Immunoconjugates and Methods

Abstract

The immunoconjugate of formula (I) comprises a linker for linking an antibody targeting ligand (Ab) to a drug (D). Embodiments of such immunoconjugates are useful for delivering a drug to selected cells or tissues, for example, for the treatment of cancer. Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I)

Description

Technical Field

[0001] Incorporation by reference to any priority application Any application for which foreign or domestic priority is claimed in the application data sheet filed herewith, such as U.S. Provisional Application No. 63 / 369,464, filed Jul. 26, 2022, is hereby expressly incorporated by reference herein in accordance with 37 CFR 1.57 and Rules 4.18 and 20.6.

[0002] Statement of sequences This application includes an electronically submitted sequence listing, which is hereby incorporated by reference in its entirety. This sequence listing, created on Jul. 21, 2023, is named ZENO144.xml and is approximately 56 kb in size.

Background Art

[0003] This application relates to conjugates comprising a linker for linking an antibody-targeted ligand to a cell-killing moiety (such as a drug), methods of making such conjugates, and methods of using such conjugates to deliver a cell-killing moiety to selected cells or tissues, for example, for the treatment or inhibition of cancer.

[0004] Description A number of antibody-drug conjugates (ADCs) have been developed for medical use. See, for example, Nejadmoghaddam, M. et al., “Antibody-Drug Conjugates: Possibilities and Challenges”, Avicenna J Med Biotech 11(1), 3-23 (2019). The antibody in an ADC functions as a targeting agent to deliver a drug to selected cells or tissues such as cancer cells or tumors. In the United States, the U.S. Food and Drug Administration (FDA) has approved several ADC formulations, including inotuzumab ozogamicin (trade name BESPONSA®), gemtuzumab ozogamicin (trade name MYLOTARG®), brentuximab vedotin (trade name ADCETRIS®), and ado-trastuzumab emtansine (trade name KADCYLA®).

[0005] U.S. Patent No. 10,155,821 discloses an ADC in which an anti-tumor compound is conjugated to an anti-HER2 antibody via a linker. See also U.S. Patent Application Publication No. 2020 / 0385486 and U.S. Patent Application Publication No. 2019 / 0077880. Trastuzumab deruxtecan is an example of an ADC in which an anti-HER2 antibody (trastuzumab) is linked to an anti-tumor compound (deruxtecan) via a cleavable maleimide tetrapeptide linker. The FDA has approved a formulation known as fam-trastuzumab deruxtecan-nxki (trade name ENHERTU®) for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer who have received two or more previous anti-HER2-based regimens in a metastatic setting. Figure 1 shows a manner in which a linker is thought to connect an antibody (mAb) to a drug moiety.

[0006] FDA approval represents a milestone in the ongoing development of therapeutic ADCs. However, there remains a need for improved ADCs that can help address the long-standing need for additional options for treating cancer and / or delivering a therapeutic payload to selected cells or tissues.

SUMMARY OF THE INVENTION

[0007] Some embodiments provide an immunoconjugate of formula (I) comprising an antibody or antigen-binding fragment thereof (Ab), a drug moiety (D), and a linker that links Ab to D. In one embodiment, the immunoconjugate of formula (I) comprises a drug moiety of formula (II).

[0008] One embodiment provides an immunoconjugate having formula (I), Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I) Wherein, Ab can be an antibody or an antigen-binding fragment thereof, L 1 is,

[0009]

Chemical formula

[0010]

Chemical formula

[0011]

Chemical formula

[0012]

Chemical formula

[0013] In one embodiment, D in formula (I) may be a drug moiety of formula (II) having the following structure,

[0014]

Chemical formula

[0015] One embodiment provides a compound of formula (IV) having the following structure or a pharmaceutically acceptable salt thereof,

[0016]

Chemical Formula

[0017] One embodiment provides a pharmaceutical composition comprising an immunoconjugate described herein, a drug compound described herein, or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0018] ​One embodiment provides a method of treating cancer or a tumor, comprising administering to a subject having the cancer or tumor an effective amount of an immunoconjugate described herein, a drug compound described herein, or a pharmaceutically active salt thereof, or a pharmaceutical composition described herein.

[0019] One embodiment provides the use of an effective amount of an immunoconjugate described herein, a drug compound described herein, or a pharmaceutically active salt thereof, or a pharmaceutical composition described herein, in the manufacture of a medicament for treating cancer or a tumor.

[0020] Some embodiments provide a conjugate of formula (III) comprising a functional group M1, a drug moiety (D), and a linker connecting M1 to D. In one embodiment, the conjugate of formula (III) comprises a drug moiety of formula (II).

[0021] One embodiment provides a conjugate having formula (III), Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III) Wherein, Mi is

[0022]

Chemical formula

[0023]

Chemical formula

[0024]

Chemical formula

[0025]

Chemical formula

[0026] One embodiment provides a process for generating an immunoconjugate, comprising reacting an effective amount of a thiol-functionalized antibody or an antigen-binding fragment thereof with a conjugate as described herein under reaction conditions effective to form the immunoconjugate described herein.

[0027] One embodiment provides an immunoconjugate, pharmaceutical composition, method of treatment, use, or manufacturing process as described herein, wherein Ab is an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof a) comprises a VH CDR1 having an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, A VHCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, and a heavy chain comprising a VHCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, and b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8, a VLCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of AAS, and a light chain comprising a VLCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, comprising, wherein said antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0028] One embodiment provides an immunoconjugate, pharmaceutical composition, treatment method, use, or manufacturing process described herein, wherein Ab is an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is a) a heavy chain comprising a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, a VHCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, and a VHCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22, a VLCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAY, and a light chain comprising a VLCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24, comprising, The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0029] One embodiment provides an immunoconjugate, pharmaceutical composition, treatment method, use, or manufacturing process described herein, wherein Ab is an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29, a VHCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a VHCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31, and a heavy chain, b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36, a VLCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAS, and a VLCDR3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38, and a light chain, and the antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0030] These embodiments and other embodiments are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0031]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27A

Figure 27B

Figure 28A

Figure 28B

Figure 29A

Figure 29B

Figure 29C

Figure 29D

Figure 29E

Figure 29F

Figure 29G

Figure 29H

Figure 30

Figure 31

Figure 32

Mode for Carrying Out the Invention

[0032] Definitions Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referred to in this specification are incorporated by reference in their entirety unless otherwise specifically noted. In the event that there are multiple definitions for a term in this specification, the definitions in this section shall control unless otherwise specifically noted.

[0033] As used herein, "conjugate" is a compound comprising two or more substances (e.g., an antibody, a linker moiety, and / or a drug moiety) linked together by a chemical bond. Examples of conjugates include antibody-drug conjugates (which may optionally include a linker moiety), drug-linker conjugates, and antibody-linker conjugates. An "immunoconjugate" is a conjugate that includes an immunological substance such as an antibody.

[0034] As used herein, an "antibody" (Ab) is a protein produced by the immune system, or a synthetic variant thereof, that binds to a specific site on a cell or tissue. An "antigen-binding fragment" (Fab) is a portion of an antibody that binds to a specific antigen. A monoclonal antibody is a type of synthetic antibody. In cancer treatment, monoclonal antibodies can directly kill cancer cells, they can block the development of tumor blood vessels, or they can help the immune system kill cancer cells.

[0035] Whenever a group is described as being "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "unsubstituted or substituted", in the case of substitution, the substituent may be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group may be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, amino, mono-substituted amine group, di-substituted amine group, mono-substituted amine(alkyl) group, and di-substituted amine(alkyl), each independently selected from one or more (plural possible) of the groups.

[0036] As used herein, "C" where "a" and "b" are integers a -C b " refers to the number of carbon atoms in the group. The indicated group can contain "a" to "b" (including a and b) carbon atoms. Thus, for example, the "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified, the broadest scope described in these definitions is assumed.

[0037] When it is described that two "R" groups are "joined together", the atoms to which the R groups are attached can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle. For example, but not limited to, ortho R on the phenyl ring 1 and R 2 substituents are shown to be -O-(CR 5 R 6 ) m -O-, such that when R 1 and R 2 are "joined together" to form a ring, -O-(CR 5 R 6 ) m -O- is covalently bonded to the phenyl ring at the R 1 and R 2 positions to form a heterocycle:

[0038]

Chemical Structure

[0039] As used herein, the term "alkyl" refers to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight-chain. Examples of branched-chain alkyl groups include, but are not limited to, iso-propyl, sec-butyl, t-butyl, etc. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, etc. An alkyl group may have from 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as "1 to 30" refers to each integer within the given range; for example, "1 to 30 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and may consist of up to 30 carbon atoms, and this definition also applies when the term "alkyl" appears without a specified numerical range). An alkyl group may also be an intermediate-sized alkyl having from 1 to 12 carbon atoms. An alkyl group may also be a lower alkyl having from 1 to 6 carbon atoms. An alkyl group may be either substituted or unsubstituted. An alkyl group is typically monovalent, unless the context dictates otherwise. For example, one of ordinary skill in the art would recognize that C1-C6 alkyl is divalent in the formula: -(C1-C6 alkyl)-X 2 as recognized herein.

[0040] As used herein, the term "alkylene" refers to a divalent, fully saturated, straight-chain aliphatic hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene. The alkylene group may be

[0041]

Chem.

[0042]

Chem.

[0043] [Chemical formula] ) and can be substituted by replacing one or more hydrogens of the lower alkylene group and / or by replacing both hydrogens on the same carbon.

[0044] As used herein, the term "alkenyl" refers to a monovalent straight-chain or branched-chain radical containing a carbon-carbon double bond and having 2 to 20 carbon atoms, including but not limited to 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. The alkenyl group may be either unsubstituted or substituted.

[0045] As used herein, the term "alkynyl" refers to a monovalent straight-chain or branched-chain radical containing a carbon-carbon triple bond and having 2 to 20 carbon atoms, including but not limited to 1-propynyl, 1-butynyl, 2-butynyl, etc. The alkynyl group may be either unsubstituted or substituted.

[0046] As used herein, the term "halogen atom" or "halogen" means any one of the radiation-stable atoms in Group 7 of the Periodic Table of the Elements, such as fluorine, chlorine, bromine, and iodine.

[0047] As used herein, "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl, tri-haloalkyl, and polyhaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl, and pentafluoroethyl. Haloalkyl may be substituted or unsubstituted.

[0048] As used herein, "haloalkenyl" refers to an alkenyl group in which one or more hydrogen atoms have been replaced by a halogen (e.g., mono-haloalkenyl, di-haloalkenyl, tri-haloalkenyl, and polyhaloalkenyl).

[0049] As used herein, "haloalkynyl" refers to an alkynyl group in which one or more hydrogen atoms have been replaced by a halogen (e.g., mono-haloalkynyl, di-haloalkynyl, tri-haloalkynyl, and polyhaloalkynyl).

[0050] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms have been replaced by a halogen (e.g., mono-haloalkoxy, di-haloalkoxy, and tri-haloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. Haloalkoxy may be substituted or unsubstituted.

[0051] As used herein, "heterocyclyl" or "heteroaricyclic" refers to monocyclic, bicyclic, and tricyclic systems having from 3 to 18 members, including 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered rings, wherein the carbon atoms together with from 1 to 5 heteroatoms form the above ring systems. The heterocycle may optionally contain one or more unsaturated bonds, provided that the unsaturated bonds are positioned such that a completely delocalized π-electron system does not occur throughout the entire ring. The heteroatoms are elements other than carbon, and include, for example, but are not limited to, oxygen, sulfur, and nitrogen. The heterocycle may further contain one or more carbonyl or thiocarbonyl functional groups so that this definition includes oxo- and thio-based systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When the ring consists of two or more polycycles, the rings may be linked to each other in a fused, bridged, or spiro form. As used herein, the term "fused" refers to two rings sharing two atoms and one bond. As used herein, the term "bridged heterocyclyl" or "bridged heteroaricyclic" refers to a compound in which the heterocyclyl or heteroaricyclic contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings sharing one atom, and the two rings are not linked by a bridge. The heterocyclyl group and the heteroaricyclic group can contain from 3 to 30 atoms in the ring(s), from 3 to 20 atoms in the ring(s), from 3 to 10 atoms in the ring(s), from 3 to 8 atoms in the ring(s), or from 3 to 6 atoms in the ring(s). For example, 5 carbon atoms and 1 heteroatom, 4 carbon atoms and 2 heteroatoms, 3 carbon atoms and 3 heteroatoms, 4 carbon atoms and 1 heteroatom, 3 carbon atoms and 2 heteroatoms, 2 carbon atoms and 3 heteroatoms, 1 carbon atom and 4 heteroatoms, 3 carbon atoms and 1 heteroatom, or 2 carbon atoms and 1 heteroatom. Additionally, any nitrogen in the heteroaricyclic may be quaternized. The heterocyclyl group or the heteroaricyclic group may be substituted or unsubstituted.Examples of such "heterocyclyl" or "heteroaryclyl" groups include, but are not limited to, 1,3-dioxine, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, azepane, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiomorpholine, thiomorpholine sulfoxide, thiomorpholine sulfone, and their benzofused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, and / or 3,4-methylenedioxyphenyl). Examples of spiroheterocyclyl groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane, and 2-azaspiro[3.4]octane.

[0052] When the number of substituents is not specified (e.g., haloalkyl, haloalkenyl, haloalkynyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may include one or more of the same or different alkoxy groups containing 1, 2, or 3 atoms.

[0053] As used herein, a radical refers to a species having a single unpaired electron such that a species containing the radical can be covalently bonded to another species. Thus, in this regard, a radical is not necessarily a free radical. Rather, a radical refers to a particular portion of a larger molecule. The term "radical" can be used interchangeably with the term "group".

[0054] The term "pharmaceutically acceptable salt" refers to salts of a compound that do not provide a significant irritation to the organism to which it is administered and do not inactivate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutically acceptable salts can be obtained by reacting the compound with an inorganic acid, such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid (such as 2,3-dihydroxypropyl dihydrogen phosphate), or an organic acid, such as aliphatic or aromatic carboxylic acid or sulfonic acid, such as formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, benzoic acid, salicylic acid, 2-oxopentanedioic acid, or naphthalenesulfonic acid. Pharmaceutically acceptable salts can also be obtained by reacting the compound with a base to form salts, such as ammonium salts, alkali metal salts, such as sodium, potassium, or lithium salts, alkaline earth metal salts, such as calcium or magnesium salts, carbonate salts, bicarbonate salts, salts of organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts having amino acids such as arginine and lysine. With respect to the compound of formula (I), one of ordinary skill in the art will understand that when a salt is formed by protonation of a nitrogen-based group (e.g., NH2), the nitrogen-based group can associate with a positive charge (e.g., NH2 can become NH3 + ), and the positive charge can be balanced by a counterion having a negative charge (such as Cl - ).

[0055] In any of the compounds described herein that have one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, it is understood that each center may independently be of the R configuration, the S configuration, or a mixture thereof. Accordingly, the compounds provided herein may be enantiomerically pure compounds, enantiomerically enriched compounds, racemic mixtures, diastereomerically pure compounds, diastereomerically enriched compounds, or stereoisomeric mixtures. As used herein, whenever the stereochemistry of a compound is "optionally assigned", it refers to a stereocenter of the (R) or (S) configuration, and the compound may be the enantiomer opposite to that shown. In addition, in any of the compounds described herein that have one or more double bonds that can give rise to geometric isomers that can be defined as E or Z, it is understood that each double bond may independently be E, Z, or a mixture thereof. Similarly, it is understood that all tautomeric forms are also intended to be included in any of the compounds described.

