 H NMR(400MHz,DMSO-d6)δ7.92(d,J=10.7Hz,1H),7.41(s,1H),5.63–5.38(m,4 H),5.22(s,1H),4.25–4.00(m,1H),3.60–3.48(m,2H),3.33(m,1H),3.15–3. 06(m,1H),2.76(t,J=14.0Hz,1H),2.45(s,3H),2.21(s,2H),1.90(dt,J=22. 4,7.1Hz,3H),1.71–1.57(m,1H),1.44–1.08(m,7H),0.90(t,J=7.20Hz,3H).;

[0238] Example 15:

[0239] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((2R)-hydroxycyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 15-P1 or 15-P2)

[0240] as well as

[0241] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((2R)-hydroxycyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 15-P2 or Compound P1)

[0242] Reaction route:

[0243] Steps:

[0244] Step A: Compound 1-1 (30.0 mg, 0.07 mmol), cyclohexene oxide (1 mL), and ACN (0.5 mL) were added to a 10 mL sealed tube; the reaction system was then sealed in an 80°C oil bath for 16 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was cooled to room temperature, diluted with ethyl acetate (50 mL), and washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to afford compounds 15-P1 (5.0 mg, 16.7% yield) and 15-P2 (2.4 mg, 8.0% yield).

[0245] Compound 15-P1:

[0246] LCMS (ESI) m / z: 534.2 [M+H] + .

[0247] HPLC conditions: 0-13 min, 10-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 7.202 min.

[0248] 1 H NMR (400MHz, MeOD) δ7.77(d,J=10.2Hz,1H),7.64(s,1H),5.61(d,J=16.3Hz,1H),5.49( s,2H),5.41(d,J=16.2Hz,1H),5.22(s,1H),3.56(d,J=6.2Hz,1H),3.37(s,1H),2.90(d ,J=14.9Hz,1H),2.50(s,1H),2.49(s,3H),2.35(s,1H),2.21–2.11(m,1H),1.99–1.93( m,4H),1.69(s,3H),1.46(dd,J=24.9,12.8Hz,3H),1.28(s,2H),1.01(t,J=7.3Hz,3H).

[0249] Compound 15-P2:

[0250] LCMS (ESI) m / z: 534.2 [M+H] + .

[0251] HPLC conditions: 0-13 min, 10-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 7.817 min.

[0252] 1 H NMR (400MHz, DMSO-d6) δ7.74(d,J=10.7Hz,1H),7.35(d,J=6.2Hz,1H),7.31(s,1H ),6.52(s,1H),5.43(s,3H),4.58(d,J=35.7Hz,2H),3.22(d,J=11.5Hz,2H),3.07( s,1H),2.38(s,3H),2.26–2.15(m,1H),2.09–1.97(m,3H),1.86(m,3H),1.63(m,2H ),1.50–1.40(m,1H),1.32(d,J=14.9Hz,2H),1.16(d,J=12.1Hz,2H),0.87(m,3H).

[0253] Example 16:

[0254] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-methoxycyclohexyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 16)

[0255] Reaction route:

[0256] Steps:

[0257] Step A: Dissolve compound 1-1 (35.0 mg, 0.11 mmol), 2-methoxycyclohexanone (120.0 mg, 0.55 mmol), and sodium cyanoborohydride (143.0 mg, 1.11 mmol) in methanol (1 mL). The reaction system was moved to an 80°C oil bath and stirred for 16 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of water (10 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL x 3) and saturated sodium chloride solution (10 mL x 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to afford the title compound (40.0 mg, 80.0% yield).

[0258] LCMS (ESI) m / z: 548.3 [M+H] + .

[0259] 1 H NMR (400MHz, DMSO-d6) δ7.82-7.66 (m, 1H), 7.38 (d, J = 79.5Hz, 1H), 6.50 (s, 1H),5.47–5.20(m,3H),4.69(q,J=11.8Hz,1H),4.34(s,1H),3.26(s,3H),3. 02(m,4H),2.76(s,1H),2.33(d,J=4.6Hz,3H),2.18(s,1H),2.12(d,J=6.8Hz ,4H),1.95–1.80(m,1H),1.70(s,2H),1.33–1.13(m,5H),0.91-0.81(m,3H);

[0260] Example 17:

[0261] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-hydroxybenzyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 17)

[0262] Reaction route:

[0263] Steps:

[0264] Step A: Dissolve compound 1-1 (30.0 mg, 0.07 mmol), compound 17-1 (87.7 mg, 0.41 mmol), and sodium cyanoborohydride (25.7 mg, 0.41 mmol) in dichloromethane (3 mL); stir the mixture at room temperature for 5 h. After LCMS monitoring showed the disappearance of the starting material, water (20 mL) was added to quench the reaction solution. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (10 mL × 3). The filtrate was then concentrated under reduced pressure and dissolved in N,N-dimethylformamide (3 mL). The product was purified by preparative HPLC using 0-100% acetonitrile / water (0.1% formic acid) and lyophilized to afford compound 17-2 (15.0 mg, 28.8% yield).

[0265] LCMS (ESI) m / z: 632.2 [M+H] + .

[0266] 1 H NMR(400MHz, DMSO-d6)δ9.31–8.91(m,1H),7.89(d,J=10.9Hz,1H),7.59–7.23(m,7H),7.05(d, J=8.2Hz,2H),6.56(s,1H),5.54(s,1H),5.45(s,2H),5.38(s,1H),5.13(s,2H),4.58–4.39(m,1H),4.37–4 .12(m,1H),2.67(s,3H),2.42(s,4H),2.33(s,1H),2.26–2.10(m,1H),1.88(s,2H),0.87(t,J=7.2Hz,3H).

[0267] Step B: In a microwave tube, compound 17-2 (15 mg, 0.02 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL). NH4COOH (4.5 mg, 0.07 mmol) and Pd / C (3.0 mg) were added. The reaction was microwaved at 60°C for 2 h. After 2 h, the reaction solution was cooled to room temperature and quenched by the addition of water (50 mL). The mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined and washed with saturated brine (50 mL x 3). The filtrate was then concentrated under reduced pressure. The concentrated organic phase was dissolved in N,N-dimethylformamide and purified by preparative HPLC using 0-100% acetonitrile / water (0.1% formic acid). The product was then lyophilized to afford the title compound (3.2 mg, 36.8% yield).

[0268] LCMS (ESI) m / z: 542.2 [M+H] + .

[0269] 1 H NMR(400MHz,MeOD)δ7.78(d,J=10.5Hz,1H),7.78(d,J=10.5Hz,1H),7.65(s,1H), 7.37(d,J=8.5Hz,2H),6.87(d,J=8.6Hz,2H),5.59(d,J=16.6Hz,1H),5.46–5.19( m,3H),4.47(s,1H),4.32(s,1H),3.48(s,1H),3.13(s,1H),2.96–2.76(m,1H),2. 48(s,3H),2.37–2.27(m,1H),1.97(dd,J=7.4,3.7Hz,2H),1.01(t,J=7.4Hz,3H).

[0270] Example 18:

[0271] Preparation of 2-(aminooxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)acetamide (Compound 18)

[0272] Reaction route:

[0273] Steps:

[0274] Step A: Dissolve compound 1-1 (30.0 mg, 0.07 mmol), compound 18-1 (19.8 mg, 0.10 mmol), and N,N-diisopropylethylamine (44.6 mg, 0.35 mmol) in dichloromethane (3 mL). Add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (52.5 mg, 0.14 mmol). Stir and react overnight at room temperature. After LCMS monitoring shows the disappearance of the starting material, the reaction solution is cooled to room temperature and quenched by adding water (20 mL). The mixture is extracted with ethyl acetate (50 mL x 3). The organic phases are combined and washed with saturated brine (10 mL x 3). The filtrate is then concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain crude compound 18-2 (15.0 mg, yield 24.8%).

[0275] LCMS (ESI) m / z: 609.2 [M+H] + .

[0276] Step B: Compound 18-2 (15.0 mg) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and trifluoroacetic acid (2 mL) was slowly added. The reaction was allowed to react at room temperature for 3 h. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The concentrated organic phase was dissolved in N,N-dimethylformamide (3 mL) and purified by preparative HPLC in 0-100% acetonitrile / water (0.1% formic acid). The residue was lyophilized to afford the title compound (1.0 mg, 28.2% yield).

[0277] LCMS (ESI) m / z: 509.2 [M+H].

[0278] 1H NMR (400MHz, MeOD) δ8.64–8.36(m,1H),7.77–7.50(m,2H),5.72(s,1H),5.55(t,J=15.6Hz,1H),5.36(d,J=17.9Hz,3H),5.17(d,J=18.7Hz,2H ), 2.39 (d, J = 39.5Hz, 3H), 2.35–2.26 (m, 1H), 2.19 (t, J = 7.6Hz, 2H), 1.99 (d, J = 30.6Hz, 3H), 1.76 (d, J = 6.3Hz, 1H), 1.60 (s, 2H), 1.00 (s, 2H).

[0279] Example 19:

[0280] Preparation of ((1S,9S)-9-ethyl-5-fluoro-1-((4-fluoro-2-hydroxyphenyl)amino)-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (Compound 19)

[0281] Reaction route:

[0282] Steps:

[0283] Step A: Compound 1-1 (20.0 mg, 0.04 mmol), 2-benzyloxy-4-fluorophenylboronic acid (40.0 mg, 0.16 mmol), and copper acetate (20.0 mg, 0.1 mmol) were dissolved in dichloromethane (10 mL) and acetonitrile (5 mL). The reaction was stirred at 65°C in a sealed system for 36 hours. After LCMS monitoring showed the disappearance of the starting material, dichloromethane (10 mL) was added to the reaction solution, filtered, and finally concentrated under reduced pressure. The product was then purified by Prep-HPLC in 0-100% acetonitrile / water (0.1% ammonia) to provide compound 19-1 (2.0 mg, 7.8% yield).

[0284] LCMS (ESI) m / z: 636.2 [M+H] +

[0285] Step B: Dissolve compound 19-1 (4.0 mg, 0.01 mmol) in anhydrous dichloromethane (5 mL) and add trifluoromethanesulfonic acid (0.01 mL) dropwise at room temperature. Stir the reaction at 25°C for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. Prep-HPLC was then performed using 0-100% acetonitrile / water (0.1% ammonia) to give the title compound (1.0 mg, 31.0% yield).

[0286] LCMS (ESI) m / z: 546.2 [M+H] + .

[0287] 1 H NMR (400MHz, MeOD) δ7.74-7.61(m,2H),6.80(d,J=5.5Hz,1H),6.65-6.44(m,2H),5.55(d,J=16.3Hz ,2H),5.33(d,J=4.9Hz,3H),2.44(d,J=1.7Hz,3H),2.19(s,2H),2.03(s,2H),1.01(t,J=7.5Hz,4H).

[0288] Example 20:

[0289] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indeno[1,2-b]quinolin-1-yl)-2-hydroxyethane-1-sulfonamide (Compound 20)

[0290] Reaction route:

[0291] Steps:

[0292] Step A: Compound 1-1 (50.0 mg, 0.12 mmol) and triethylamine (48.0 mg, 0.48 mmol) were dissolved in N,N-dimethylformamide (2 mL). 2-Methoxy-1-ethylsulfonyl chloride (111 mg, 0.70 mmol) was added with stirring. The mixture was stirred at room temperature in an anhydrous and oxygen-free atmosphere for 6 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of water (10 mL). The mixture was diluted with dichloromethane (50 mL), washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by large-plate HPLC (developing solvent: dichloromethane / methanol = 20 / 1) to afford compound 20-1 (15.0 mg, 30.0% yield).

[0293] LCMS (ESI) m / z: 558.2 [M+H] + .

[0294] Step B: Compound 20-1 (15.0 mg, 0.027 mmol) was dissolved in dichloromethane (2 mL), cooled to 0°C, and BBr3 (3 drops) was added dropwise; after 2 h, LCMS showed little product and a large amount of starting material remaining; the temperature was lowered again and BBr3 (3 drops) was added. After 2 h, LCMS showed an increase in product and a decrease in starting material; the temperature was lowered again and BBr3 (3 drops) was added. After 1 h, LCMS showed an increase in product and a decrease in starting material. After 2 h, LCMS showed no significant increase in product or decrease in starting material, but an increase in impurities, and the reaction was stopped. The reaction solution was directly concentrated and then purified by preparative HPLC in 0-100% acetonitrile / water (0.1% trifluoroacetic acid) and lyophilized to give the title compound (2.0 mg, 13.3% yield).

[0295] LCMS (ESI) m / z: 544.2 [M+H] + .

[0296] 1 H NMR(400MHz,MeOD)δ7.63(d,J=8.4Hz,2H),5.59(dd,J=17.9,7.5Hz,2H),5.49 (s,1H),5.45–5.31(m,3H),5.20(dd,J=10.8,6.5Hz,2H),4.05(t,J=6.2Hz,2H) ,3.50(t,J=6.3Hz,2H),3.26–3.09(m,2H),2.41(d,J=13.5Hz,4H),2.22–2.16 (m,1H),2.05–2.00(m,1H),1.96(dd,J=7.4,3.9Hz,1H),1.01(t,J=7.4Hz,3H).

[0297] Example 21:

[0298] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[2,1-g]quinoline-10,13-dione (Compound 21)

[0299] Reaction route:

[0300] Steps:

[0301] Step A: Compound 1-1 (10.0 mg, 0.02 mmol) and triethylamine (4.7 mg, 0.05 mmol) were added to dichloromethane (10 mL). The mixture was stirred at room temperature for 10 minutes, followed by the addition of phenyl chloroformate (7.2 mg, 0.05 mmol). The reaction was stirred at room temperature under argon for 16 hours. After LCMS monitoring showed no change in product content, the reaction solution was quenched by adding water (10 mL). The mixture was diluted with dichloromethane (30 mL) and washed sequentially with water (10 mL x 3) and saturated sodium chloride solution (10 mL x 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified on a silica gel plate to afford compound 21-2 (5.0 mg, 39.2% yield).

[0302] LCMS (ESI) m / z: 556.2 [M+H] + .

[0303] Step B: Compound 21-2 (15.0 mg, 0.03 mmol) and triethylamine (4.7 mg, 0.05 mmol) were added to 1,4-dioxane (10 mL), followed by aminoacetaldehyde dimethyl acetal (5.7 mg, 0.05 mmol) and 4-dimethylaminopyridine (6.6 mg, 0.05 mmol). The reaction system was stirred at 100°C under argon for 4 hours. After LCMS monitoring showed no change in product content, the reaction solution was quenched by adding water (10 mL). The mixture was diluted with dichloromethane (10 mL) and washed with water (10 mL x 3). The aqueous phases were combined, and the crude product was lyophilized. The resulting crude product was purified on a silica gel plate to obtain compound 21-3 (8.0 mg, 52.2% yield).

[0304] LCMS (ESI) m / z: 567.1 [M+H] + .

[0305] 1 H NMR (400MHz, Methanol-d4) δ8.52(s,1H),7.61(d,J=8.2Hz,1H),5.56(d,J=16.3Hz,1H),5.44–5.31(m,3H),5.18(d,J=19.1Hz,1H),4.61–4.40(m,1H ),3.41(s,6H),3.36(d,J=5.2Hz,2H),3.21(d,J=28.1Hz,2H),2.40(s,3H) ,2.19(t,J=7.7Hz,2H),1.95(dd,J=7.3,3.2Hz,2H),1.00(t,J=7.4Hz,3H).

[0306] Step C: Compound 21-3 (8.0 mg, 0.01 mmol) and formic acid (2.6 mg, 0.06 mmol) were added to acetonitrile (10 mL). The reaction system was stirred at 70°C under argon for 3 hours. After LCMS monitoring showed no change in product content, the reaction solution was concentrated and the crude product was purified by silica gel column chromatography to obtain the title compound (1.8 mg, 25.4% yield).

[0307] LCMS (ESI): m / z: 503.1 [M+H] + .

[0308] 1 H NMR (400MHz, DMSO-d6) δ10.41(d,J=1.4Hz,1H),7.82(d,J=11.2Hz,1H),7.3 1(s,1H),6.53(d,J=10.4Hz,2H),5.78–5.69(m,1H),5.38(s,2H),4.96(dd,J =14.5,4.3Hz,1H),4.24(dd,J=17.6,1.7Hz,1H),2.42(s,3H),2.02–1.96(m ,2H),1.85(dd,J=7.7,7.1Hz,2H),1.47-1.45(m,3H),0.86(d,J=1.0Hz,3H).

[0309] Example 22:

[0310] Preparation of ((1S,9S)-1-(2,4-dioxoimidazolin-1-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[2,1-g]quinoline-10,13-dione (Compound 22)

[0311] Reaction route:

[0312] Steps:

[0313] Step A: Dissolve compound 1-1 (30.0 mg, 0.07 mmol) and ethyl chloroacetate (33.6 mg, 0.28 mmol) in dichloromethane (3 mL). Slowly add triethylamine (2 mL) in anhydrous and oxygen-free conditions, and stir at room temperature for 5 h. After LCMS monitoring shows the disappearance of the starting material, quench the reaction solution with water (20 mL). The mixture is extracted with dichloromethane (50 mL × 3). The lower organic phases are combined, washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue is purified by silica gel chromatography (developing solvent: dichloromethane / methanol = 10 / 1) to obtain compound 22-2 (12.0 mg, 41.6% yield).

[0314] LCMS (ESI) m / z: 522.20 [M+H] + .

[0315] Step B: Compound 22-2 (10.0 mg, 0.02 mmol) was added to a 10% hydrochloric acid solution, followed by the addition of potassium cyanate (6.2 mg, 0.08 mmol), and the mixture was heated under reflux at 90°C with stirring for 3 h. After completion of the reaction, the reaction solution was purified by prep-HPLC in 0-100% acetonitrile / water (0.1% trifluoroacetic acid) and lyophilized to obtain the title compound (1.8 mg, yield 17.6%).

[0316] LCMS (ESI) m / z: 519.10 [M+H] + .

[0317] 1 H NMR (400MHz, CD3OD) δ7.74–7.60(m,2H),5.75–5.69(m,1H),5.57(dd,J=16.3,3 .1Hz,1H),5.39(d,J=5.7Hz,1H),5.28–5.19(m,1H),5.09(dd,J=18.5,4.2Hz,1 H),4.12(dd,J=17.6,6.7Hz,1H),3.78(dd,J=17.5,11.0Hz,1H),2.45(s,3H),2 .18(d,J=7.7Hz,4H),2.03(s,3H),1.95(d,J=5.6Hz,1H),1.00(t,J=7.4Hz,3H).

[0318] Example 23:

[0319] Preparation of (1S,9S)-1-(2,5-dioxoimidazolin-1-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[2,1-g]quinoline-10,13-dione (Compound 23)

[0320] Reaction route:

[0321] Steps:

[0322] Step A: Dissolve compound 1-1 (20.0 mg, 0.05 mmol) and compound 23-2 (12.0 mg, 0.09 mmol) in chloroform (10 mL). Stir and react at room temperature for 2 hours under argon protection. After LCMS analysis shows the reaction is complete, the chloroform in the reaction solution is concentrated under reduced pressure. Add hydrochloric acid (5 mL) and ethanol (5 mL), and react at 80°C under argon protection for 16 hours. After LCMS analysis shows the reaction is complete, add 5 mol / L sodium hydroxide solution to adjust the pH to 7-8, and extract with dichloromethane (10 mL x 3). The lower organic phases are combined and dried over anhydrous sodium sulfate. Finally, the organic phases are concentrated under reduced pressure and dried to obtain the crude compound, which is purified by silica gel column chromatography to obtain the title compound (5.0 mg, yield 21.0%).

[0323] LCMS (ESI) m / z: 519.1 [M+H] + .

[0324] 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),7.79(d,J=11.0Hz,1H),7.31(d,J=1.6Hz,1H),6.53(s,1H),5.65(s,1H),5.40(s,2H),5.06(d,J=18.3Hz,1H ),4.92(d,J=18.5Hz,1H),4.08–3.89(m,2H),3.26–3.08(m,2H),2.39(s ,3H),2.32–2.19(m,1H),2.07–1.71(m,3H),0.87(td,J=7.2,3.1Hz,3H).

[0325] Example 24:

[0326] Preparation of 3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizin[1,2-b]quinolin-1-yl)-1-hydroxy-1-methylurea (Compound 24)

[0327] Reaction route:

[0328] Step A:

[0329] Compound 1-1 (30.0 mg, 0.07 mmol), N,N-diisopropylethylamine (0.2 mL), and N,N'-carbonyldiimidazole (22.0 mg, 0.14 mmol) were dissolved in tetrahydrofuran (2 mL) and stirred at room temperature for 30 min. Methylhydroxylamine hydrochloride (12.0 mg, 0.14 mmol) and N,N-diisopropylethylamine (0.2 mL) were then added and allowed to react at room temperature for 18 hours. LCMS monitoring indicated the reaction was complete. The mixture was diluted with ethyl acetate (50 mL) and washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to afford the title compound (1.5 mg, 4.4% yield).

[0330] LCMS (ESI) m / z: 509.2 [M+H] + .

[0331] 1 H NMR(400MHz,DMSO-d6)δ9.90–9.70(m,1H),8.46(s,1H),7.76(d,J=11.1Hz,1H ),7.69(d,J=9.3Hz,1H),7.32(s,1H),6.61(d,J=52.8Hz,1H),5.43(s,2H),5.3 9–5.27(m,2H),5.10(d,J=18.8Hz,1H),3.12(s,3H),2.39(s,3H),2.19(s,2H) ,2.01(d,J=7.9Hz,1H),1.91–1.84(m,1H),1.46(s,1H),0.87(m,J=7.2Hz,3H).

[0332] Example 25:

[0333] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-((S)-3,3,3-trifluoro-2-hydroxypropyl)amino)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 25-P1 or Compound 25-P2)

[0334] as well as

[0335] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-((R)-3,3,3-trifluoro-2-hydroxypropyl)amino)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 25-P2 or Compound 25-P1)

[0336] Reaction route:

[0337] Steps:

[0338] Step A: Compound 1-1 (30.0 mg, 0.07 mmol) and 1,1,1-trifluoro-2,3-epoxypropane (0.8 mL) were dissolved in acetonitrile (0.2 mL). The reaction system was sealed in a 70°C oil bath for 16 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by adding water (100 mL). The mixture was diluted with ethyl acetate (500 mL), washed sequentially with water (100 mL × 3) and saturated sodium chloride solution (100 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC using 0-100% acetonitrile / water (0.1% hydrochloric acid) to afford Compound 25-P1 (26.8 mg, 8.0% yield) and Compound 25-P2 (20.1 mg, 6.0% yield).

[0339] Compound 25-P1:

[0340] LCMS (ESI) m / z: 548.2 [M+H] + .

[0341] HPLC conditions: 0-13 min, 10-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 6.732 min.

[0342] 1 H NMR (400MHz, CD3OD) δ7.74(d,J=10.9Hz,1H),7.62(s,1H),5.57(m,2H),5.41(m,2H),5.05(s,1H),4.69(s,1H),3.65(s,1H),3.35(d,J= 15.3Hz,2H),3.23–3.12(m,1H),2.81(s,1H),2.47(s,3H),2.43–2.32(m,1H),1.96(d,J=7.3Hz,2H),1.29(s,1H),1.00(t,J=7.2Hz,3H).

[0343] Compound 25-P2:

[0344] LCMS (ESI) m / z: 548.2 [M+H] + .

[0345] HPLC conditions: 0-13 min, 10-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 6.194 min.

[0346] 1 H NMR (400MHz, CD3OD) δ7.73(d,J=10.3Hz,1H),7.63(s,1H),5.55(m,2H),5.47–5.36(m,2H),5.09(s,1H),4.51(s,1H),3.60(s,1H),3 .40(s,2H),3.25–3.19(m,1H),2.89–2.64(m,1H),2.46(s,3H),2.42–2.36(m,1H),1.95(s,2H),1.29(s,1H),1.01(t,J=6.8Hz,3H).

[0347] Example 26:

[0348] Preparation of (Z)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[2,1-g]quinolin-1-yl)-N'-hydroxyacetimidamide (Compound 26)

[0349] Reaction route:

[0350] Steps:

[0351] Step A: Compound 26-1 (1.0 g, 16.93 mmol) was dissolved in N,N-dimethylformamide (20 mL), and N-chlorosuccinimide (983.2 mg, 16.93 mmol) was added. The mixture was stirred at 50°C under argon for 2 hours. After the reaction was complete as indicated by a spectrophotometer, the reaction solution was quenched by adding water (100 mL). The mixture was extracted with dichloromethane (50 mL × 3). The lower organic phases were combined, washed with saturated brine (10 mL × 3), and then dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1) to obtain compound 26-2 (200.0 mg, yield 12.6%).

[0352] Step B: Dissolve compound 1-1 (20.0 mg, 0.05 mmol) in dichloromethane (20 mL), add compound 26-2 (13.0 mg, 0.09 mmol), and react at room temperature under argon for 4 h. After completion of the reaction, LCMS analysis indicated that the dichloromethane in the reaction solution was concentrated under reduced pressure to remove the residue. The residue was purified by prep-HPLC in 0-100% acetonitrile / water (0.1% trifluoroacetic acid) and lyophilized to obtain the title compound (1.9 mg, 8.0% yield).

[0353] LCMS (ESI) m / z: 493.1 [M+H] + .

[0354] 1 H NMR(400MHz,DMSO-d6)δ9.14(d,J=4.3Hz,1H),8.77(s,1H),8.41(s,1H),7.76 (d,J=10.6Hz,1H),7.31(s,1H),6.51(s,1H),6.36–6.17(m,1H),5.41-5.40(m, 1H),5.23(d,J=19.5Hz,1H),5.06(s,1H),3.09(s,2H),2.38(s,3H),2.05(d,J= 29.4Hz,2H),1.95(d,J=21.5Hz,3H),1.89–1.85(m,2H),0.87(d,J=7.3Hz,3H).

[0355] Example 27:

[0356] Preparation of (Z)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[2,1-g]quinolin-1-yl)-N',2-dihydroxyacetimidamide (Compound 27)

[0357] Reaction route:

[0358] Steps:

[0359] Step A: Dissolve compound 27-1 (1.0 g, 6.00 mmol) and hydroxylamine hydrochloride (1.9 g, 9.00 mmol) in tetrahydrofuran (10 mL), and slowly add 5 mL of pyridine. Stir the reaction system at room temperature overnight. After LCMS monitoring shows the disappearance of the starting material, cool the reaction solution to room temperature and quench it with water (10 mL). The mixture is diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL x 3) and saturated sodium chloride solution (10 mL x 1). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue is purified by prep-HPLC using 0-100% acetonitrile / water (0.1% trifluoroacetic acid) to afford compound 27-2 (750.0 mg, 75.0% yield) as a colorless oil.

[0360] LCMS (ESI) m / z: 166.1 [M+H] + :.

[0361] Step B: Dissolve compound 27-2 (160.0 mg, 0.97 mmol) and N-chlorosuccinimide (128.2 mg, 0.97 mmol) in N,N-dimethylformamide (3 mL) and stir at 50°C for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by adding water (50 mL). The mixture was diluted with ethyl acetate (50 mL) and washed sequentially with water (50 mL × 3) and saturated sodium chloride solution (30 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was crude compound 27-3 (140.0 mg, 87.0% yield).