[0056] If a compound disclosed herein has a valence that is unfilled, it is to be understood that that valence is filled with hydrogen or an isotope thereof, for example, hydrogen-1 (protium) and hydrogen-2 (deuterium). The compounds described herein may include all isotopes of the atoms that occur in the intermediates or final compounds. Isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0057] It is understood that the compounds described herein may be labeled with isotopes. Substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as an increase in in vivo half-life or a reduction in the required dosage. Each chemical element represented in the compound structure may include any isotope of that element. For example, in the compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position in the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium), hydrogen-2 (deuterium), and hydrogen-3 (tritium). Accordingly, references to compounds herein include all possible isotopic forms unless the context clearly indicates otherwise.

[0058] It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, including different crystal packing arrangements of the same elemental composition of the compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvated forms having pharmaceutically acceptable solvents such as water, ethanol. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain either a stoichiometric or non-stoichiometric amount of the solvent and may be formed during the crystallization process with a pharmaceutically acceptable solvent such as water, ethanol. A hydrate is formed when the solvent is water, or an alcoholate is formed when the solvent is an alcohol. In addition, the compounds provided herein can exist in both unsolvated and solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0059] It is understood that when ranges of values are provided, the upper and lower limits, and each value intervening between the upper and lower limits of that range, are included within the embodiments.

[0060] The terms and phrases used in this application, and variations thereof, particularly those in the appended claims, should be construed as non - limiting and non - restrictive unless otherwise expressly stated. By way of example, the term "including" should be construed to mean "including without limitation", "including but not limited to", etc. As used herein, the term "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or non - limiting, and does not exclude additional, unrecited elements or method steps. The term "having" should be construed to mean "having at least". The term "including" should be construed to mean "including but not limited to". The term "example" is used to provide illustrative instances rather than an exhaustive or limiting list of the items under consideration. The use of terms such as "preferably", "preferred", "desired", or "desirable", and words of similar meaning, is not intended to imply that a particular feature is critical, essential, or even important to its structure or function, but rather is simply intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Additionally, the term "comprising" shall be construed as a synonym for the phrases "having at least" or "including at least". When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0061] Regarding the use of substantially any plural and / or singular terms in this specification, those skilled in the art can appropriately convert from plural to singular and / or from singular to plural according to the context and / or application. Various singular / plural exchanges may be explicitly described in this specification for clarity. The indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain means are recited in mutually different dependent claims does not indicate that combinations of these means cannot be used to obtain an advantage. Any reference signs in the claims should not be construed as limiting their scope.

[0062] Compound The various embodiments disclosed herein are compounds of formula (IV) having the following structure or pharmaceutically acceptable salts thereof,

[0063]

Chemical formula

[0064] Various embodiments disclosed herein are a compound of formula (IV*) having the following structure or a pharmaceutically acceptable salt thereof,

[0065]

Chemical formula

[0066] In various embodiments, R 1 and R 2 of formula (IV) or formula (IV*) may each independently be hydrogen, halogen, -CN, -OR 5 -NR 5 R 6 ​, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 haloalkyl, a substituted or unsubstituted O-(C1-C6 alkyl), a substituted or unsubstituted O-(C1-C6 haloalkyl), and [(CY2) p O(CY2) q t CY3, or a substituted or unsubstituted O-(CR 5 R 6 ) m -O- and can be selected therefrom, whereby R 1 and R 2 can together form a ring. In one embodiment, at least one of R 1 and R 2 can be hydrogen. In one embodiment, at least one of R 1 and R 2 can be halogen. For example, in one embodiment, at least one of R 1 and R 2 can be fluoro. In one embodiment, R 1 and R 2 can each be fluoro. In another embodiment, R 1 can each be fluoro and R 2 can be hydrogen. In yet another embodiment, R 1 can each be hydrogen and R 2 can be fluoro. In one embodiment, at least one of R 1 and R 2 can be chloro. In one embodiment, one of R 1 and R 2 can be fluoro and the other of R 1 and R 2 can be chloro. In one embodiment, R 1 can be chloro and R 2 can be fluoro. In one embodiment, at least one of R 1 and R 2 can be -CN. In one embodiment, at least one of R 1 and R 2 can be -OR 5 , wherein R 5 ​can be a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 1 and R 2 at least one of can be methoxy. In one embodiment, R 1 and R 2 can be a substituted or unsubstituted O-(CR 5 R 6 ) m -O-, whereby R 1 and R 2 can together form a ring. In one embodiment, one of R 1 and R 2 can be a substituted or unsubstituted O-(CH2)-O, whereby R 1 and R 2 can together form 1,3-dioxolane.

[0067] In one embodiment, at least one of R 1 and R 2 of formula (IV) or formula (IV*) can be -NR 5 R 6 , wherein R 5 and R 6 can each individually be a substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl.

[0068] In one embodiment, at least one of R 1 and R 2 of formula (IV) or formula (IV*) can be a substituted or unsubstituted C1-C6 alkyl. In one embodiment, at least one of R 1 and R 2 can be C1-C3 alkyl. For example, in one embodiment, at least one of R 1 and R 2 can be methyl. In one embodiment, at least one of R 1 and R 2 can be C1-C3 alkyl and the other can be halogen. For example, in one embodiment, R1 and R 2 at least one of which may be methyl and the other may be fluoro. In one embodiment, R 1 may be methyl and the other R 2 may be fluoro.

[0069] In one embodiment, R of formula (IV) or formula (IV*) 1 and R 2 at least one of which may be substituted or unsubstituted C1-C6 haloalkyl. For example, in one embodiment, R 1 and R 2 at least one of which may be difluoromethyl. In one embodiment, R 1 and R 2 at least one of which may be substituted or unsubstituted O-(C1-C6 alkyl). For example, in one embodiment, R 1 and R 2 at least one of which may be methoxy. In one embodiment, R 1 and R 2 at least one of which may be -[(CY2) p O(CY2) q t CY3. In one embodiment, R 1 and R 2 may be substituted or unsubstituted O-(CR 5 R 6 ) m -O-, whereby R 1 and R 2 together form a ring that can form a heterocyclic ring by covalently bonding the terminal of -O-(CR 5 R 6 ) m -O- to the phenyl ring at the R 1 and R 2 positions of formula (IV) or formula (IV*).

[0070] In one embodiment, one of R 1 and R 2 of formula (IV) or formula (IV*) may be hydrogen and the other of R 1 and R 2 may be halogen. In one embodiment, R of formula (IV) or formula (IV*)​1 and R 2 one of which may be hydrogen, and R 1 and R 2 the other may be fluoro. In one embodiment, R in formula (IV) or formula (IV*) 1 may be fluoro, and R 2 may be hydrogen. In one embodiment, R in formula (IV) or formula (IV*) 1 may be hydrogen, and R 2 may be fluoro. In one embodiment, R 1 and R 2 one of which may be hydrogen, and R 1 and R 2 the other may be substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 and R 2 one of which may be hydrogen, and R 1 and R 2 the other may be substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 one of which may be hydrogen, and R 1 and R 2 the other may be substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 both may be hydrogen. In one embodiment, R 1 and R 2 both may not be hydrogen.

[0071] In one embodiment, R in formula (IV) or formula (IV*) 1 and R 2 one of which may be halogen, and R 1 and R 2 the other may be substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 and R 2 one of which may be halogen, and R 1 and R 2 the other may be substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 one of which may be halogen, and R 1 and R 2The other may be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 may both independently be halogen. In one embodiment, R 1 and R 2 may also not be halogen.

[0072] In one embodiment, one of R 1 and R 2 in formula (IV) or formula (IV*) may be a substituted or unsubstituted C1-C6 alkyl, and the other of R 1 and R 2 may be a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, one of R 1 and R 2 may be a substituted or unsubstituted C1-C6 alkyl, and the other of R 1 and R 2 may be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, both of R 1 and R 2 may independently be a substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 and R 2 may also not be a substituted or unsubstituted C1-C6 alkyl.

[0073] In one embodiment, one of R 1 and R 2 in formula (IV) or formula (IV*) may be a substituted or unsubstituted C1-C6 haloalkyl, and the other of R 1 and R 2 may be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, both of R 1 and R 2 may independently be a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 may also not be a substituted or unsubstituted C1-C6 haloalkyl.

[0074] In one embodiment, one of R 1 and R 2One of them may be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 may both independently be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 may not be a substituted or unsubstituted O-(C1-C6 alkyl either. In one embodiment, R 1 and R 2 may be a substituted or unsubstituted O-(CR 5 R 6 ) m -O-, whereby R 1 and R 2 may together form a ring. In various embodiments, R 1 and R 2 may each independently be selected from hydrogen, fluoro, methoxy, methyl, difluoromethyl, and O-(CH2)-O-, whereby R 1 and R 2 may together form a ring.

[0075] In various embodiments, R 3 of formula (IV) may be hydrogen, -OR 5 , -NR 5 R 6 , a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C2-C6 alkenyl, a substituted or unsubstituted C2-C6 alkynyl, a substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 3 may be hydrogen. In one embodiment, R 3 may be -OR 5 such as -OCH3. In one embodiment, R 3 may be -NR 5 R 6 , wherein R 5 and R 6 ​is each independently optionally substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached can form an optionally substituted 4- or 5-membered heterocyclyl. In one embodiment, R 3 is optionally substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3 can be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 3 can be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 3 is optionally substituted or unsubstituted C2-C6 alkenyl. For example, in one embodiment, R 3 can be -CH=CH2. In another embodiment, R 3 can be -CH2CH=CH2. In one embodiment, R 3 is optionally substituted or unsubstituted C2-C6 alkynyl. For example, in one embodiment, R 3 can be -C≡CH. In one embodiment, R 3 is -[(CY2) p O(CY2) q t CY3, where each Y can independently be H or halogen.

[0076] In various embodiments, R 4 in formula (IV) is hydrogen, -OR 5 , -NR 5 R 6 , optionally substituted or unsubstituted C1-C6 alkyl, optionally substituted or unsubstituted C2-C6 alkenyl, optionally substituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 4 can be hydrogen. In one embodiment, R 4 can be -OR 5 such as -OCH3. In one embodiment, R​​4 is -NR 5 R 6 and may be, where R 5 and R 6 may each independently be substituted or unsubstituted C1 - C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached may form a substituted or unsubstituted 4 - or 5 - membered heterocyclyl. In one embodiment, R 4 may be substituted or unsubstituted C1 - C6 alkyl. For example, in one embodiment, R 4 may be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 4 may be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 4 may be substituted or unsubstituted C2 - C6 alkenyl. For example, in one embodiment, R 4 may be -CH=CH2. In another embodiment, R 4 may be -CH2CH=CH2. In one embodiment, R 4 may be substituted or unsubstituted C2 - C6 alkynyl. For example, in one embodiment, R 4 may be -C≡CH. In one embodiment, R 4 is -[(CY2) p O(CY2) q t CY3 and each Y may independently be H or halogen.

[0077] In various embodiments, R 3 of formula (IV*) may be selected from hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1 - C6 alkyl, substituted or unsubstituted 4 - or 5 - membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 3 ​​can be hydrogen. In one embodiment, R 3 can be -OR such as -OCH3 5 In one embodiment, R 3 can be -NR 5 R 6 wherein R 5 and R 6 can each independently be substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl. In one embodiment, R 3 can be substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3 can be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 3 can be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 3 is -[(CY2) p O(CY2) q t CY3 where each Y can independently be H or halogen.

[0078] In various embodiments, R 4 of formula (IV*) can be hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 4 can be hydrogen. In one embodiment, R 4 can be -OR such as -OCH3 5 In one embodiment, R 4 can be -NR 5 R 6 wherein R 5 and R 6 ​​is each independently optionally substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached may form an optionally substituted 4- or 5-membered heterocyclyl. In one embodiment, R 4 is optionally substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 4 is methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 4 is -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 4 is -[(CY2) p O(CY2) q t CY3, where each Y is independently H or halogen.

[0079] In some embodiments, one of R 3 and R 4 in formula (IV) or formula (IV*) may be hydrogen, and the other of R 3 and R 4 may be optionally substituted or unsubstituted C1-C6 alkyl. In other embodiments, one of R 3 and R 4 may be hydrogen, and the other of R 3 and R 4 may be -NR 5 R 6 . In some embodiments, one of R 3 and R 4 may be CH3, provided that both of R 3 and R 4 cannot be -CH3. For example, in some embodiments, one of R 3 and R 4 may be -CH3, and the other of R 3 and R 4 may be optionally substituted or unsubstituted C1-C6 alkyl that cannot be -CH3, such as -CH2OH or CH2CH2OH. In some embodiments, one of R 3 and R 4 may be -CH3, and the other of R 3 and R​4 On the other hand, it may be -NR 5 R 6 as possible.

[0080] In some embodiments, R of formula (IV) or formula (IV*) 3 and / or R 4 may be -NR 5 R 6 as possible. For example, in one embodiment, R 5 and R 6 may each individually be substituted or unsubstituted C1-C6 alkyl (-CH3, -CH2CH2OH, or

[0081]

Chemical formula

[0082]

Chemical formula

[0083]

Chemical formula

[0084] In some embodiments, R of formula (IV) or formula (IV*) 3 and / or R 4is -[(CY2) p O(CY2) q t which may be CY3. For example, in some embodiments, one of R 3 and R 4 may be -CH2OH or CH2CH2OH, and the other of R 3 and R 4 may be -CH2OCH3.

[0085] In some embodiments, R 3 and R 4 of formula (IV) or formula (IV*) may together with the carbon atom to which they are attached form an unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl substituted with an optionally substituted C1-C3 alkyl. For example, in some embodiments, R 3 and R 4 together with the carbon atom to which they are attached may be

[0086]

Chemical formula

[0087]

Chemical formula

[0088] In one embodiment, one of R 3 and R 4 of formula (IV) or formula (IV*) may be hydrogen, and the other of R 3 and R 4 ​The other can be -CH2OH. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -CH3 for one of them, and R 3 and R 4 can be -CH2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -OCH3 for one of them, and R 3 and R 4 can be -CH2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -C2H5 for one of them, and R 3 and R 4 can be -CH2OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be -CH=CH2 for one of them, and R 3 and R 4 can be -CH2OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be -C≡CH for one of them, and R 3 and R 4 can be -CH2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be hydrogen for one of them, and R 3 and R 4 can be -(CH2)2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -CH3 for one of them, and R 3 and R 4 can be -(CH2)2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -OCH3 for one of them, and R 3 and R 4 can be -(CH2)2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -C2H5 for one of them, and R 3 and R 4The other can be -(CH2)2OH. In one embodiment, R of formula (IV) 3 and R 4 can be -CH=CH2 for one of them, and R 3 and R 4 can be -(CH2)2OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be -C≡CH for one of them, and R 3 and R 4 can be -(CH2)2OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be hydrogen for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -CH3 for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -OCH3 for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be -C2H5 for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be -CH=CH2 for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be -C≡CH for one of them, and R 3 and R 4 can be -(CH2)3OH for the other. In one embodiment, R of formula (IV) 3 and R 4 can be hydrogen for one of them, and R 3 and R 4The other can be --CH2CH=CH2. In one embodiment, R of formula (IV) 3 and R 4 can be hydrogen, and R 3 and R 4 can be -C≡CH. In one embodiment, both R 3 and R 4 can be -CH2OH. In one embodiment, one of R 3 and R 4 of formula (IV) or formula (IV*) can be hydrogen, and R 3 and R 4 can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) or formula (IV*) can be -CH3, and R 3 and R 4 can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) or formula (IV*) can be -OCH3, and R 3 and R 4 can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) or formula (IV*) can be -C2H5, and R 3 and R 4 can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) can be -CH=CH2, and R 3 and R 4 can be -(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) can be -CH=CH2, and R 3 and R 4 can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 of formula (IV) can be -C≡CH, and R 3 and R 4The other can be -CH2O(CH2)2OH. In one embodiment, R of formula (IV) or formula (IV*) 3 and R 4 can be either hydrogen or CH3, and R 3 and R 4 the other can be

[0089]

Chemical formula

[0090]

Chemical formula

[0091]

Chem.