[0362] LCMS (ESI) m / z: 200.63 [M+H] + .

[0363] Step C: Dissolve compound 1-1 (40.0 mg, 0.10 mmol), 27-3 (55.0 mg, 0.30 mmol), and triethylamine (5 mL) in dichloromethane (2 mL). Stir the reaction at room temperature for 3 hours. After LCMS monitoring indicated the disappearance of the starting material, cool the reaction to room temperature and quench with water (50 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (50 mL × 3) and saturated sodium chloride solution (30 mL × 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC using 0-100% acetonitrile / water (0.1% ammonia) to afford compound 27-4 (15.0 mg, 37.5% yield).

[0364] LCMS (ESI) m / z: 599.22 [M+H] + .

[0365] 1 1 H NMR(400MHz, CDCl3)δ7.55(d,J=27.0Hz,2H),7.47–7.30(m,4H),7.24–7.13(m,H),5.52(s,2H),5.08(s,1H),4.85–4.44(m,3H) ),3.15(s,1H),2.93(s,1H),2.37(s,3H),2.17(s,1H),1.27(s,2H),1.25(s,3H),1.23–1.18(m,1H),1.02(s,3H),0.88(s,1H).

[0366] Step D: Dissolve compound 27-4 (30.0 mg, 0.1 mmol) and trifluoromethanesulfonic acid (0.5 mL) in dichloromethane (2 mL), and stir the reaction at room temperature for 3 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by adding water (10 mL). The mixture was diluted with ethyl acetate (30 mL), washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC using 0-100% acetonitrile / water (0.1% ammonia) to afford the title compound (7.0 mg, 46.7% yield).

[0367] LCMS (ESI) m / z: 509.18 [M+H] + .

[0368] 1H NMR (400MHz, CD3OD) δ7.72–7.39(m,2H),5.52(ddd,J=15.6,13.9,5.0Hz,2H),5.40–5.23(m,3H),4. 80(dd,J=27.5,12.8Hz,2H),3.41(dd,J=17.0,3.9Hz,1H),3.22(dd,J=17.5,12.2Hz,1H),2.69–2.57 (m, 1H), 2.42 (d, J = 5.7Hz, 3H), 1.93 (dd, J = 14.7, 7.2Hz, 2H), 1.31 (d, J = 21.7Hz, 2H), 0.98 (t, J = 7.4Hz, 3H).

[0369] Example 28:

[0370] Preparation of (Z)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[2,1-g]quinolin-1-yl)-N'-hydroxyacetimidamide (Compound 28)

[0371] Reaction route:

[0372] Steps:

[0373] Step A: Dissolve compound 28-1 (1.0 g, 6.28 mmol) and hydroxylamine hydrochloride (654.8 mg, 9.42 mmol) in methanol (5 mL). Slowly add pyridine (1.5 mL) and stir overnight at room temperature. After TLC indicates complete reaction, quench the reaction mixture with water (30 mL). The mixture is extracted with dichloromethane (50 mL × 3). The lower organic phases are combined, washed with saturated brine (10 mL × 3), and dried over anhydrous sodium sulfate. Finally, the solvent is evaporated under reduced pressure to obtain crude compound 28-2 (600.0 mg, 60% yield).

[0374] LCMS (ESI): m / z [M+H + ]calcd for C7H 14 N2O3:174.20,mass found:no mass.

[0375] 1H NMR (400MHz, MeOD) δ7.43 (ddd, J=7.7, 4.4, 1.4Hz, 1H), 3.93 (d, J=3.7Hz, 1H), 3.74 (d, J=5.2Hz, 1H), 1.44 (d, J=2.2Hz, 9H).

[0376] Step B: Compound 28-2 (200.0 mg, 1.15 mmol) was dissolved in N,N-dimethylformamide (3 mL), and N-chlorosuccinimide (154.4 mg, 1.15 mmol) was added. The mixture was reacted at 50°C for 3 hours. After TLC showed that the reaction was complete, the reaction solution was quenched by adding water (30 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 3), and then dried over anhydrous sodium sulfate. Finally, the organic phase was concentrated under reduced pressure to obtain crude compound 28-3 (100.0 mg, yield 50.0%), which was used directly in the next step without further purification.

[0377] LCMS (ESI): m / z [M+H + ]calcd for C7H 13 ClN2O3:208.64,mass found:no mass.

[0378] Step C: Compound 1-1 (20.0 mg, 0.05 mmol) and compound 28-3 (28.7 mg, 0.14 mmol) were dissolved in dichloromethane (5 mL), and triethylamine (0.5 mL) was slowly added. The mixture was stirred at room temperature under anhydrous and oxygen-free conditions for 5 h. After LCMS analysis showed the reaction was complete, the dichloromethane in the reaction solution was concentrated under reduced pressure to remove it. The residue was purified by prep-HPLC in 0-100% acetonitrile / water (0.1% ammonia) and lyophilized to obtain compound 28-4 (8.0 mg, 40% yield).

[0379] LCMS (ESI) m / z: 608.3 [M+H] + .

[0380] Step D: Compound 28-4 (8.0 mg, 0.01 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was slowly added. The mixture was stirred at room temperature under anhydrous and oxygen-free conditions for 6 hours. After completion of the reaction, the dichloromethane in the reaction solution was concentrated under reduced pressure by LCMS. The residue was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% ammonia) and lyophilized to afford the title compound (1.1 mg, 16.7% yield).

[0381] LCMS (ESI) m / z: 608.3 [M+H] + .

[0382] 1 H NMR(400MHz, DMSO-d6)δ9.98(d,J=3.0Hz,1H),8.41(s,3H),7.79(d,J=10.6Hz,1H),7.33(s,3H),6.53(s,1H),5.57–5.24(m,4H ),5.05(s,1H),3.96(d,J=7.9Hz,2H),3.19(d,J=10.6Hz,2H),2.39(s,3H),2.28(s,1H),2.10(s,1H),1.87(s,2H),0.88(s,3H).

[0383] Example 29:

[0384] Preparation method of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-(hydroxymethyl)benzyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[2,1-g]quinoline-10,13-dione (Compound 29)

[0385] Reaction route:

[0386] Steps:

[0387] Step A: Compound 1-1 (30.0 mg, 0.07 mmol), p-hydroxymethylbenzaldehyde (57.1 mg, 0.42 mmol), and sodium cyanoborohydride (26.0 mg, 0.42 mmol) were dissolved in N,N-dimethylformamide (2 mL). The reaction was stirred at room temperature for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of water (10 mL). The mixture was diluted with ethyl acetate (50 mL), washed sequentially with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to yield the title compound (10.0 mg, 33.3% yield).

[0388] LCMS (ESI) m / z: 556.2 [M+H] + .

[0389] 1H NMR (400MHz, CD3OD) δ7.63–7.55(m,2H),7.49–7.35(m,4H),5.56(d,J=16.4Hz,1H),5.37(d,J= 16.3Hz,1H),5.24(t,J=12.3Hz,1H),5.08(d,J=19.1Hz,1H),4.62(s,1H),4.58(s,1H),4.23(s, 1H),4.08(d,J=12.7Hz,1H),3.95(d,J=13.2Hz,1H),3.23–3.03(m,1H),2.40(d,J=7.3Hz,3H), 2.34(d,J=5.1Hz,1H),2.19(s,1H),1.95(m,2H),1.31(d,J=18.0Hz,2H),1.00(t,J=7.4Hz,3H).

[0390] Example 30:

[0391] Preparation of N-((S)-9-benzyl-1-(4-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)methylphenyl)-5,8,11,14-tetraoxy-2-oxo-2-oxy-4,7,10,13-tetraaza-15-yl)-6-2,5-dioxy-2-dihydro-2-dioxy-1H-pyrancarboxamide (Compound 30)

[0392] Reaction route:

[0393] Steps:

[0394] Step A: Compound LKO (100.0 mg, 0.25 mmol) and 4-hydroxymethylbenzaldehyde (110.0 mg, 0.80 mmol) were dissolved in anhydrous dichloromethane (5 mL). PPTs (20.0 mg, 0.08 mmol) was added, and the reaction system was stirred at room temperature for 16 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was quenched by adding water (20 mL). The mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. Purification by column chromatography (eluent: dichloromethane / methanol = 25 / 1) afforded compound 30-2 (135.0 mg, 38.0% yield).

[0395] LCMS (ESI) m / z: 462 [M+18] + .

[0396] Step B: Dissolve compound 1-1 (30.0 mg, 0.06 mmol) and compound 30-2 (90.0 mg, 0.18 mmol) in anhydrous N,N-dimethylformamide (1 mL). Add sodium cyanoborohydride (25.0 mg, 0.32 mmol) at room temperature, and stir the reaction at 25°C for 2 hours. After LCMS monitoring shows the disappearance of the starting material, quench the reaction by adding water (20 mL). The mixture is extracted with dichloromethane (10 mL x 3). The combined organic phases are washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. Purification by column chromatography (eluent: dichloromethane / methanol = 25 / 1) affords compound 30-3 (12.0 mg, 23% yield).

[0397] LCMS (ESI) m / z: 864.4 [M+H] + .

[0398] Step C: Compound 30-3 (13.0 mg, 0.02 mmol) and piperidine (0.01 mL) were dissolved in anhydrous N,N-dimethylformamide (0.7 mL), and the reaction system was stirred at 0°C for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The mixture was purified by column chromatography (eluent: dichloromethane / methanol = 20 / 1) to obtain compound 30-4 (8.9 mg, yield 93.3%).

[0399] LCMS (ESI) m / z: 642.3 [M+H] + .

[0400] Step D: Compound 30-4 (8.9 mg, 0.01 mmol) and compound LK1 (9.9 mg, 0.021 mmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.9 mg, 0.021 mmol) and N,N-diisopropylethylamine (0.01 mL) were added at 0°C, and the reaction system was stirred at 0°C for 2 hours.

[0401] After LCMS monitoring showed the disappearance of the starting material, water (20 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with dichloromethane (10 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 3), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The product was purified by prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to give the title compound (1.4 mg, 6.5% yield).

[0402] LCMS (ESI) m / z: 1096.5 [M+H] +

[0403] 1 H NMR (400MHz, DMSO-d6) δ8.58(s,1H),8.31(s,2H),8.08(m,3H),7.74(d,J=10.9Hz,1H),7.42(d,J=7.8Hz,2H),7.30–7. 14(m,6H),6.98(s,1H),6.51(s,1H),5.33(m,3H),4.62(d,J=7.0Hz,2H),4.47(m,2H),4.22(s,1H),3.99(d,J=13.5Hz, 1H),3.88(d,J=13.3Hz,2H),3.79–3.53(m,12H),3.05-2.98(m,2H),2.85–2.63(m,2H),2.38(s,3H),2.30-2.24(m,2H) ,2.07–1.94(m,2H),1.87(d,J=8.4Hz,2H),1.46(dd,J=14.7,7.8Hz,4H),1.16(d,J=8.8Hz,2H),0.86(d,J=6.8Hz,3H).

[0404] Example 31:

[0405] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-(hydroxymethyl)-4H-1,2,4-triazol-4-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[2,1-g]quinoline-10,13-dione (Compound 31)

[0406] Reaction route:

[0407] Steps:

[0408] Step A: Dissolve compound 1-1 (2.2 mg, 0.01 mmol), hydroxyacetohydrazide (13 mg, 0.02 mmol), and N,N-dimethylformamide dimethyl acetal (17.8 mg, 0.015 mmol) in acetonitrile (2 mL). Heat under reflux for 3 hours. After evaporating the acetonitrile to dryness, add acetic acid (0.1 mL). Heat to 100°C overnight. After LCMS monitoring indicated the disappearance of the starting material, the reaction mixture was cooled to room temperature and analyzed by prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) to yield the title compound (2.1 mg, 80.8% yield).

[0409] LCMS (ESI) m / z: 518.2 [M+H] + .

[0410] 1 H NMR (400MHz, DMSO-d6) δ8.38 (s, 1H), 7.87 (d, J = 10.8Hz, 1H), 7.31 (s, 1H), 7.28-6.93 (m, 1H), 6.53(d,J=6.8Hz,1H),6.27-6.20(m,1H),5.87-5.84(m,1H),5.42-5.24(m,3H),5.04(d,J=19.0Hz,1H),4.86(d,J=5.6Hz,1H) ,3.62(q,J=5.0Hz,1H),2.98-2.97(m,1H),2.43(s,3H),2.00(q,J=7.1Hz,2H),1.91-1.78(m,J=6.7Hz,2H),0.87-0.83(m,3H).

[0411] Example 32:

[0412] Preparation of (1S,9S)-1-((S)-2,3-dihydroxypropyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 32)

[0413] Reaction route:

[0414] Steps:

[0415] Step A: Dissolve compound 1-1 (20.0 mg, 0.05 mmol) and glycidol (3 mL) in ACN (3 mL). The mixture was sealed in a microwave tube and stirred at room temperature overnight. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was quenched by adding water (20 mL). The mixture was extracted with dichloromethane (30 mL x 3). The organic phases were combined, washed with saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC using 0-100% acetonitrile / water (0.1% formic acid) and lyophilized to afford the title compound (1.5 mg, 7.4% yield).

[0416] LCMS (ESI) m / z: 510.20 [M+H] + .

[0417] 1 H NMR(400MHz,CD3OD)δ8.37(s,1H),7.62(s,1H),7.59(s,1H),5.58(d,J=16.3Hz,1H) ,5.51(d,J=19.0Hz,1H),5.42–5.35(m,2H),5.33(s,1H),4.39(s,2H),3.81(s,1H),3 .59(d,J=5.6Hz,2H),3.14–3.10(m,1H),3.08(d,J=3.8Hz,1H),2.78(dd,J=11.9,7.5 Hz,1H),2.41(s,3H),2.33(s,2H),1.96(dd,J=7.4,3.8Hz,2H),1.01(t,J=7.4Hz,3H)

[0418] Example 33:

[0419] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizin[1,2-b]quinolin-1-yl)cyanamide (Compound 33-INT)

[0420] as well as

[0421] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinolin-1-yl)-3-hydroxyguanidine (Compound 33)

[0422] Reaction route:

[0423] Steps:

[0424] Step A: Compound 1-1 (20.0 mg, 0.05 mmol) was dissolved in methanol (10 mL), cooled to 0°C, and sodium acetate (11.3 mg, 0.14 mmol) and cyanogen bromide (14.6 mg, 0.14 mmol) were added under argon. The mixture was heated to room temperature and stirred for 6 hours. After LCMS showed that the starting material had reacted completely, the reaction solution was quenched by adding water (20 mL). The mixture was extracted with dichloromethane (50 mL × 3). The lower organic phases were combined and dried over anhydrous sodium sulfate. Finally, the organic phases were concentrated under reduced pressure and purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% formic acid) to obtain compound 33-2 (4.0 mg, yield 18.9%).

[0425] LCMS (ESI) m / z: 461.1 [M+H] + .

[0426] 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=10.9Hz,1H),7.76(d,J=5.5Hz,1H),7.35(s,1H),6.55(s,1H),5.45(t,J=9.3Hz,3H),5.36(d,J=19.3Hz,1H),5 .03(d,J=4.5Hz,1H),3.21–3.15(m,2H),2.40(s,3H),2.31–2.24(m,1H) ,2.00(dt,J=12.6,7.9Hz,1H),1.91–1.81(m,2H),0.89(t,J=7.2Hz,3H).

[0427] Step B: Compound 33-2 (16.0 mg, 0.04 mmol) was dissolved in anhydrous ethanol (10 mL), and triethylamine (10.6 mg, 0.11 mmol) and hydroxylamine hydrochloride (7.3 mg, 0.11 mmol) were added. The mixture was heated to 80°C and stirred for 6 hours. After LCMS showed that the starting material had reacted completely, the reaction solution was quenched by adding water (10 mL). The mixture was extracted with dichloromethane (50 mL x 3). The lower organic phases were combined and dried over anhydrous sodium sulfate. Finally, the organic phases were concentrated under reduced pressure and purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (1.4 mg, 7.8% yield).

[0428] LCMS (ESI) m / z: 494.1 [M+H] +

[0429] 1 H NMR(400MHz,DMSO-d6)δ10.81(s,1H),10.03(s,1H),8.49(s,1H),8.10(s ,1H),7.83(d,J=10.8Hz,1H),7.35(s,1H),6.54(s,1H),5.43(s,2H),5.35 (d,J=4.9Hz,1H),5.25(s,1H),3.11(dd,J=8.9,5.4Hz,2H),2.54(s,1H), 2.41(s,3H),2.34–2.16(m,3H),1.92–1.82(m,2H),0.88(t,J=7.3Hz,3H).

[0430] Example 34:

[0431] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizin[1,2-b]quinolin-1-yl)-3-hydroxypropane-1-sulfonamide (Compound 34)

[0432] Reaction route:

[0433] Steps:

[0434] Step A: Dissolve compound 34-1 (2.5 g, 16.30 mmol) and sodium sulfite (2.0 g, 16.30 mmol) in water (75 mL). Heat the reaction system to 100°C and stir for 24 hours. The reaction solution is lyophilized to obtain crude compound 34-2 (4.0 g), which is used directly in the next step.

[0435] Step B: Compound 34-2 (0.5 g, crude) was dissolved in phosphorus oxychloride (15 mL), the reaction system was heated to 110 °C and stirred for 3 hours, and then concentrated under reduced pressure to give compound 34-3 (1.0 g, crude), which was used directly in the next step.

[0436] Step C: Compound 34-3 (150.0 mg, crude product) was dissolved in dichloromethane (2 mL), and compound 1-1 (15.0 mg, 0.03 mmol) and triethylamine (0.25 mL) were added, and the mixture was reacted at room temperature under argon protection for 4 hours.

[0437] After LCMS detection showed that the dichloromethane in the reaction solution was completed, it was concentrated under reduced pressure to remove the methylene chloride. The residue was purified by preparative liquid chromatography under the conditions of 0-100% acetonitrile / water (0.1% formic acid) and lyophilized to obtain compound 34-Me (2.5 mg, yield 13.0%).

[0438] LCMS (ESI) m / z: 572.2 [M+H] + .

[0439] 1 H NMR(400MHz,MeOD)δ8.11(s,1H),7.73-7.58(m,2H),5.61-5.57(m,2H),5.48-5.41(m,2H),3.74-3.40(m,4H),3 .36(s,3H),3.27-3.10(m,3H),2.45(s,3H),2.22-2.04(m,4H),2.01-1.91(m,3H),0.99(dt,J=7.6,5.5Hz,3H).

[0440] Step D: Compound 34-Me (2.5 mg, 0.01 mmol) was dissolved in anhydrous dichloromethane (2.5 mL). Boron tribromide (0.01 mL) was added dropwise at 0°C. The reaction system was warmed to room temperature and stirred for 6 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure and purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% trifluoroacetic acid) to provide the title compound (1.2 mg, 53.8% yield).

[0441] LCMS (ESI) m / z: 558.1 [M+H] + .

[0442] 1 H NMR (400MHz, DMSO-d6) δ7.84(dd,J=34.6,9.7Hz,2H),7.32(s,1H),7.28-6.93(m,3H),6.52(d,J=9.2Hz,1H),4.72(s,1H),3. 51(dd,J=28.2,14.8Hz,5H),3.21-3.16(m,2H),2.39(s,3H),2.25(d,J=5.8Hz,2H),1.96-1.78(m,4H),0.88(t,J=7.3Hz,3H).

[0443] Example 35:

[0444] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinolin-1-yl)-4-(hydroxymethyl)benzamide (Compound 35)

[0445] Reaction route:

[0446] Compound 1-1 (30.0 mg, 0.07 mmol), p-hydroxymethylbenzoic acid (63.0 mg, 0.41 mmol), N,N-diisopropylethylamine (36.0 mg, 0.28 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (53.0 mg, 0.14 mmol) were dissolved in N,N-dimethylformamide (2 mL) in sequence; the reaction system was stirred at room temperature for 2 hours.

[0447] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was cooled to room temperature and quenched by the addition of water (10 mL). The mixture was diluted with ethyl acetate (50 mL) and washed sequentially with water (50 mL × 3) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was dissolved in N,N-dimethylformamide and purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (10.0 mg, 33.3% yield).

[0448] LCMS (ESI) m / z: 570.59 [M+H] + .

[0449] 1 H NMR (400MHz, DMSO-d6) δ8.98(d,J=8.4Hz,1H),7.86(dd,J=34.8,9.6Hz,3H),7.49–7.24(m,2H),6.52(s,1H),5.79(dd,J=13.0,6.6Hz,1H),5. 47–4.98(m,6H),4.55(s,2H),3.20–3.11(m,2H),2.42(s,3H),2.28–2. 24(m,1H),2.02–1.94(m,1H),1.90–1.73(m,2H),0.85(d,J=7.4Hz,3H).

[0450] Example 36:

[0451] Preparation of (Z)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-N'-hydroxycyclopropylamidine (Compound 36)

[0452] Reaction route:

[0453] Steps:

[0454] Step A: Dissolve cyclopropanecarboxaldehyde (1.0 g, 14.3 mmol) and hydroxylamine hydrochloride (1.49 g, 21.4 mmol) in methanol (5 mL), then slowly add pyridine (1.5 mL), and stir the reaction at room temperature overnight. Add water (20 mL) to the reaction solution to quench the reaction. The mixture is extracted with dichloromethane (50 mL × 3), and the lower organic phases are combined. The organic phases are first washed with saturated brine (10 mL × 3), and then dried over anhydrous sodium sulfate. Finally, the organic phase is concentrated under reduced pressure. The resulting product 36-2 (839.0 mg, white solid, crude product) is directly used in the next step.

[0455] Step B: 36-2 (839.0 mg, 9.90 mmol) was dissolved in N,N-dimethylformamide, and N-chlorosuccinimide (1.3 g, 9.86 mmol) was added with stirring at room temperature. The mixture was heated under reflux at 50°C with stirring for 3 hours. Water (20 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (50 mL×3). The upper organic phases were combined, washed with saturated brine (10 mL×3), and then dried over anhydrous sodium sulfate. Finally, the organic phase was concentrated under reduced pressure to obtain compound 36-3 (1.3 g, crude product), which was directly used in the next step.

[0456] Step C: Compound 36-3 (14.3 mg, 0.12 mmol) and compound 1-1 (30.0 mg, 0.06 mmol) were dissolved in 5 mL of dichloromethane. Triethylamine (0.02 mL) was added dropwise with stirring. The mixture was reacted at room temperature for 4 h. The solvent was evaporated to dryness by rotary evaporation. The title compound (2.7 mg, 6.0% yield) was obtained by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% ammonia water) to give the title compound (2.7 mg, 6.0% yield).

[0457] LCMS (ESI) m / z: 519.3 [M+H] +

[0458] 1H-NMR(400MHz,MeOD):7.60(d,J=12.3Hz,1H),7.10(d,J=22.1Hz,2H),5.57-5.49(m,3H),5.37-5.33(m ,2H),5.22-5.17(m,1H),2.41(s,3H),2.22-2.14(m,3H),1.99(dd,J=30.1,7.8Hz,4H),1.00(t,J=7.2Hz 3H),0.89(d,J=7.1Hz,5H).

[0459] Example 37:

[0460] Preparation of 1-cyclopropyl-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-1-hydroxyurea (Compound 37)

[0461] Reaction route:

[0462] Steps:

[0463] Step A: Sodium tungstate (120.0 mg, 0.37 mmol) and urea peroxide (200.0 mg, 2.13 mmol) were added to a solution of cyclopropylamine (200.0 mg, 3.51 mmol) in diethyl ether (15 mL). The mixture was stirred at room temperature under argon for 2 hours. After TLC monitoring indicated complete reaction, dichloromethane (50 mL) was added to the reaction solution, which was then dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to yield crude compound 37-2 (200.0 mg). Proceed directly to the next step.

[0464] Step B: Compound 37-2 (30.0 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL). Triethylamine (0.5 mL) and N,N'-carbonyldiimidazole (162.0 mg, 1.00 mmol) were added and stirred for 1 hour. Compound 1-1 (50.0 mg, 0.68 mmol) was then added. The mixture was reacted at room temperature under argon for 3 hours. After LCMS analysis indicated completion of the reaction, the dichloromethane in the reaction solution was concentrated under reduced pressure to remove it. The residue was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% trifluoroacetic acid) and lyophilized to afford the title compound (1.0 mg, 3.5% yield).

[0465] LCMS (ESI) m / z: 535.2 [M+H] + .

[0466] 1 H NMR(400MHz,MeOD)δ8.49(s,1H),7.78-7.52(m,3H),5.60-5.58(m,2H),2.69(s,2H),2.52-2.49(m,3H),2.41(s, 1H),2.21-217(m,2H),1.96-1.94(m,4H),1.70-1.65(m,2H),1.60(s,1H),1.02-0.97(m,2H),0.95-0.88(m,2H).

[0467] Example 38:

[0468] Preparation of 3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-1-hydroxy-1-phenylurea (Compound 38)

[0469] Reaction route:

[0470] Steps:

[0471] Step A: Compound 1-1 (27.0 mg, 0.07 mmol) was dissolved in 5 mL of dichloromethane. Triethylamine (13.0 mg, 0.13 mmol, 0.01 mL) was slowly added dropwise with stirring until the mixture became clear. N,N'-carbonyldiimidazole (20.0 mg, 0.13 mmol) was slowly added. After stirring at room temperature for 1 h, phenylhydroxylamine (8.0 mg, 0.1 mmol) was slowly added and stirred at room temperature for 4 h. The reaction solution was quenched with water and washed three times with dichloromethane (20 mL). The organic phases were combined and washed three times with water (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The crude product was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% ammonia) to obtain the title compound (2.6 mg, 8.0% yield).

[0472] LCMS (ESI) m / z: 571.2 [M+H] + .

[0473] 1H NMR (400MHz, DMSO-d6): δ10.40(s,1H),7.98(d,J=8.7Hz,1H),7.78(d,J=11.1Hz, 1H),7.64(d,J=7.6Hz,1H),7.36-7.30(m,2H),7.19(s,1H),7.09(s,1H),6.51(s, 1H),5.41(s,2H),5.37(s,1H),5,32(t,J=4.6Hz,2H),5.21(d,J=19.3Hz,1H),2.4 0(s,3H),2.30(d,J=25.4Hz,2H),1.99(s,2H),1.86(s,2H),0.85(d,J=7.0Hz,3H).

[0474] Example 39:

[0475] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-N'-hydroxycarboxamidine (Compound 39)

[0476] Reaction route:

[0477] Steps:

[0478] Step A: Formaldehyde oxime (300.0 mg, 6.67 mmol) was dissolved in N,N-dimethylformamide (3 mL), and N-chlorosuccinimide (889.0 mg, 6.67 mmol) was added. The mixture was stirred at 50°C under argon for 2 hours. After TLC indicated complete reaction, the reaction solution was quenched by adding water (100 mL). The mixture was extracted with dichloromethane (50 mL × 3). The lower organic phases were combined, washed with saturated brine (10 mL × 3), and dried over anhydrous sodium sulfate. The organic phases were concentrated under reduced pressure to obtain crude compound 39-2 (400.0 mg), which was directly carried to the next step.

[0479] Step B: Dissolve compound 1-1 (20.0 mg, 0.05 mmol) in dichloromethane (5 mL). Add triethylamine (0.01 mL) and compound 39-2 (40 mg, crude product). React at room temperature under argon for 4 hours. After LCMS analysis indicated completion of the reaction, the dichloromethane was removed from the reaction solution under reduced pressure. The residue was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% ammonia) and lyophilized to afford the title compound (1.0 mg, 4.1% yield).

[0480] LCMS (ESI) m / z: 479.1 [M+H] +

[0481] 1 H NMR (400MHz, MeOD) δ7.69-7.63(m,1H),7.35(s,2H),5.58(d,J=16.2Hz,1H),5.42(d,J=11.8Hz,1H),5.36-5.32(m,2H ),5.10(s,1H),4.62(s,1H),2.44(s,3H),2.20(dd,J=15.4,7.8Hz,3H),2.01(d,J=22.4Hz,3H),0.75(d,J=7.3Hz,3H).

[0482] Example 40:

[0483] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-N'-hydroxy-2,6-dimethylbenzamidine (Compound 40)

[0484] Reaction route:

[0485] Steps:

[0486] Step A: Add 2,6-dimethylbenzaldehyde (1.0 g, 7.45 mmol) and hydroxylamine hydrochloride (621.0 mg, 8.94 mmol) to a reaction flask. Add a mixed solvent of ethanol (10 mL) and water (5 mL), then add sodium hydroxide (358.0 mg, 8.94 mmol). Stir and react at room temperature overnight. Add dilute hydrochloric acid to the reaction solution to adjust the pH to 5-6. Extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 40-2 (1.1 g), which is used directly in the next step.

[0487] MS (ESI) M / Z: 150.0 [M+H] + .

[0488] Step B: Add compound 40-2 (100.0 mg, 0.67 mmol) and N-chlorosuccinimide (89.0 mg, 0.67 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 40-3 (120.0 mg), which is used directly in the next step.

[0489] MS (ESI) M / Z: 184.0 [M+H] + .

[0490] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 40-3 (26.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (5.0 mg, yield 7.8%).

[0491] MS (ESI) M / Z: 583.2 [M+H] + .

[0492] 1 H NMR(400MHz,Chloroform-d)δ7.67-7.50(m,2H),7.21-7.02(m,3H),5.72-5.63(m ,1H),5.55(t,J=19.5Hz,1H),5.37-5.31(m,1H),5.31-5.17(m,2H),4.56-4.42(m ,1H),3.22-3.14(m,1H),2.94-2.83(m,1H),2.50-2.38(m,6H),2.34(s,3H),2.06 -1.96(m,2H),1.87(q,J=7.3Hz,2H),1.62(d,J=7.9Hz,1H),1.01(t,J=6.7Hz,3H).

[0493] Example 41:

[0494] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-N'-hydroxyisonicotinimide (Compound 41)

[0495] Reaction route:

[0496] Steps:

[0497] Step A: Add 4-formylpyridine (1.0 g, 9.34 mmol) and hydroxylamine hydrochloride (779.0 mg, 11.21 mmol) to a reaction flask, followed by a mixture of ethanol (10 mL) and water (5 mL), and then sodium hydroxide (448.0 mg, 11.21 mmol). Stir the mixture at room temperature overnight. Add dilute hydrochloric acid to adjust the pH to 5-6, and extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 41-2 (1.1 g), which is used directly in the next step.

[0498] MS (ESI) M / Z: 123.0 [M+H] + .

[0499] Step B: Add compound 41-2 (100.0 mg, 0.82 mmol) and N-chlorosuccinimide (109.0 mg, 0.82 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for testing. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction solution to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 41-3 (120.0 mg), which is used in the next step without further purification.

[0500] MS (ESI) M / Z: 157.0 [M+H] + .

[0501] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 41-3 (22.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (19.0 mg, yield 24.6%).

[0502] MS (ESI) M / Z: 556.2 [M+H] + .

[0503] 1 H NMR(400MHz,Chloroform-d)δ8.81–8.73(m,2H),7.69(d,J=10.4Hz,1H),7.64–7 .59(m,2H),7.59–7.54(m,1H),5.80–5.70(m,1H),5.63–5.53(m,1H),5.37–5.28( m,2H),5.27–5.20(m,1H),4.87–4.71(m,1H),3.28–3.17(m,1H),3.06–2.91(m,1 H),2.40(s,3H),2.01(d,J=6.3Hz,2H),1.92–1.85(m,2H),1.03(t,J=6.5Hz,3H).

[0504] Example 42:

[0505] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-fluoro-N'-hydroxybenzamidine (Compound 42)

[0506] Reaction route:

[0507] Steps:

[0508] Step A: Add 2-fluorobenzaldehyde (1.0 g, 8.06 mmol) and hydroxylamine hydrochloride (672.0 mg, 9.67 mmol) to a reaction flask, followed by a mixture of ethanol (10 mL) and water (5 mL), and then sodium hydroxide (387.0 mg, 9.67 mmol). Stir the mixture at room temperature overnight. Add dilute hydrochloric acid to adjust the pH to 5-6, and extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 42-2 (1.1 g), which is used directly in the next step.

[0509] MS (ESI) M / Z: 140.0 [M+H] + .

[0510] Step B: Add compound 42-2 (100.0 mg, 0.72 mmol) and N-chlorosuccinimide (96.0 mg, 0.72 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C, stir and reflux for 2 hours. Samples are taken for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). The organic layers are combined, dried (over anhydrous sodium sulfate), filtered, and concentrated to afford crude compound 42-3 (120.0 mg), which is used directly in the next step.

[0511] MS (ESI) M / Z: 174.0 [M+H] + .

[0512] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 42-3 (24.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (5.0 mg, yield 7.9%).

[0513] MS (ESI) M / Z: 573.2 [M+H] + .

[0514] 1H NMR(400MHz,Chloroform-d)δ7.79-7.39(m,4H),7.26-7.10(m,2H),5.75–5.50(m,2H),5.47–5.22(m,2H),5.20–5.08(m,1H),4.63(d,J=55.3Hz ,1H),3.21(d,J=16.7Hz,1H),2.92(d,J=20.2Hz,1H),2.34(d,J=21.6Hz ,3H),2.26–2.06(m,2H),1.95–1.71(m,2H),0.98(t,J=8.5,7.3Hz,3H).

[0515] Example 43:

[0516] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-N',4-dihydroxybenzamidine (Compound 43)

[0517] Reaction route:

[0518] Steps:

[0519] Step A: Add 4-hydroxybenzaldehyde (1.0 g, 8.19 mmol) and hydroxylamine hydrochloride (683.0 mg, 9.83 mmol) to a reaction flask, followed by a mixture of ethanol (10 mL) and water (5 mL), and then sodium hydroxide (383.0 mg, 9.83 mmol). Stir the mixture at room temperature overnight. Add dilute hydrochloric acid to adjust the pH to 5-6, and extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 43-2 (1.0 g), which is used directly in the next step.

[0520] MS (ESI) M / Z: 138.0 [M+H] + .

[0521] Step B: Add compound 43-2 (100.0 mg, 0.73 mmol) and N-chlorosuccinimide (97.0 mg, 0.73 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 43-3 (80.0 mg), which is used directly in the next step.

[0522] MS (ESI) M / Z: 172.2 [M+H] + .

[0523] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 43-3 (16.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (17.0 mg, yield 31.7%).

[0524] MS (ESI) M / Z: 571.2 [M+H] + .

[0525] 1 H NMR(400MHz, Methanol-d4)δ7.71–7.64(m,1H),7.60(dd,J=8.6,3.6Hz,1H),7.49(d,J=14.0Hz, 1H),7.34(dd,J=10.8,3.8Hz,1H),7.04(d,J=8.5Hz,1H),6.97(d,J=8.6Hz,1H),5.62–5.45(m,2H ),5.41–5.23(m,2H),5.21–5.08(m,1H),5.00–4.88(m,1H),3.30–3.09(m,1H),2.98–2.74(m,1H) ,2.41–2.32(m,1H),2.30–2.24(m,3H),2.23–2.15(m,1H),1.99–1.88(m,2H),1.03–0.95(m,3H).

[0526] Example 44:

[0527] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-4-fluoro-N'-hydroxybenzamidine (Compound 44)

[0528] Reaction route:

[0529] Steps:

[0530] Step A: Add 4-fluorobenzaldehyde (1.0 g, 8.06 mmol) and hydroxylamine hydrochloride (672.0 mg, 9.67 mmol) to a reaction flask, followed by a mixture of ethanol (10 mL) and water (5 mL), and then sodium hydroxide (387.0 mg, 9.67 mmol). Stir the mixture at room temperature overnight. Add dilute hydrochloric acid to adjust the pH to 5-6, and extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 44-2 (1.0 g), which is used directly in the next step.

[0531] MS (ESI) M / Z: 140.0 [M+H] + .

[0532] Step B: Add compound 44-2 (100.0 mg, 0.72 mmol) and N-chlorosuccinimide (96.0 mg, 0.72 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 44-3 (100.0 mg), which is used directly in the next step.

[0533] MS (ESI) M / Z: 174.0 [M+H] + .

[0534] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 44-3 (24.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of a new spot, and LCMS monitoring showed the formation of a product ion peak. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title product (13.0 mg, yield 24.2%).

[0535] MS (ESI) M / Z: 573.2 [M+H] + .

[0536] 1 H NMR(400MHz,Chloroform-d)δ8.00–7.81(m,1H),7.79–7.64(m,1H),7.63–7.3 5(m,2H),7.36–7.28(m,1H),7.26–7.08(m,1H),5.89–5.53(m,2H),5.51–5.32 (m,1H),5.31–5.00(m,2H),4.95–4.70(m,1H),3.33–3.15(m,1H),2.98–2.79( m,1H),2.33(s,3H),2.29–2.08(m,2H),1.96–1.78(m,2H),1.06–0.87(m,3H).

[0537] Example 45:

[0538] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-N'-hydroxy-4-methylbenzamidine (Compound 45)

[0539] Reaction route:

[0540] Steps:

[0541] Step A: Add 4-methylbenzaldehyde (1.0 g, 8.32 mmol) and hydroxylamine hydrochloride (694.0 mg, 9.98 mmol) to a reaction flask, followed by a mixture of ethanol (10 mL) and water (5 mL), and then sodium hydroxide (399.0 mg, 9.98 mmol). Stir the mixture at room temperature overnight. Add dilute hydrochloric acid to adjust the pH to 5-6, then extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 45-2 (1.0 g), which is used directly in the next step.

[0542] MS (ESI) M / Z: 136.0 [M+H] + .

[0543] Step B: Add compound 45-2 (100.0 mg, 0.74 mmol) and N-chlorosuccinimide (99.0 mg, 0.74 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 45-3 (100.0 mg), which is used directly in the next step.

[0544] MS (ESI) M / Z: 170.0 [M+H] + .

[0545] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to a reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring to dissolve, compound 45-3 (24.0 mg, 0.14 mmol) was added. The reaction mixture was stirred at room temperature overnight. TLC indicated the formation of a new spot, and LCMS monitoring revealed the formation of a product ion peak. The reaction mixture was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (31.0 mg, 58.0% yield).

[0546] MS (ESI) M / Z: 569.2 [M+H] + .

[0547] 1H NMR(400MHz,Chloroform-d)δ7.65–7.54(m,2H),7.53–7.39(m,2H),7.29(d,J =7.8Hz,1H),7.22(d,J=7.8Hz,1H),5.78–5.56(m,2H),5.36–5.21(m,1H),5.2 0–5.02(m,1H),4.90–4.67(m,1H),3.28–3.12(m,1H),2.96–2.83(m,1H),2.41 –2.29(m,3H),2.34(s,3H),2.13(s,2H),1.91–1.73(m,2H),1.03–0.86(m,3H).

[0548] Example 46:

[0549] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2,6-difluoro-N'-hydroxybenzamidine (Compound 46)

[0550] Reaction route:

[0551] Steps:

[0552] Step A: Add 2,5-difluorobenzaldehyde (1.0 g, 7.04 mmol) and hydroxylamine hydrochloride (587.0 mg, 8.45 mmol) to a reaction flask. Add a mixed solvent of ethanol (10 mL) and water (5 mL), then add sodium hydroxide (338.0 mg, 8.45 mmol). Stir and react at room temperature overnight. Add dilute hydrochloric acid to the reaction solution to adjust the pH to 5-6. Extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 46-2 (1.0 g), which is used directly in the next step.

[0553] MS (ESI) M / Z: 158.0 [M+H] + .

[0554] Step B: Add compound 46-2 (100.0 mg, 0.64 mmol) and N-chlorosuccinimide (85.0 mg, 0.64 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C and stir under reflux. After 2 hours, take a sample for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). Combine the organic layers, dry them (over anhydrous sodium sulfate), filter, and concentrate to obtain crude compound 46-3 (90.0 mL), which is used directly in the next step.

[0555] MS (ESI) M / Z: 192.2 [M+H] + .

[0556] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 46-3 (27.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (10.0 mg, yield 18.0%).

[0557] MS (ESI) M / Z: 591.2 [M+H] + .

[0558] 1 H NMR(400MHz,Chloroform-d)δ7.62–7.58(m,1H),7.56–7.47(m,2H),7.11(t,J=8.4Hz,2H),5.68–5.52(m,2H),5.37–5.23(m,1H),5.22–5.05( m,2H),4.65–4.47(m,1H),3.26–3.18(m,1H),2.95–2.86(m,1H),2.35( s,3H),2.31–2.23(m,2H),1.87(q,J=6.8Hz,2H),1.01(t,J=7.3Hz,3H).

[0559] Example 47:

[0560] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-N'-hydroxy-2-trifluoromethylbenzamidine (Compound 47)

[0561] Reaction route:

[0562] Steps:

[0563] Step A: Add 2-trifluoromethylbenzaldehyde (1.0 g, 5.74 mmol) and hydroxylamine hydrochloride (480.0 mg, 6.89 mmol) to a reaction flask. Add a mixed solvent of ethanol (10 mL) and water (5 mL), then add sodium hydroxide (280.0 mg, 6.89 mmol). Stir and react at room temperature overnight. Add dilute hydrochloric acid to the reaction solution to adjust the pH to 5-6. Extract with ethyl acetate (20 mL x 2). The organic phase is dried and concentrated to obtain the crude product 47-2 (1.0 g), which is used directly in the next step.

[0564] MS (ESI) M / Z: 190.0 [M+H] + .

[0565] Step B: Add compound 47-2 (100.0 mg, 0.53 mmol) and N-chlorosuccinimide (71.0 mg, 0.53 mmol) to a dry reaction flask, dissolve in N,N-dimethylformamide (5 mL), and stir until uniform. Heat in an oil bath to 50°C, stir and reflux for 2 hours. Samples are taken for analysis. TLC analysis shows a new spot, confirming the completion of the reaction. Cool the reaction mixture to room temperature. Add water (20 mL) and extract with ethyl acetate (20 mL x 2). The organic layers are combined, dried (over anhydrous sodium sulfate), filtered, and concentrated to afford crude compound 47-3 (100.0 mg), which is used directly in the next step.

[0566] MS (ESI) M / Z: 224.2 [M+H] + .

[0567] Step C: Compound 1-1 (50.0 mg, 0.09 mmol) was added to the reaction flask, followed by N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (30.0 mg, 0.23 mmol). After stirring and dissolving, compound 47-3 (32.0 mg, 0.14 mmol) was added. The reaction solution was stirred at room temperature overnight. TLC showed the formation of new spots, and LCMS monitoring showed the formation of product ion peaks. The reaction solution was purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (8.0 mg, yield 13.7%).

[0568] MS (ESI) M / Z: 623.0 [M+H] + .

[0569] 1 H NMR(400MHz,Chloroform-d)δ7.96–7.81(m,2H),7.80–7.71(m,1H),7.70–7.63(m,1H),7.60(s, 1H),7.57–7.49(m,1H),5.88–5.70(m,1H),5.70–5.58(m,1H),5.52–5.33(m,2H),5.29–5.12(m,1H),4.72–4.59 (m,1H),3.19–3.09(m,1H),2.88–2.78(m,1H),2.31(s,3H),2.28–2.17(m,2H),1.89–1.77(m,2H),0.95(t,3H).

[0570] Example 48:

[0571] Preparation of 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-3-hydroxyurea (Compound 48)

[0572] Reaction route:

[0573] Steps:

[0574] Step A: Dissolve compound 1-1 (150.0 mg, 0.34 mmol), N,N-diisopropylethylamine (0.2 mL), and N,N'-carbonyldiimidazole (165.4 mg, 1.02 mmol) in N,N-dimethylformamide (5 mL) and stir at room temperature for 30 minutes. Then add hydroxylamine hydrochloride (70.9 mg, 1.02 mmol) and react at room temperature for 6 hours. LCMS monitoring shows that the reaction is complete. The reaction solution is purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (120.0 mg, yield 70.5%).

[0575] MS (ESI) M / Z: 495.0 [M+H] + .

[0576] 1 H NMR(400MHz,DMSO-d6)δ8.76(s,1H),8.73(s,1H),7.74(d,J=10.9Hz,1H),7 .40(d,J=9.0Hz,1H),7.30(s,1H),6.50(s,1H),5.40(d,J=10.3Hz,3H),5.2 9(d,J=19.1Hz,1H),5.13(d,J=19.0Hz,1H),3.26–3.18(m,1H),3.14–3.01( m,1H),2.37(s,3H),2.19(m,2H),1.93–1.79(m,2H),0.87(t,J=7.4Hz,3H).

[0577] Example 49:

[0578] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-N'-hydroxypropylamine (Compound 49)

[0579] Reaction route:

[0580] Steps:

[0581] Step A: Compound 49-1 (100.0 mg, 1.36 mmol) was dissolved in N,N-dimethylformamide (1.5 mL), and N-chlorosuccinimide (182.0 mg, 1.36 mmol) was added. The mixture was stirred at 50°C under argon for 2 hours. After monitoring showed that the reaction of the starting material was complete, the reaction solution was quenched by adding water (100 mL). The mixture was extracted with dichloromethane (50 mL × 3). The lower organic phases were combined and washed with saturated brine (10 mL × 3). The organic phases were dried and concentrated to obtain the crude product 49-2 (100.0 mg), which was used directly in the next step.

[0582] Step B: Compound 49-2 (3.0 mg, 0.28 mmol) was dissolved in dichloromethane (2 mL), and compound 1-1 (3.0 mg, 0.002 mmol) was added. The mixture was reacted at room temperature under argon for 4 hours. After LCMS analysis showed that the reaction was complete, the solvent was concentrated under reduced pressure and the reaction solution was purified by preparative liquid chromatography in 0-100% acetonitrile / water (0.1% formic acid) to obtain the title compound (1.3 mg, yield 36.7%).

[0583] MS (ESI) M / Z: 507.3 [M+H] + .

[0584] 1 H NMR(400MHz,MeOD)δ8.49(s,1H),7.78–7.52(m,2H),5.58–5.51(m,2H),5.38–5.34(m,1H),5.23–5.20(m,2H),4.57(s,1H),2.69( s,2H),2.52–2.49(m,3H),2.41(s,1H),2.21–2.17(m,1H),1.96–1.94(m,3H),1.60(s,1H),1.02–0.97(m,3H),0.95–0.88(m,3H).

[0585] Example 50:

[0586] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-N'-hydroxybenzimide (Compound 50)

[0587] Reaction route:

[0588] Steps:

[0589] Step A: Compound 50-1 (500.0 mg, 4.14 mmol) was dissolved in N,N-dimethylformamide (10 mL), and N-chlorosuccinimide (552.8 mg, 4.14 mmol) was added. The mixture was stirred at 50°C under argon for 2 hours. After monitoring indicated that the reaction was complete, the reaction solution was quenched by adding water (100 mL). The mixture was extracted with dichloromethane (50 mL × 3). The lower organic phases were combined, washed with saturated brine (10 mL × 3), and then dried over anhydrous sodium sulfate. Finally, the organic phase was concentrated under reduced pressure and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1) to obtain compound 50-2 (200.0 mg, yield 12.6%).

[0590] Step B: Dissolve compound 1-1 (30.0 mg, 0.06 mmol) in dichloromethane (5 ml), add compound 50-2 (9.0 mg, 0.08 mmol), and react at room temperature under argon protection for 4 hours.

[0591] After LCMS showed that the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and purified by preparative liquid chromatography under 0-100% acetonitrile / water (0.1% formic acid) to give the title compound (1.2 mg, yield 3.2%).

[0592] MS (ESI) M / Z: 555.3 [M+H] + .

[0593] 1 H NMR(400MHz,DMSO-d6)δ10.67(s,1H),7.74(m,2H),7.52(m,3H),7.32(d,J =2.7Hz,1H),7.30–7.00(m,1H),5.53–5.46(m,1H),5.44(s,2H),5.40–5.2 3(m,1H),4.97–4.76(s,1H),3.21(m,1H),2.93(dd,J=12.8,6.5Hz,1H),2. 42(m,1H),2.33(s,3H),2.20(m,1H),1.93–1.81(m,2H),0.92–0.84(m,3H).

[0594] Example 51:

[0595] Preparation of stereoisomers of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-4-fluoro-N'-hydroxybenzamidine (Compound 44), Compound 44-P1 and Compound 44-P2

[0596] Steps:

[0597] Compound 44 obtained in Example 44 was subjected to chiral separation to obtain compound 44-P1 and compound 44-P2;

[0598] Compound 44-P1:

[0599] LCMS (ESI) M / Z: 573.6 [M+H] + .

[0600] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 7.068 min.

[0601] 1 H NMR(400MHz,MeOD)δ7.84(s,2H),7.50(s,1H),7.38-7.15(m,3H),5.56(d,J=18.5Hz,2H),5.28(dd,J=43.8,17.4Hz,2H) ,4.81-4.77(m,2H),3.18(d,J=16.7Hz,1H),2.75(s,1H),2.27(s,4H),2.09(s,1H),1.95(d,J=6.6Hz,2H),1.01(s,3H).

[0602] Compound 44-P2:

[0603] LCMS (ESI) M / Z: 573.2 [M+H] + .

[0604] HPLC conditions: 0-13 min, 3-95% acetonitrile / water (0.1% trifluoroacetic acid), 13-16 min, 95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 7.385 min.

[0605] 1H NMR(400MHz,MeOD)δ7.65(d,J=23.5Hz,2H),7.46(s,2H),7.35-7.16(m,3H),5.60(d,J=12Hz,2H),5.22(dd ,J=36.3,17.6Hz,3H),2.87(s,1H),2.24(s,3H).2.10(d,J=6.7Hz,3H),1.93(s,2H),1.08(t,J=7.1Hz,3H).

[0606] Example 52:

[0607] Preparation of stereoisomers of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2,6-difluoro-N'-hydroxybenzamidine (Compound 46), Compound 46-P1 and Compound 46-P2

[0608] Steps:

[0609] The compound 46 obtained in Example 46 was subjected to chiral separation to obtain stereoisomers, compound 46-P1 and compound 46-P2, respectively:

[0610] Compound 46-P1:

[0611] LCMS (ESI) M / Z: 591.1 [M+H] + .

[0612] HPLC conditions: 0-2 min, 10-30% acetonitrile / water (0.1% trifluoroacetic acid), 2-12 min, 30-95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 8.048 min.

[0613] 1 H NMR (400MHz, MeOD) δ7.70-7.50(m,3H),7.19(t,J=8.4Hz,2H),5.64(dd,J=42.7,17.7Hz,2H),5.38(dd,J=17.9,8.0Hz,2H),4.65(d ,J=4.8Hz,1H),3.27-3.22(m,1H),2.86(s,1H),2.43-2.27(m,4H),2.15(s,1H),1.97(dd,J=7.4,3.5Hz,2H),1.02(t,J=7.4Hz,3H).

[0614] Compound 46-P2:

[0615] LCMS (ESI) M / Z: 591.2 [M+H] + .

[0616] HPLC conditions: 0-2 min, 10-30% acetonitrile / water (0.1% trifluoroacetic acid), 2-12 min, 30-95% acetonitrile / water (0.1% trifluoroacetic acid). Retention time Rt = 8.142 min.

[0617] 1 H NMR(400MHz,MeOD)δ7.56-7.36(m,3H),7.07(t,J=8.3Hz,2H),5.56(dd,J=53.4,17.9Hz,2H),5.26(dd,J=17.8,10.9Hz,2H),4 .55(d,J=5.4Hz,1H),3.16(s,1H),2.82-2.17(m,4H),2.14-2.00(m,1H),1.86(dt,J=10.2,7.0Hz,2H),0.90(t,J=7.3Hz,3H).

[0618] Example 53:

[0619] Preparation of 5-chloro-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-3-fluoro-N'-hydroxypyridinimidazole (Compound 53)

[0620] Synthesis route:

[0621] Steps:

[0622] Step A: Dissolve compound 53-1 (271.0 mg, 1.70 mmol), hydroxylamine hydrochloride (153.0 mg, 2.20 mmol), and sodium hydroxide (152.0 mg, 2.50 mmol) in water (1.5 mL) and methanol (5 mL). Stir the mixture at 25°C for 1.5 h. Add concentrated hydrochloric acid until the pH is approximately 6. Concentrate to dryness, then add water (20 mL). Extract with dichloromethane (10 mL x 3). Combine the organic phases, wash with saturated brine (10 mL x 3), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to yield compound 53-2 (90.0 mg, crude product).

[0623] LCMS (ESI) M / Z: 175.2 [M+H]+ .

[0624] Step B: Dissolve compound 53-2 (45.0 mg, 0.25 mol) and N-chlorosuccinimide (34.5 mg, 0.25 mol) in N,N-dimethylformamide (1 mL), stir and react at 40°C for 2 h, then cool to room temperature, add compound 1-1 (15.0 mg, 0.035 mmol) to the reaction solution obtained in step 1, and then add triethylamine (72.0 mg, 0.72 mmol), and then react at room temperature for 2 hours.

[0625] After LCMS monitoring indicated completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL), washed with saturated sodium chloride solution (15 mL x 2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (purification conditions: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% over 13 minutes; detection wavelength: 254 nm) to obtain the title compound 53 (4.1 mg, yield 22.2%).

[0626] LCMS (ESI) M / Z: 608.0 [M+H] + .

[0627] 1 H NMR (400MHz, DMSO-d6) δ10.36(s,1H),8.57(s,1H),8.42(s,2H),8.25(d,J=9 .2Hz,1H),7.74(d,J=10.8Hz,1H),7.32(s,1H),6.94(t,J=14.7Hz,1H),6.54( s,1H),5.61-5.29(m,2H),4.77(s,1H),2.94(s,1H),2.33(s,3H),2.16(d,J= 24.5Hz,2H),2.01(d,J=6.9Hz,1H),1.94-1.83(m,2H),0.89(t,J=7.3Hz,3H).

[0628] Example 54:

[0629] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizine[1,2-b]quinolin-1-yl)-N'-hydroxy-2-oxopropionamide (Compound 54)

[0630] Synthesis route:

[0631] Steps:

[0632] Step A: Dissolve compound 54-1 (30.0 mg, 0.35 mmol) and N-chlorosuccinimide (56.0 mg, 0.35 mmol) in anhydrous N,N-dimethylformamide (1 mL) and stir at 40°C for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was transferred to a container at room temperature, and compound 1-1 (20.0 mg, 0.05 mmol) was added to the reaction solution. Triethylamine (72.0 mg, 0.72 mmol) was then added, and the reaction was continued at room temperature for 2 hours.