[0092]

Chem.

[0093]

Chem.

[0094] In various embodiments, R in formula (IV) or formula (IV*) 7 is H, -COR 8 , -CO2R 8 , or (CO)-NHR 8 , wherein R 8 is described elsewhere herein. In one embodiment, R 7 can be H. In one embodiment, R 7 can be -COR 8 . In one embodiment, R 7 can be -CO2R 8 . In one embodiment, R 7 can be -(CO)-NHR 8 .

[0095] In various embodiments, R in formula (IV) or formula (IV*) 8 is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or [(CY2)p O(CY2) q t CY3, where the variables p, q, t, and Y are described elsewhere in this specification. In one embodiment, R 8 can be a substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 8 can be a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 8 is -[(CY2) p O(CY2) q t CY3.

[0096] In various embodiments, m in formula (IV) or formula (IV*) can be 1 or 2. In one embodiment, m can be 1. In another embodiment, m can be 2.

[0097] In various embodiments, n 4 and n 5 can each individually be 0, 1, or 2, provided that both n 4 and n 5 cannot both be 0. In one embodiment, both n 4 and n 5 can be 1. In one embodiment, n 4 can be 0 and n 5 can be 1. In one embodiment, n 4 can be 0 and n 5 can be 2. In one embodiment, n 4 can be 1 and n 5 can be 0. In one embodiment, n 4 can be 2 and n 5 can be 0.

[0098] ​​In various embodiments, each Y of formula (IV) or formula (IV*) can individually be H or a halogen. In one embodiment, each Y can be hydrogen. In one embodiment, -CY2 can be -CH2. In one embodiment, -CY3 can be -CH3. In one embodiment, -CY3 can be -CHF2. In one embodiment, -CY3 can be -CH2F. In one embodiment, -CY3 can be -CF3.

[0099] In various embodiments, each p of formula (IV) or formula (IV*) can individually be 1, 2, 3, 4, 5, or 6. In one embodiment, p can be 1. In one embodiment, p is 2.

[0100] In various embodiments, each q of formula (IV) or formula (IV*) can individually be 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q can be 1. In one embodiment, q can be 2.

[0101] In various embodiments, each t of formula (IV) or formula (IV*) can individually be 1, 2, 3, 4, 5, or 6. In one embodiment, t can be 1. In one embodiment, p can be t.

[0102] In various embodiments, R 1 and R 2 can each individually be hydrogen, a halogen, and unsubstituted C1-C6 alkyl, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, such that R 1 and R 2 can together form a ring, and R 3 and R 4 can each individually be hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that R 3 ​and R 4 Neither is hydrogen, or R 3 and R 4 together with the carbon atom to which they are attached may form an unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl substituted with unsubstituted C1-C3 alkyl, R 5 and R 6 may each individually be unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached may form a substituted or unsubstituted 4- or 5-membered heterocyclyl, n 4 may be 2, n 5 may be 0, each Y may be H, each m may be 1, p may be 1, q may be 2, t may be 1, R 7 may be H. In various embodiments, R 1 and R 2 may each individually be selected from hydrogen, halogen, and unsubstituted C1-C6 alkyl, R 3 and R 4 may each individually be hydrogen, -OR 5 -NR 5 R 6 substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that neither R 3 and R 4 is hydrogen, or R 3 and R 4 together with the carbon atom to which they are attached may form an unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl substituted with unsubstituted C1-C3 alkyl, R 5 and R 6 may each individually be unsubstituted C1-C6 alkyl, or R 5 and R 6 ​can, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted 4- or 5-membered heterocyclyl, and n 4 can be 2, and n 5 can be 0, each Y can be H, each m can be 1, p can be 1, q can be 2, t can be 1, and R 7 can be H.

[0103] In various embodiments, the present disclosure provides a compound of formula (IV) and / or formula (IV*), or a compound that can be represented by a structure selected from the following, or a pharmaceutically acceptable salt thereof.

[0104]

Chemical formula

[0105]

Chemical formula

[0106]

Chemical formula

[0107]

Chemical formula

[0108]

Chemical formula

[0109]

Chemical formula

[0110]

Chemical formula

[0111]

Chemical formula

[0112] In various embodiments, the compound of formula (IV) and / or formula (IV*) can be represented by a structure selected from the following, or a pharmaceutically acceptable salt thereof.

[0113] [Chemical formula]

[0114] [Chemical formula]

[0115] [Chemical formula]

[0116] In various embodiments, the compound of formula (IV) and / or formula (IV*) can be represented by a structure selected from the following, or a pharmaceutically acceptable salt thereof.

[0117] [Chemical formula]

[0118] [Chemical formula]

[0119] In various embodiments, the compound of formula (IV) and / or formula (IV*) can be represented by a structure selected from the following, or a pharmaceutically acceptable salt thereof.

[0120] [Chemical formula]

[0121] [Chemical formula]

[0122]

Chem.

[0123] Conjugate (toxin-linker or linker-payload) Various embodiments disclosed herein relate to a conjugate of formula (III) having the following structure. Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III)

[0124] In various embodiments, Mi of formula (III)

[0125]

Chem.

[0126] In various embodiments, L of formula (III) 2 may not be present,

[0127]

Chem.

[0128]

Chem.

[0129] [Chemical formula] can be. In one embodiment, L of formula (III) 2 is

[0130] [Chemical formula] can be.

[0131] In various embodiments, Z of formula (III) 1 and Z 2 can each independently be hydrogen, halogen, NO2, -O-(C1-C6 alkyl), or C1-C6 alkyl. In one embodiment, Z 1 and Z 2 at least one of can be hydrogen. In one embodiment, Z 1 and Z 2 at least one of can be halogen. In one embodiment, Z 1 and Z 2 at least one of can be NO2. In one embodiment, Z 1 and Z 2 at least one of can be -O-(C1-C6 alkyl). For example, in one embodiment, Z 1 and Z 2 at least one of can be methoxy. In one embodiment, Z 1 and Z 2 at least one of can be C1-C6 alkyl. For example, in one embodiment, Z 1 and Z 2 at least one of can be methyl.

[0132] In various embodiments, L of formula (III) 3 is -(CH2)n 1 -C(=O)- or (CH2CH2O)n 1 -(CH2)n 1 C(=O)- and in the formula, each n 1 can independently be an integer from 0 to 12. In one embodiment, L 3 can be -(CH2)n 1 -C(=O)-. For example, in one embodiment, L 3 can be -C(=O)-. In one embodiment, L 3 can be -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-. For example, in one embodiment, L 3 can be -CH2C(=O)-. In embodiments, n 1 can be an integer from 1 to 12 such as 1 to 6 or 1 to 3.

[0133] In various embodiments, L of formula (III) 4 can be a tetrapeptide residue. For example, in one embodiment, L 4 can be a tetrapeptide residue selected from SEQ ID NO: 43 GGFG (gly-gly-phe-gly), SEQ ID NO: 44 EGGF (glu-gly-gly-phe), SEQ ID NO: 45 SGGF (ser-gly-gly-phe), and SEQ ID NO: 46 KGGF (lys-gly-gly-phe).

[0134] In various embodiments, L of formula (III) 5 is absent or may be [NH(CH2)n 2 n 3 - where in the formula, n 2 can be an integer from 0 to 6 and n 3 can be an integer from 0 to 2. In one embodiment, L 5 may be absent. In one embodiment, L 5 can be -[NH(CH2)n 2 n 3 -. For example, in one embodiment, L 5can be -NH-. In another embodiment, L 5 can be -NHCH2-.

[0135] In various embodiments, L of formula (III) 6 is absent or

[0136]

Chemical formula

[0137]

Chemical formula

[0138] In various embodiments, L of formula (III) 7 may be absent,

[0139]

Chemical formula

[0140]

Chemical formula

[0141]

Chemical formula

[0142]

Chemical formula

[0143] [Chemical formula] It may be. In various embodiments, D in the conjugate of formula (III) may be a drug moiety described herein (e.g., under the heading "Drug Moiety" below or under the heading "Compound" above). In various embodiments, D in the conjugate of formula (III) may be a compound of formula (II). In various embodiments, D in the conjugate of formula (III) may be a compound of formula (II*). In various embodiments, D in the conjugate of formula (III) may be a compound of formula (IV). In various embodiments, D in the conjugate of formula (III) may be a compound of formula (IV*). In one embodiment, D may be a cytotoxic anti-cancer drug moiety.

[0144] In various embodiments, the conjugate of formula (III) may be represented by a structure selected from the following.

[0145] [Chemical formula]

[0146] [Chemical formula]

[0147] [Chemical formula]

[0148] [Chemical formula]

[0149] [Chemical formula]

[0150] In various embodiments, the conjugate of formula (III) can be represented by a structure selected from the following.

[0151] [Chemical formula]

[0152] [Chemical formula]

[0153] [Chemical formula]

[0154] [Chemical formula]

[0155] [Chemical formula]

[0156] In various embodiments, the conjugate of formula (III) can be represented by a structure selected from the following.

[0157] [Chemical formula]

[0158] [Chemical formula]

[0159] [Chemical formula]

[0160] Drug moiety In various embodiments, D in the immunoconjugate of formula (I) or the conjugate of formula (III) can be a drug moiety. The drug moiety can be any compound of formula (IV) or formula (IV*) described herein having appropriate modifications (e.g., those described above under the heading "Compounds"), whereby the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - is connected to D. For example, in various embodiments, the drug moiety D is a compound of formula (II) having the following structure,

[0161]

Chemical formula

[0162] In an alternative embodiment, the drug moiety D in the immunoconjugate of formula (I) or the conjugate of formula (III) is a compound of formula (II*) having the following structure,

[0163] ​ [Chemical formula] In the formula, R 1 and R 2 are each independently hydrogen, halogen, -CN, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted -O-(C1-C6 alkyl), substituted or unsubstituted -O-(C1-C6 haloalkyl), -[(CY2) p O(CY2) q t CY3, and substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, and may be selected from the group consisting of; R 1 and R 2 together form a ring, R 3 and R 4 are each independently hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, and may be selected from the group consisting of; provided that both of R 3 and R 4 cannot be hydrogen, or R 3 and R 4 together with the carbon atom to which they are attached may form a substituted or unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl optionally substituted with a substituted or unsubstituted C1-C3 alkyl, or one of R 3 and R 4 is substituted or unsubstituted -(C1-C6 alkyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted -(C1-C6 alkenyl)-X​​2 、 substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 、 substituted or unsubstituted -(C1-C6 alkynyl)-X 2 、 or substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 may be, X 2 is -OR 9 、 -SR 9 、 or -NHR 9 may be, R 5 and R 6 may each independently be substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 may together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl, n 4 and n 5 may each independently be 0, 1, or 2, provided that n 4 and n 5 cannot both be 0, each Y may independently be H or halogen, each m may independently be 1 or 2, each p may independently be 1, 2, 3, 4, 5, or 6, each q may independently be 0, 1, 2, 3, 4, 5, or 6, each t may independently be 1, 2, 3, 4, 5, or 6, R 7 is H, -COR 8 、 -CO2R 8 、 -(CO)-NHR 8 、 L 4 、 L 5 、 L 6 、 or L 7 may be, R 8 is substituted or unsubstituted C1-C6 alkyl-X 3 、 substituted or unsubstituted C1-C6 haloalkyl-X 3 、 or -[(CY2) p O(CY2) q ​t CY2-X 3 can be, R 9 is H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 can be, provided that R 7 and R 9 exactly one of which is L 4 , L 5 , L 6 , or L 7 can be, and each X 3 can individually be a compound that is -H, -OH, -SH, or -NH2.

[0164] One of ordinary skill in the art will understand that the compounds of formula (II) can be connected via R 3 or R 4 (when defined to include X 2 , and thus R 9 ) or via R 7 to the linker -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -.

[0165] In various embodiments, R 1 and R 2 of formula (II) or formula (II*) are each individually hydrogen, halogen, -CN, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted O-(C1-C6 alkyl), substituted or unsubstituted -O-(C1-C6 haloalkyl), and [(CY2) p O(CY2) q t CY3, or substituted or unsubstituted -O-(CR 5 R​6 ) m can be selected from -O-, whereby R 1 and R 2 can together form a ring. In one embodiment, at least one of R 1 and R 2 can be hydrogen. In one embodiment, at least one of R 1 and R 2 can be halogen. For example, in one embodiment, at least one of R 1 and R 2 can be fluoro. In one embodiment, R 1 and R 2 can each be fluoro. In another embodiment, R 1 can each be fluoro, and R 2 can be hydrogen. In yet another embodiment, R 1 can each be hydrogen, and R 2 can be fluoro. In one embodiment, at least one of R 1 and R 2 can be chloro. In one embodiment, one of R 1 and R 2 can be fluoro, and the other of R 1 and R 2 can be chloro. In one embodiment, R 1 can be chloro, and R 2 can be fluoro. In one embodiment, at least one of R 1 and R 2 can be -CN. In one embodiment, at least one of R 1 and R 2 can be -OR 5 , where R 5 can be substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, at least one of R 1 and R 2 can be methoxy. In one embodiment, R 1 and R 2 can be substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, whereby R 1 and R2 can combine together to form a ring. In one embodiment, R 1 and R 2 one of them can be substituted or unsubstituted -O-(CH2)-O-, whereby R 1 and R 2 can combine together to form 1,3-dioxolane.

[0166] In one embodiment, at least one of R 1 and R 2 in formula (II) or formula (II*) can be -NR 5 R 6 , wherein R 5 and R 6 can each independently be substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can combine with the nitrogen atom to which they are attached to form a substituted or unsubstituted 4- or 5-membered heterocyclyl.

[0167] In one embodiment, at least one of R 1 and R 2 in formula (II) or formula (II*) can be substituted or unsubstituted C1-C6 alkyl. In one embodiment, at least one of R 1 and R 2 can be C1-C3 alkyl. For example, in one embodiment, at least one of R 1 and R 2 can be methyl. In one embodiment, at least one of R 1 and R 2 can be C1-C3 alkyl and the other can be halogen. For example, in one embodiment, at least one of R 1 and R 2 can be methyl and the other can be fluoro.

[0168] In one embodiment, at least one of R 1 and R 2 in formula (II) or formula (II*) can be substituted or unsubstituted C1-C6 haloalkyl. For example, in one embodiment, at least one of R 1 and R 2At least one of them can be difluoromethyl. In one embodiment, R 1 and R 2 At least one of them can be substituted or unsubstituted -O-(C1-C6 alkyl). For example, in one embodiment, R 1 and R 2 At least one of them can be methoxy. In one embodiment, R 1 and R 2 At least one of them can be -[(CY2) p O(CY2) q t CY3. In one embodiment, R 1 and R 2 can each independently be substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, whereby R 1 and R 2 together form a ring in which the terminal of -O-(CR 5 R 6 ) m -O- is covalently bonded to the phenyl ring at the R 1 and R 2 positions of formula (II) or formula (II*) to form a heterocyclic ring.

[0169] In one embodiment, one of R 1 and R 2 of formula (II) or formula (II*) can be hydrogen, and the other of R 1 and R 2 can be halogen. In one embodiment, one of R 1 and R 2 of formula (II) or formula (II*) can be hydrogen, and the other of R 1 and R 2 can be fluoro. In one embodiment, R 1 of formula (II) or formula (II*) can be fluoro, and R 2 can be hydrogen. In one embodiment, R 1 of formula (II) or formula (II*) can be hydrogen, and R 2 can be fluoro. In one embodiment, one of R 1 and R 2 can be hydrogen, and R​1 and R 2 The other can be substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 and R 2 One of them can be hydrogen, and R 1 and R 2 The other can be substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 One of them can be hydrogen, and R 1 and R 2 The other can be substituted or unsubstituted -O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 Both can be hydrogen. In one embodiment, R 1 and R 2 do not have to be hydrogen either.

[0170] In one embodiment, one of R 1 and R 2 in formula (II) or formula (II*) can be halogen, and R 1 and R 2 The other can be substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 and R 2 One of them can be halogen, and R 1 and R 2 The other can be substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 One of them can be halogen, and R 1 and R 2 The other can be substituted or unsubstituted -O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 Both can independently be halogen. In one embodiment, R 1 and R 2 do not have to be halogen either.

[0171] In one embodiment, one of R 1 and R 2 in formula (II) or formula (II*) can be substituted or unsubstituted C1-C6 alkyl, and R 1 and R2 On the other hand, it may be a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 and R 2 One of them may be a substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 The other of them may be a substituted or unsubstituted -O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 Both may independently be a substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 1 Nor R 2 May not be a substituted or unsubstituted C1-C6 alkyl either.

[0172] In one embodiment, one of R 1 and R 2 in formula (II) or formula (II*) may be a substituted or unsubstituted C1-C6 haloalkyl, and R 1 and R 2 The other of them may be a substituted or unsubstituted O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 Both may independently be a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 1 Nor R 2 May not be a substituted or unsubstituted C1-C6 haloalkyl either.

[0173] In one embodiment, one of R 1 and R 2 in formula (II) or formula (II*) may be a substituted or unsubstituted -O-(C1-C6 alkyl). In one embodiment, R 1 and R 2 Both may independently be a substituted or unsubstituted -O-(C1-C6 alkyl). In one embodiment, R 1 Nor R 2 May not be a substituted or unsubstituted -O-(C1-C6 alkyl either. In one embodiment, R 1 and R 2 May be a substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, whereby R1 and R 2 can combine to form a ring. In various embodiments, R 1 and R 2 can each independently be selected from hydrogen, fluoro, methoxy, methyl, difluoromethyl, and O-(CH2)-O-, such that R 1 and R 2 can combine to form a ring.

[0174] In various embodiments, R of formula (II) 3 is hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 3 can be hydrogen. In one embodiment, R 3 can be -OR 5 such as -OCH3. In one embodiment, R 3 can be -NR 5 R 6 , wherein R 5 and R 6 can each independently be substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can combine with the nitrogen atom to which they are attached to form a substituted or unsubstituted 4- or 5-membered heterocyclyl. In one embodiment, R 3 can be substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3 can be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 3 can be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 3 ​can be a substituted or unsubstituted C2-C6 alkenyl. For example, in one embodiment, R 3 can be -CH=CH2. In another embodiment, R 3 can be -CH2CH=CH2. In one embodiment, R 3 can be a substituted or unsubstituted C2-C6 alkynyl. For example, in one embodiment, R 3 can be -C≡CH. In one embodiment, R 3 is -[(CY2) p O(CY2) q t CY3, where each Y can be individually H or halogen.

[0175] In various embodiments, R of formula (II) 4 is hydrogen, -OR 5 , -NR 5 R 6 , a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C2-C6 alkenyl, a substituted or unsubstituted C2-C6 alkynyl, a substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 4 can be hydrogen. In one embodiment, R 4 can be -OR 5 such as -OCH3. In one embodiment, R 4 can be -NR 5 R 6 , where R 5 and R 6 can each individually be a substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl. In one embodiment, R 4 can be a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 4 ​​can be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 4 can be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 4 can be substituted or unsubstituted C2-C6 alkenyl. For example, in one embodiment, R 4 can be -CH=CH2. In another embodiment, R 4 is -CH2CH=CH2. In one embodiment, R 4 can be substituted or unsubstituted C2-C6 alkynyl. For example, in one embodiment, R 4 can be -C≡CH. In one embodiment, R 4 is -[(CY2) p O(CY2) q t CY3, where each Y can individually be H or halogen.

[0176] In various embodiments, R of formula (II*) 3 is selected from hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot both be hydrogen. In one embodiment, R 3 can be hydrogen. In one embodiment, R 3 can be -OR 5 such as -OCH3. In one embodiment, R 3 can be -NR 5 R 6 , where R 5 and R 6 can each individually be substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 can together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl. In one embodiment, R​​3 may be a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3 may be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 3 may be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 3 is -[(CY2) p O(CY2) q t CY3, and each Y may individually be H or halogen.

[0177] In various embodiments, R of formula (II*) 4 is hydrogen, -OR 5 , -NR 5 R 6 , a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen. In one embodiment, R 4 may be hydrogen. In one embodiment, R 4 may be -OR 5 such as -OCH3. In one embodiment, R 4 is -NR 5 R 6 , wherein R 5 and R 6 may each individually be a substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 may together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl. In one embodiment, R 4 may be a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 4 may be methyl, -CH2OH, or CH2CH2OH. As another example, in one embodiment, R 4 ​​can be -(CH2)3OH or CH2O(CH2)2OH. In one embodiment, R 4 is -[(CY2) p O(CY2) q t CY3, and each Y can individually be H or halogen.

[0178] In some embodiments, one of R 3 and R 4 in formula (II) or formula (II*) can be hydrogen, and the other of R 3 and R 4 can be substituted or unsubstituted C1-C6 alkyl. In other embodiments, one of R 3 and R 4 can be hydrogen, and the other of R 3 and R 4 can be -NR 5 R 6 In some embodiments, one of R 3 and R 4 can be -CH3, provided that both of R 3 and R 4 cannot be -CH3. For example, in some embodiments, one of R 3 and R 4 can be -CH3, and the other of R 3 and R 4 can be substituted or unsubstituted C1-C6 alkyl that cannot be -CH3, such as -CH2OH or CH2CH2OH. In some embodiments, one of R 3 and R 4 can be CH3, and the other of R 3 and R 4 can be -NR 5 R 6 In some embodiments, R

[0179] and / or R 3 in formula (II) or formula (II*) can be -NR 4 R 5 R 6 described herein. For example, in one embodiment, R 5 and R 6 ​is, individually, a substituted or unsubstituted C1-C6 alkyl (e.g., -CH3, -CH2CH2OH, or

[0180]

Chemical formula

[0181]

Chemical formula

[0182]

Chemical formula

[0183] In some embodiments, R 3 and / or R 4 of formula (II) or formula (II*) can be -[(CY2) p O(CY2) q t CY3 such as -CH2OCH3. For example, in some embodiments, one of R 3 and R 4 can be -CH2OH or CH2CH2OH, and the other of R 3 and R 4 can be -CH2OCH3.

[0184] In some embodiments, R 3 and R 4 ​can, together with the carbon atom to which they are attached, form a 4-, 5- or 6-membered heterocyclyl optionally substituted with an unsubstituted 4-, 5- or 6-membered heterocyclyl or an optionally substituted C1-C3 alkyl. For example, in some embodiments, R 3 and R 4 can, together with the carbon atom to which they are attached,

[0185]

Chemical formula

[0186]

Chemical formula

[0187] In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be hydrogen, and the other of R 3 and R 4 can be -CH2OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -CH3, and the other of R 3 and R 4 can be -CH2OH. In one embodiment, one of R 3 and R 4 in formula (IV) or formula (IV*) can be -OCH3, and the other of R 3 and R 4 can be -CH2OH. In one embodiment, one of R 3 and R 4One of them is -C2H5, and R 3 and R 4 The other can be -CH2OH. In one embodiment, R of formula (II) 3 and R 4 One of them can be -CH=CH2, and R 3 and R 4 The other can be -CH2OH. In one embodiment, R of formula (II) 3 and R 4 One of them can be -C≡CH, and R 3 and R 4 The other can be -CH2OH. In one embodiment, R of formula (II) or formula (II*) 3 and R 4 One of them can be hydrogen, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (II) or formula (II*) 3 and R 4 One of them can be -CH3, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (II) or formula (II*) 3 and R 4 One of them can be -OCH3, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (II) or formula (II*) 3 and R 4 One of them can be -C2H5, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (IV) 3 and R 4 One of them can be -CH=CH2, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (II) 3 and R 4 One of them can be -C≡CH, and R 3 and R 4 The other can be -(CH2)2OH. In one embodiment, R of formula (II) or formula (II*) 3 and R 4One of them can be hydrogen, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -CH3, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -OCH3, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -C2H5, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, one of R 3 and R 4 in formula (II) can be -CH=CH2, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, one of R 3 and R 4 in formula (II) can be -C≡CH, and R 3 and R 4 The other can be -(CH2)3OH. In one embodiment, R 3 and R 4 Both can be -CH2OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be hydrogen, and R 3 and R 4 The other can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -CH3, and R 3 and R 4 The other can be -CH2O(CH2)2OH. In one embodiment, one of R 3 and R 4 in formula (II) or formula (II*) can be -OCH3, and R 3 and R4 The other is -CH2O(CH2)2OH. In one embodiment, R in formula (II) or formula (II*) 3 and R 4 can be -C2H5 for one of them, and R 3 and R 4 The other is -CH2O(CH2)2OH. In one embodiment, R in formula (II) 3 and R 4 can be -CH=CH2 for one of them, and R 3 and R 4 The other can be -CH2O(CH2)2OH. In one embodiment, R in formula (IV) 3 and R 4 can be -C≡CH for one of them, and R 3 and R 4 The other can be -CH2O(CH2)2OH. In one embodiment, R in formula (II) or formula (II*) 3 and R 4 can be hydrogen or CH3 for one of them, and R 3 and R 4 The other is

[0188]

Chemical formula

[0189]

Chem.

[0190]

Chem.

[0191]

Chem.

[0192] can be substituted or unsubstituted -(C1-C6 alkyl)-X 3 and R 4 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted -(C1-C6 alkenyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 , substituted or unsubstituted -(C1-C6 alkynyl)-X 2 , and substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 , and 2may be selected from. In various embodiments, X 2 is -OR 9 -SR 9 or NHR 9 and R 9 is H, -COR 8 -CO2R 8 -(CO)-NHR 8 L 4 L 5 L 6 or L 7 wherein exactly one of R 7 and R 9 can be L 4 L 5 L 6 or L 7 In such embodiments, the compound of formula (II) has R 3 or R 4 each containing X 2 and R 9 being L 4 L 5 L 6 or L 7 and can be connected via R 3 or R 4 to the linker -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - In one embodiment, R 3 or R 4 can be substituted or unsubstituted -(C1-C6 alkyl)-X 2 In one embodiment, R 3 or R 4 can be substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 In one embodiment, R 3 or R 4 can be substituted or unsubstituted -(C1-C6 alkenyl)-X 2 In one embodiment, R 3 or R 4 can be substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 In one embodiment, R 3 or R4 is a substituted or unsubstituted -(C1-C6 alkynyl)-X 2 and can be. In one embodiment, R 3 or R 4 is a substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 and can be. R 3 or R 4 is X 2 In each such embodiment that contains, R 9 is L 4 L 5 L 6 or L 7 is provided as an option, and thus, the compound of formula (II) is provided with the option of connecting it through R 3 or R 4 to the linker -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -.

[0193] In various embodiments, R 7 of formula (II) or formula (II*) can be H, -COR 8 -CO2R 8 -(CO)-NHR 8 L 4 L 5 L 6 or L 7 and each R 8 can individually be a substituted or unsubstituted C1-C6, alkyl-X 3 a substituted or unsubstituted C1-C6, haloalkyl-X 3 or [(CY2) p O(CY2) q t CY2-X 3 and can be. In one embodiment, R 7 can be H. In one embodiment, R 7 can be -COR 8 . In one embodiment, R 7 can be -CO2R 8 . In one embodiment, R 7 can be -(CO)-NHR​8 It can be. Those skilled in the art will recognize that R 7 can be H, -COR 8 , -CO2R 8 , or (CO)-NHR 8 In the case where it can be, the connection of the compound of formula (II) or formula (II*) to the linker -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - can be via R 3 or R 4 (thus R 9 ), as will be understood.

[0194] In various embodiments, each R of formula (II) or formula (II*) 8 can individually be a substituted or unsubstituted C1-C6 alkyl-X 3 , a substituted or unsubstituted C1-C6 haloalkyl-X 3 , or [(CY2) p O(CY2) q t CY2-X 3 wherein X 3 can be -H, -OH, -SH, or NH2. In one embodiment, each R 8 can individually be a substituted or unsubstituted C1-C6 alkyl-X 3 . In one embodiment, each R 8 can individually be a substituted or unsubstituted C1-C6 haloalkyl-X 3 . In one embodiment, each R 8 can individually be -[(CY2) p O(CY2) q t CY2-X 3 .

[0195] In various embodiments, X of formula (II) or formula (II*) 2 can be -OR 9 , -SR 9 , or NHR 9 , where R 9 is H, -COR 8 ​​, -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 may be. In one embodiment, X 2 may be -OR 9 In one embodiment, X 2 may be -SR 9 In one embodiment, X 2 may be -NHR 9 may be.

[0196] In various embodiments, R of formula (II) or formula (II*) 9 may be H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 may be, wherein R 8 may be substituted or unsubstituted C1-C6 alkyl-X 3 , substituted or unsubstituted C1-C6 haloalkyl-X 3 , or -[(CY2) p O(CY2) q t CY2-X 3 may be. In one embodiment, R 9 may be H. In one embodiment, R 9 may be -COR 8 In one embodiment, R 9 may be -CO2R 8 In one embodiment, R 9 may be -(CO)-NHR 8 may be. Those skilled in the art will appreciate that when R 9 may be H, -COR 8 , -CO2R 8 , or (CO)-NHR 8 may be, the linker -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 ​- The connection of the compound of formula (II) thereto can be via R as described elsewhere in this specification. 7 It will be understood that this can be done.