[0633] After LCMS monitoring indicated completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL) and washed with saturated sodium chloride solution (15 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure to yield 78 mg of crude compound 54. The crude product was purified by preparative HPLC (purification conditions: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% over 13 minutes; detection wavelength: 254 nm) to afford the title compound 54 (1.6 mg, 8.2% yield).

[0634] LCMS (ESI) M / Z: 521.2 [M+H] + .

[0635] 1 HNMR(400MHz,MeOD)δ7.57(s,2H),5.54(d,J=16.3Hz,1H),5.32(dt,J=31.7,13.5Hz,4H),3.05(dd,J=31.2,23.8Hz, 2H), 2.45 (s, 3H), 2.37 (d, J = 8.9Hz, 2H), 2.18 (dd, J = 18.4, 10.5Hz, 3H), 2.03 (d, J = 6.3Hz, 2H), 0.89 (d, J = 6.9Hz, 3H).

[0636] Example 55:

[0637] Preparation of (Z)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]indolizine[1,2-b]quinolin-1-yl)-N'-hydroxy-2-oxo-2-phenylacetimide (Compound 55)

[0638] Synthesis route:

[0639] Steps:

[0640] Step A: Compound 55-1 (40.0 mg, 0.27 mmol) and N-chlorosuccinimide (35.7 mg, 0.27 mmol) were dissolved in N,N-dimethylformamide (1 mL), and the reaction solution was stirred at 40°C for 2 hours.

[0641] After LCMS monitoring showed that the starting material disappeared, the reaction solution was transferred to a container at room temperature, and compound 1-1 (15.0 mg, 0.03 mmol) was added to the reaction solution, and triethylamine (72.0 mg, 0.72 mmol) was added, and then reacted at room temperature for 2 hours.

[0642] After LCMS monitoring indicated completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL) and washed with saturated sodium chloride solution (15 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure to yield 56 mg of crude compound 55. The crude product was purified by preparative HPLC (purification conditions: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% over 13 minutes; detection wavelength: 254 nm) to afford the title compound 55 (1.3 mg, 8.6% yield).

[0643] LCMS (ESI) M / Z: 583.3 [M+H] + .

[0644] 1 H NMR(400MHz, CDCl3)δ9.19(s,1H),8.75–8.52(m,1H),7.86(d,J=7.3Hz,2H),7.66(d,J=42.6Hz,3H),7.49(s,2H),5.71(s,1H),5.58–4.9 5(m,3H),3.24(s,1H),2.45(s,3H),2.41–2.32(m,1H),2.29–2.17(m,1H),2.00(s,2H),1.88(s,2H),1.64(s,1H),1.02(t,J=7.1Hz,3H).

[0645] Example 56:

[0646] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-((R)-1-hydroxyethyl)benzyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 56)

[0647] Synthesis route:

[0648] Steps:

[0649] Step A: Compound 56-1 (1.0 g, 5.62 mmol) was dissolved in tetrahydrofuran (20 mL), cooled to 0°C, and then lithium aluminum hydride (854.0 mg, 22.47 mmol) was slowly added. The reaction was allowed to proceed at room temperature for 4 hours.

[0650] LCMS monitoring indicated the reaction was complete. Water (0.9 mL) and 2 mol / L sodium hydroxide solution (0.9 mL) were then added dropwise to quench the reaction. The mixture was filtered through celite, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to yield the racemic product (500.0 mg, 58.9% yield). 200.0 mg was then separated by supercritical fluid chromatography (SCFC) using the following conditions: mobile phase: supercritical carbon dioxide and 0.1% diethylamine in ethanol; flow rate: 3 mL / min; gradient: 15% 0.1% diethylamine in ethanol over 0.5 min; then, 15% to 40% 0.1% in ethanol over 5 min; then, 40% 0.1% in ethanol over 2.5 min. Detection wavelength: 214 nm; retention time: Rt = 2.460 min) to yield compound 56-2 (90.0 mg, 26.5% yield).

[0651] LCMS (ESI) M / Z: 251.2 [M+H] + .

[0652] Step B: Compound 56-2 (50.0 mg, 0.33 mmol) was dissolved in acetonitrile (3 mL), and then copper bromide (74.0 mg, 0.33 mmol), N-methylimidazole (55.0 mg, 0.66 mmol) and 2,2,6,6-tetramethylpiperidinyl-N-oxyl radical (52.0 mg, 0.33 mmol) were added, and the reaction was stirred at room temperature for 1 hour.

[0653] LCMS monitoring indicated the reaction was complete. The mixture was diluted with ethyl acetate (20 mL) and washed sequentially with saturated sodium bicarbonate solution (10 mL × 1 time) and saturated sodium chloride solution (10 mL × 1 time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to afford compound 56-3 (10.0 mg, 20% yield).

[0654] Step C: Compound 1-1 (10.0 mg, 0.02 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and then compound 56-3 (10.0 mg, 0.07 mmol) was added, followed by sodium cyanoborohydride (10.0 mg, 0.16 mmol), and the reaction was stirred at room temperature overnight.

[0655] LCMS monitoring indicated the reaction was complete. The mixture was diluted with ethyl acetate (20 mL) and washed sequentially with water (10 mL × 3 times) and saturated sodium chloride solution (10 mL × 1 time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (ACN / 0.1% NH4HCO3 and water) to afford the title compound 56 (1.1 mg, 8.5% yield).

[0656] LCMS (ESI) M / Z: 570.3 [M+H] + .

[0657] 1 H NMR (400MHz, DMSO-d6) δ8.32(s,1H),7.87(d,J=7.9Hz,1H),7.74(d,J=11.0Hz,1H),7.58(d,J =7.9Hz,1H),7.40(m,2H),7.30(d,J=6.7Hz,2H),6.53(s,1H),5.44(s,1H),5.31(m,2H),5.11( s,1H),4.72(d,J=6.2Hz,1H),4.23(s,1H),3.88(m,1H),3.02(d,J=16.7Hz,2H),2.39(s,3H), 2.27(s,1H),2.08(s,1H),1.94–1.80(m,2H),1.33(m,3H),1.24(s,1H),0.88(t,J=7.2Hz,3H).

[0658] Example 57:

[0659] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((4-((S)-1-hydroxyethyl)benzyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 57)

[0660] Synthesis route:

[0661] Step A: Compound 57-1 (1.0 g, 5.62 mmol) was dissolved in tetrahydrofuran (20 mL), cooled to 0°C, and then lithium aluminum hydride (854.0 mg, 22.47 mmol) was slowly added. The reaction was allowed to proceed at room temperature for 4 hours.

[0662] LCMS monitoring indicated the reaction was complete. Water (0.9 mL) and 2N sodium hydroxide solution (0.9 mL) were then added dropwise to quench the reaction. The mixture was filtered through celite, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the racemic product (500.0 mg, 58.9% yield). This was then resolved by supercritical fluid chromatography (SCFC) (mobile phase: supercritical carbon dioxide and 0.1% diethylamine ethanol solution; flow rate: 3 mL / min; gradient: 0.1% diethylamine ethanol at 15% over 0.5 min; then 0.1% diethylamine ethanol at 15% to 40% over 5 min; then 0.1% diethylamine ethanol at 40% over 2.5 min. Detection wavelength: 214 nm. Retention time: Rt = 2.636 min) to afford compound 57-2 (200.0 mg, 23.6% yield).

[0663] LCMS (ESI) M / Z: 251.2 [M+H] + .

[0664] Step B: Compound 57-2 (200.0 mg, 1.32 mmol) was dissolved in acetonitrile (10 mL), and then copper bromide (223.0 mg, 1.32 mmol), N-methylimidazole (82.0 mg, 2.64 mmol) and 2,2,6,6-tetramethylpiperidinyl-N-oxyl radical (156.0 mg, 1.32 mmol) were added, and the reaction was stirred at room temperature for 1 hour.

[0665] LCMS monitoring showed that the reaction was complete. The mixture was diluted with ethyl acetate (50 mL) and washed sequentially with saturated sodium bicarbonate solution (20 mL × 1 time) and saturated sodium chloride solution (20 mL × 1 time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound 57-3 (80.0 mg, yield 40.0%).

[0666] Step C: Compound 1-1 (30.0 mg, 0.07 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and then compound 57-3 (30.0 mg, 0.20 mmol) was added, followed by sodium cyanoborohydride (30.0 mg, 0.48 mmol), and the reaction was stirred at room temperature overnight.

[0667] LCMS monitoring indicated the reaction was complete. The mixture was diluted with ethyl acetate (50 mL) and washed sequentially with water (20 mL × 3 times) and saturated sodium chloride solution (20 mL × 1 time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (ACN / 0.1% NH4HCO3 and water) to afford the title compound 57 (19.0 mg, 48.7% yield).

[0668] LCMS (ESI) M / Z: 570.3 [M+H] + .

[0669] 1 H NMR (400MHz, DMSO-d6) δ7.74(d,J=10.5Hz,1H),7.38(d,J=5.4Hz,2H),7.30(d,J=3.6Hz,3H), 6.52(d,J=4.6Hz,2H),5.40(d,J=25.4Hz,3H),5.25(d,J=19.6Hz,1H),5.09(s,1H),4.79–4.64 (m,1H),4.22(s,1H),3.94(d,J=51.8Hz,1H),3.26–3.19(m,1H),3.04(s,1H),2.38(s,3H),2. 32–2.22(m,1H),2.08(s,1H),1.92–1.79(m,2H),1.31(d,J=6.4Hz,4H),0.87(t,J=7.2Hz,3H).

[0670] Example 58:

[0671] Preparation of (1S,9S)-1-((2-cyclopropyl-2-hydroxyethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[2,1-g]quinoline-10,13-dione (Compound 58)

[0672] Synthesis route:

[0673] Steps:

[0674] Step A: Add compound 1-1 (50.0 mg, 0.10 mmol) to N,N-dimethylformamide (2 mL), add triethylamine (0.3 mL), stir for 5 min, then add 1-cyclopropyl-2-bromoethanone (157.0 mg, 1.00 mmol), and stir at 50 ° C under argon protection for 2 hours.

[0675] After LCMS showed that the raw material reaction was complete, water (20 mL) was added to the reaction solution to quench it. The mixed solution was extracted with ethyl acetate (20 mL × 3 times), and the lower organic phases were combined and dried over anhydrous sodium sulfate. Finally, the organic phase was concentrated under reduced pressure and pulled dry with an oil pump to obtain compound 58-1 (50.0 mg, yield 78.3%).

[0676] LCMS (ESI) M / Z: 518.2 [M+H] + .

[0677] Step B: Compound 58-1 (50.0 mg, 0.10 mmol) was dissolved in tetrahydrofuran (2 mL), and borane-tetrahydrofuran solution (0.3 mL) and (R)-2-methyl-CBS-oxazaborolidine (0.3 mL) were added, and the mixture was reacted at -80°C under argon protection for 2 hours.

[0678] After LCMS detection showed that the reaction was completed, dichloromethane (50 mL) was added to the reaction solution, washed with NH4Cl (20 mL), and then dried over anhydrous sodium sulfate. Finally, the organic phase was concentrated under reduced pressure and pulled dry with an oil pump. After preparative purification and lyophilization, the title compound 58 (1.7 mg, yield 3.5%) was obtained.

[0679] LCMS (ESI) M / Z: 520.3 [M+H] + .

[0680] 1H NMR (400MHz, CDCl3) δ7.73–7.35(m,2H),7.16(s,1H),5.28(d,J=17.0Hz,1H),5.14(d,J=19.2Hz,1H),5.00(d,J=17.7Hz,1H),4.73(dd,J= 54.3, 23.6Hz, 1H), 4.21 (s, 1H), 3.88 (s, 1H), 3.33–2.80 (m, 2H), 2.28 (t, J = 22.2Hz, 4H), 1.92 (dd, J = 43.4, 37.7Hz, 7H), 1.34–0.84 (m, 8H).

[0681] Example 59:

[0682] Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-4-hydroxybenzenesulfonamide (Compound 59)

[0683] Synthesis route:

[0684] Steps:

[0685] Step A: Compound 1-1 (4.4 mg, 0.01 mmol), compound 59-1 (23.0 mg, 0.12 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (11.4 mg, 0.06 mmol) were added to a dichloromethane (2 mL) reaction solution, followed by 4-dimethylaminopyridine (7.2 mg, 0.06 mmol). The mixture was reacted at room temperature for 16 hours. After LCMS showed that the reaction of the starting materials was complete, water (20 mL) was added to quench the reaction solution. The mixture was extracted with dichloromethane (20 mL × 3), and the lower organic phases were combined and dried over anhydrous sodium sulfate. Finally, the organic phases were concentrated under reduced pressure to completely remove the solvent to obtain the crude product.

[0686] The crude product was purified by HPLC (purification conditions were as follows: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% in 13 minutes; detection wavelength: 254 nm) to give the title compound 59 (1.1 mg, yield 12%).

[0687] LCMS (ESI) M / Z: 592.15 [M+H] + .

[0688] 1H NMR (400MHz, MeOD) δ7.78(d,J=8.7H,2H),7.66-7.56(m,2H),6.94(d,J-8.7Hz,2H),5.57(d,J=16.3Hz,1H),5.36(dd,J =17.9,10.6Hz,2H),5.28(s,2H),3.08(dd,J=47.2,14.0Hz,2H),2.38(s,3H),2.10-1.93(m,4H),1.01(t,J=7.4Hz,3H).

[0689] Example 60:

[0690] Preparation of (E)-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-3-(4-hydroxyphenyl)acrylamide (Compound 60)

[0691] Synthesis route:

[0692] Steps:

[0693] Step A: Compound 1-1 (21.0 mg, 0.05 mmol), compound 60-1 (13.9 mg, 0.10 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (19.0 mg, 0.01 mmol) were added to the reaction solution of N,N-dimethylformamide (3 mL), and then 4-dimethylaminopyridine (12.0 mg, 0.01 mmol) was added, and then the reaction was carried out at room temperature for 16 hours.

[0694] The reaction solution was filtered and purified by HPLC (purification conditions were as follows: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile increased from 10% to 95% within 13 minutes; detection wavelength: 254 nm) to obtain the title compound 60 (12.0 mg, yield 43.0%).

[0695] LCMS (ESI) M / Z: 557.1 [M+H] + .

[0696] 1H NMR (400MHz, MeOD) δ7.78-7.51(m,3H),7.33(s,1H),6.78(d,J=8.8Hz,1H),5.82(s,1H),5.49(d,J=16.3Hz,1H),5.34-5 .24(m,2H),5.12(d,J=19.3Hz,1H),3.18-3.26(m,2H),2.47-2.30(m,5H),1.92(d,J=7.2Hz,2H),0.98(t,J=7.3Hz,3H).

[0697] Example 61:

[0698] Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-oxy-1,2-dihydropyridine-3-carboxamide (Compound 61)

[0699] Synthesis route:

[0700] Steps:

[0701] Step A: Compound 1-1 (20.0 mg, 0.05 mmol), compound 61-1 (12.7 mg, 0.09 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (17.5 mg, 0.09 mmol) were added to the reaction solution of N,N-dimethylformamide (3 mL), and then 4-dimethylaminopyridine (11.2 mg, 0.09 mmol) was added, and then the reaction was carried out at room temperature for 16 hours.

[0702] The reaction solution was filtered and purified by HPLC (purification conditions were as follows: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile increased from 10% to 95% within 13 minutes; detection wavelength: 254 nm) to obtain the title compound 61 (2.0 mg, yield 7.8%).

[0703] LCMS (ESI) M / Z: 557.3 [M+H] + .

[0704] 1H NMR (400MHz, DMSO-d6) δ12.51(s,1H),10.51(d,J=8.7Hz,1H),8.43(d,J=7.3Hz,1H), 7.95(d,J=32.3Hz,1H),7.79(dd,J=29.0,7.9Hz,2H),7.30(s,1H),6.78-6.26(m,1H), 5.77(d,J=8.8Hz,1H),5.52-5.17(m,3H),3.21(d,J=24.2Hz,2H),2.67(s,1H),2.43(s ,3H),2.33-2.23(m,2H),2.03-1.94(m,1H),1.87-1.77(m,1H),0.85(t,J=7.2Hz,3H).

[0705] Example 62:

[0706] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-hydroxy-1,3-dioxoisooctanol-2-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 62)

[0707] Synthesis route:

[0708] Steps:

[0709] Step A: Compound 1-1 (13.0 mg, 0.03 mmol) and compound 62-1 (16.0 mg, 0.3 mmol) were dissolved in acetic acid (2 mL) and stirred at 130° C. for 4 hours. After LCMS showed that the reaction was complete, the mixture was concentrated under reduced pressure to completely remove the solvent to obtain a crude product.

[0710] The crude product was purified by HPLC (purification conditions were as follows: mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile increased from 10% to 95% within 13 minutes; detection wavelength: 254 nm) to give the title compound 62 (2.3 mg, yield 17.2%).

[0711] LCMS (ESI) M / Z: 582.3 [M+H] + .

[0712] 1H NMR (400MHz, MeOD) δ7.75-7.52(m,3H),7.38(d,J=7.0Hz,1H),7.21(d,J=8.5Hz,1H),5.95(s,1H),5.45(d,J=16.3Hz,1 H),5.27(d,J=16.2Hz,2H),5.03(d,J=18.4Hz,2H),3.18(s,2H),2.45(s,5H),1.98-1.82(m,2H),0.96(t,J=7.4Hz,3H).

[0713] Example 63:

[0714] Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(N-methylsulfonylamino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 63)

[0715] Synthesis route:

[0716] Steps:

[0717] Step A: Compound 1-1 (30.0 mg, 0.07 mmol), triethylamine (21.0 mg, 0.21 mmol), N-methylaminosulfonyl chloride (15.0 mg, 0.12 mmol) and anhydrous dichloromethane (2 mL) were added to a 10 mL single-necked bottle; the reaction system was then stirred at room temperature for 2 hours.

[0718] LCMS monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, diluted with dichloromethane (20 mL), and washed successively with water (10 mL × 1 time) and saturated sodium chloride solution (10 mL × 1 time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (ACN / 0.1% TFA and water) to give the title compound 63 (2.4 mg, yield 6.6%).

[0719] LCMS (ESI) M / Z: 529.3 [M+H] + .

[0720] 1H NMR (400MHz, DMSO-d6) δ7.79(d,J=10.8Hz,1H),7.62(d,J=8.0Hz,1H),7.31(s,1H),7.12–7.04(m,1H),6.53(s,1H),5.42(d, J=8.3Hz,3H),4.88(s,1H),3.13(s,3H),2.58(d,J=4.2Hz,3H),2.38(s,3H),2.27(s,2H),1.87(s,2H),0.87(d,J=6.9Hz,3H).

[0721] Example 64:

[0722] Preparation of (S)-2-cyclopropyl-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-(hydroxyamino)acetamide (Compound 64-P1 or Compound 64-P2)

[0723] as well as

[0724] Preparation of (R)-2-cyclopropyl-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-(hydroxyamino)acetamide (Compound 64-P2 or Compound 64-P1)

[0725] Synthesis route:

[0726] Steps:

[0727] Step A: Dissolve hydroxylamine hydrochloride (1.2 g, 18.10 mmol) in dichloromethane (50.0 mL) at 0°C. Slowly add triethylamine (2.2 g, 21.72 mmol) to the solution and stir for 30 minutes. Compound 64-1 (1.5 g, 7.24 mmol) is then added to the reaction mixture and stirred at 45°C for 12 hours.

[0728] After LCMS monitoring showed the product was generated, the reaction solution was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to give compound 64-2 (490.0 mg, yield 42.6%).

[0729] MS (ESI) M / Z: 160.2 [M+H] + .

[0730] Step B: Dissolve compound 64-2 (490.0 mg, 3.08 mmol) in methanol (15.0 mL) at room temperature. Then, add acetic acid (554.4 mg, 9.24 mmol) and benzaldehyde (979.4 mg, 9.24 mmol) to the solution. The reaction system is then stirred at room temperature for 16 hours.

[0731] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure, and water (20 mL) was added to the resulting residue to quench the reaction. The mixture was extracted with ethyl acetate (20 mL × 3 times), and the organic phases were combined and washed with saturated brine (15 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1) to give compound 64-3 (650.0 mg, yield 85.4%).

[0732] MS (ESI) M / Z: 248.2 [M+H] + .

[0733] Step C: Dissolve compound 64-3 (650.0 mg, 2.63 mmol) in methanol (7.5 mL) and water (7.5 mL) at room temperature. Then, add lithium hydroxide monohydrate (220.9 mg, 5.26 mmol) to the solution. The reaction system is then stirred at room temperature for 2 hours.

[0734] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. Water (15 mL) was added to the resulting residue, and the pH was adjusted to 4 at 0°C. The mixture was then extracted with dichloromethane (20 mL × 3 times), and the organic phases were combined and washed with saturated brine (15 mL × 2 times). Finally, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide compound 64-4 (410.0 mg, 78.1% yield).

[0735] MS (ESI) M / Z: 220.2 [M+H] + .

[0736] Step D: Compound 64-4 (410.0 mg, 1.86 mmol) and compound 1-1 (493.8 mg, 0.93 mmol) were dissolved in N,N-dimethylformamide (9.0 mL) at room temperature under nitrogen. The solution was then cooled to 0°C, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (706.8 mg, 1.86 mmol) and N,N-diisopropylethylamine (599.9 mg, 4.65 mmol) were added sequentially. The reaction system was then stirred at 0°C for 30 minutes.

[0737] After LCMS monitoring showed the disappearance of the starting material, water (40 mL) was added to the reaction solution to quench the mixture. The mixture was extracted with ethyl acetate (15 mL × 3 times), and the organic phases were combined and washed with saturated brine (25 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to provide compound 64-5 (400.0 mg, yield 73.1%).

[0738] MS (ESI) M / Z: 637.2 [M+H] + .

[0739] Step E: Dissolve compound 64-5 (400.0 mg, 0.63 mmol) in methanol (6.0 mL) at room temperature. Then, cool the solution to 0°C and add hydroxylamine hydrochloride (217.2 mg, 3.15 mmol). Stir the reaction system at room temperature for 2 hours.

[0740] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to obtain compound 64-P1 (170.0 mg, yield 49.2%) and compound 64-P2 (80.0 mg, yield 23.2%).

[0741] Compound 64-P1

[0742] LCMS (ESI) M / Z: 549.0 [M+H] + .

[0743] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 6.639 min.

[0744] 1H NMR(400MHz,DMSO-d6)δ8.30(d,J=8.8Hz,1H),7.81(d,J=11.2Hz,1H),7.33(s,1H),7.30 (s,1H),6.51(s,1H),5.83(s,1H),5.67-5.58(m,1H),5.41(s,2H),5.29(d,J=19.2Hz,1H ),5.21(d,J=18.8Hz,1H),3.24-3.14(m,2H),2.67(d,J=8.8Hz,1H),2.41(s,3H),2.23-2 .06(m,2H),1.95-1.75(m,2H),0.92-0.79(m,4H),0.57-0.35(m,3H),0.29-0.18(m,1H).

[0745] Compound 64-P2

[0746] LCMS (ESI) M / Z: 549.0 [M+H] + .

[0747] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 6.729 min.

[0748] 1 H NMR(400MHz,DMSO-d6)δ8.30(d,J=8.8Hz,1H),7.81(d,J=10.8Hz,1H),7.33(s,1H),7.30 (s,1H),6.51(s,1H),5.84(s,1H),5.67-5.56(m,1H),5.41(s,2H),5.29(d,J=19.2Hz,1H ),5.21(d,J=19.2Hz,1H),3.24-3.14(m,2H),2.67(d,J=8.8Hz,1H),2.41(s,3H),2.22-2 .07(m,2H),1.92-1.77(m,2H),0.93-0.78(m,4H),0.57-0.35(m,3H),0.29-0.20(m,1H).

[0749] Example 65:

[0750] Preparation of 3-chloro-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxamide (Compound 65)

[0751] Synthesis route:

[0752] Steps:

[0753] Step A: Compound 65-1 (187.0 mg, 1.00 mmol) was dissolved in MeOH (4 mL), and NaOH (120.0 mg, 3.00 mmol) was added at 0°C. The reaction was allowed to proceed at room temperature for 12 hours.

[0754] LCMS monitoring indicated the reaction was complete. The reaction solution was adjusted to pH 5-6 with dilute HCl, diluted with ethyl acetate (20 mL), and washed sequentially with water (10 mL × 1) and saturated sodium chloride solution (10 mL × 1). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 65-2 (50 mg, 28.8% yield).

[0755] Step B: Compound 65-2 (30.0 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (3 mL), and then 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (52.0 mg, 0.10 mmol), N,N-diisopropylethylamine (0.3 mL) and compound 1-1 (20.0 mg, 0.05 mmol) were added thereto, and the reaction was carried out at room temperature for 12 hours.

[0756] After LCMS monitoring showed that the reaction was complete, the product was diluted with ethyl acetate (20 mL), washed sequentially with water (10 mL × 1 time) and saturated sodium chloride solution (10 mL × 1 time), the organic phases were combined, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography (purification conditions are as follows, mobile phase: 0.1% formic acid aqueous solution and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% within 13 minutes; detection wavelength: 254 nm) to give the title compound 65 (1.6 mg, yield 5.8%).

[0757] LCMS (ESI) M / Z: 591.2 [M+H] + .

[0758] 1H NMR (400MHz, MeOD) δ7.70–7.62(m,3H),6.72(d,J=8.9Hz,1H),5.81(d,J=5.7Hz,1H),5.61(t,J=17.3Hz, 2H),5.40–5.31(m,3H),3.28–3.21(m,3H),2.47–2.39(m,6H),1.99–1.92(m,2H),1.00(t,J=7.4Hz,3H).

[0759] Example 66:

[0760] Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-3-methyl-6-oxo-1,6-dihydropyridine-2-carboxamide (Compound 66)

[0761] Synthesis route:

[0762] Steps:

[0763] Step A: Dissolve 3-methyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid (16.0 mg, 0.10 mmol) in N,N-dimethylformamide (4 mL). Then, add 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (52.0 mg, 0.10 mmol), N,N-diisopropylethylamine (0.5 mL), and compound 1-1 (20.0 mg, 0.05 mmol). The reaction is allowed to proceed at room temperature for 12 hours.

[0764] LCMS monitoring indicated the reaction was complete. The product was diluted with dichloromethane (20 mL) and washed sequentially with water (10 mL x 1) and saturated sodium chloride solution (10 mL x 1). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by preparative high-performance liquid chromatography (HPLC) (purification conditions: mobile phase: 0.1% aqueous formic acid and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% over 13 minutes; detection wavelength: 254 nm) to afford the title compound 66 (1.9 mg, 7.2% yield).

[0765] LCMS (ESI) M / Z: 571.2 [M+H] + .

[0766] 1H NMR (400MHz, MeOD) δ7.73–7.62(m,2H),7.53(d,J=8.8Hz,1H),6.64(d,J=9.8Hz,1H),5.80(s,1H),5.54(t,J=17.2Hz,2H ),5.34(s,2H),2.45(s,3H),2.40(s,3H),2.19(t,J=7.6Hz,2H),2.03(s,2H),1.99–1.89(m,2H),1.00(t,J=7.4Hz,3H).