[0197] In various embodiments, R of formula (II) or formula (II*) 9 can be L 4 , L 5 , L 6 or L 7 . In one embodiment, R 9 can be L 4 . In one embodiment, R 9 can be L 5 . In one embodiment, R 9 can be L 6 . In one embodiment, R 9 can be L 7 . One of ordinary skill in the art will appreciate that when R 9 is L 4 , L 5 , L 6 or L 7 , the connection of the compound of formula (II) or formula (II*) to linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 can be via R 3 or R 4 as described elsewhere in this specification. In one embodiment, exactly one of R 7 and R 9 is L 4 , L 5 , L 6 or L 7 , in which case the covalent bond can link the drug D to linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - and thus to Mi.

[0198] In various embodiments, each X of formula (II) or formula (II*) 3can each independently be -H, -OH, -SH, or NH2. In one embodiment, X 3 can be -H. In one embodiment, X 3 can be -OH. In one embodiment, X 3 can be -SH. In one embodiment, X 3 can be -NH2.

[0199] In various embodiments, m of formula (II) or formula (II*) can be 1 or 2. In one embodiment, m can be 1. In another embodiment, m can be 2.

[0200] In various embodiments, n 4 and n 5 of formula (II) or formula (II*) can each independently be 0, 1, or 2, provided that both n 4 and n 5 cannot both be 0. In one embodiment, both n 4 and n 5 can be 1. In one embodiment, n 4 can be 0 and n 5 can be 1. In one embodiment, n 4 can be 0 and n 5 can be 2. In one embodiment, n 4 can be 1 and n 5 can be 0. In one embodiment, n 4 can be 2 and n 5 can be 0.

[0201] In various embodiments, each Y of formula (II) or formula (II*) can each independently be H or halogen. In one embodiment, each Y can be hydrogen. In one embodiment, -CY2 can be -CH2. In one embodiment, -CY3 can be -CH3. In one embodiment, -CY3 can be -CHF2. In one embodiment, -CY3 can be -CH2F. In one embodiment, -CY3 can be -CF3.

[0202] In various embodiments, each p of formula (II) or formula (II*) can individually be 1, 2, 3, 4, 5, or 6. In one embodiment, p can be 1. In one embodiment, p can be 2.

[0203] In various embodiments, each q of formula (II) or formula (II*) can individually be 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q can be 1. In one embodiment, q can be 2.

[0204] In various embodiments, each t of formula (II) or formula (II*) can individually be 1, 2, 3, 4, 5, or 6. In one embodiment, t can be 1. In one embodiment, p can be t.

[0205] In various embodiments, R 1 and R 2 can each individually be hydrogen, halogen, and unsubstituted C1-C6 alkyl, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, such that R 1 and R 2 can together form a ring, and R 3 and R 4 can each individually be hydrogen, -OR 5 , -NR 5 R 6 , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 cannot be hydrogen, or R 3 and R 4 can together with the carbon atom to which they are attached form an unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl substituted with unsubstituted C1-C3 alkyl, or one of R 3 and R 4 is unsubstituted -(C1-C6 alkyl)-X​2 or unsubstituted -(C1-C6 alkenyl)-X 2 may be, and X 2 is -OR 9 may be, and R 5 and R 6 may each individually be unsubstituted C1-C6 alkyl, or R 5 and R 6 may together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl, and n 4 may be 2, and n 5 may be 0, each Y may be H, each m may be 1, p may be 1, q may be 2, t may be 1, and R 7 may be H, and R 9 may be H, provided that 7 and R 9 exactly one of 4 L 5 L 6 L 7 or L 1 is. In various embodiments, R 2 and R 3 may each individually be selected from the group consisting of hydrogen, halogen, and unsubstituted C1-C6 alkyl, and R 4 and R 5 may each individually be selected from the group consisting of hydrogen, -OR 5 R 6 substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and [(CY2) p O(CY2) q t CY3, provided that both R 3 and R 4 are not hydrogen, or R 3 and R 4 may together with the carbon atom to which they are attached form an unsubstituted 4-, 5- or 6-membered heterocyclyl, or a 4-, 5- or 6-membered heterocyclyl substituted with unsubstituted C1-C3 alkyl, or one of R 3 and R 4 is unsubstituted -(C1-C6 alkyl)-X 2 ​or unsubstituted -(C1-C6 alkenyl)-X 2 may be, and X 2 is -OR 9 may be, R 5 and R 6 may each independently be unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached may form a substituted or unsubstituted 4- or 5-membered heterocyclyl, n 4 may be 2, n 5 may be 0, each Y may be H, each m may be 1, p may be 1, q may be 2, t may be 1, R 7 may be H, R 9 may be H, provided that 7 and R 9 exactly one of 4 L 5 L 6 L 7 or L 2 is present. In some embodiments including those of this paragraph, L 3 need not be present, L 1 may be -(CH2)n 1 -C(=O)-, n 4 may be a tetrapeptide residue, L 5 may be -[NH(CH2)n 2 n 3 -, n 2 may be 1, n 3 may be 1, L 6 need not be present, L 7 need not be present, and n may be 1. In other embodiments including those of this paragraph, L 2 is

[0206]

Chemical formula

[0207]

Chemical formula

[0208] In some embodiments, a portion of the drug moiety can be attached to L 4 , L 5 , L 6 , or L 7 as described herein. By way of example, the portion of the drug moiety attached to L 4 , L 5 , L 6 , or L 7 can be R 3 or R 4 as described herein. In some embodiments, L 4 , L 5 , L 6or L 7 The moiety of the drug moiety connected to may be -CH2-O- or CH2CH2-O-. In other embodiments, L 4 L 5 L 6 or L 7 The moiety of the drug moiety connected to may be a monocyclic amine having an -O- substitution, wherein the -O- may be connected directly to the cyclic amine or via an alkylene. Examples of monocyclic amines having a hydroxy directly connected to the cyclic amine include

[0209]

Chemical formula

[0210]

Chemical formula

[0211]

Chemical formula

[0212]

Chemical formula

[0213]

Chem.

[0214]

Chem.

[0215]

Chem.

[0216]

Chem.

[0217] Immunoconjugate (antibody-drug conjugate) Various embodiments disclosed herein relate to immunoconjugates of formula (I) having the following structure. Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I)

[0218] In various embodiments, L of formula (III) 1 is

[0219]

Chemical formula

[0220] In various embodiments, L of formula (III) 2 may not be present,

[0221]

Chemical formula

[0222]

Chemical formula

[0223]

Chemical formula

[0224]

Chemical formula

[0225] In various embodiments, Z of formula (III) 1 and Z 2 may each independently be hydrogen, halogen, NO2, -O-(C1-C6 alkyl), or C1-C6 alkyl. In one embodiment, at least one of Z 1 and Z 2 may be hydrogen. In one embodiment, at least one of Z 1 and Z 2 may be halogen. In one embodiment, at least one of Z 1 and Z 2 may be NO2. In one embodiment, at least one of Z 1 and Z 2 may be -O-(C1-C6 alkyl). For example, in one embodiment, at least one of Z 1 and Z 2 may be methoxy. In one embodiment, at least one of Z 1 and Z 2 may be C1-C6 alkyl. For example, in one embodiment, at least one of Z 1 and Z 2 may be methyl.

[0226] In various embodiments, L of formula (III)3 is -(CH2)n 1 -C(=O)- or (CH2CH2O)n 1 -(CH2)n 1 C(=O)- and in the formula, each n 1 can independently be an integer from 0 to 12. In one embodiment, L 3 can be -(CH2)n 1 -C(=O)-. For example, in one embodiment, L 3 can be -C(=O)-. In one embodiment, L 3 can be -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-. For example, in one embodiment, L 3 can be -CH2C(=O)-. In an embodiment, n 1 can be an integer from 1 to 12 such as 1 to 6 or 1 to 3.

[0227] In various embodiments, L of formula (III) 4 can be a tetrapeptide residue. For example, in one embodiment, L 4 can be a tetrapeptide residue selected from SEQ ID NO: 43 GGFG (gly - gly - phe - gly), SEQ ID NO: 44 EGGF (glu - gly - gly - phe), SEQ ID NO: 45 SGGF (ser - gly - gly - phe), and SEQ ID NO: 46 KGGF (lys - gly - gly - phe).

[0228] In various embodiments, L of formula (III) 5 can be absent or can be [NH(CH2)n 2 n 3 -, in the formula, n 2 can be an integer from 0 to 6 and n 3 can be an integer from 0 to 2. In one embodiment, L 5 may be absent. In one embodiment, L 5 can be -[NH(CH2)n 2 n 3 -. For example, in one embodiment, L 5 can be -NH-. In another embodiment, L 5can be -NHCH2-.

[0229] In various embodiments, L of formula (III) 6 may not be present, or

[0230]

Chemical formula

[0231]

Chemical formula

[0232] In various embodiments, L of formula (III) 7 may not be present,

[0233]

Chemical formula

[0234]

Chemical formula

[0235]

Chemical formula

[0236]

Chemical formula

[0237]

Chemical formula

[0238] In various embodiments, D in the immunoconjugate of formula (I) can be a drug moiety described herein (e.g., under the heading "Drug Moiety" above or under the heading "Compound" above). In various embodiments, D in the conjugate of formula (III) can be a compound of formula (II). In various embodiments, D in the conjugate of formula (III) can be a compound of formula (II*). In various embodiments, D in the conjugate of formula (III) can be a compound of formula (IV). In various embodiments, D in the conjugate of formula (III) can be a compound of formula (IV*). In one embodiment, D can be a cytotoxic anti-cancer drug moiety. In one embodiment, the drug moiety can be exatecan.

[0239] In various embodiments, Ab in the immunoconjugate of formula (I) can be an antibody or an antigen-binding fragment thereof. In one embodiment, Ab can specifically bind to human receptor tyrosine kinase-like orphan receptor 1 (ROR1), Her2, TROP2, or Her3. In one embodiment, Ab can bind to the cancer cell surface. In one embodiment, Ab can be an anti-HER2 antibody or an antigen-binding fragment thereof.

[0240] In various embodiments, the immunoconjugate of formula (I) can be selected from the following.

[0241]

Chemical formula

[0242]

Chemical formula

[0243] In various embodiments, the immunoconjugate of formula (I) can be selected from the following.

[0244]

Chemical formula

[0245]

Chemical formula

[0246]

Chemical formula

[0247]

Chemical formula

[0248] In various embodiments, the immunoconjugate of formula (I) can be selected from the following.

[0249]

Chemical formula

[0250]

Chemical formula

[0251]

Chemical formula

[0252] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) is an antibody or an antibody-binding fragment thereof, and the antibody or the antibody-binding fragment thereof (i) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 or (ii) comprises VHCDR1 of the amino acid sequence of SEQ ID NO: 1, (iii) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 or (iv) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (v) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3 or (vi) a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and b) a light chain, (vii) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8 or (viii) a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 8, (ix) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of AAS or (x) a VLCDR2 comprising the amino acid sequence of AAS, and (xi) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 or (xii) a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 10, and The antibody or an antibody binding fragment thereof may be an antibody or an antibody binding fragment thereof that specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0253] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) may be (a) an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NO: 5, 7, 12, or 14 or (b) an antibody or an antibody binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 5, 7, 12, or 14.

[0254] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) may be (a) an amino acid sequence having at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NO: 5, 7, 12, or 14 or (b) an antibody or an antibody binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 5, 7, 12, or 14.

[0255] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) can be (a) any one amino acid sequence of SEQ ID NO: 5, 7, 12, or 14 or (b) an antibody or an antibody binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 5, 7, 12, or 14.

[0256] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) is an antibody or an antibody binding fragment thereof, wherein the antibody or the antibody binding fragment thereof (i) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or (ii) VHCDR1 comprising the amino acid sequence of SEQ ID NO: 15, (iii) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 or (iv) VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and (v) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17 or (vi) VHCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and b) a light chain, wherein (vii) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22 or (viii) VLCDR1 comprising the amino acid sequence of SEQ ID NO: 22, (ix) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAY or (x) VLCDR2 comprising the amino acid sequence of DAY, and (xi) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24 or (xii) VLCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and including a light chain, the antibody or the antigen-binding fragment thereof can be an antibody or an antibody binding fragment thereof that specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0257] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) can be (a) an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NO: 19, 21, 26, or 28 or (b) an antibody or an antibody-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 19, 21, 26, or 28.

[0258] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) can be (a) an amino acid sequence having at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NO: 19, 21, 26, or 28 or (b) an antibody or an antibody-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 19, 21, 26, or 28.

[0259] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) can be (a) any one of the amino acid sequences of SEQ ID NO: 19, 21, 26, or 28 or (b) an antibody or an antibody-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 19, 21, 26, or 28.

[0260] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) is an antibody or an antibody-binding fragment thereof, wherein the antibody or the antibody-binding fragment thereof (i) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29 or (ii) comprises VHCDR1 comprising the amino acid sequence of SEQ ID NO: 29, (iii) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 30 or (iv) comprises VHCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and (v) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31 or (vi) comprises VHCDR3 comprising the amino acid sequence of SEQ ID NO: 31, and b) a light chain, (vii) has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36 or (viii) comprises VLCDR1 comprising the amino acid sequence of SEQ ID NO: 36, (ix) An amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAS or (x) VLCDR2 containing the amino acid sequence of DAS, and (xi) An amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38 or (xii) a light chain comprising VLCDR3 containing the amino acid sequence of SEQ ID NO: 38, The antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0261] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) may be (a) an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 33, 35, 40, or 42 or (b) an antibody or antibody-binding fragment thereof containing the amino acid sequence of SEQ ID NOs: 33, 35, 40, or 42.

[0262] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) may be (a) an amino acid sequence having at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 33, 35, 40, or 42 or (b) an antibody or antibody-binding fragment thereof containing the amino acid sequence of SEQ ID NOs: 33, 35, 40, or 42.

[0263] In one embodiment, any Ab of the aforementioned immunoconjugates of formula (I) may be (a) any one of the amino acid sequences of SEQ ID NOs: 33, 35, 40, or 42 or (b) an antibody or antibody-binding fragment thereof containing the amino acid sequence of SEQ ID NOs: 33, 35, 40, or 42.

[0264] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a closely associated non-covalent association. In this arrangement, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. Even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen) can have the ability to recognize and bind the antigen, although in some cases with a lower affinity than the entire binding site. Similarly, as used herein with respect to the use of the term CDR, in some alternative forms, any one or more of the CDRs can differ from the CDR sequences recited in the sequence listing by one amino acid, for example, a conservative amino acid substitution.

[0265] Pharmaceutical composition Some embodiments described herein relate to pharmaceutical compositions that can include an effective amount of one or more compounds described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0266] The term "pharmaceutical composition" refers to a mixture of one or more compounds and / or salts disclosed herein and other chemical components such as diluents or carriers. Pharmaceutical compositions facilitate the administration of the compounds to a living being. Pharmaceutical compositions can also be obtained by reacting the compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutical compositions are usually tailored to a particular intended route of administration.

[0267] The term "physiologically acceptable" defines a carrier, diluent, or excipient that does not inhibit the biological activity and properties of a compound and does not cause appreciable harm or impairment to the animal to which the composition is intended for delivery.

[0268] As used herein, "carrier" refers to a compound that facilitates the uptake of a compound into a cell or tissue. For example, but not limited to, dimethyl sulfoxide (DMSO) is a commonly utilized carrier that facilitates the uptake of many organic compounds into the cells or tissues of interest.

[0269] As used herein, "diluent" refers to a component in a pharmaceutical composition that lacks significant pharmacological activity but is necessary or desirable in a pharmaceutical composition. For example, a diluent can be used to increase the volume of a potent drug whose mass is too small for manufacture and / or administration. It can also be a liquid for dissolving a drug administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution such as phosphate-buffered saline that mimics the pH and isotonicity of human blood.

[0270] As used herein, "excipient" refers to an essentially inert substance that is added to a pharmaceutical composition to provide, among other things, volume, consistency, stability, binding ability, lubrication, disintegration ability, etc. to the composition. For example, stabilizers such as antioxidants and metal chelating agents are excipients. In one embodiment, the pharmaceutical composition includes an antioxidant and / or a metal chelating agent. A "diluent" is a type of excipient.

[0271] The pharmaceutical compositions described herein can be administered to human patients as a pharmaceutical composition by itself or mixed with other active ingredients, or carriers, diluents, excipients, or combinations thereof, as in a combination therapy. Suitable formulations depend on the route of administration selected. Techniques for formulating and administering the compounds described herein are known to those of ordinary skill in the art.

[0272] The pharmaceutical compositions disclosed herein can be manufactured in a manner known per se, for example, by conventional mixing, dissolving, granulating, sugar coating, pulverizing, emulsifying, encapsulating, entrapping, or tablet-making processes. Further, the active ingredient is contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein can be provided as salts with pharmaceutically compatible counterions.

[0273] A plurality of administration techniques for compounds, salts, and / or compositions exist in the art, including, but not limited to, oral, rectal, pulmonary, topical, aerosol, injection, infusion, and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In some embodiments, the compounds of formula (I) or pharmaceutically acceptable salts thereof can be administered orally.

[0274] Compounds, salts, and / or compositions can also be administered more locally than systemically, for example, often as depot or sustained release formulations, by directly injecting or implanting the compound at the site of the disease. Further, in targeted drug delivery systems, for example, the compound can be administered in liposomes coated with a targeting ligand for a particular cell or tissue type. The liposomes target the target cell or tissue and are selectively taken up by the target cell or tissue.

[0275] The composition may be provided in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may comprise a metal or plastic foil such as, for example, a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the form of the drug for human or veterinary administration. Such notice may be, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. A composition which may contain the compounds and / or salts described herein formulated in a pharmaceutically acceptable carrier may also be prepared for the treatment of a disease indicative of a need for treatment, placed in an appropriate container, and labeled.

[0276] Therapeutic Use and Methods Some embodiments described herein relate to methods of treating cancer or tumors described herein that may include administering to a subject having cancer or a tumor a pharmaceutical composition comprising an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) or a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof). Other embodiments described herein relate to the use of a pharmaceutical composition comprising an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) or a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for treating cancer or tumors described herein. Still other embodiments described herein relate to a pharmaceutical composition comprising an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) or a compound described herein (e.g., an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof) for treating cancer or tumors described herein.

[0277] Examples of cancers and tumors include, but are not limited to, lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, or sarcoma.

[0278] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates such as fish, crustaceans, reptiles, etc., particularly mammals. "Mammal" includes, but is not limited to, primates such as mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, monkeys, chimpanzees, and apes, particularly humans. In some embodiments, the subject can be a human. In some embodiments, the subject can be a pediatric and / or infant, e.g., a pediatric or infant having a fever. In other embodiments, the subject can be an adult.

[0279] As used herein, the terms "treat", "treating", "treatment", "therapeutic", and "therapy" do not necessarily mean a complete cure or elimination of a disease or illness. Any alleviation of any undesirable sign or symptom of a disease or illness can be considered treatment and / or therapy to any extent. Further, treatment can include an act that can worsen the overall well-being or appearance of a subject.

[0280] The terms "therapeutically effective amount" and "effective amount" are used to indicate the amount of an active compound or pharmaceutical that elicits the indicated biological or pharmaceutical response. For example, a therapeutically effective amount of a compound, salt, or composition can be the amount necessary to prevent, reduce, or alleviate a disease or disorder or its symptoms, or to extend the survival time of a subject being treated. This response can occur in a tissue, system, animal, or human and includes the alleviation of the signs or symptoms of the disease or disorder being treated. Determination of an effective amount is well within the ability of one of ordinary skill in the art, considering the disclosure provided herein. The therapeutically effective amount of a compound disclosed herein required as a dosage depends on the route of administration, the type of animal being treated, including a human, and the physical characteristics of the particular animal being considered. The dosage can be adjusted to achieve the desired effect but depends on factors such as body weight, diet, co-administered drugs, and other factors recognized by one of ordinary skill in the medical arts.

[0281] For example, an effective amount of a compound is an amount that results in (a) a reduction, alleviation, or disappearance of one or more symptoms caused by cancer, (b) a reduction in tumor size, (c) the elimination of a tumor, and / or (d) the long-term disease stabilization (growth arrest) of a tumor. In the treatment of lung cancer (such as non-small cell lung cancer), a therapeutically effective amount is an amount that reduces or eliminates cough, shortness of breath, and / or pain.

[0282] The amount of an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof required for use in a treatment varies not only with the particular compound or salt selected but also with the route of administration, the nature and / or symptoms of the disease or disorder being treated, and the age and condition of the patient, and ultimately is at the discretion of the attending physician or clinician. In the case of administration of a pharmaceutically acceptable salt, the dosage can be calculated as the free base. As will be understood by one of ordinary skill in the art, in certain circumstances, it may be necessary to administer a compound disclosed herein in amounts exceeding, or far exceeding, the dosage ranges described herein, particularly to effectively and aggressively treat an invasive disease or disorder.

[0283] However, generally, appropriate dosages are, in many cases, in the range of about 0.05 mg / kg to about 10 mg / kg. For example, appropriate dosages can be in the range of about 0.10 mg / kg to about 7.5 mg / kg per day of body weight, such as about 0.15 mg / kg to about 5.0 mg / kg per day of the recipient's body weight, about 0.2 mg / kg to 4.0 mg / kg per day of the recipient's body weight, or any amount in between. The compound can be administered in unit dosage forms containing, for example, from 1 to 500 mg, 10 to 100 mg, 5 to 50 mg, or any amount in between of the active ingredient per unit dosage form.

[0284] Desirable dosages can conveniently be presented as a single dosage, or as divided dosages administered at appropriate intervals, for example, as two, three, four, or more sub-doses per day. The sub-doses themselves can be further divided, for example, into several separate and more widely spaced administrations.

[0285] As will be readily apparent to those skilled in the art, the useful in vivo dosage administered and the particular mode of administration will vary depending on the age, body weight, severity of pain, mammalian species being treated, the particular compound being used, and the particular use for which these compounds are used. The determination of an effective dosage level, i.e., the dosage level necessary to achieve the desired result, can be accomplished by those skilled in the art using routine methods, such as human clinical trials, in vivo studies, and in vitro studies. For example, the useful dosage of an immunoconjugate compound of formula (I), a drug compound of formula (IV) or formula (IV*), or a pharmaceutically acceptable salt thereof can be determined by comparing their in vitro activity and in vivo activity in animal models. Such comparisons can be made by comparison to established drugs such as cisplatin and / or gemcitabine.

[0286] The dosage and interval can be individually adjusted to provide a plasma level of the active moiety sufficient to maintain the modulating effect or the minimum effective concentration (MEC). The MEC varies for each compound and can be estimated from in vivo and / or in vitro data. The dosage required to achieve the MEC depends on the individual characteristics and the route of administration. However, plasma concentrations can be determined using an HPLC assay or a bioassay. The dosing interval can also be determined using the MEC value. The compositions should be administered using regimens that maintain plasma levels above the MEC for between 10% and 90% of the time, preferably between 30% and 90% of the time, and most preferably between 50% and 90% of the time. In the case of topical administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0287] It should be noted that the attending physician knows how and when to terminate, interrupt, or adjust the administration due to toxicity or organ dysfunction. Conversely, the attending physician will also know to adjust the treatment to a higher level if the clinical response is inappropriate (excluding toxicity). The magnitude of the dosage administered in the management of the target disorder varies depending on the severity of the disease or condition being treated and the route of administration. The severity of the disease or condition can be partially evaluated, for example, by standard prognostic assessment methods. Furthermore, the dosage and possibly the dosing frequency also vary according to the age, weight, and response of the individual patient. Programs comparable to those discussed above can be used in veterinary medicine.

[0288] The compounds, salts, and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicity of a particular compound, or a subset of compounds sharing a particular chemical moiety, can be established by determining its in vitro toxicity against mammalian, preferably human cell lines. The results of such studies often predict toxicity in animals such as mammals, or more specifically in humans. Alternatively, the toxicity of a particular compound in an animal model such as a mouse, rat, rabbit, dog, or monkey can be determined using known methods. The efficacy of a particular compound can be established using any of several well-recognized methods such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, one of ordinary skill in the art can be guided by the state of the art for selecting an appropriate model, dosage, route of administration, and / or regimen.

[0289] Synthesis The drug compounds of formula (II), formula (II*), formula (IV), or formula (IV*), or pharmaceutically acceptable salts thereof, can be made in various ways by one of ordinary skill in the art using known techniques guided by the detailed teachings provided herein. For example, in one embodiment, the drug compounds of formula (II), formula (II*), formula (IV), or formula (IV*) are prepared according to the general schemes shown in FIGS. 2-4.

[0290] The conjugates of formula (III) can be made in various ways by one of ordinary skill in the art using known techniques guided by the detailed teachings provided herein. A general scheme for making the conjugates of formula (III) is provided in FIG. 7B, where the variables L 2 , L 3 , L 4 , L 5 , L 6 , and L 7 are as defined herein for formula (III), and R 1 , R 2 , R 3 , R 4 , R 7 , n 4 , and n5 is as defined herein for formula (II), formula (II*), formula (IV), or formula (IV*), A 1 , A 2 , A 3 , A 4 , and R x are defined within FIG. 7B itself.

[0291] The immunoconjugates of formula (I) can be prepared in various ways by those skilled in the art using known techniques guided by the detailed teachings provided herein. For example, in one embodiment, the immunoconjugates of formula (I) are prepared according to the general scheme shown in Figure 7A. Briefly, the antibody is prepared for reduction (forming cysteine residue Cys-SH) by adding Tris buffer at pH 8.5, mM EDTA, and subsequently adding TCEP. After adding DMA and gently mixing with the reduced antibody solution to achieve a final 10% v / v during conjugation, a stock solution containing the toxin-linker, i.e., the compound of formula (III), in DMA is added and gently mixed. The bioconjugation is allowed to proceed overnight at 20 °C for approximately 16 - 20 hours. The process for preparing the immunoconjugates of formula (I) is described in more detail in Example 30. In one embodiment, the process for generating the immunoconjugates described herein comprises reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with the conjugate described herein under reaction conditions effective to form the immunoconjugate. In one embodiment, the process comprises (i) forming a thiol-functionalized antibody or its antigen-binding fragment by reducing the antibody or its antigen-binding fragment, and (ii) adding the conjugate to the thiol-functionalized antibody or its antigen-binding fragment. In one embodiment, step (i) of this process comprises reducing the thiol-functionalized antibody or its antigen-binding fragment at about pH 8.5 in the presence of (2-carboxyethyl)phosphine (TCEP) and ethylenediaminetetraacetic acid (EDTA). In one embodiment, step (ii) of this process comprises incubating at about 20 °C for at least 16 hours by adding the conjugate to the thiol-functionalized antibody or its antigen-binding fragment in the presence of dimethylacetamide (DMA).

Example

[0292] Further embodiments, which are not intended to limit the claims in any way, are disclosed in more detail in the following examples.

[0293] Example 1 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-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 (1-16) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-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 (1-17) (Figure 8)

[0294]

Chemical formula

[0295] 1-Bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2): To a stirred solution of 2-fluoro-1-methyl-4-nitro-benzene (1-1) (1.00 kg, 6.45 mol) in H2SO4 (1.25 L) and heptane (5.00 L), 1-bromopyrrolidine-2,5-dione (2.29 kg, 12.9 mol) was added portionwise at 65 °C over 6 hours. After stirring at 65 °C for 1 hour, this was quenched with 3.1 kg of ice, extracted with ethyl acetate (2 × 650 mL), washed with 10% Na2SO3 (3 × 650 mL), saturated NaHCO3 (2 × 650 mL), dried over Na2SO4, filtered, and concentrated. Standing at room temperature for 16 hours formed a yellow solid, which was filtered, washed with cold heptane (1.20 L), and dried under vacuum to give 1-bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2) (238 g, yield 16%). 1 1H NMR (400 MHz, CDCl3) δ ppm 8.25 (t, J = 1.7 Hz, 1H), 7.87 (dd, J = 8.8, 2.2 Hz, 1H), 2.43 (d, J = 2.4 Hz, 3H).

[0296] 3-Bromo-5-fluoro-4-methyl-aniline (1-3): To a stirred solution of 1-bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2) (100 g, 427 mmol) in ethyl acetate (1.50 L), Pt / C (10 wt%, 10.0 g) was added under an argon atmosphere, purged three times with H2, and stirred at 60 °C for 4 h under H2 (15 psi). After replacing the H2 atmosphere with argon, this was filtered through a pad of celite, the filter cake was washed with ethyl acetate (500 mL), and the combined filtrates were concentrated and dried under vacuum to give 3-bromo-5-fluoro-4-methyl-aniline (1-3) (94.7 g, purity 71%, yield 77%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 6.79 (s, 1H), 6.50 (dd, J = 11.7, 2.1 Hz, 1H), 2.10 (d, J = 2.1 Hz, 3H). 19 F NMR (376 MHz, CD3OD) δ ppm -112.13. LCMS (ESI+) m / z: [[MH]] + , 205.8.