[0767] Example 67:

[0768] Preparation of (N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-5-methyl-6-oxo-1,6-dihydropyridine-2-carboxamide (Compound 67)

[0769] Synthesis route:

[0770] Compound 5-methyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid (16.0 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (4 mL), and then 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.0 mg, 0.08 mmol) and N,N-diisopropylethylamine (0.5 mL) were added. After reacting at room temperature for 0.5 hours, compound 1-1 (20.0 mg, 0.05 mmol) was added and the reaction was allowed to proceed at room temperature for 2 hours.

[0771] LCMS monitoring indicated the reaction was complete. The product was diluted with dichloromethane (20 mL) and washed sequentially with water (10 mL × 1) and saturated sodium chloride solution (10 mL × 1). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by preparative high-performance liquid chromatography (HPLC) (purification conditions: mobile phase: 0.1% aqueous formic acid and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 95% over 13 minutes; detection wavelength: 254 nm) to afford the title compound 67 (2.1 mg, 8.0% yield).

[0772] LCMS (ESI) M / Z: 571.4 [M+H] + .

[0773] 1H NMR(400MHz,MeOD)δ7.66(d,J=10.8Hz,1H),7.61(s,1H),7.51(d,J=7.0Hz,1H),7.12(s,1H),5.91–5.79(m,1H),5.50(d, J=16.3Hz,1H),5.35–5.12(m,3H),3.25(s,2H),2.54–2.27(m,5H),2.17(s,3H),2.00–1.87(m,2H),0.98(t,J=7.4Hz,3H).

[0774] Example 68:

[0775] Preparation of 2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)amino)acetonitrile (Compound 68)

[0776] Reaction route:

[0777] Steps:

[0778] Step A: Dissolve compound 1-1 (50.0 mg, 0.09 mmol) in N,N-dimethylformamide (0.45 mL) at room temperature. Cool the solution to 0°C and add N,N-diisopropylethylamine (46.0 mg, 0.36 mmol) and 2-chloroacetonitrile (10.0 mg, 0.14 mmol) sequentially. The reaction mixture is stirred at 80°C for 16 hours.

[0779] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (purification conditions: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm). The product was collected and lyophilized under reduced pressure to obtain the title compound 68 (14.9 mg, 23.0% yield).

[0780] LCMS (ESI) M / Z: 475.0 [M+H] + .

[0781] 1H NMR (400MHz, DMSO-d6) δ7.76(d,J=10.8Hz,1H),7.32(s,1H),6.53(s,1H),5.45(d,J=18.8Hz,1H),5.44(s,2H),5.36(d,J=18.8Hz,1H) ,4.35(brs,1H),3.97-3.75(m,2H),3.22-2.98(m,3H),2.42-2.31(m,4H),2.11-1.98(m,1H),1.97-1.78(m,2H),0.88(t,J=7.2Hz,3H).

[0782] Example 69:

[0783] Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-sulfamide (Compound 69)

[0784] Reaction route:

[0785] Steps:

[0786] Step A: Dissolve compound 1-1 (150.0 mg, 0.28 mmol) in N,N-dimethylformamide (3.0 mL) at room temperature under nitrogen. Cool the solution to 0°C, add triethylamine (141.4 mg, 1.40 mmol), and stir for 10 minutes. Then, add aminosulfonyl chloride (64.4 mg, 0.56 mmol), warm to room temperature, and continue stirring for 1 hour.

[0787] After LCMS monitoring indicated product formation, the reaction solution was directly purified by reverse-phase chromatography. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 0% to 50% over 30 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 69 (15.3 mg, 10.6% yield).

[0788] LCMS (ESI) M / Z: 515.0 [M+H] + .

[0789] 1H NMR (400MHz, DMSO-d6) δ7.78 (d, J = 11.2Hz, 1H), 7.46-7.26 (m, 2H), 6.97 (s, 2H), 6.52 (s, 1H), 5.56-5.35 (m, 4H), 5. 00-4.86(m,1H),3.27-3.21(m,1H),3.15-3.05(m,1H),2.43-2.21(m,5H),1.95-1.79(m,2H),0.88(t,J=7.4Hz,3H).

[0790] Example 70:

[0791] Preparation of 1-ethyl-3-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(2-hydroxyethyl)urea (Compound 70)

[0792] Reaction route:

[0793] Steps:

[0794] Step A: Dissolve compound 1-1 (50.0 mg, 0.09 mmol) in N,N-dimethylformamide (0.45 mL) at room temperature. Cool the solution to 0°C and add N,N'-carbonyldiimidazole (29.0 mg, 0.18 mmol) and N,N-diisopropylethylamine (58.0 mg, 0.45 mmol) sequentially. Stir for 1 hour. Then, add 2-ethylaminoethane-1-ol (8.0 mg, 0.09 mmol) and continue stirring for 30 minutes.

[0795] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 70 (7.9 mg, 15.3% yield).

[0796] LCMS (ESI) M / Z: 551.1 [M+H] + .

[0797] 1H NMR(400MHz, DMSO-d6)δ7.76(d,J=11.2Hz,1H),7.31(s,1H),6.87(d,J=8.0Hz,1H) ,6.51(s,1H),5.49-5.34(m,3H),5.23(d,J=19.2Hz,1H),5.17(d,J=19.2Hz,1H),4. 87(brs,1H),3.56-3.47(m,2H),3.45-3.38(m,2H),3.26-3.03(m,4H),2.39(s,3H), 2.20-2.10(m,2H),1.94-1.78(m,2H),1.08(t,J=7.0Hz,3H),0.87(t,J=7.2Hz,3H).

[0798] Example 71:

[0799] Preparation of 3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-1-hydroxy-1-methylguanidine (Compound 71)

[0800] Reaction route:

[0801] Steps:

[0802] Step A: Dissolve compound 33-1 (50.0 mg, 0.11 mmol) in N,N-dimethylformamide (1 mL) at room temperature. Cool the solution to 0°C and add N-methylhydroxylamine (27.0 mg, 0.32 mmol) and triethylamine (66.0 mg, 0.66 mmol) sequentially. Stir the reaction mixture for 5 minutes.

[0803] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 71 (6.4 mg, 11.0% yield).

[0804] LCMS (ESI) M / Z: 508.0 [M+H] + .

[0805] 1H NMR (400MHz, DMSO-d6) δ8.30 (s, 2H), 7.80 (d, J = 10.8Hz, 1H), 7.33 (s, 1H), 5.60-5.14 (m, 5H), 3.30 (s, 3H), 3. 17-3.05(m,2H),2.40(s,3H),2.32-2.22(m,1H),2.19-2.11(m,1H),1.95-1.78(m,2H),0.88(t,J=7.2Hz,3H).

[0806] Example 72:

[0807] Preparation of 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)urea (Compound 72)

[0808] Reaction route:

[0809] Steps:

[0810] Step A: Dissolve compound 1-1 (70.0 mg, 0.13 mmol) in N,N-dimethylformamide (1 mL) at room temperature. Cool the solution to 0°C and add N,N-carbonyldiimidazole (42.0 mg, 0.26 mmol) and triethylamine (66.0 mg, 0.65 mmol) sequentially. Stir the reaction mixture for 40 minutes, then add aqueous ammonia (9.0 mg, 0.26 mmol). Stir the mixture for 20 minutes.

[0811] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: methanol from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 72 (12.2 mg, 20.0% yield).

[0812] LCMS (ESI) M / Z: 479.0 [M+H] + .

[0813] 1H NMR (400MHz, DMSO-d6) δ7.78(d,J=10.8Hz,1H),7.31(s,1H),6.73(d,J=8.8Hz,1H),6.52(s,1H),5.68(s,2H),5.43(s ,2H),5.38-5.19(m,3H),3.20-3.11(m,2H),2.40(s,3H),2.23-2.08(m,2H),1.95-1.80(m,2H),0.88(t,J=7.2Hz,3H).

[0814] Example 73:

[0815] Preparation of 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-3-(2-hydroxyethyl)urea (Compound 73)

[0816] Reaction route:

[0817] Steps:

[0818] Step A: Dissolve compound 1-1 (75.0 mg, 0.14 mmol) and N,N-diisopropylethylamine (109.4 mg, 0.85 mmol, 0.15 mL) in N,N-dimethylformamide (1.5 mL) at 0°C. Add N,N-carbonyldiimidazole (34.3 mg, 0.21 mmol) and stir for 30 minutes. Then, add 2-aminoethane-1-ol (17.2 mg, 0.28 mmol) and continue stirring for 1 hour.

[0819] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SunFire Prep C18OBD 10 μm, 19 mm × 150 mm; mobile phase: water (containing 0.1% formic acid) and acetonitrile; flow rate: 20 mL / min; gradient: 40% to 55% acetonitrile over 15 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 73 (9.8 mg, 13.3% yield).

[0820] LCMS (ESI) M / Z: 523.3 [M+H] + .

[0821] 1H NMR(400MHz,DMSO-d6)δ7.76(d,J=11.2Hz,1H),7.30(s,1H),6.74(d,J=9.2 Hz,1H),6.51(s,1H),6.01(t,J=5.6Hz,1H),5.42(s,2H),5.39-5.28(m,2H), 5.21(d,J=19.2Hz,1H),4.70(brs,1H),3.49-3.40(m,2H),3.22-3.06(m,4H ),2.38(s,3H),2.23-2.06(m,2H),1.94-1.75(m,2H),0.87(t,J=7.2Hz,3H).

[0822] Example 74:

[0823] Preparation of 3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-1-(2-hydroxyethyl)-1-methylurea (Compound 74)

[0824] Reaction route:

[0825] Steps:

[0826] Step A: Dissolve compound 1-1 (75.0 mg, 0.14 mmol) and N,N-diisopropylethylamine (109.4 mg, 0.85 mmol) in N,N-dimethylformamide (1.5 mL) at 0°C. Add N,N-carbonyldiimidazole (34.3 mg, 0.21 mmol) to the solution and stir for 30 minutes. Then, add 2-(methylamino)ethane-1-ol (24.2 mg, 0.28 mmol) and continue stirring for 1 hour.

[0827] After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was directly purified by preparative HPLC. Purification conditions were as follows: preparative column: SunFire Prep C18OBD 10 μm, 19 mm × 150 mm; mobile phase: water (containing 0.1% formic acid) and acetonitrile; flow rate: 20 mL / min; gradient: 40% to 55% acetonitrile over 15 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 74 (8.6 mg, 11.3% yield).

[0828] LCMS (ESI) M / Z: 537.5 [M+H] + .

[0829] 1 H NMR (400MHz, DMSO-d6) δ7.76(d,J=10.8Hz,1H),7.31(s,1H),6.81(d,J=8.4Hz,1H),5.50-5.34(m,3 H),5.23(d,J=18.8Hz,1H),5.16(d,J=18.8Hz,1H),4.78(t,J=5.2Hz,1H),3.59-3.47(m,2H),3.41- 3.35(m,1H),3.29-3.17(m,2H),3.15-3.04(m,1H),2.92(s,3H),2.39(s,3H),2.21-2.10(m,2H),1.93-1.78(m,2H),0.87(t,J=7.2Hz,3H).

[0830] Example 75:

[0831] Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-1-methylpiperidine-2-carboxamide (Compound 75)

[0832] Reaction route:

[0833] Steps:

[0834] Step A: Dissolve compound 1-1 (70.0 mg, 0.13 mmol) in N,N-dimethylformamide (1 mL) at room temperature. The solution was then cooled to 0°C and (S)-1-methylpiperidine-2-carboxylic acid (24.0 mg, 0.17 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (59.0 mg, 0.16 mmol), and N,N-diisopropylethylamine (84.0 mg, 0.65 mmol) were added sequentially. The reaction mixture was stirred for 5 minutes.

[0835] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 75 (7.31 mg, 10.0% yield).

[0836] LCMS (ESI) M / Z: 561.2 [M+H] + .

[0837] HPLC conditions: 0-0.5 min, 5% acetonitrile / water (0.05% trifluoroacetic acid), 0.5-9 min, 5-95% acetonitrile / water (0.05% trifluoroacetic acid), 9-12 min, 95% acetonitrile / water (0.05% trifluoroacetic acid). Retention time Rt = 4.837 min. 1 H NMR (400MHz, DMSO-d6) δ8.46(d,J=8.4Hz,1H),7.80(d,J=10.8Hz,1H),7.31(s,1H),6. 52(s,1H),5.62-5.51(m,1H),5.43(s,2H),5.26(d,J=18.8Hz,1H),5.08(d,J=18.8Hz,1 H),3.21-3.12(m,2H),2.88-2.78(m,1H),2.49-2.45(m,1H),2.40(s,3H),2.20-2.07( m,5H),2.00-1.81(m,3H),1.79-1.42(m,5H),1.25-1.08(m,1H),0.88(t,J=7.4Hz,3H).

[0838] Example 76:

[0839] Preparation of (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-1-methylpiperidine-2-carboxamide (Compound 76)

[0840] Reaction route:

[0841] Steps:

[0842] Step A: Dissolve compound 1-1 (70.0 mg, 0.13 mmol) in N,N-dimethylformamide (1 mL) at room temperature. The solution was then cooled to 0°C and (S)-1-methylpiperidine-2-carboxylic acid (24.0 mg, 0.17 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (59.0 mg, 0.16 mmol), and N,N-diisopropylethylamine (84.0 mg, 0.65 mmol) were added sequentially. The reaction mixture was stirred for 5 minutes.

[0843] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 76 (6.5 mg, 9.0% yield).

[0844] LCMS (ESI) M / Z: 561.1 [M+H] + .

[0845] HPLC conditions: 0-0.5 min, 5% acetonitrile / water (0.05% trifluoroacetic acid), 0.5-9 min, 5-95% acetonitrile / water (0.05% trifluoroacetic acid), 9-12 min, 95% acetonitrile / water (0.05% trifluoroacetic acid). Retention time Rt = 4.933 min.

[0846] 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=8.8Hz,1H),7.78(d,J=10.8Hz,1H),7.31(s,1H),6. 52(s,1H),5.63-5.53(m,1H),5.42(s,2H),5.20(d,J=20.8Hz,1H),5.13(d,J=18.8Hz,1 H),3.20-3.15(m,2H),2.90-2.81(m,1H),2.57-2.52(m,1H),2.39(s,3H),2.25-2.07( m,5H),2.00-1.76(m,4H),1.75-1.38(m,4H),1.29-1.12(m,1H),0.88(t,J=7.4Hz,3H).

[0847] Example 77:

[0848] (R)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-2-(hydroxy(methyl)amino)acetamide (Compound 77-P1 or Compound 77-P2) and ( Preparation of S)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]naza[1,2-b]quinolin-1-yl)-2-(hydroxy(methyl)amino)acetamide (Compound 77-P2 or Compound 77-P1)

[0849] Reaction route:

[0850] Steps:

[0851] Step A: Dissolve compound 77-1 (2.0 g, 9.66 mmol) in dichloromethane (45 mL) at room temperature. Cool the solution to 0°C and add triethylamine (3.4 g, 28.98 mmol) and methylhydroxylamine hydrochloride (1.2 g, 14.40 mmol) sequentially. Stir the reaction at room temperature for 16 hours.

[0852] After LCMS monitoring showed the disappearance of the starting material, dichloromethane (10 mL) was added to the reaction mixture for dilution, and the mixture was washed with saturated sodium chloride solution (25 mL x 2). It was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified on a silica gel column (eluent: dichloromethane / methanol = 20 / 1) to afford compound 77-2 (590.0 mg, 25.0% yield).

[0853] LCMS (ESI) M / Z: 174.0 [M+H] + .

[0854] Step B: Dissolve compound 77-2 (590.0 mg, 3.41 mmol) in methanol (10 mL) at room temperature. Cool the solution to 0°C and add lithium hydroxide (214.0 mg, 5.12 mmol) and water (7 mL) sequentially. Stir the reaction at room temperature for 3 hours.

[0855] After LCMS monitoring showed the disappearance of the starting material, dilute hydrochloric acid was added dropwise to the reaction solution at 0°C to adjust the pH to 4. The solution was then concentrated under reduced pressure to obtain a crude product of compound 77-3 (740.0 mg, yield 151.0%).

[0856] LCMS (ESI) M / Z: 146.0 [M+H] + .

[0857] Step C: Dissolve compound 77-3 (600.0 mg, 0.46 mmol) in N,N-dimethylformamide (3.8 mL) at room temperature. Then, cool the solution to 0°C and add compound 1-1 (200.0 mg, 0.38 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (175.0 mg, 0.46 mmol) in this order. Stir the reaction mixture for 20 minutes.

[0858] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254nm. The product was collected and lyophilized under reduced pressure to obtain a mixture of compound 77-P1 and compound 77-P2. The mixture was then subjected to TLC (methanol / dichloromethane = 1:15) to obtain the title compound 77-P1 (21.4mg, 19.0% yield) and the title compound 77-P2 (1.4mg, 1.0% yield).

[0859] Compound 77-P1:

[0860] LCMS (ESI) M / Z: 563.1 [M+H] + .

[0861] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 7.036 min.

[0862] 1H NMR(400MHz,DMSO-d6)δ8.13-7.92(m,2H),7.79(d,J=11.2Hz,1H),7.32(s,1H),6.52 (s,1H),5.57-5.48(m,1H),5.43(s,2H),5.23(s,2H),3.21-3.11(m,2H),2.59(s,3H) ,2.39(s,3H),2.26-2.16(m,2H),2.15-2.04(m,1H),1.93-1.79(m,2H),1.05-0.94(m ,1H),0.88(t,J=7.4Hz,3H),0.65-0.54(m,1H),0.51-0.40(m,1H),0.39-0.25(m,2H).

[0863] Compound 77-P2:

[0864] LCMS (ESI) M / Z: 563.2 [M+H] + .

[0865] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 7.012 min.

[0866] 1 H NMR (400MHz, DMSO-d6) δ8.14-8.02(m,2H),7.79(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1 H),5.66-5.54(m,1H),5.42(s,2H),5.29(d,J=19.2Hz,1H),5.22(d,J=19.2Hz,1H),3.23- 3.07(m,2H),2.62(s,3H),2.40(s,3H),2.23(d,J=9.6Hz,1H),2.20-2.10(m,2H),1.94-1. 76(m,2H),1.08-0.94(m,1H),0.87(t,J=7.4Hz,3H),0.77-0.54(m,2H),0.47-0.26(m,2H).

[0867] Example 78:

[0868] Preparation of (R)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-2-hydroxypropionamide (Compound 78)

[0869] Reaction route:

[0870] Steps:

[0871] Step A: Dissolve compound 78-1 (200.0 mg, 1.90 mmol) in 1,4-dioxane (5.0 mL) and water (1.6 mL) at room temperature. Cool the solution to 0°C, add sodium bicarbonate (478.8 mg, 5.7 mmol), and stir for 10 minutes. Then, add di-tert-butyl dicarbonate (621.3 mg, 2.85 mmol) and continue stirring for 16 hours.

[0872] After LCMS monitoring indicated product formation, the reaction mixture was quenched by the addition of water (12 mL). The mixture was adjusted to pH 4-5 with 1 M dilute hydrochloric acid and extracted with ethyl acetate (10 mL × 3 times). The organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 78-2 (400.0 mg, 102.5% yield).

[0873] LCMS (ESI) M / Z: 228.2 [M+Na] + .

[0874] Step B: Compound 78-2 (150.0 mg, 0.73 mmol) was dissolved in N,N-dimethylformamide (3.5 mL) at room temperature. The solution was then cooled to 0°C, and compound 1-1 (150.0 mg, 0.28 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (159.6 mg, 0.42 mmol), and N,N-diisopropylethylamine (252.8 mg, 1.96 mmol) were added sequentially. The reaction was stirred at 0°C for 15 minutes. After LCMS monitoring indicated the disappearance of the starting material, the reaction mixture was quenched by the addition of water (20 mL). The mixture was extracted with ethyl acetate (7 mL x 3 times), and the organic phases were combined and washed with saturated brine (15 mL x 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to give compound 78-3 (50.0 mg, yield 10.9%).

[0875] LCMS (ESI) M / Z: 523.4 [M-99].

[0876] Step C: At room temperature under nitrogen protection, compound 78-3 (50.0 mg, 0.08 mmol) was dissolved in 4 mol / L hydrochloric acid-ether solution (0.4 mL) and dichloromethane (0.8 mL), and the reaction system was stirred at room temperature for 2 hours.

[0877] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered, and the resulting solid was dissolved in water and lyophilized under reduced pressure to obtain the title compound 78 (8.2 mg, yield 19.6%).

[0878] LCMS (ESI) M / Z: 523.0 [M+H] + .

[0879] 1 H NMR (400MHz, DMSO-d6) δ8.79(d,J=8.8Hz,1H),8.21(s,3H),7.72(d,J=10.8Hz, 1H),7.29(s,1H),5.63-5.52(m,1H),5.41(s,2H),5.22(d,J=18.8Hz,1H),5.02( d,J=18.8Hz,1H),4.38(dd,J=9.2,3.2Hz,1H),3.36-3.19(m,2H),3.17-3.04(m ,2H),2.36(s,3H),2.26-2.12(m,2H),1.94-1.77(m,2H),0.87(t,J=7.2Hz,3H).

[0880] Example 79:

[0881] Preparation of (R)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-3-hydroxypropylamine (Compound 79)

[0882] Reaction route:

[0883] Steps:

[0884] Step A: Compound 1-1 (75.0 mg, 0.14 mmol) and N,N-diisopropylethylamine (109.4 mg, 0.85 mmol, 0.15 mL) were dissolved in N,N-dimethylformamide (3 mL) at 0°C. (tert-Butyloxycarbonyl)-D-serine (34.8 mg, 0.17 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (69.8 mg, 0.18 mmol) were then added to the solution. The reaction was stirred for 1 hour.

[0885] After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was directly purified by preparative HPLC. Purification conditions were as follows: preparative column: SunFire Prep C18OBD 10 μm, 19 mm × 150 mm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 20 mL / min; gradient: 40% to 55% acetonitrile over 15 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain compound 79-1 (50.0 mg, white solid, 56.9% yield).

[0886] LCMS (ESI) M / Z: 623.5 [M+H] + .

[0887] Step B: Compound 79-1 (50.0 mg, 0.08 mmol) was dissolved in dichloromethane (3 mL) at 0°C, and then 4 mol / L hydrochloric acid-ether solution (1 mL) was added. The reaction system was then stirred for 1 hour.

[0888] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in water (1 mL) at 0°C and the pH was adjusted to 8 with 25% wt aqueous ammonia. The mixture was then purified by preparative HPLC. The purification conditions were as follows: preparative column: SunFire Prep C18OBD 10um 19mm×150mm; mobile phase: water (containing 0.1% formic acid) and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 40% to 55% over 15 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 79 (24.7 mg, white solid, yield 58.7%).

[0889] LCMS (ESI) M / Z: 523.3 [M+H] + .

[0890] 1H NMR(400MHz,DMSO-d6)δ8.48(s,1H),7.80(d,J=10.8Hz,1H),7.31(s,1H),6.52(s,1H),5.57(brs,1H),5.43(s,2H),5.23(s,2H),3 .64-3.53(m,3H),3.37-3.34(m,2H),3.21-3.13(m,2H),2.41(s,3H),2.27-2.06(m,2H),1.93-1.78(m,2H),0.87(t,J=7.4Hz,3H).

[0891] Example 80:

[0892] Preparation of (2R,3R)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]nazo[1,2-b]quinolin-1-yl)-3-hydroxybutylamine (Compound 80)

[0893] Reaction route:

[0894] Steps:

[0895] Step A: Dissolve compound 80-1 (300.0 mg, 2.52 mmol) in 1,4-dioxane (6.0 mL) and water (2.0 mL) at room temperature. Cool the solution to 0°C, add sodium bicarbonate (635.0 mg, 7.56 mmol), and stir for 10 minutes. Then, add di-tert-butyl dicarbonate (824.0 mg, 3.78 mmol), warm to room temperature, and continue stirring for 16 hours.

[0896] After LCMS monitoring indicated product formation, the reaction mixture was quenched by the addition of water (12 mL). The mixture was adjusted to pH 4-5 with 1 M dilute hydrochloric acid and extracted with ethyl acetate (10 mL × 3 times). The organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 80-2 (320.0 mg, 58.0% yield).

[0897] LCMS (ESI) M / Z: 242.0 [M+Na] + .

[0898] Step B: Dissolve compound 80-2 (100 mg, 0.46 mmol) in N,N-dimethylformamide (2.5 mL) at room temperature. Cool the solution to 0°C and add compound 1-1, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (209.7 mg, 0.55 mmol), and N,N-diisopropylethylamine (178.0 mg, 1.38 mmol) in sequence. Stir the reaction at 0°C for 15 minutes.

[0899] After LCMS monitoring showed the disappearance of the starting material, water (15 mL) was added to the reaction mixture to quench the reaction. The mixture was extracted with ethyl acetate (5 mL × 3 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to provide compound 80-3 (150.0 mg, yield 51.2%).

[0900] LCMS (ESI) M / Z: 637.8 [M+H] + .

[0901] Step C: At room temperature under nitrogen protection, compound 80-3 (50.0 mg, 0.08 mmol) was dissolved in 4 mol / L hydrochloric acid-ether solution (0.4 mL) and dichloromethane (0.8 mL), and the reaction system was stirred at room temperature for 2 hours.

[0902] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered and the obtained solid was dissolved in deionized water and lyophilized under reduced pressure to obtain the title compound 80 (10.7 mg, yield 24.9%).

[0903] LCMS (ESI) M / Z: 537.0 [M+H] + .

[0904] 1H NMR (400MHz, DMSO-d6) δ8.97(d,J=8.4Hz,1H),8.17(s,3H),7.84(d,J=10.8Hz,1H),7.3 3(s,1H),5.44(s,2H),5.40(d,J=19.2Hz,1H),5.29(d,J=18.8Hz,1H),4.12-4.03(m,1H ),3.96-3.85(m,1H),3.31-3.21(m,1H),3.18-3.04(m,1H),2.42(s,3H),2.29-2.17(m, 1H),2.17-2.02(m,1H),1.96-1.80(m,2H),1.15(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[0905] Example 81:

[0906] Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-(hydroxyamino)butanamide (Compound 81)

[0907] Reaction route:

[0908] Steps:

[0909] Step A: Dissolve tert-butyl ((tert-butoxycarbonyl)oxy)carbamate (500.0 mg, 2.10 mmol) in N,N-dimethylformamide (9 mL) at 0°C under nitrogen. Add sodium hydride (128.0 mg, 3.20 mmol, 60% content) to the solution, warm to room temperature, and stir for 30 minutes. Then, lower the temperature to 0°C and slowly add compound 81-1 (489 mg, 2.7 mmol). Stirring continues for 16 hours.

[0910] After LCMS monitoring indicated product formation, the reaction mixture was quenched by the addition of water (10 mL). The mixture was extracted with ethyl acetate (10 mL × 3 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent volume ratio: methanol / dichloromethane = 1 / 10) to afford compound 81-2 (360.0 mg, 72% yield).

[0911] MS (ESI) M / Z: 356.0 [M+Na]+ .

[0912] Step B: Compound 81-2 (200.0 mg, 0.60 mmol) was dissolved in methanol (4 mL) and water (1 mL) at room temperature, followed by the addition of lithium hydroxide (29.0 mg, 1.20 mmol), and the reaction mixture was stirred for 30 minutes.

[0913] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was adjusted to pH 3-4 with 1M dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL × 3 times). The organic phases were combined and washed with saturated brine (10 mL × 2 times). Then, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 81-3 (60.0 mg, yield 30.0%).

[0914] MS (ESI) M / Z: 242.0 [M+Na-Boc] + .

[0915] Step C: Dissolve compound 81-3 (60.0 mg, 0.19 mmol) in N,N-dimethylformamide (1 mL) at room temperature. The solution was then cooled to 0°C and compound 1-1 (101.0 mg, 0.19 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (108.0 mg, 0.29 mmol), and N,N-diisopropylethylamine (147.0 mg, 1.14 mmol) were added sequentially. The reaction mixture was stirred for 30 minutes.

[0916] After LCMS monitoring showed the disappearance of the starting material, water (20 mL) was added to the reaction solution to quench the mixture. The mixture was extracted with ethyl acetate (10 mL × 3 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The organic phases were then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to provide compound 81-4 (40.0 mg, yield 67.0%).

[0917] MS (ESI) M / Z: 737.2 [M+H] + .

[0918] Step D: Compound 81-4 (40.0 mg, 0.05 mmol) was dissolved in dichloromethane (0.27 mL) at room temperature, then the solution was cooled to 0°C, 4 mol / L hydrochloric acid-ethyl acetate solution (0.2 mL) was added, and the reaction system was stirred for 30 minutes.

[0919] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 81 (4.8 mg, 16.5% yield).

[0920] MS (ESI) M / Z: 537.2 [M+H] + .

[0921] 1 H NMR(400MHz,DMSO-d6)δ8.42(d,J=8.8Hz,1H),7.76(d,J=10.8Hz,1H),7.30(s,1 H),7.11(s,1H),6.52(s,1H),5.61-5.50(m,1H),5.42(s,2H),5.27-5.08(m,2H) ,3.21-3.11(m,2H),2.73(t,J=7.0Hz,2H),2.38(s,3H),2.22(t,J=7.4Hz,2H),2 .16-2.06(m,2H),1.94-1.79(m,2H),1.78-1.68(m,2H),0.87(t,J=7.24Hz,3H).

[0922] Example 82:

[0923] Preparation of (S)-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpyrrolidine-2-carboxamide (Compound 82)

[0924] Reaction route:

[0925] Steps:

[0926] Step A: Compound 82-1 (500.0 mg, 4.30 mmol) was dissolved in methanol (22 mL) at room temperature. 10% wt palladium / carbon (25.0 mg) and 37% wt formaldehyde (0.36 mL) were added to the solution in sequence. The reaction was then hydrogenated and stirred under a hydrogen atmosphere for 2 hours.

[0927] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered and the filtrate was concentrated under reduced pressure. The residue was slurried with methyl tert-butyl ether, filtered, and the solid was collected to obtain compound 82-2 (446.0 mg, yield 89.2%).

[0928] MS (ESI) M / Z: 130.2 [M+H] + .

[0929] Step B: Compound 82-2 (82.0 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (0.75 mL) at room temperature. The solution was then cooled to 0°C and 1-1 (20.0 mg, 0.15 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (85.5 mg, 0.23 mmol), and N,N-diisopropylethylamine (97.0 mg, 0.75 mmol) were added sequentially. The reaction system was then stirred for 30 minutes.

[0930] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: Preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 82 (5.0 mg, 25.1% yield).

[0931] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 7.818 min. MS (ESI) M / Z: 547.0 [M+H] + .

[0932] 1H NMR (400MHz, DMSO-d6) δ8.47(d,J=8.8Hz,1H),7.77(d,J=10.8Hz,1H),7.30( s,1H),6.51(s,1H),5.56-5.47(m,1H),5.42(s,2H),5.17(s,2H),3.23-3.05 (m,3H),3.04-2.97(m,1H),2.94-2.88(m,1H),2.38(s,3H),2.33(s,3H),2.3 1-2.23(m,1H),2.19-2.07(m,3H),1.93-1.68(m,5H),0.88(t,J=7.4Hz,3H).

[0933] Example 83:

[0934] Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpyrrolidine-2-carboxamide (Compound 83)

[0935] Reaction route:

[0936] Steps:

[0937] Step A: Dissolve compound 1-1 (82.0 mg, 0.15 mmol) in N,N-dimethylformamide (0.75 mL) at room temperature. The solution was then cooled to 0°C and compound 83-1 (20.0 mg, 0.15 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (85.5 mg, 0.23 mmol), and N,N-diisopropylethylamine (97.0 mg, 0.75 mmol) were added sequentially. The reaction mixture was stirred for 30 minutes.

[0938] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: Preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 83 (6.1 mg, 7.4% yield).

[0939] MS (ESI) M / Z: 547.0 [M+H] +.

[0940] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 7.925 min.

[0941] 1 H NMR (400MHz, DMSO-d6) δ8.56(d,J=8.0Hz,1H),7.77(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1H),5.62-5.50(m,1H),5.42(s,2H),5.18(d,J=1 8.8Hz,1H),5.11(d,J=18.8Hz,1H),3.26-2.94(m,5H),2.40(s,3H),2. 38(s,3H),2.30-2.10(m,3H),2.03-1.71(m,5H),0.87(t,J=7.2Hz,3H).

[0942] Example 84:

[0943] Preparation of (S)-N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylazetidine-2-carboxamide (Compound 84)

[0944] Reaction route:

[0945] Steps:

[0946] Step A: Dissolve compound 1-1 (90 mg, 0.17 mmol) in N,N-dimethylformamide (1 mL) at room temperature. Cool the solution to 0°C and add 1-methylazetidine-2-carboxylic acid (20 mg, 0.17 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (99 mg, 0.26 mmol), and N,N-diisopropylethylamine (110 mg, 0.85 mmol) sequentially. Stir the reaction mixture for 30 minutes.

[0947] After LCMS monitoring indicated the disappearance of the starting material, the resulting reaction solution was purified by preparative HPLC. Purification conditions were as follows: preparative column: SNAP Ultra C18 30g 25μm; mobile phase: 0.1% formic acid in water and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 15% to 50% over 18 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure to obtain the title compound 84 (12.5 mg, 13.8% yield).

[0948] MS (ESI) M / Z: 533.0 [M+H] + .

[0949] HPLC conditions: 0-0.5 min, 10% acetonitrile / water (10 mM ammonium bicarbonate), 0.5-9.5 min, 10-95% acetonitrile / water (10 mM ammonium bicarbonate), 9.5-11.5 min, 95% acetonitrile / water (10 mM ammonium bicarbonate). Retention time Rt = 7.377 min.

[0950] 1 H NMR (400MHz, DMSO-d6) δ8.57(d,J=9.2Hz,1H),7.77(d,J=11.2Hz,1H),7.30(s,1H),6. 51(s,1H),5.61-5.48(m,1H),5.41(s,2H),5.21(d,J=18.8Hz,1H),5.09(d,J=18.8Hz, 1H),3.60(t,J=8.2Hz,1H),3.26-3.18(m,1H),3.18-3.07(m,1H),2.97-2.87(m,1H),2 .38(s,3H),2.35(s,3H),2.31-2.04(m,5H),1.92-1.78(m,2H),0.87(t,J=7.4Hz,3H).

[0951] Example 85:

[0952] Preparation of 3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxy-1-isopropylurea (Compound 85)

[0953] Reaction route:

[0954] Steps:

[0955] Step A: Dissolve compound 1-1 (200.0 mg, 0.46 mmol) in N,N-dimethylformamide (10 mL). Slowly add triethylamine (178.4 mg, 1.38 mmol, 0.23 mL) dropwise with stirring until the system becomes clear. Slowly add N,N'-carbonyldiimidazole (223.8 mg, 1.38 mmol). After stirring at room temperature for 1 h, slowly add isopropylhydroxylamine hydrochloride (103.7 mg, 1.38 mmol) and stir at room temperature for 4 h. The reaction solution was quenched with water and washed three times with dichloromethane (20 mL). The organic phases were combined and washed three times with water (20 mL) and saturated brine (20 mL), respectively, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The crude product was purified by preparative liquid chromatography under (0-100%) acetonitrile / water (0.1% formic acid) to give compound 85 (150.0 mg, yield 60.9%).

[0956] LCMS (ESI) m / z: 537.0 [M+H] + .

[0957] 1 H NMR (400MHz, Methanol-d4) δ7.62(s,1H),7.55(d,J=10.8Hz,1H),5.57(d,J=16.3Hz,1H),5.44(dd, J=11.2,4.7Hz,1H),5.37(d,J=11.3Hz,1H),5.32(d,J=14.0Hz,1H),4.92(d,J=19.0Hz,1H),4.47(h ept,J=6.7Hz,1H),3.38–3.32(m,1H),3.17–3.09(m,1H),2.38(s,3H),2.36(d,J=4.6Hz,1H),2.22– 2.13(m,1H),1.99–1.91(m,2H),1.30(d,J=6.7Hz,3H),1.19(d,J=6.6Hz,3H),1.00(t,J=7.4Hz,3H).

[0958] Example 86

[0959] Preparation of (2R)-1-(4-((S)-2-(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutanamide)propionamide)benzyl)-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)carbamoyl)-1-methylpiperidin-1-ol (Compound 86)

[0960] Reaction route:

[0961] Steps:

[0962] Step A: Compound 86-1 (1.0 g, 3.47 mmol) was dissolved in dichloromethane (25.0 mL) at room temperature under nitrogen. The solution was then cooled to 0°C, and (4-aminophenyl)methanol (430.5 mg, 3.50 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (865.0 mg, 3.50 mmol) were added sequentially. The reaction system was stirred for 12 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was concentrated under reduced pressure and filtered. The filter cake was rinsed with dichloromethane (40 mL x 2). The solid was collected and dried under reduced pressure to afford compound 86-2 (1.4 g, 102.6% yield).

[0963] MS (ESI) M / Z: 383.0 [M-55].

[0964] Step B: Compound 86-2 (600.0 mg, 1.52 mmol) was dissolved in 4 M hydrochloric acid-1,4-dioxane solution (7.5 mL) and dichloromethane (15.0 mL) at room temperature under nitrogen. The reaction was stirred for 2 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was concentrated under reduced pressure to afford the title compound 86-3 (420.0 mg, 88.8% yield).

[0965] MS (ESI) M / Z: 312.0 [M+H] + .

[0966] Step C: Compound 86-3 (390.0 mg, 1.25 mmol) was dissolved in N,N-dimethylformamide (6.0 mL) at room temperature under nitrogen. The solution was then cooled to 0°C, and 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (390.0 mg, 1.25 mmol) and N,N-diisopropylethylamine (483.8 mg, 3.75 mmol) were added sequentially. The reaction system was stirred at room temperature for 2 hours. After LCMS monitoring showed the disappearance of the starting material, water (30 mL) was added to quench the reaction solution. The mixture was extracted with ethyl acetate (10 mL x 3 times). The organic phases were combined and washed with saturated brine (25 mL x 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10) to give compound 86-4 (450.0 mg, yield 71.4%).

[0967] MS (ESI) M / Z: 505.0 [M+H] + .

[0968] Step D: Compound 86-4 (28.0 mg, 0.05 mmol) was dissolved in N-methylpyrrolidone (0.5 mL) at room temperature under nitrogen. The solution was then cooled to 0°C and (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)-1-methylpiperidine-2-carboxamide (33.6 mg, 0.06 mmol) and sodium iodide (22.5 mg, 0.15 mmol) were added sequentially. The reaction system was then stirred at room temperature for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 10). The resulting crude product was further purified by preparative thin layer chromatography to give the title compound 86 (4.7 mg, yield 9.1%).

[0969] MS (ESI) M / Z: 1029.2 [M] + .

[0970] 1H NMR (400MHz, DMSO-d6) δ10.17(s,0.8H),10.12(s,0.2H),9.31(d,J=8.0Hz,0.8H),9.15(d,J=8.0Hz,0.2H),8.21(d,J=6.8Hz,1H),7. 92-7.75(m,2H),7.69(d,J=8.4Hz,1.6H),7.62(d,J=8.8Hz,0.4H),7.46(d,J=8.4Hz,1.6H),7.37(d,J=8.4Hz,0.4H),7.34(s,0.7H),7 .31(s,0.3H),7.20(s,0.5H),7.00(s,2H),6.65(s,0.5H),6.55(s,0.8H),6.53(s,0.2H),5.68-5.57(m,1H),5.48-5.28(m,4H),5.01( d,J=13.2Hz,1H),4.92(d,J=12.8Hz,1H),4.42-4.30(m,1H),4.26-4.21(m,0.8H),4.19-4.10(m,1H),3.99-3.95(m,0.2H),3.68-3.57 (m,2H),3.25-3.15(m,2H),3.09-2.92(m,4H),2.43(s,3H),2.28-2.08(m,6H),2.04-1.92(m,3H),1.88-1. 76(m,3H),1.65-1.56(m,1H),1.54-1.42(m,4H),1.37-1.29(m,4H),1.20-1.11(m,2H),0.93-0.79(m,9H).

[0971] Example 87:

[0972] Preparation of (2R)-1-(4-((S)-2-(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)benzyl)-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,9,13,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]nazo[1,2-b]quinolin-1-yl)carbamoyl)-1-methylpyrrolidine-1-cation (Compound 87)

[0973] Reaction route:

[0974] Steps:

[0975] Step A: Compound 1-1 (136.0 mg, 0.26 mmol) was dissolved in N,N-dimethylformamide (1.3 mL) at 0°C. Methyl-D-proline (50.0 mg, 0.39 mmol), 2-(7-benzotriazole oxide)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (146.0 mg, 0.39 mmol), and N,N-diisopropylethylamine (165.0 mg, 1.28 mmol) were then added to the solution. The reaction was stirred at room temperature for 10 minutes. After LCMS monitoring indicated the disappearance of the starting material, water (30 mL) was added to quench the reaction mixture. The mixture was extracted with ethyl acetate (60 mL x 3 times). The organic phases were combined and washed with saturated sodium chloride solution (20 mL x 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 96 / 4) to give compound 87-2 (84.0 mg, yield 54.5%).

[0976] MS (ESI) M / Z: 547.10 [M+H] + .

[0977] Step B: Compound 87-2 (42.0 mg, 0.08 mmol) was dissolved in N-methylpyrrolidone (0.6 mL) at 0°C. N-(((S)-1-((((S)-1-(4-(chloromethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (70.0 mg, 0.14 mmol) and sodium iodide (58.0 mg, 0.39 mmol) were then added to the above solution. The reaction system was then stirred at room temperature for 16 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was purified by preparative thin-layer chromatography to give the title compound 87 (16.8 mg, yield 19.7%).

[0978] MS (ESI) M / Z: 1016.50 [M+H] + .

[0979] 1H NMR (400MHz, DMSO-d6) δ10.43(s,1H),9.98(s,0.4H),9.77(s,0.6H),8.28(t,J=6.0Hz,1H),7.88(t,J=7.2 Hz,1H),7.80(d,J=11.2Hz,1H),7.77-7.67(m,2H),7.50(d,J=8.4Hz,1H),7.44(d,J=8.4Hz,1H),7.31(s,0. 6H),7.30(s,0.4H),7.00(s,2H),6.54(s,0.6H),6.53(s,0.4H),5.70-5.54(m,1H),5.51-5.20(m,4H),4.9 0(s,0.4H),4.74-4.56(m,2H),4.55-4.31(m,1.6H),4.22-4.09(m,1H),3.91-3.81(m,0.4H),3.75-3.64(m, 0.6H),3.54-3.44(m,0.6H),3.41-3.37(m,1.4H),3.32-3.11(m,3H),3.07(s,1.8H),2.95(s,1.2H),2.70-2.58(m,1H),2.4 1(s,3H),2.37-2.05(m,7H),2.02-1.78(m,3H),1.58-1.41(m,4H),1.36-1.27(m,3H),1.19-1.12(m,2H),0.92-0.78(m,9H).

[0980] Example 88:

[0981] Preparation of (2R)-1-(4-((S)-2-(S)-2-[(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-1-methylpyrrolidine-1-ol (Compound 88)

[0982] Reaction route:

[0983] Steps:

[0984] Step A: Compound 88-1 (189.0 mg, 0.33 mmol) was dissolved in dichloromethane (0.9 mL) at room temperature. Thionyl chloride (314.0 mg, 2.64 mmol) was then added dropwise to the reaction mixture. The reaction mixture was stirred at 0°C for 3 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction mixture was concentrated under reduced pressure. The crude product, compound 88-2 (219.1 mg, crude product), was obtained and carried on to the next step without further purification.

[0985] MS (ESI) M / Z: 591.2 [M+H] + .

[0986] Step B: Compound 88-2 (75.0 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (0.5 mL) at room temperature. (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpyrrolidine-2-carboxamide (32.0 mg, 0.06 mmol) and potassium iodide (68.0 mg, 0.41 mmol) were then added to the solution. The reaction was stirred at room temperature for 3 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (15 mL). The mixture was extracted with ethyl acetate (3 mL × 3 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 88 (12.5 mg, 18.9% yield).

[0987] MS (ESI) M / Z: 1101.5 [M+H] + .

[0988] 1H NMR (400MHz, DMSO-d6) δ10.26(s,1H),9.44(d,J=8.4Hz,0.5H),9.28(d,J=8.0Hz,0.5H),8.15(t,J=7.4Hz,1H),7.91-7.62(m,4H),7.52-7.40 (m,2H),7.32(s,1H),7.00(d,J=1.6Hz,2H),6.55(d,J=2.8Hz,1H),6.18-6.00(m,1H),5.68-5.56(m,1H),5.50-5.14(m,6H),4.65-4.48(m,2H ),4.46-4.29(m,2H),4.22-4.10(m,1H),3.90-3.84(m,0.5H),3.68-3. 63(m,0.5H),3.57-3.50(m,2H),3.26-3.19(m,2H),3.08(s,2H),3.02- 2.91(m,3H),2.68-2.58(m,1H),2.42(s,3H),2.16-2.07(m,5H),2.02- 1.79(m,4H),1.72-1.40(m,8H),1.23-1.11(m,2H),0.90-0.75(m,9H).

[0989] Example 89:

[0990] Preparation of (2R)-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-1-methyl-1-(4-((S)-2-(S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-carboxamido)butanamido)propanamido)benzyl)pyrrolidine-1-cation (Compound 89)

[0991] Reaction route:

[0992] Steps:

[0993] Step A: Compound 89-1 (200 mg, 0.64 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoic acid (174.2 mg, 0.65 mmol) were dissolved in N,N-dimethylformamide (3.0 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (291.8 mg, 0.77 mmol) and N,N-diisopropylethylamine (247.7 mg, 1.92 mmol) were then added to the solution. The reaction was stirred at 0°C for 0.5 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (15 mL). The mixture was extracted with ethyl acetate (3 mL x 3 times). The organic phases were combined and washed with saturated brine (10 mL x 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain compound 89-2 (300.0 mg, yield 83.5%).

[0994] MS (ESI) M / Z: 561.2 [M+H] + .

[0995] Step B: Compound 89-2 (40.0 mg, 0.07 mmol) was dissolved in N,N-dimethylformamide (0.5 mL) at room temperature. (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpyrrolidine-2-carboxamide (39.5 mg, 0.07 mmol) and potassium iodide (69.1 mg, 0.42 mmol) were then added to the solution. The reaction was stirred at room temperature for 3 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (15 mL). The mixture was extracted with ethyl acetate (3 mL × 3 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 89 (11.3 mg, 15.1% yield).

[0996] MS (ESI) M / Z: 1072.2 [M] + .

[0997] 1H NMR (400MHz, DMSO-d6) δ10.22(s,1H),9.37(d,J=7.6Hz,0.5H),9.23(d,J=8.4Hz,0.5 H),9.12(d,J=2.4Hz,2H),8.27(t,J=6.8Hz,1H),7.94(t,J=8.0Hz,1H),7.82(dd,J=10 .8,2.8Hz,1H),7.71(d,J=8.4Hz,1H),7.67(d,J=8.8Hz,1H),7.48(d,J=8.4Hz,1H),7. 44(d,J=8.4Hz,1H),7.31(d,J=1.6Hz,1H),6.54(d,J=3.2Hz,1H),5.68-5.57(m,1H),5 .48-5.28(m,3.5H),5.19(d,J=18.8Hz,0.5H),4.63-4.50(m,2.5H),4.42-4.32(m,1. 5H),4.25-4.16(m,1H),3.91-3.83(m,0.5H),3.72-3.60(m,1.5H),3.41(s,3H),3.24- 3.15(m,2H),3.08(s,1.8H),2.93(s,1.2H),2.68-2.55(m,2H),2.41(s,3H),2.37-2.2 3(m,4H),2.22-2.06(m,3H),2.04-1.76(m,6H),1.36-1.27(m,3H),0.93-0.78(m,9H).

[0998] Example 90:

[0999] Preparation of (2R)-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-1-methyl-1-(4-(((S)-2-(S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)butanamido)-5-ureidopentaamido)benzyl)pyrrolidin-1-ium (Compound 90)

[1000] Reaction route:

[1001] Steps:

[1002] Step A: Compound 90-1 (300.0 mg, 0.79 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (233.0 mg, 0.87 mmol) were dissolved in N,N-dimethylformamide (4.0 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (331.0 mg, 0.87 mmol) and N,N-diisopropylethylamine (306.0 mg, 2.37 mmol) were then added to the solution. The reaction was stirred for 0.5 h. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (40 mL). The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined and washed with saturated brine (20 mL x 2). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain compound 90-2 (205.1 mg, yield 41.2%).

[1003] MS (ESI) M / Z: 630.3 [M+H] + .

[1004] Step B: Compound 90-2 (140.0 mg, 0.22 mmol) was dissolved in N-methylpyrrolidone (4.4 mL) at room temperature. Suspended thionyl chloride (131.1 mg, 1.10 mmol) was slowly added dropwise to the solution. The reaction system was stirred for 2 hours. After LCMS monitoring showed the disappearance of the starting material, water (15 mL) was added to the reaction solution to quench the mixture. The mixture was extracted with ethyl acetate (20 mL × 3 times). The organic phases were combined and washed with saturated brine (20 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was slurried with diethyl ether (15 mL) and concentrated under reduced pressure to afford compound 90-3 (105.2 mg, 73.6% yield).

[1005] MS (ESI) M / Z: 648.3 [M+H] + .

[1006] Step C: Dissolve compound 90-3 (100.0 mg, 0.15 mmol) in N,N-dimethylformamide (1.5 mL) at room temperature. Potassium iodide (174 mg, 1.05 mmol) was then added to the solution and stirred for 10 minutes. (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpyrrolidine-2-carboxamide (82 mg, 0.15 mmol) was then added and stirring continued for 16 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give a crude product, which was then purified by preparative TLC to give compound 90 (19.3 mg, yield 11.1%).

[1007] MS (ESI) M / Z: 1158.4 [M] + .

[1008] 1H NMR (400MHz, DMSO-d6) δ10.42 (s, 0.55H), 10.40 (s, 0.45H), 9.96 (d, J = 8.4Hz, 0.45H), 9.6 3(d,J=8.0Hz,0.55H),9.12(s,2H),8.42(s,1H),8.40-8.32(m,1H),8.02(d,J=8.8Hz,1H), 7.82(d,J=10.8Hz,1H),7.72(d,J=8.4Hz,1H),7.69(d,J=8.8Hz,1H),7.53-7.38(m,2H),7. 32(s,0.55H),7.31(s,0.45H),6.56(s,1H),6.24(s,1H),5.66-5.56(m,1H),5.50(s,0.8H) ,5.49(s,1.2H),5.45-5.28(m,3.5H),5.21(d,J=18.8Hz,0.5H),4.69-4.29(m,4H),4.25-4 .19(m,1H),3.93-3.84(m,0.5H),3.68-3.50(m,2.5H),3.26-3.14(m,2H),3.08(s,2H),3.0 1-2.92(m,3H),2.65-2.53(m,3H),2.41(s,3H),2.38-2.23(m,4H),2.22-2.06(m,2H),2.04 -1.93(m,1H),1.90-1.77(m,4H),1.74-1.55(m,2H),1.49-1.32(m,2H),0.91-0.80(m,9H).

[1009] Example 91:

[1010] Preparation of N-((S)-13-benzyl-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-9,12,15,18-tetraoxo-6-oxo-3,8,11,14,17-pentaazanonan-19-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound 91)

[1011] Reaction route:

[1012] Steps:

[1013] Step A: Compound 91-1 (2.0 g, 22.45 mmol) was dissolved in dichloromethane (56 mL) at 0°C. Triethylamine (4.5 g, 44.90 mmol) and 9-fluorenylmethyl chloroformate (8.7 g, 33.68 mmol) were then added sequentially to the solution. The reaction was stirred at room temperature for 4 hours. After LCMS monitoring indicated the disappearance of the starting material, the reaction mixture was quenched with water (60 mL). The mixture was extracted with dichloromethane (50 mL x 3). The organic phases were combined and washed with saturated brine (50 mL x 2). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 3 / 97) to afford compound 91-2 (4.6 g, yield 66.4%).

[1014] MS (ESI) M / Z: 312.3 [M+H] + .

[1015] Step B: Compound 91-2 (3.1 g, 9.98 mmol) was dissolved in dry dichloromethane (41 mL) at room temperature. Methyl (2-(((benzyloxy)carbonyl)amino)acetamido)acetate (2.3 g, 8.32 mmol) and pyridinium 4-methylbenzenesulfonate (3.4 g, 12.48 mmol) were then added to the solution. The reaction was stirred at 50°C for 16 hours.

[1016] After LCMS monitoring indicated the disappearance of the starting material, dichloromethane (40 mL) was added to the reaction solution. The mixture was washed with water (40 mL × 3 times) and then with saturated brine (40 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 4 / 96) to provide compound 91-3 (1.8 g, yield 39.8%).

[1017] MS (ESI) M / Z: 554.2 [M+Na].

[1018] Step C: Compound 91-3 (1.8 g, 3.31 mmol) was dissolved in dichloromethane (16 mL) at room temperature. Diethylamine (1.0 g, 13.26 mmol) was then added to the solution. The reaction system was stirred for 16 hours.

[1019] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 13 / 87) to obtain compound 91-4 (560.0 mg, yield 55.6%).

[1020] MS (ESI) M / Z: 310.4 [M+H] + .

[1021] Step D: Compound (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (105.0 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (1.2 mL) at 0°C. N,N'-carbonyldiimidazole (58.0 mg, 0.36 mmol) was then added to the solution and stirred for 0.5 hours. Compound 91-4 (89.0 mg, 0.29 mmol) was then slowly added and stirred for 20 minutes. After LCMS monitoring showed the disappearance of the starting material, water (20 mL) was added to the reaction mixture to quench the mixture. The mixture was extracted with ethyl acetate (20 mL × 3 times), and the organic phases were combined and washed with saturated brine (40 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 4 / 96) to provide compound 91-5 (165.0 mg, yield 88.1%).

[1022] MS (ESI) M / Z: 793.2 [M+Na].