[0297] N-(3-Bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4): To a solution of 3-bromo-5-fluoro-4-methyl-aniline (1-3) (300 g, 1.47 mol) in ethyl acetate (4.5 L) at 15 °C, triethylamine (420, 3.01 mol) and acetic anhydride (179 mL, 1.91 mol) were added. After stirring at 15 °C for 12 h, this was quenched with saturated NH4Cl (2.5 L), extracted with ethyl acetate (3 × 850 mL), the combined organic layers were washed with brine, dried over Na2SO4, concentrated, and the residue was purified by silica gel column chromatography eluting with 15% ethyl acetate in petroleum ether to give N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (200 g, yield 55%). 1 H NMR (400 MHz, CD3OD) δ ppm 7.59 (t, J = 1.5 Hz, 1H), 7.41 (dd, J = 11.6, 2.0 Hz, 1H), 2.26 (d, J = 2.2 Hz, 3H), 2.11 (s, 3H). 1919F NMR (376 MHz, CD3OD) δ ppm -112.95. LCMS (ESI+) m / z: [M+H] + 247.8

[0298] (E)-4-(5-Acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (1-5): To a stirred solution of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (200 g, 813 mmol) in tetrahydrofuran (1.00 L) and water (200 mL) were added diisopropylethylamine (566 mL, 3.25 mol), tris-o-tolylphosphane (49.5 g, 163 mmol), but-3-enoic acid (168 g, 1.95 mol), and Pd(OAc)2 (18.2 g, 81.3 mmol) under N2. After stirring at 75 °C for 16 h, this was diluted with water (350 mL), the pH was adjusted to 2 with 3N HCl, and filtered through a pad of celite. The mixture was extracted with ethyl acetate (650 mL) and water (650 mL), and after separation, the aqueous layer was extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 70% ethyl acetate in petroleum ether to give (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (1-5) (100 g, 49% yield). 1 1H NMR (400 MHz, DMSO-d6) δ ppm 12.32 (brs, 1H), 10.02 (s, 1H), 7.48 (ddd, J = 12.0, 8.3, 1.6 Hz, 1H), 7.39 (s, 1H), 7.08 - 6.98 (m, 1H), 6.94 - 6.83 (m, 1H), 6.75 - 6.50 (m, 1H), 6.13 (td, J = 15.7, 7.2 Hz, 1H), 5.69 (d, J = 15.5 Hz, 1H), 3.59 (ddd, J = 6.5, 4.1, 2.5 Hz, 1H), 3.56 - 3.46 (m, 1H), 3.31 - 3.21 (m, 1H), 2.16 - 2.06 (m, 3H), 2.05 - 1.99 (m, 3H). 1919F NMR (376 MHz, DMSO-d6) δ ppm -115.38. LCMS (ESI+) m / z: [M+H] + 252.0

[0299] 4-(5-Acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (1-6): To a stirred solution of (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (1-5) (100 g, 398 mmol) in methanol (1.5 L) was added Pd / C (30.0 g, 39.8 mmol, 10 wt%, 0.10 equiv) under an argon atmosphere. The mixture was purged with H2 three times and stirred at 35 °C for 12 h under H2 (15 psi). After replacing the H2 atmosphere with argon, the mixture was filtered through a pad of celite, and the filter cake was washed with methanol (2 L). The combined filtrates were concentrated and dried under vacuum to give 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (1-6) (82.5 g, 82% yield), which was used in the next step without further purification. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 9.98 (s, 1H), 7.42 (dd, J = 12.2, 1.7 Hz, 1H), 7.04 (s, 1H), 2.59 - 2.53 (m, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.09 (d, J = 1.8 Hz, 3H), 2.01 (s, 3H), 1.73 - 1.67 (m, 2H). 19 19F NMR (376 MHz, DMSO-d6) δ ppm -115.64. LCMS (ESI+) m / z: [M+H] + 254.0

[0300] N-(3-Fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7): To a stirred solution of 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (1-6) (110 g, 434 mmol) in trifluoroacetic acid (330 mL) at 0 °C was added trifluoroacetic anhydride (121 mL, 869 mmol). After stirring at 15 °C for 15 h, the reaction mixture was poured into a 50% aqueous acetonitrile solution (2 L) at 0 °C and stirred at 0 °C for 0.5 h. The resulting yellow suspension was adjusted to pH 7 with 25% NaOH at 0 °C, and the resulting solid was filtered, washed with water (350 mL) and methyl tert-butyl ether (700 mL), dried, triturated with methyl tert-butyl ether (200 mL), filtered, and dried to give N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7) (89.9 g, 88% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.18 (s, 1H), 8.28 (d, J = 13.2 Hz, 1H), 2.89 (t, J = 6.1 Hz, 2H), 2.68 - 2.60 (m, 2H), 2.18 - 2.08 (m, 6H), 1.99 (quin, J = 6.4 Hz, 2H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -103.89. LCMS (ESI+) m / z: [M+H] + 236.0。

[0301] N-(7-((Dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8): A stirred solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7) (100 mg, 0.426 mmol) in 1,1-dimethoxy-N,N-dimethylmethanamine (1 mL) was heated at 110 °C for 5 h, then cooled to 15 °C, concentrated, and the residue was purified by silica gel column chromatography eluting with 70% ethyl acetate in petroleum ether to give N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8) (96 mg, 77% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 12.88 (brs, 1H), 8.35 (d, J = 13.0 Hz, 1H), 7.73 (s, 1H), 3.17 (s, 6H), 2.75 - 2.84 (m, 4H), 2.20 (s, 3H), 2.16 (d, J = 1.8 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ ppm -106.40. LCMS (ESI+) m / z: [MH] + 264.1. Note: 1-8 was converted to 1-9 under LCMS conditions.

[0302] N-(3-Fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9): To a stirred solution of N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (1-8) (49.0 mg, 0.168 mmol) in dichloromethane (1 mL) at 15 °C was added 1 N HCl (1 mL). After stirring at 15 °C for 15 h, it was extracted with dichloromethane (3 × 2 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and dried under vacuum to give N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9) (30.0 mg, 67% yield). 11H NMR (400 MHz, CDCl3) δ ppm 13.99 (d, J = 11.1 Hz, 1H), 11.85 (s, 1H), 8.40 (d, J = 12.8 Hz, 1H), 7.45 (d, J = 11.1 Hz, 1H), 2.81 - 2.87 (m, 2H), 2.48 (t, J = 6.8 Hz, 2H), 2.24 (s, 3H), 2.18 (d, J = 1.8 Hz, 3H). 19 19F NMR (376 MHz, CDCl3) δ ppm -101.81. LCMS (ESI+) m / z: [MH] + 264.1.

[0303] N-(3-Fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-10): To a stirred solution of N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9) (10.0 g, 38.0 mmol) in dichloroethane (100 mL) and acetic acid (1 mL) at 20 °C was added sodium triacetoxyborohydride (9.66 g, 45.5 mmol) portionwise. After stirring at 30 °C for 12 h, the reaction was quenched with water (100 mL), extracted with dichloromethane (3 × 150 mL), the combined organic layers were washed with water, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 33% - 66% ethyl acetate in petroleum ether to give N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-10) (7.25 g, 72% yield). 1 1H NMR (400 MHz, CDCl3) δ ppm 12.13 (brs, 1H), 8.44 (d, J = 12.9 Hz, 1H), 3.81 - 3.98 (m, 2H), 3.07 - 3.09 (m, 1H), 2.67 - 2.90 (m, 3H), 2.24 (s, 3H), 2.10 - 2.18 (m, 4H), 1.83 - 1.97 (m, 1H). 19 19F NMR (376 MHz, CDCl3) δ ppm -101.14. LCMS (ESI+) m / z: [MH]+ 266.1。

[0304] N-(7-((allyloxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-11): To a stirred solution of N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-10) (11.8 g, 44.7 mmol) and 3-iodoprop-1-ene (20.3 mL, 222 mmol) in acetonitrile (236 mL) at 25 °C was added Ag2O (30.9 g, 133 mmol). After stirring at 25 °C for 12 h, the mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 7% ethyl acetate in petroleum ether to give N-(7-((allyloxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-11) (3.80 g, yield 28%). 1 1H NMR (400 MHz, CDCl3) δ ppm 12.22 (s, 1H), 8.43 (d, J = 12.9 Hz, 1H), 5.83 - 6.02 (m, 1H), 5.07 - 5.40 (m, 2H), 4.05 (d, J = 5.6 Hz, 2H), 3.88 (dd, J = 9.5, 4.5 Hz, 1H), 3.70 (dd, J = 9.5, 7.1 Hz, 1H), 3.02 - 3.05 (m, 1H), 2.72 - 2.87 (m, 2H), 2.34 - 2.36 (m, 1H), 2.22 (s, 3H), 2.15 (d, J = 1.6 Hz, 3H), 1.89 - 2.04 (m, 1H). 19 19F NMR (376 MHz, CDCl3) δ ppm -101.93. LCMS (ESI+) m / z: [M+H] + 306.2。

[0305] 2-((allyloxy)methyl)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-12): To a stirred solution of N-(7-((allyloxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-11) (3.80 g, 12.5 mmol) in methanol (152 mL) at 20 °C was added 2N HCl (152 mL). After stirring at 60 °C for 2 h, the reaction mixture was cooled to 0 °C, the pH was adjusted to 7 with saturated NaHCO3, and the mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 2% ethyl acetate in petroleum ether to give 2-((allyloxy)methyl)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-12) (2.60 g, 79% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 6.49 (brs, 2H), 6.19 (d, J = 11.6 Hz, 1H), 5.94 (m, 1H), 5.30 (dq, J = 17.2, 1.6 Hz, 1H), 5.20 (dq, J = 10.4, 1.3 Hz, 1H), 3.98 - 4.10 (m, 2H), 3.92 (dd, J = 9.5, 4.4 Hz, 1H), 3.67 (dd, J = 9.5, 7.8 Hz, 1H), 2.98 (dt, J = 17.3, 4.6 Hz, 1H), 2.65 - 2.80 (m, 2H), 2.27 - 2.37 (m, 1H), 2.06 (d, J = 1.6 Hz, 3H), 1.92 - 1.86 (m, 1H). 19 F NMR (376 MHz, CDCl3) δ ppm -106.56. LCMS (ESI+) m / z: [MH] + 264.2.

[0306] (9S)-1-((allyloxy)methyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-14): 2-((allyloxy)methyl)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-12) (100 mg, 0.380 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (100 mg, 0.380 mmol), and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinan 2,4,6-trioxide (1.13 mL, 1.90 mmol, 50% wt in ethyl acetate) were heated at 60 °C for 10 h. Nineteen additional reactions were set up as above, and once cooled to room temperature, all 20 reaction mixtures were combined for workup and purification. The combined reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (4 × 200 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 5% - 80% ethyl acetate in petroleum ether to afford (9S)-1-((allyloxy)methyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-14) (310 mg, 5.4% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 7.71 (brd, J = 10.6 Hz, 1H), 7.62 (s, 1H), 5.63 - 6.04 (m, 2H), 4.99 - 5.57 (m, 5H), 3.94 (brd, J = 5.4 Hz, 2H), 3.66 - 3.79 (m, 2H), 3.47 - 3.64 (m, 2H), 3.14 (brd, J = 3.1 Hz, 1H), 2.91 - 3.07 (m, 1H), 2.28 - 2.54 (m, 4H), 2.03 - 2.14 (m, 1H), 1.83 - 1.97 (m, 2H), 1.00 - 1.09 (m, 3H). 19 F NMR (376 MHz, CDCl3) δ ppm -110.59. LCMS (ESI+) m / z: [MH] + 491.0.

[0307] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-15): To a stirred solution of (9S)-1-((allyloxy)methyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-14) (360 mg, 0.734 mmol) in tetrahydrofuran (18 mL) at 25 °C was added zinc(II) chloride (130 mg, 0.954 mmol), and the mixture was stirred at 25 °C for 0.25 h. Subsequently, tetrakis(triphenylphosphine)palladium (212 mg, 0.183 mmol) was added, and the mixture was stirred for 0.25 h. Subsequently, tributylstannane (3.88 mL, 14.7 mmol) was added. After stirring at 50 °C for 1.5 h, the reaction mixture was cooled to 20 °C, quenched with water (30 mL), extracted with ethyl acetate (4 × 30 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and dried under vacuum to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-15) (110 mg, yield 33%). LCMS (ESI+) m / z: + 451.1。

[0308] (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-16) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-17): (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-15) (110 mg, 0.244 mmol) was subjected to SFC (equipment: Waters SFC80 preparative SFC, column: Phenomenex-Cellulose-2 (250 mm * × 30 mm, 10 um), mobile phase: A was CO2 and B was EtOH, gradient: B% = 55% isocratic elution mode, flow rate: 80 g / min, wavelength: 220 nm, column temperature: 40 °C, system back pressure: 100 bar), to obtain (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-16) (15.0 mg, yield 4.5%) (Compound 1-16 may be the opposite stereoisomer of that shown) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (1-17) (10.1 mg, yield 3.1%) (Compound 1-17 may be the opposite stereoisomer of that shown). Note: The stereochemistry in the F ring is arbitrarily assigned.

[0309] 1-16: 11H NMR (400 MHz, DMSO-D6) δ ppm 7.75 (broad doublet, J = 11.1 Hz, 1H), 7.30 (singlet, 1H), 6.53 (singlet, 1H), 5.27 - 5.52 (multiplet, 4H), 5.02 (broad triplet, J = 5.4 Hz, 1H), 3.67 (triplet, J = 6.1 Hz, 2H), 3.46 (broad doublet, J = 0.9 Hz, 1H), 2.92 - 3.19 (multiplet, 2H), 2.33 - 2.43 (multiplet, 4H), 1.75 - 2.02 (multiplet, 3H), 0.87 (broad triplet, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.87. LCMS (ESI+) m / z: [MH] + 451.1

[0310] 1 - 17: 1 1H NMR (400 MHz, DMSO-D6) δ ppm 7.75 (doublet, J = 11.1 Hz, 1H), 7.30 (singlet, 1H), 6.52 (singlet, 1H), 5.27 - 5.50 (multiplet, 4H), 5.02 (triplet, J = 5.7 Hz, 1H), 3.66 (triplet, J = 6.4 Hz, 2H), 3.46 (broad doublet, J = 3.5 Hz, 1H), 2.94 - 3.18 (multiplet, 2H), 2.37 (singlet, 4H), 1.78 - 1.99 (multiplet, 3H), 0.87 (triplet, J = 7.3 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.86. LCMS (ESI+) m / z: C 25 H 24 F N2O5 + calculated for [MH] + : 451.1, found: 451.1

[0311] Example 2 (S)-9-Ethyl-5-fluoro-9-hydroxy-1,1-bis(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (2 - 20) (Figure 9)

[0312]

Chemical Structure

[0313] N-(3-Fluoro-7,7-bis(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-18): To a stirred solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7) (5.00 g, 21.3 mmol) in acetonitrile (80 mL) were added sodium carbonate (451 mg, 4.25 mmol) and aqueous formaldehyde solution (10.4 g, 128 mmol). After stirring at 40 °C for 15 h, the reaction was quenched with water (30 mL), extracted with dichloromethane (3 × 30 mL), the combined organic layers were washed with water, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 5% - 70% ethyl acetate in petroleum ether to give a solid, which was triturated with tert-butyl methyl ether (10 mL) at 20 °C for 30 min to afford N-(3-fluoro-7,7-bis(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-18) (3.00 g, 47% yield). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 11.98 - 12.25 (m, 1H), 8.29 (dd, J = 13.2, 3.6 Hz, 1H), 4.70 (t, J = 5.2 Hz, 1H), 4.54 (brd, J = 7.6 Hz, 1H), 3.65 - 3.81 (m, 2H), 3.40 - 3.56 (m, 2H), 2.93 (brt, J = 5.6 Hz, 2H), 2.05 - 2.19 (m, 8H). 19 19F NMR (400 MHz, DMSO-D6) δ ppm -104.42. LCMS (ESI+) m / z: 296.1.