[1023] Step E: Compound 91-5 (45.0 mg, 0.10 mmol) was dissolved in methanol (4 mL) at room temperature. 10% wt palladium on carbon (22.5 mg) was then added to the solution. The reaction was stirred under a hydrogen atmosphere for 1 hour. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered and the filtrate was concentrated under reduced pressure. The resulting residue, 91-6 (30.0 mg, 81.1% yield), was used directly in the next step without further purification.

[1024] MS (ESI) M / Z: 637.3 [M+H] + .

[1025] Step F: Compound 91-6 (30.0 mg, 0.05 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycyl-L-phenylalanine (25.0 mg, 0.05 mmol) were dissolved in N,N-dimethylformamide (1.0 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (20.0 mg, 0.05 mmol) and N,N-diisopropylethylamine (12.0 mg, 0.09 mmol) were then added to the solution. The reaction was stirred for 0.5 h.

[1026] After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (25 mL). The mixture was extracted with ethyl acetate (10 mL × 3 times), and the organic phases were combined and washed with saturated brine (20 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by preparative HPLC and lyophilized under reduced pressure to give the title compound 91 (11.0 mg, 22.2% yield).

[1027] MS (ESI) M / Z: 1091.3 [M+H] + .

[1028] 1 H NMR (400MHz, DMSO-d6) δ8.51(t,J=5.8Hz,1H),8.29(t,J=5.6Hz,1H),8.11(d,J=8.0Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.6Hz,1H),7 .76(d,J=10.8Hz,1H),7.31(s,1H),7.27-7.13(m,5H),6.99(s,1.5H),6.80(d,J=8.4Hz,1H),6.51(s,1H),5.48-5.03(m,5H),4.60-4.41(m ,3H),3.81-3.61(m,6H),3.55-3.48(m,2H),3.41-3.34(m,4H),3.28- 3.17(m,2H),3.15-2.99(m,3H),2.84-2.74(m,1H),2.38(s,3H),2.22 -2.13(m,2H),2.09(t,J=7.4Hz,2H),1.92-1.77(m,2H),1.57-1.36(m,4H),1.25-1.13(m,2H),1.08(t,J=6.8Hz,3H),0.87(t,J=7.2Hz,3H).

[1029] Example 92a

[1030] 1-Cyclopropyl-3-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]nazo[1,2-b]quinolin-1-yl-)1-(2-hydroxyethyl)urea

[1031] Reaction route:

[1032] Steps:

[1033] Step A: At 0°C, compound 1-1 (75 mg, 0.14 mmol) and N,N-diisopropylethylamine (109.42 mg, 0.85 mmol, 0.15 mL) were dissolved in N,N-dimethylformamide (1.5 mL). The solution was then cooled to 0°C and di(1H-imidazol-1-yl)methanone (45.76 mg, 0.28 mmol) was added sequentially. The mixture was stirred for 30 minutes. 2-(Cyclopropylamino)ethan-1-ol (28.54 mg, 0.28 mmol) was then added and stirring continued for 1 hour. After LCMS monitoring indicated the disappearance of the starting material, the reaction solution was directly purified by preparative HPLC to afford the title compound 92a (9.81 mg, 13.3% yield).

[1034] MS (ESI) M / Z: 563.4 [M+H] + .

[1035] 1 H NMR (400MHz, DMSO-d6) δ7.77(d,J=10.8Hz,1H),7.32(s,1H),6.91(d,J=8.8Hz,1H),6.52 (s,1H),5.50-5.39(m,3H),5.31(d,J=19.2Hz,1H),5.17(d,J=18.8Hz,1H),4.73(t,J=5.4 Hz,1H),3.66-3.42(m,3H),3.29-3.20(m,3H),3.15-3.06(m,1H),2.60-2.57(m,1H),2.3 9(s,3H),2.27-2.16(m,2H),1.95-1.78(m,2H),0.87(t,J=7.4Hz,3H),0.84-0.64(m,4H).

[1036] Example 92:

[1037] Preparation of N-((S)-12-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound 92)

[1038] Reaction route:

[1039] Steps:

[1040] Step A: Dissolve compound 92-1 (2.0 g, 19.63 mmol) in dichloromethane (60.0 mL) at 0°C. Then, add triethylamine (4.54 g, 44.90 mmol) and 9-fluorenylmethyl chloroformate (8.71 g, 33.68 mmol) to the solution. The reaction mixture is stirred at room temperature for 4 hours.

[1041] After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched by adding water (60 mL). The mixture was extracted with dichloromethane (50 mL × 3 times), and the organic phases were combined and washed with saturated brine (50 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to provide compound 92-2 (5.1 g, 80.1% yield).

[1042] MS (ESI) M / Z: 324.2 [M+H] + .

[1043] Step B: Compound 92-2 (2.3 g, 7.12 mmol) was dissolved in dry dichloromethane (35 mL) at room temperature. Methyl (2-(((benzyloxy)carbonyl)amino)acetamido)acetate (2.33 g, 8.32 mmol) and pyridinium 4-methylbenzenesulfonate (3.36 g, 12.48 mmol) were then added to the solution. The reaction was stirred at 50°C for 16 hours.

[1044] After LCMS monitoring indicated the disappearance of the starting material, dichloromethane (40 mL) was added to the reaction solution. The mixture was washed with water (40 mL × 3 times) and then with saturated brine (40 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 4 / 96) to afford compound 92-3 (1.7 g, 43.8% yield).

[1045] MS (ESI) M / Z: 567.2 [M+Na] + .

[1046] Step C: Dissolve compound 92-3 (1.7 g, 3.13 mmol) in dichloromethane (16 mL) at room temperature. Then, add diethylamine (1.0 g, 13.26 mmol) to the solution. The reaction system is then stirred at room temperature for 12 hours.

[1047] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 13 / 87) to obtain compound 92-4 (230.0 mg, yield 22.8%).

[1048] MS (ESI) M / Z: 322.2 [M+H] + .

[1049] Step D: Compound 1-1 (60.5 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (1.5 mL) at 0°C. N,N'-carbonyldiimidazole (58.0 mg, 0.36 mmol) was then added to the solution, and stirring continued for 0.5 hours. Compound 92-4 (100.0 mg, 0.30 mmol) was then added. The reaction system was stirred for 20 minutes. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was quenched by addition of water (20 mL). The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined and washed with saturated brine (40 mL x 2). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 4 / 96) to afford compound 92-5 (45.1 mg, 38.4% yield).

[1050] MS (ESI) M / Z: 805.2 [M+Na] + .

[1051] Step E: Compound 92-5 (45 mg, 0.057 mmol) was dissolved in methanol (2.0 mL) at room temperature. Palladium on carbon (23 mg) was then added to the solution. The reaction was stirred under a hydrogen atmosphere for 1 hour. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to give compound 92-6 (38.0 mg, crude product), which was used directly in the next step without further purification.

[1052] MS (ESI) M / Z: 649.2 [M+H] + .

[1053] Step F: Compound 92-6 (30.0 mg, 0.046 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycyl-L-phenylalanine (25.1 mg, 0.052 mmol) were dissolved in N,N-dimethylformamide (1.0 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (20.0 mg, 0.052 mmol) and N,N-diisopropylethylamine (12.0 mg, 0.094 mmol) were then added to the solution. The reaction was stirred at 0°C for 0.5 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was quenched with water (25 mL). The mixture was extracted with ethyl acetate (10 mL × 3 times), and the organic phases were combined and washed with saturated brine (20 mL × 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 92 (6.3 mg, 12.4% yield).

[1054] MS (ESI) M / Z: 1103.4 [M+H] + .

[1055] 1H NMR (400MHz, DMSO-d6) δ8.59(t,J=6.4Hz,1H),8.35(t,J=5.8Hz,1H),8.19(d,J =8.4Hz,1H),8.12(t,J=5.6Hz,1H),8.07(d,J=5.2Hz,1H),7.76(d,J=11.2Hz,1 H),7.32(s,1H),7.26-7.18(m,4H),7.18-7.12(m,1H),6.99(s,2H),6.94(d,J= 8.4Hz,1H),6.52(s,1H),5.49-5.37(m,3H),5.30(d,J=18.8Hz,1H),5.18(d,J= 18.8Hz,1H),4.68-4.54(m,2H),4.53-4.43(m,1H),3.80-3.48(m,8H),3.30-3. 20(m,2H),3.14-3.01(m,2H),2.87-2.77(m,1H),2.63-2.54(m,1H),2.39(s,3H ),2.26-2.15(m,2H),2.10(t,J=7.4Hz,2H),2.04-1.95(m,1H),1.92-1.78(m,2 H),1.52-1.39(m,4H),1.22-1.12(m,2H),0.90-0.78(m,5H),0.73-0.61(m,2H).

[1056] Example 93:

[1057] Preparation of (S)-N-(2-((2-(1-cyclopropyl-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)ureido)ethoxy)methyl)amino)-2-oxoethyl)-2-(2-(2-(3-(2-(methylsulfonyl)pyrimidin-5-yl)prop-2-yn-1-yl)oxy)acetamido)acetamido)-3-phenylpropanamide (Compound 93)

[1058] Reaction route:

[1059] Steps:

[1060] Step A: Compound 93-1 (50.0 mg, 0.44 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL). 5-Bromo-2-methylthiopyrimidine (104.3 mg, 0.44 mmol), tetrakis(triphenylphosphine)palladium (5.1 mg, 0.0044 mmol), cuprous iodide (0.42 mg, 0.0022 mmol), and triethylamine (89.1 mg, 0.88 mmol) were added. The reaction system was vigorously refluxed at 95°C under nitrogen for 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and flash purified. The prepared solutions were combined and lyophilized to obtain compound 93-2 (20.0 mg, 17.1% yield).

[1061] MS (ESI) M / Z: 239.0 [M+H] + .

[1062] Step B: Compound 92-6 (100.0 mg, 0.15 mmol) was dissolved in ultra-dry N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (0.048 mL) was added. Fmoc-glycyl-glycyl-L-phenylalanine (97.1 mg, 0.19 mmol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (114.1 mg, 0.30 mmol) were added. The reaction system was stirred at this temperature for 1 h. After completion of the reaction, the reaction solution was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to give compound 93-3 (100.0 mg, 57.1% yield).

[1063] MS (ESI) M / Z: 1132.2 [M+H] + .

[1064] Step C: Disperse compound 93-3 (100.0 mg, 0.088 mmol) in dichloromethane (8 mL) and add diethylamine (2 mL). Stir the reaction system at room temperature for 2 h. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 93-4 (55.0 mg, 68.3% yield).

[1065] MS (ESI) M / Z: 910.2 [M+H] + .

[1066] Step D: Compound 93-4 (55.0 mg, 0.060 mmol) was dissolved in ultra-dry N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (0.18 mL) was added. 93-2 (21.4 mg, 0.090 mmol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (27.4 mg, 0.072 mmol) were added, and the reaction system was stirred at this temperature for 2 h. After completion of the reaction, the reaction solution was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 93-5 (35.0 mg, yield 51.3%).

[1067] MS (ESI) M / Z: 1130.3 [M+H] + .

[1068] Step E: Dissolve compound 93-5 (17.5 mg, 0.015 mmol) in dichloromethane (3 mL) and add m-chloroperbenzoic acid (15.53 mg, 0.090 mmol). Stir the reaction at room temperature for 5 h. After completion, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to afford compound 93 (7.5 mg, 44.1% yield).

[1069] MS (ESI) M / Z: 1162.1 [M+H] + .

[1070] Example 94:

[1071] Preparation of N-((S)-12-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-4-((2-(methylsulfonyl)pyrimidine)-5-sulfonylamino)butanamide (Compound 94)

[1072] Reaction route:

[1073] Steps:

[1074] Step A: Dissolve compound 94-1 (3.5 g, 33.9 mmol) in toluene (50 mL), add benzyl alcohol (23.8 g, 220 mmol) and p-toluenesulfonic acid (7.6 g, 44.12 mmol), and reflux the reaction system overnight at 130°C under nitrogen. After completion, cool the reaction mixture to room temperature to precipitate a large amount of crude product. This product was then added to diethyl ether and stirred to precipitate a white flocculent solid, which was then filtered. The solid was washed twice with diethyl ether and then ethyl acetate, and dried to yield the p-toluenesulfonate salt of compound 94-2 (7.5 g, crude product).

[1075] Step B: Dissolve compound 94-2 (0.5 g, 2.59 mmol) in tetrahydrofuran (20 mL), add triethylamine (1.19 mL), cool to -78°C, and add 2-chloro-pyrimidine-5-sulfonyl chloride (0.55 g, 2.59 mmol). Stir the reaction system at this temperature for 0.5 h. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 94-3 (300 mg, 31.3% yield).

[1076] MS (ESI) M / Z: 368.0 [M+H] + .

[1077] Step C: Compound 94-3 (160 mg, 0.432 mmol) was dissolved in N,N-dimethylformamide (10 mL), and NaSCH3 (57 mg, 0.865 mmol) was added. The reaction system was stirred at 80°C for 3 h. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain 94-4 (120.0 mg, 72.2% yield).

[1078] MS (ESI) M / Z: 382.3 [M+H] + .

[1079] Step D: Compound 94-4 (90 mg, 0.235 mmol) was dissolved in tetrahydrofuran (5 mL) and water (5 mL). LiOH (8.47 mg, 0.353 mmol) was added and the reaction was stirred at room temperature for 2 h. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 94-5 (40 mg, 58.2% yield).

[1080] MS (ESI) M / Z: 292.2 [M+H] + .

[1081] Step E: Compound 94-5 (30 mg, 0.035 mmol) was dissolved in N,N-dimethylformamide (2 mL). Compound 93-4 (13 mg, 0.042 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (20.35 mg, 0.053 mmol), and N,N-diisopropylethylamine (13.83 mg, 0.107 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 94-6 (20 mg, 50.3% yield).

[1082] MS (ESI) M / Z: 1183.3 [M+H] + .

[1083] Step F: Dissolve compound 94-6 (20.0 mg, 0.017 mmol) in dichloromethane (2 mL) and add m-chloroperbenzoic acid (9.29 mg, 0.053 mmol). Stir the reaction at room temperature for 1 hour. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to yield compound 94 (3.1 mg, 18.8% yield).

[1084] MS (ESI) M / Z: 1215.5 [M+H] + .

[1085] 1H NMR (400MHz, DMSO) δ9.40 (s, 2H), 8.58 (t, J=6.5Hz, 1H), 8.45 (s, 1H), 8.34 (t, J= 5.7Hz,1H),8.20-8.12(m,2H),8.06(t,J=5.5Hz,1H),7.76(d,J=10.8Hz,1H),7.3 2(s,1H),7.23-7.18(m,4H),6.94(d,J=8.5Hz,1H),6.53(s,1H),5.47-5.36(m,3 H),5.30(d,J=19.2Hz,1H),5.18(d,J=19.0Hz,1H),4.66-4.54(m,2H),4.51-4.43 (m,1H),3.75-3.71(m,2H),3.66(d,J=5.4Hz,2H),3.61-3.50(m,6H),3.46(s,3H ),3.08(dd,J=19.8,8.9Hz,3H),2.92(dd,J=13.2,8.1Hz,3H),2.85-2.76(m,2H), 2.38(s,3H),2.19-2.15(m,2H),1.91-1.80(m,2H),1.64(dd,J=14.4,7.5Hz,2H) ,1.29-1.20(m,2H),0.87(t,J=7.1Hz,3H),0.84-0.75(m,2H),0.72-0.62(m,2H).

[1086] Example 95:

[1087] Preparation of (S)-N-(2-((2-(1-cyclopropyl-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)ureido)ethoxy)methyl)amino)-2-oxoethyl)-2-(2-(2-(3-((2-methylsulfonyl)pyrimidinyl)-5-sulfonylamino)propionamido)acetamido)acetamido)acetamido)-3-phenylpropionamide (Compound 95)

[1088] Reaction route:

[1089] Steps:

[1090] Step A:

[1091] Compound Int-1 (4 g, 44.90 mmol) was dissolved in toluene (50 mL), and BnOH (31.35 g, 289.90 mmol) and TsOH (10.02 g, 58.19 mmol) were added. The reaction system was refluxed at 130° C. overnight (a water separator was required) under nitrogen protection.

[1092] After the reaction was complete, the reaction mixture was cooled to room temperature to precipitate a large amount of crude product, which was then added to diethyl ether and stirred to precipitate a white flocculent solid, which was then filtered. The solid was washed twice with diethyl ether and twice with ethyl acetate, and dried to obtain a crude p-toluenesulfonate salt of product 95-1 (15 g, 50% purity).

[1093] Step B:

[1094] Compound 95-1 (0.6 g, 1.67 mmol) was dissolved in THF (20 mL), and TEA (1.19 mL) was added. The temperature was cooled to -78°C, followed by the addition of compound Int-2 (1.0 g, 4.69 mmol). The reaction system was stirred at this temperature for 0.5 h. After completion, the reaction solution was diluted with THF and then purified via Prep-Flash (ACN / 0.1% FA and water) to afford compound 95-2 (300 mg, 50.4% yield).

[1095] MS (ESI) M / Z: 356.2 [M+H] + .

[1096] Step C:

[1097] Compound 95-2 (150 mg, 0.42 mmol) was dissolved in DMF (10 mL), and NaSCH3 (59 mg, 0.84 mmol) was added. The reaction system was stirred at 80°C for 3 h. After completion of the reaction, the reaction solution was purified by Prep-Flash purification (ACN / 0.1% FA and water) to obtain compound 95-3 (60 mg, yield: 38.7%).

[1098] MS (ESI) M / Z: 366.1 [M+H] + .

[1099] Step D:

[1100] Compound 95-3 (50 mg, 0.14 mmol) was dissolved in THF (10 mL), and Pd / C (15 mg) was added. The reaction system was purged with H₂ three times, and then stirred at 50°C for 5 h. After completion of the reaction, the reaction solution was filtered and purified via Prep-Flash (ACN / 0.1% FA and water) to afford compound 95-4 (20 mg, 53.3% yield).

[1101] MS (ESI) M / Z: 276.0 [MH] - .

[1102] Step E:

[1103] Compound In-3 (42 mmol, 15 g), raw material 4 (46 mmol, 12 g), and HATU (46 mmol, 17.7 g) were added to a round-bottom flask, and 250 mL of DMF and 21 mL of DIEA were added, and the mixture was stirred at room temperature for 15 hours. 200 mL of ethyl acetate was added to the reaction solution, washed once with 300 mL of water and twice with 200 mL of saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After complete dissolution with dichloromethane, the mixture was concentrated under reduced pressure until a large amount of solid precipitated. 300 mL of petroleum ether was added, filtered, and the filter cake was rinsed with a mixed solution of dichloromethane and petroleum ether. The filter cake was compound 95-5 (28 g, yield: 78.1%).

[1104] MS (ESI) M / Z: 580.5 [M+Na] + .

[1105] Step F:

[1106] Compound 95-5 (8.97 mmol, 5 g) was added to a round-bottom flask and dissolved in 45 mL of dichloromethane. Then, 9 mL of diethylamine was added and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the reaction mixture was dissolved in DMF and purified by Prep-HPLC (ACN / 0.1% TFA and water) to afford compound 95-6 (2.04 g, yield: 67.83%).

[1107] MS (ESI) M / Z: 336.2 [M+H] + .

[1108] Step G:

[1109] Compound 95-4 (90 mg, 0.32 mmol), compound 95-6 (128.8 mg, 0.38 mmol), and HATU (243.3 mg, 0.64 mmol) were placed in a 25 mL single-necked flask. Dry DMF (4 mL) was added, followed by DIEA (0.21 mL, 1.28 mmol). The reaction system was incubated at room temperature under argon for 2 h. After completion, the reaction solution was filtered, and the filtrate was flash purified (ACN / 0.1% TFA and water) to afford compound 95-7 (150 mg, 77% yield).

[1110] MS (ESI) M / Z: 595.2 [M+H]+ .

[1111] Step H:

[1112] Compound 95-7 (140 mg, 0.24 mmol) was added to a 100 mL single-necked flask, followed by DCM (20 mL) and trifluoroacetic acid (5 mL). The reaction system was incubated at room temperature for 2 hours under argon. After completion of the reaction, the solvent was evaporated to dryness at low temperature, and the crude product was flash purified (ACN / 0.1% TFA and water) to afford compound 95-8 (90 mg, 71% yield).

[1113] MS (ESI) M / Z: 539.1 [M+H] + .

[1114] 1 H NMR (400MHz, DMSO) δ12.72(s,1H),8.92(d,J=8.5Hz,2H),8.21(t,J=5.7Hz,1 H),8.13(d,J=8.0Hz,1H),8.04(t,J=5.8Hz,1H),7.96(t,J=5.7Hz,1H),7.30- 7.24(m,2H),7.24-7.16(m,3H),4.41(td,J=8.7,5.1Hz,1H),3.77-3.59(m,4 H), 3.08-3.00 (m, 3H), 2.90-2.83 (m, 1H), 2.58 (s, 3H), 2.34 (t, J = 7.1Hz, 2H).

[1115] Step I: Compound 95-8 (90.5 mg, 0.17 mmol), compound 92-6 (90.0 mg, 0.14 mmol), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (106.4 mg, 0.28 mmol) were added to a 25 mL single-necked flask. Dried N,N-dimethylformamide (4 mL) was added, followed by N,N-diisopropylethylamine (0.09 mL, 0.56 mmol). The reaction system was incubated at room temperature under argon for 2 hours. After completion of the reaction, the crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 95-9 (113.1 mg, 69.4% yield).

[1116] MS (ESI) M / Z: 1169.4 [M+H] + .

[1117] Step J: Compound 95-9 (77.0 mg, 0.066 mmol) and m-chloroperbenzoic acid (68.3 mg, 0.40 mmol) were added to a 25 mL single-necked flask. Dichloromethane (20 mL) was added and the reaction system was incubated at room temperature for 2 hours under argon. After completion of the reaction, the dichloromethane was drained at room temperature. The crude product was dissolved in N,N-dimethylformamide (2 mL) and purified by preparative HPLC to afford compound 95 (15.1 mg, yield: 19.2%).

[1118] MS (ESI) M / Z: 1201.9 [M+H] + .

[1119] 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),8.53(d,J=17.0Hz,2H),8.34(s,2H),8.23(s,1H),8.13(s,1H),8.03 (s,1H),7.76(d,J=10.4Hz,1H),7.32(s,1H),7.19(d,J=20.3Hz,5H),6.87(d,J=36.1Hz,1H),6.53(s,1H) ,5.43(s,3H),5.23(dd,J=47.2,19.4Hz,2H),4.55(d,J=39.3Hz,3H),3.60(dd,J=69.4,34.1Hz,13H),3.2 0-2.94(m,6H),2.78(s,1H),2.34(d,J=32.1Hz,5H),2.20(s,2H),1.86(s,2H),0.87(s,5H),0.69(s,2H).

[1120] Example 96:

[1121] Preparation of N-((S)-12-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (Compound 96)

[1122] Reaction route:

[1123] Steps:

[1124] Step A: Glycylglycyl-L-phenylalanine (200.0 mg, 0.72 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoic acid (192.0 mg, 0.72 mmol) were dissolved in N,N-dimethylformamide (3.5 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (328.3 mg, 0.86 mmol) and N,N-diisopropylethylamine (278.6 mg, 2.16 mmol) were then added to the solution, and stirring was continued for 0.5 hour. After LCMS monitoring showed the disappearance of the starting material, the reaction solution was purified by reverse phase chromatography to obtain 150 mg of compound 96-1.

[1125] MS (ESI) M / Z: 530.2 [M+H] + .

[1126] Step B: Compound 96-1 (53 mg, 0.10 mmol) and compound 92-6 (32.5 mg, 0.05 mmol) were dissolved in N,N-dimethylformamide (1.0 mL) at 0°C. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (19.4 mg, 0.15 mmol) were then added to the solution. The reaction was stirred at 0°C for 0.5 hours. After LCMS monitoring indicated the disappearance of the starting material, water (15 mL) was added to the reaction mixture to quench the mixture. The mixture was extracted with ethyl acetate (3 mL x 3 times). The organic phases were combined and washed with saturated brine (10 mL x 2 times). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 96 (42.3 mg, yield: 36.5%).

[1127] MS (ESI) M / Z: 1160.2 [M+H] + .

[1128] 1H NMR (400MHz, DMSO-d6) δ9.09 (s, 2H), 8.53 (t, J = 6.6Hz, 1H), 8.37-8.25 (m, 1H), 8.18(t,J=5.6Hz,1H),8.12(d,J=8.0Hz,1H),8.05(t,J=5.4Hz,1H),7.75(d,J=1 0.8Hz,1H),7.32(s,1H),7.29-7.09(m,5H),6.89(d,J=8.4Hz,1H),6.51(s,1H) ,5.53-5.34(m,3H),5.28(d,J=18.8Hz,1H),5.15(d,J=19.2Hz,1H),4.70-4.55( m,2H),4.54-4.45(m,1H),3.82-3.65(m,5H),3.63-3.50(m,4H),3.40(s,3H),3 .30-3.19(m,1H),3.14-2.99(m,2H),2.79(dd,J=13.8,9.8Hz,1H),2.61-2.53(m ,3H),2.38(s,3H),2.32(t,J=7.4Hz,2H),2.27-2.14(m,2H),1.94-1.74(m,4H), 1.31-1.22(m,1H),0.87(t,J=7.2Hz,3H),0.83-0.74(m,2H),0.73-0.63(m,2H).

[1129] Example 97:

[1130] Preparation of N-((S)-12-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-3-((2-(methylsulfonyl)pyrimidin-5-yl)ethynyl)bicyclo[1.1.1]pentane-1-carboxamide (Compound 97)

[1131] Reaction route:

[1132] Steps:

[1133] Step A: Oxalyl chloride (422.0 mg, 3.31 mmol) was added to a vial. Dissolved in 6 mL of anhydrous dichloromethane, the mixture was cooled to -78°C and stirred for 5 minutes. A solution of dimethyl sulfoxide in dichloromethane (520.0 mg, 6.62 mmol, 1.4 mL of dichloromethane) was then added dropwise. The mixture was stirred at -78°C for 15 minutes. A solution of compound 97-1 in dichloromethane (400.0 mg, 2.56 mmol, 2.6 mL of dichloromethane) was then added dropwise. The mixture was stirred at -78°C for 30 minutes. Triethylamine (1550.0 mg, 15.32 mmol) was then added and stirred for a further 30 minutes. After completion of the reaction, the mixture was slowly warmed to room temperature and extracted with water and dichloromethane. The organic phase was collected and dried over saturated brine and anhydrous sodium sulfate. The organic phase was collected and the solvent removed under reduced pressure to obtain a crude colorless oil containing compound 97-2, which was used directly in the next reaction.

[1134] Step B: The crude product containing compound 97-2 from the previous step was dissolved in 12 mL of methanol, and potassium carbonate (1060.0 mg, 7.68 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (736.2 mg, 3.9 mmol) were added. The mixture was stirred at room temperature overnight. After the reaction was complete, 20 mL of ether and 20 mL of deionized water were added to the system and stirred until clear. The system was extracted with ether and water, and the organic phase was collected and dried over saturated brine and anhydrous sodium sulfate. The organic phase was collected and the solvent was removed under reduced pressure to obtain compound 97-3 (380.0 mg, crude product), which was used directly in the next step without further purification.

[1135] 1 H NMR (400MHz, CDCl3) δ3.67(s,3H),2.33(s,6H),2.11(s,1H).

[1136] Step C: Compound 97-3 (380.0 mg, crude), 5-bromo-2-methylthiopyrimidine (778.2 mg, 3.78 mmol), tetrakistriphenylphosphine palladium (28.1 mg, 0.024 mmol), and cuprous iodide (10.0 mg, 0.05 mmol) were added to a vial. After nitrogen purging three times, anhydrous tetrahydrofuran and 0.7 mL of triethylamine were added, and the mixture was refluxed at 95°C for 18 hours. After completion of the reaction, the solvent was mostly removed under reduced pressure, and the product was purified by preparative HPLC. Purification conditions were as follows: preparative column: YMC-Actus Triart C18 20.0 mm x 150 mm; mobile phase: water and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile from 10% to 70% over 10 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure. Compound 97-4 (330.0 mg, yield 46.8%) was obtained.

[1137] MS (ESI) M / Z: 275.3 [M+H] + .

[1138] Step D: Compound 97-4 (80.0 mg, 0.18 mmol) was placed in a vial and dissolved in 2 mL of methanol and 2 mL of deionized water. Lithium hydroxide (20.0 mg, 0.83 mmol) was then added and stirred at room temperature for 2 hours. 0.1 M aqueous hydrochloric acid was added dropwise to the system to adjust the pH to approximately 5. The mixture was extracted three times with ethyl acetate. The organic phase was collected and the solvent was removed under reduced pressure to afford compound 97-5 (64.0 mg, 84.2% yield).

[1139] MS (ESI) M / Z: 261.3 [M+H] + .

[1140] Step E: Compound 97-5 (15.0 mg, 0.058 mmol), compound 93-4 (52.0 mg, 0.057 mmol), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (26 mg, 0.068 mmol) were added to a vial, the atmosphere was purged with nitrogen three times, 0.5 mL of anhydrous N,N-dimethylformamide was added to dissolve it, and N,N-diisopropylethylamine (9.0 mg, 0.069 mmol) was added and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by high-performance thin-layer silica gel chromatography. The product was collected and lyophilized under reduced pressure to obtain compound 97-6 (10.1 mg, yield 15.1%).

[1141] MS (ESI) M / Z: 1152.2 [M+H] + .

[1142] Step F: Compound 97-6 (10.1 mg, 0.009 mmol) was added to a vial, replaced with nitrogen three times, and then dissolved with 1 mL of anhydrous dichloromethane. Subsequently, m-chloroperbenzoic acid (6.0 mg, 0.035 mmol) was slowly added at 0°C, and then reacted at room temperature for 6 hours. The reaction solution was spin-dried at low temperature, dissolved in N,N-dimethylformamide, and purified by preparative high-performance liquid chromatography. The purification conditions were as follows: preparative column: YMC-Actus Triart C18 20.0 mm*150 mm; mobile phase: water and acetonitrile; flow rate: 20 mL / min; gradient: acetonitrile increased from 10% to 70% within 10 minutes; detection wavelength: 254 nm. The product was collected and lyophilized under reduced pressure. The target compound 97 (2.8 mg, yield 27.2%) was obtained.

[1143] MS (ESI) M / Z: 1184.2 [M+H] + .

[1144] 1 H NMR (400MHz, DMSO) δ9.14(s,2H),8.53(s,1H),8.42(s,1H),8.31(s,1H),8.19(s,1H),8.13(d,J=8.8Hz,1H),8.05(s,1H),7.77 (d,J=11.4Hz,1H),7.32(s,1H),7.22(d,J=6.0Hz,5H),6.89(d,J=8.0Hz,1H),6.52(s,1H),5.43(s,2H),5.32(s,1H),5.24(d,J= 23.5Hz,1H),4.60(s,2H),4.50(s,1H),3.73(s,2H),3.67(d,J=5.9Hz,2H),3.60(s,1H),3.56(s,2H),3.50(s,1H),3.41(s,4H), 3.07(s,2H),2.78(s,1H),2.35(s,6H),2.20(s,2H),2.04-1.97(m,3H),1.86(s,3H),1.46(s,2H),0.86(dd,J=13.6,7.1Hz,7H).

[1145] Biological activity test

[1146] Biological Test Example 1: BxPC-3 and FaDu Cytotoxicity Test of Compounds

[1147] Experimental method 1: After washing with DPBS, human in situ pancreatic adenocarcinoma cells BxPC-3 (ATCC, catalog number: CRL-1687) and human pharyngeal squamous cell carcinoma cells FaDu (ATCC, catalog number: HTB-43) were digested with 0.25% Trypsin-EDTA trypsin for about 3 minutes. The digestion was terminated with complete cell culture medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the cells were adjusted to the desired density. 100 μL was added to each well, 5000 cells / well, and the cell plate was placed in a 37°C, 5% CO2 cell culture incubator for 12-16 hours.

[1148] A blank control group and a test sample group were set up in the experiment. The cell plate was removed from the incubator to observe the cell adhesion status. After the cells adhered, 100 μL of sample (starting at 10 μM, 4-fold dilution, 10 concentrations) was added to each well, gently shaken, and placed in the incubator for incubation.

[1149] After incubation for 48 h, 130 μL of supernatant was carefully discarded, and 70 μL / well of CellTiter-Glo™ (Promega, catalog number: G7572) working solution was added. The cells were gently shaken to lyse the cells, and the plate was read on a microplate reader.

[1150] The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate = (1-sample well / control well) × 100%.

[1151] Data analysis: GraphPad Prism 8.0 software was used to plot the logarithm of sample concentration as the horizontal axis and the cytotoxicity percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC value of each test sample. 50 The test results of the compounds of the present disclosure are shown in Table 1-1.

[1152] Table 1-1. Inhibitory activity of example compounds on cell proliferation

[1153] Note: ND stands for Not Detected; A stands for IC 50 <1nmol / L; B indicates IC 50 1-10nmol / L; C represents IC 50 10-50nmol / L; D represents IC 50 50-100nmol / L; E represents IC 50 100-500nmol / L; F represents IC 50 >500nmol / L.

[1154] Experimental method 2:

[1155] Human in situ pancreatic adenocarcinoma cells BxPC-3 (ATCC, Catalog No.: CRL-1687) and human pharyngeal squamous cell carcinoma cells FaDu (ATCC, Catalog No.: HTB-43) were washed with PBS and then digested with 0.25% Trypsin-EDTA for approximately 3 minutes. Digestion was terminated with complete cell culture medium and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the cells were adjusted to the desired density. 50 μL of the culture medium was added to each well, resulting in a density of 3000 cells / well. The cell plates were incubated in a 37°C, 5% CO2 cell culture incubator for 12-16 hours.

[1156] The experiment included a control group, a positive group, and a test group. Compound SN38 (7-ethyl-10-hydroxycamptothecin) was used as the positive control. The cell plates were removed from the incubator to observe cell attachment. Once cells had attached, 50 μL of sample (starting at 10 μM, diluted 5-fold, and 9 concentrations) was added to each well. The plates were gently shaken and placed back into the incubator for incubation.

[1157] After 3 days of incubation, 100 μL / well CellTiter-Glo was added to each well. TM (Promega, Cat. No. G7572) working solution, shake gently to lyse the cells, and read the plate on a microplate reader.

[1158] The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate = (1-sample well / control well) × 100%.

[1159] Data analysis: GraphPad Prism 7.0 software was used to plot the logarithm of sample concentration as the horizontal axis and the cytotoxicity percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC values ​​of each test sample. 50 The test results are shown in Table 1-2.

[1160] Table 1-2 Inhibitory activity of the example compounds on BxPC3 and Fadu cell proliferation

[1161] The SN38 structure is as follows:

[1162] The above test data indicate that the compounds of the present disclosure have good cell proliferation inhibitory activity in BxPC3 and Fadu.

[1163] Biological test example 2: MDA-MB-468, NCI-N87, KPL-4 and SW48 cell cytotoxicity test of the compound

[1164] Experimental materials and reagents:

[1165] 96-well transparent flat-bottom white plate (Corning, #3610)

[1166] RPMI1640 culture medium (Gibco, #A10491-01)

[1167] DMEM medium (Gibco, #11995-065)

[1168] Fetal Bovine Serum (Sigma-Aldrich, #F8318-500ML / #F8687-500ML)

[1169] Luminescent Cell Viability Assay (Promega, #G7572 / G7571)

[1170] Experimental methods:

[1171] Human breast cancer cells MDA-MB-468 (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Catalog No. TCHu136), human gastric cancer cells NCI-N87 (ATCC, Catalog No. CRL-5822), human breast cancer cells KPL-4 (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60379), and human colon cancer cells SW48 (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60018) were washed with PBS and trypsinized with 0.25% Trypsin-EDTA for approximately 3-10 minutes. Digestion was terminated with complete cell culture medium, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the desired cell density was adjusted. 90.0 μL of the culture medium was added to each well, resulting in 3000 cells / well. The cell plates were incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[1172] The experiment included a DMSO control group, a positive control group, and a test article group. Compounds SN38 (7-ethyl-10-hydroxycamptothecin) and Dxd were used as positive controls. The cell plates were removed from the incubator to observe cell attachment. After cell attachment, 10.00 μL of sample (starting at 10 μM, diluted 5-fold, and 9 concentrations) was added to each well. The plates were gently shaken and placed back into the incubator for incubation.

[1173] After incubation for 3 days, add 100.0 μL / well CellTiter-Glo TM (Promega, Cat. No. G7572 / G7571) working solution, gently shake to lyse the cells, and read the plate on a microplate reader after 10 minutes.

[1174] The calculation formula of cell proliferation inhibition rate is: cell proliferation inhibition rate = (1-RLU 供试品孔 / RLU DMSO对照孔 )×100%.

[1175] Data analysis: GraphPad Prism 7.0 software was used to plot the logarithm of sample concentration as the horizontal axis and the cytotoxicity percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC values ​​of each test sample. 50 value.

[1176] Table 2 Inhibitory activity of the example compounds on the proliferation of MDA-MB-468, NCI-N87, KPL-4 and SW48 cells

[1177] Note: / indicates not tested.

[1178] The Dxd structure is as follows:

[1179] The above experimental data show that the compounds of the embodiments of the present disclosure have good cell proliferation inhibitory activity, especially the compound of Example 70 has significantly better cell proliferation inhibitory activity than Dxd in SW48 cells, and the compound of Example 92a has significantly better cell proliferation inhibitory activity than Dxd in NCI-N87, KPL-4 and MDA-MB-468 cells.

[1180] Biological Test Example 3: Cytotoxicity Test of Compounds in NCI-H1975, LS174T, NCI-H441, and NCI-H1373 Cells

[1181] Experimental materials and reagents:

[1182] 96-well transparent flat-bottom white plate (Corning, #3610)

[1183] RPMI1640 culture medium (Gibco, #A10491-01)

[1184] EMEM medium (ATCC, 30-2003)

[1185] DMEM medium (Gibco, #11995-065)

[1186] Fetal Bovine Serum (Sigma-Aldrich, #F8687-500ML)

[1187] Luminescent Cell Viability Assay (Promega, #G7572 / G7571)

[1188] Experimental methods:

[1189] Human non-small cell lung cancer NCI-H1975 (ATCC, Catalog No.: CRL-5908), human colorectal cancer cells LS174T (ATCC, Catalog No.: CL-188), human lung adenocarcinoma cells NCI-H441 (ATCC, Catalog No.: CRM-HTB-174), and human lung cancer cells NCI-H1373 (Nanjing Kebai Biotechnology Co., Ltd., Catalog No.: CBP60162) were washed with PBS and trypsinized with 0.25% Trypsin-EDTA for approximately 3-10 minutes. Digestion was terminated with complete cell culture medium, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the cells were adjusted to the desired density. 90.0 μL of cell suspension was added to each well, 3000 cells / well, and the cell plate was incubated overnight in a 37°C, 5% CO2 cell culture incubator.

[1190] A DMSO control group, a positive control group, and a test article group were set up in the experiment, with compound Dxd used as the positive control compound. The cell plate was removed from the incubator to observe the cell attachment status. After the cells attached, 10.00 μL of sample (starting at a final concentration of 10 μM, diluted 5-fold, and 9 concentrations) was added to each well. The plate was gently shaken and placed in the incubator for incubation.

[1191] After incubation for 3 days, add 100.0 μL / well CellTiter-Glo TM (Promega, catalog number: G7572 / G7571) working solution, shake on a constant temperature shaker to lyse the cells, and read the plate on a microplate reader after 10 minutes.

[1192] The calculation formula of cell proliferation inhibition rate is: cell proliferation inhibition rate = (1-RLU 供试品孔 / RLU DMSO对照孔 )×100%.

[1193] Data analysis: GraphPad Prism 8.0 software was used to plot the sample concentration log value as the horizontal axis and the inhibition percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC of each test sample. 50 value.

[1194] Table 3 Inhibitory activity of the example compounds on the proliferation of NCI-H1975, LS174T, NCI-H441, and NCI-H1373 cells

[1195] Conclusion: As can be seen from Table 3, the activity of the compound in Example 92a on NCIH1975, LS174T, and NCI-H1373 cell lines is significantly better than that of Yangshen Dxd.

Claims

1. A compound represented by formula I, or a pharmaceutically acceptable salt thereof: in, R1 is selected from H, -CN, -CH2CN, C 1-4 Alkyl, C 1-4 alkyl halide; R2 is selected from -L1-Rx; L1 is selected from chemical bonds, C 1-3 Alkylene, carbonyl, -C(=NH)-, -CH(CN)-, -S(=O)2-; Rx is selected from C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl-carbonyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl, -N(OH)-Ry, -C(Rz)=N(OH), -CH(Rb)2, -N(Rb)2, The 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl, Ry, Rz may be optionally substituted by 1, 2, 3, or 4 Ra; the Ry is independently selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl, 5-7 membered aryl, benzyloxy-C 1-4 Alkyl; said Rz is independently selected from C 1-4 Alkyl, C 1-4 Alkyl-carbonyl, benzyloxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl, benzoyl, 3-6 membered cycloalkyl, 5-7 membered aryl, 5-7 membered heteroaryl; the Ra is independently selected from halogen atoms, hydroxyl, oxo (=O), amino, hydroxy-C 1-4 Alkyl, benzyloxy, benzyloxy-C 1-4 Alkyl, C 1-4 Alkyl, C 3-5 Cycloalkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy; said Rb is independently selected from H, hydroxyl, hydroxyamino, C 1-4 Alkyl, C 3-5 Cycloalkyl, hydroxyamino-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, N-methyl-N-hydroxyamino, NH2-C 1-4 Alkyl-carbonyl, preferably H, methyl, ethyl, cyclopropyl, hydroxy, hydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, N-methyl-N-hydroxyamino, hydroxymethyl, hydroxyethyl, aminoacetyl, 3-aminopropionyl; Can be substituted by 1-3 Rc; said Rc is selected from C 1-4 Alkyl, amino-C 1-4 Alkyl, preferably methyl, aminomethyl, 1-aminoethyl; Or R1, R2 and the N atom to which they are commonly connected form a 4-membered heterocycloalkyl, a 5-membered heterocycloalkyl or a 6-membered heterocycloalkyl; the 4-membered heterocycloalkyl is preferably azetidine, the 5-membered heterocycloalkyl is preferably azacyclopentane, and the 6-membered heterocycloalkyl is preferably piperidine; and the heterocycloalkyl formed by R1, R2 and the N atom to which they are commonly connected may be optionally substituted by 1, 2, 3, or 4 Rb; the Rb is independently selected from C 1-4 Alkyl, C 1- 4-haloalkyl, hydroxy, hydroxy-C 1-4 Alkyl groups are preferably methyl, ethyl and hydroxyl groups.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from H, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, trichloromethyl; more preferably H.

3. The compound of formula I according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, characterized in that: L1 C 1-3 The alkylene group is selected from methylene, ethylene, and n-propylene, preferably methylene.

4. The compound of formula I according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, characterized in that: The C 1- 4. Alkyl is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl.

5. The compound of formula I according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, characterized in that: The 3-6 membered cycloalkyl group is independently selected from cyclobutane, cyclopentane and cyclohexane; the 3-6 membered heterocycloalkyl group is preferably azetidine, azopentane and azohexane.

6. The compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The 5-7 membered aryl group is phenyl; the 5-7 membered heteroaryl group is preferably pyridyl.

7. The compound of formula I according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized in that: Said Ry is independently selected from H, methyl, ethyl, cyclopropyl, cyclobutyl, phenyl, and may be optionally substituted with 1, 2, 3, or 4 Ra.

8. The compound of formula I according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, characterized in that: The Rz is independently selected from methyl, ethyl, acetyl, benzyloxymethyl, aminomethyl, benzoyl, cyclopropyl, phenyl, pyridyl, and may be optionally substituted with 1, 2, 3, or 4 Ra.

9. The compound of formula I according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, characterized in that: Said Ra is independently selected from Cl, F, hydroxy, oxo (=O), amino, hydroxymethyl, 1-hydroxyethyl, benzyloxy, benzyloxymethyl, methyl, ethyl, cyclopropyl, methoxy, trifluoromethyl.

10. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from a chemical bond; Rx is selected from a 3-6 membered cycloalkyl group; the 3-6 membered cycloalkyl group is preferably cyclobutane, cyclopentane, cyclohexane, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in claim 1 or 9, and is preferably hydroxyl, benzyloxy, hydroxymethyl, benzyloxymethyl, methoxy.

11. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from C 1-3 Alkylene, preferably methylene or ethylene; Rx is selected from 5-7 membered aryl, 5-7 membered heteroaryl; the 5-7 membered aryl is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in claim 1 or 9, and is preferably hydroxyl, hydroxymethyl, 1-hydroxyethyl.

12. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from a chemical bond; Rx is selected from C 1-4 Alkyl; the C 1-4 The alkyl group is preferably ethyl or n-propyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in claim 1 or 9, and is preferably hydroxy, hydroxymethyl, trifluoromethyl, or cyclopropyl.

13. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from carbonyl; Rx is selected from C 1-4 Alkyl, 5-7 membered aryl; said C 1-4 The alkyl group is preferably ethyl or n-propyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the 5-7 membered aryl group is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in claim 1 or 9; preferably, Ra is selected from hydroxyl, hydroxymethyl, and amino.

14. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from carbonyl; Rx is selected from -N(OH)-Ry; Ry has the same meaning as in claim 1 or 7; preferably, Ry is selected from H, methyl, cyclopropyl, phenyl, and may be optionally substituted with 1, 2, 3, or 4 Ra; said Ra has the same meaning as in claim 1 or 9.

15. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from a chemical bond; Rx is selected from -C(Rz)=N(OH); Rz has the same meaning as in claim 1 or 8; preferably, Rz is selected from methyl, ethyl, acetyl, benzyloxymethyl, aminomethyl, benzoyl, cyclopropyl, phenyl, pyridyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in claim 1 or 9, preferably Cl, F, methyl, trifluoromethyl, hydroxyl, hydroxymethyl.

16. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from carbonyl; Rx is selected from -C(Rz)=N(OH); Rz has the same meaning as in claim 1 or 8; preferably, Rz is selected from methyl, trifluoromethyl, phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in claim 1 or 9.

17. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from -C(=NH)-; Rx is selected from 5-7 membered aryl, 5-7 membered heteroaryl, N(Rb)2; the 5-7 membered aryl is preferably phenyl, and may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in claim 1 or 9, preferably F, hydroxyl; the Rb is independently selected from H, hydroxyl, C 1-4 Alkyl groups are preferably independently selected from H, hydroxyl and methyl.

18. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: L1 is selected from -S(=O)2-; Rx is selected from C 1-4 Alkyl, 5-7 membered aryl, 5-7 membered heteroaryl, preferably methyl, ethyl, n-propyl, phenyl, and optionally substituted by 1, 2, 3, or 4 Ra; said Ra has the same meaning as in claim 1 or 9, preferably methyl, methoxy, hydroxy, 1-hydroxyethyl; or It is characterized in that R1 is selected from -CN or -CH2CN; R2 is selected from -L1-Rx; L1 is a chemical bond; Rx has the same meaning as in formula I, preferably H, hydroxy-C 1-4 Alkyl, hydroxy-C 1-4 Alkyl-carbonyl, more preferably H, hydroxyethyl, hydroxyacetyl; or It is characterized in that R1 is H; R2 is selected from -L1-Rx, L1 is -CH(CN)-, Rx is C 1-4 Alkyl, hydroxy-C 1-4 Alkyl; the C 1-4 Alkyl is selected from methyl and ethyl; preferably, Rx is selected from methyl and hydroxymethyl; or It is characterized in that R1 is H; R2 is selected from -L1-Rx, L1 is -S(O)2-, Rx is -N(Rb)2; the Rb is independently selected from H, hydroxyl, C 1-4 Alkyl, hydroxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl-carbonyl, C 3-5 Cycloalkyl, preferably H, methyl, hydroxy, hydroxymethyl, aminoacetyl, 3-aminopropionyl, cyclopropyl; Or it is characterized in that R1 is H, R2 is selected from -L1-Rx, L1 is carbonyl, Rx is -N(Rb)2; the Rb is independently selected from H, hydroxyl, C 1-4 Alkyl, C 3-5 Cycloalkyl, hydroxy-C 1-4 Alkyl, NH2-C 1-4 Alkyl-carbonyl, preferably H, methyl, ethyl, cyclopropyl, hydroxy, hydroxymethyl, hydroxyethyl. In a more specific embodiment, one Rb in -N(Rb)2 is selected from H, methyl, ethyl, cyclopropyl, and the other Rb is selected from H, hydroxyethyl; Or it is characterized in that R2 is -L1-Rx, L1 is a carbonyl group, and Rx is CH(Rb)2; the Rb has the same meaning as in Formula I, preferably H, methyl, cyclopropyl, hydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, N-methyl-N-hydroxyamino; in a more specific technical solution, one Rb in CH(Rb)2 is selected from H, methyl, cyclopropyl, and the other Rb is selected from hydroxyamino, hydroxyaminomethyl, hydroxyaminoethyl, and N-methyl-N-hydroxyamino; Or it is characterized in that R2 is -L1-Rx, L1 is a carbonyl group, and Rx is described may be optionally substituted with 1, 2, 3, or 4 Rc; wherein Rc is selected from C 1-4 Alkyl, amino-C 1-4 Alkyl, preferably methyl, aminomethyl, 1-amino ethyl; Alternatively, R2 is -L1-Rx, L1 is a carbonyl group, and Rx is a 3-6 membered heterocycloalkyl group; the 3-6 membered heterocycloalkyl group is preferably azetidine, azopentane, or azohexane. The 3-6 membered heterocycloalkyl group may be optionally substituted with 1, 2, 3, or 4 Ra groups; Ra has the same meaning as in Formula I, preferably methyl or ethyl. Or it is characterized in that R2 is -L1-Rx, L1 is a carbonyl group, and Rx is a pyridyl group; the pyridyl group may be optionally substituted by 1, 2, 3, or 4 Ra; the Ra has the same meaning as in formula I, preferably Cl, F, oxo (=O), methyl, hydroxyl, hydroxymethyl, or trifluoromethyl.

19. A compound represented by formula I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, or I-12, or a pharmaceutically acceptable salt thereof: in, m is selected from 1, 2, or 3; n is selected from 1, 2, 3, or 4; R1, Ra, Ry, and Rz have the same meanings as in claims 1-18.

20. The following compound or a pharmaceutically acceptable salt thereof:

21. A cytotoxic drug, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, selected from:

22. An antibody drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, selected from the structure represented by formula (IA), in: Ab is an antibody or antigen-binding fragment; Y is the linker site connected to Ab; M is a peptide residue consisting of 2 to 4 amino groups selected from phenylalanine, glycine, valine, citrulline, alanine, glutamic acid, and aspartic acid; X is a structural fragment connecting Dr and M; Dr is a cytotoxic drug fragment of right 21, which is selected from t is a value between 4 and 8.

23. The antibody drug conjugate according to claim 22, its stereoisomer or pharmaceutically acceptable salt thereof, wherein X is selected from -CH2-NH- or 24. The antibody drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to claim 22 or 23, wherein M is a peptide residue consisting of 2 to 4 amino groups selected from phenylalanine, glycine, valine, citrulline, and alanine; Preferably, M is selected from 25. The antibody drug conjugate according to any one of claims 22 to 24, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Y is -C(=O)-Y 1 -Y 2 -S-, Y 1 Selected from -(CH2) p 、-(CH2) p -NH-SO2-, -CH2-N + (CH3)2-(CH2) p -NH-SO2-, -C 2-4 Alkynylidene-NH-SO2-, -C 2-5 Alkynylidene-, -(CH2) p -OC 2-4 Alkynylidene-, -C 3-5 Cycloalkyl-C 2-4 Alkynylidene-, -(CH2) p -6-membered heterocyclic-SO2- or -C 6-10 Aryl-SO2-NH-; p is selected from 0, 1, 2, 3, 4 or 5; Y 2 is selected from 5-6 membered heteroaryl or 5-6 membered heterocyclic group.

26. The antibody drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to claim 25, wherein: Y 1 selected from -(CH2)5-, -CH2-NH-SO2-, -(CH2)2-NH-SO2-, -(CH2)3-NH-SO2-, -CH2-N + (CH3)2-(CH2)2-NH-SO2-, -C≡C-CH2-NH-SO2-, -(CH2)3-C≡C-, -CH2-O-CH2-C≡C-, 27. The antibody drug conjugate according to claim 25, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein Y 2 is selected from 5-6 membered N-containing heteroaryl or 5-6 membered N-containing heterocyclic group; preferably, Y 2 Selected from 28. The antibody drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to any one of claims 22 to 27, wherein Y is selected from 29. The antibody drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to any one of claims 22 to 28, wherein the structural unit -XMY- is selected from:

30. A cytotoxic drug-linker, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug-linker has a structure represented by formula (IB): Dr——X——M——Y′ (IB), wherein Y′ is a structure in which Y is inserted before Ab according to any one of claims 22 to 29, X, M, and Y are as defined in any one of claims 22 to 29, and Dr is a cytotoxic drug fragment according to claim 21, which is selected from Preferably, Y' is selected from: Preferably, X is selected from -CH2-NH- or Preferably, M is selected from 31. The cytotoxic drug-linker according to claim 30, its stereoisomers or pharmaceutically acceptable salts thereof, which is selected from:

32. The antibody drug conjugate according to any one of claims 22 to 29, which is selected from: The Ab is an antibody; t is a value between 4 and 8.

33. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 22 to 29 or 32 or the cytotoxic drug-linker according to claims 30 to 31, and a pharmaceutically acceptable carrier.

34. Use of the antibody drug conjugate according to any one of claims 22 to 29 or 32, the cytotoxic drug-linker according to claims 30 to 31, or the pharmaceutical composition according to claim 33 in the preparation of a drug for treating cancer.

35. A method for treating cancer, characterized in that An effective dose of the drug conjugate according to any one of claims 22 to 29 or 32, the cytotoxic drug-linker according to claims 30 to 31, or the pharmaceutical composition according to claim 33 is administered to a patient in need thereof.