[0314] 8-Amino-6-fluoro-2,2-bis(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-19): To a stirred solution of N-(3-fluoro-7,7-bis(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-18) (1.00 g, 3.39 mmol) in methanol (40 mL) was added 2N HCl (40 mL). After stirring at 60 °C for 2 h, the reaction mixture was cooled to 0 °C, quenched with ice water (10 mL), adjusted to pH 7 with saturated sodium bicarbonate, extracted with ethyl acetate (3 × 100 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 5% - 33% ethyl acetate in petroleum ether to afford 8-amino-6-fluoro-2,2-bis(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-19) (0.50 g, 58% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 6.21 (d, J = 11.6 Hz, 1H), 3.97 (d, J = 11.2 Hz, 2H), 3.74 (d, J = 11.2 Hz, 2H), 2.86 (t, J = 6.4 Hz, 2H), 2.06 (d, J = 1.6 Hz, 3H), 1.89 (t, J = 6.4 Hz, 2H). 19 F NMR (400 MHz, CDCl3) δ ppm -105.09. LCMS (ESI+) m / z: [M + H] + 254.1.

[0315] (S)-9-Ethyl-5-fluoro-9-hydroxy-1,1-bis(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (2-20): A stirred mixture of 8-amino-6-fluoro-2,2-bis(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-19) (100 mg, 0.394 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (208 mg, 0.790 mmol) in toluene (2 mL) was added with 1-butyl-3-methyl-1H-imidazol-3-ium tetrafluoroborate (2 mL). (One additional reaction was set up as above and, upon cooling to room temperature, the two reaction mixtures were combined.) After stirring at 130 °C for 16 h, this was quenched with water (5 mL), extracted with ethyl acetate (3 × 5 mL), the combined organic layers were dried over Na2SO4, filtered, concentrated, and the residue was purified by preparative TLC (SiO2, ethyl acetate / methanol = 10 / 1) to afford a solid, which was triturated with water (0.5 mL) at 20 °C for 10 min, filtered, and dried under vacuum to give (S)-9-ethyl-5-fluoro-9-hydroxy-1,1-bis(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (2-20) (7.0 mg, yield 3.6%). 1 H NMR (400 MHz, CD3OD) δ ppm 7.55 - 7.78 (m, 2H), 5.53 - 5.69 (m, 3H), 5.40 (d, J = 16.0 Hz, 1H), 4.15 (d, J = 11.6 Hz, 2H), 3.86 (d, J = 11.6 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H), 2.43 (s, 3H), 2.30 (t, J = 6.8 Hz, 2H), 1.97 (dd, J = 7.2, 5.2 Hz, 2H), 1.01 (t, J = 7.2 Hz, 3H). 19 F NMR (400 MHz, CD3OD) δ ppm -113.70. LCMS (ESI+) m / z: [MH] + 481.1.

[0316] Example 3 (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-27) and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-28) (Figure 10)

[0317]

Chemical Structure

[0318] 4-(5-Acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (3-21): To a stirred mixture of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (10.0 g, 40.6 mmol) and 2-methylbut-3-enoic acid (13.0 g, 130 mmol) in tetrahydrofuran (40 mL) and water (10 mL) were added N-ethyl-N,N-diisopropylamine (38.2 mL, 219 mmol), tris-o-tolylphosphane (2.47 g, 8.13 mmol), and diacetoxypalladium (912 mg, 4.06 mmol). After stirring at 75 °C for 5 h, the mixture was cooled to 0 °C, quenched with water (50 mL), adjusted to pH 3 with 3N HCl, filtered through a pad of celite, and the filter cake was washed with ethyl acetate. The combined filtrates were extracted with ethyl acetate (3 × 150 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether to give 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (3-21) (6.00 g, 56% yield). 11H NMR (400 MHz, DMSO-D6) δ ppm 12.38 (s, 1H), 10.02 (d, J = 4.0 Hz, 1H), 7.46 - 7.52 (m, 1H), 6.99 - 7.38 (m, 1H), 6.65 - 6.69 (m, 1H), 6.17 (dd, J = 15.6, 8.0 Hz, 1H), 3.49 (d, J = 7.2 Hz, 1H), 2.11 (dd, J = 13.2, 1.6 Hz, 3H), 2.02 (d, J = 4.8 Hz, 3H), 1.24 - 1.88 (m, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -115.68. LCMS (ESI+) m / z: [M+H] + 266.1.

[0319] 4-(5-Acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (3-22): To a degassed stirred mixture of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (3-21) (5.00 g, 18.8 mmol) in methanol (20 mL), Pd / C (10 wt%) (2.40 g) was added, purged three times with H2, and stirred at 25 °C for 10 h under H2 (15 psi). After replacing the H2 atmosphere with argon, this was filtered through a pad of celite, the filter cake was washed with methanol (300 mL), and the combined filtrates were concentrated and dried under vacuum to give 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (3-22) (4.4 g, 87% yield), which was used directly in the next step. 1 1H NMR (400 MHz, DMSO-D6) δ ppm 10.0 (s, 1H), 7.42 (dd, J = 12.4, 1.6 Hz, 1H), 7.04 (s, 1H), 2.52 - 2.58 (m, 2H), 2.24 (t, J = 6.8 Hz, 2H), 2.08 (d, J = 1.6 Hz, 3H), 2.01 (s, 3H), 1.40 - 1.65 (m, 4H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -125.77. LCMS (ESI+) m / z: [M+H] + 268.1.

[0320] N-(7-Fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (3-23): To a mixture of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (3-22) (5.00 g, 18.7 mmol) in trifluoroacetic acid (10 mL), trifluoroacetic anhydride (5.20 mL, 37.4 mmol) was added at 0 °C. After stirring at 0 °C for 2 h, the reaction mixture was quenched with ice water (100 mL), the pH was adjusted to 7 at 0 °C with 25% aqueous NaOH, extracted with ethyl acetate (3 × 200 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel flash column chromatography eluting with 8% ethyl acetate in petroleum ether to give N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (3-23) (3.50 g, 75% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.13 (s, 1H), 8.28 (d, J = 13.2 Hz, 1H), 2.93 - 3.04 (m, 1H), 2.81 - 2.92 (m, 1H), 2.63 - 2.75 (m, 1H), 2.07 - 2.18 (m, 7H), 1.74 (qd, J = 12.0, 4.8 Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -104.64. LCMS (ESI+) m / z: [MH] + 250.1.

[0321] N-(3-Fluoro-7-(hydroxymethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-24): To a stirred solution of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-23) (2.00 g, 8.04 mmol) in acetonitrile (32 mL) were added sodium carbonate (106 mg, 0.802 mmol) and aqueous formaldehyde solution (723 mg, 24.1 mmol). After stirring at 60 °C for 16 h, the reaction mixture was quenched with water (20 mL), extracted with dichloromethane (3 × 20 mL), the combined organic layers were washed with water, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel eluted with 30% ethyl acetate in petroleum ether to give N-(3-fluoro-7-(hydroxymethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-24) (1.40 g, 59% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.17 (s, 1H), 8.26 - 8.34 (m, 1H), 4.81 (t, J = 5.6 Hz, 1H), 3.69 (dd, J = 10.4, 5.6 Hz, 1H), 2.78 - 3.01 (m, 2H), 2.06 - 2.32 (m, 8H), 1.73 - 1.90 (m, 1H), 1.07 (s, 3H). LCMS (ESI+) m / z: [MH] + 280.1。

[0322] 8-Amino-6-fluoro-2-(hydroxymethyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (3-25): To a stirred solution of N-(3-fluoro-7-(hydroxymethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-24) (1.40 g, 5.00 mmol) in methanol (56 mL) was added HCl / methanol (56 mL, 2 M) at 15 °C. After stirring at 60 °C for 1 h, the reaction mixture was quenched with ice water (10 mL), the pH was adjusted to 7 with saturated sodium bicarbonate at 0 °C, extracted with ethyl acetate (6 × 100 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 15% ethyl acetate in petroleum ether to give 8-amino-6-fluoro-2-(hydroxymethyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (3-25) (1.00 g, 80% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.43 (brs, 2H), 6.34 (d, J = 12.6 Hz, 1H), 4.65 (t, J = 5.6 Hz, 1H), 3.65 (dd, J = 10.4, 5.6 Hz, 1H), 3.25 (dd, J = 10.4, 5.6 Hz, 1H), 2.64 - 2.87 (m, 2H), 2.06 - 2.08 (m, 1H), 1.98 (d, J = 1.2 Hz, 3H), 1.62 - 1.76 (m, 1H), 1.01 (s, 3H). LCMS (ESI+) m / z: [MH] + 238.1。

[0323] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetra-hydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-26): A stirred clear solution of 8-amino-6-fluoro-2-(hydroxymethyl)-2,5-dimethyl-3,4-dihydro-naphthalen-1(2H)-one (3-25) (200 mg, 0.842 mmol) in toluene (10 mL) at 130 °C was treated with (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (332 mg, 1.26 mmol) and 4-methylbenzenesulfonic acid (58.1 mg, 0.337 mmol). (Four additional reactions were set up as above and, after cooling to room temperature, all five reaction mixtures were combined for workup and purification.) After stirring at 130 °C for 12 h, the reaction mixture was concentrated and the residue was purified by silica gel column chromatography eluting with ethyl acetate to afford (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-26) (157 mg, 6.8% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (d, J = 10.8 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 6.46 - 6.56 (m, 1H), 5.35 - 5.58 (m, 4H), 5.04 (brd, J = 6.8 Hz, 1H), 3.69 - 3.89 (m, 1H), 3.60 (brd, J = 8.8 Hz, 1H), 2.96 - 3.16 (m, 2H), 2.26 - 2.41 (m, 3H), 2.17 - 2.19 (m, 1H), 1.87 - 1.92 (m, 3H), 1.32 - 1.48 (m, 3H), 0.87 (td, J = 7.2, 2.0 Hz, 3H). LCMS (ESI+) m / z: [MH] + 465.1。

[0324] (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-27) and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-28): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-26) (157 mg, 0.323 mmol) was dissolved in methanol, and separated by SFC (equipment: Waters SFC150AP preparative SFC. Column: REGIS (s,s) WHELK-O1 (250 mm * × 30 mm, 10 um), mobile phase: A was CO2 and B was EtOH, gradient: B% = 50% isocratic elution mode, flow rate: 70 g / min, wavelength: 220 nm, column temperature: 35 °C, system back pressure: 120 bar) to obtain (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-27) (18.0 mg, yield 12%) (Compound 3-27 may be the opposite stereoisomer of the one shown) and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-28) (15.0 mg, yield 10%) (Compound 3-28 may be the opposite stereoisomer of the one shown). Note: The stereochemistry in the F ring is arbitrarily assigned.

[0325] 3-27: 1 1H NMR (400 MHz, DMSO-D6) δ ppm 7.76 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.34 - 5.63 (m, 4H), 5.03 (t, J = 5.2 Hz, 1H), 3.79 (dd, J = 11.3, 5.6 Hz, 1H), 3.56 - 3.68 (m, 1H), 3.10 (brd, J = 5.6 Hz, 2H), 2.38 (s, 3H), 2.18 - 2.30 (m, 1H), 1.75 - 1.94 (m, 3H), 1.42 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -112.58. LCMS (ESI+) m / z: [M+H] + 465.1.

[0326] 3-28: 1 1H NMR (400 MHz, DMSO-D6) δ ppm 7.76 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.29 - 5.63 (m, 4H), 5.06 (t, J = 5.2 Hz, 1H), 3.81 (brdd, J = 11.2, 5.2 Hz, 1H), 3.61 (brdd, J = 11.2, 5.2 Hz, 1H), 3.03 - 3.18 (m, 2H), 2.38 (s, 3H), 2.25 (m, 1H), 1.78 - 1.94 (m, 3H), 1.40 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -112.57. LCMS (ESI+) m / z: [M+H] + 465.1.

[0327] Example 4 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-38) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-39) (Figure 11)

[0328] [Chemical formula]

[0329] Di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (4-29): To a stirred mixture of N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (3-23) (35.1 g, 140 mmol) in toluene (700 mL) was added sodium bis(trimethylsilyl)amide (309 mL, 1 M) dropwise at 0 °C under nitrogen, cooled to -40 °C, and a solution of di-tert-butyl diazene-1,2-dicarboxylate (42.1 g, 183 mmol) in toluene (350 mL) was added dropwise. After stirring at 25 °C for 4 h, the reaction mixture was cooled to 0 °C, diluted with water (1 L), extracted with ethyl acetate (3 × 500 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 20% ethyl acetate in petroleum ether to give di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (4-29) (41.0 g, 60% yield). 11H NMR (400 MHz, DMSO-D6) δ ppm 11.61 - 11.84 (m, 1H), 8.26 (d, J = 12.8 Hz, 1H), 7.96 - 8.15 (m, 1H), 2.96 - 3.16 (m, 2H), 2.68 - 2.84 (m, 1H), 2.12 (s, 4H), 2.08 (s, 3H), 1.35 - 1.46 (m, 21H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -105.29. LCMS (ESI+) m / z: C 24 H 35 F N3O6 + calculated for [MH] + : 480.2, found: 502 [MS+Na].

[0330] N-(3-Fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-31): To a solution of di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (4-29) (41.0 g, 85.5 mmol) in dichloromethane (820 mL) was added trifluoroacetic acid (410 mL) at 25 °C. The mixture was stirred at 25 °C for 1 h, and then acetone (480 mL) was added. After stirring at 25 °C for 0.5 h, the mixture was concentrated to give N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-31) (18.1 g, 66% yield over two steps). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 11.90 (s, 1H), 8.31 (d, J = 12.8 Hz, 1H), 3.05 - 3.14 (m, 1H), 2.90 - 3.02 (m, 1H), 2.11 - 2.19 (m, 7H), 2.05 - 2.08 (m, 2H), 1.98 (d, J = 2.0 Hz, 6H), 1.34 (s, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -75.04. LCMS (ESI+) m / z: C17 H 23 FN3O2 + [MH] calculated for + : 320.1, measured value: 320.1.

[0331] N-(7-Amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-32): To a mixture of N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-31) (15.1 g, 47.3 mmol) in acetic acid (302 mL) was added zinc powder (40.8 g, 624 mmol) portionwise. After stirring at 20 °C for 2 h, the reaction mixture was filtered, the filtrate was concentrated and dried under vacuum to give N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-32) (23.8 g, crude), which was used directly in the next step without further purification. 1 H NMR (400 MHz, DMSO-D6) δ ppm 11.56 (s, 1H), 8.26 - 8.30 (m, 1H), 3.01 - 3.09 (m, 2H), 2.12 - 2.19 (m, 10H), 1.43 (s, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -73.57. LCMS (ESI+) m / z: C 14 H 18 FN2O2 + [MH] calculated for + : 265.1, measured value: 265.1.

[0332] N,N’-(3-Fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide (4-33): To a stirred mixture of N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-32) (23.8 g, 73.3 mmol) in dichloromethane (414 mL) was added acetic anhydride (8.28 mL, 88.0 mmol) and triethylamine (30.6 mL, 220 mmol). After stirring at 25 °C for 12 h, the reaction mixture was quenched with water (500 mL), extracted with dichloromethane (3 × 200 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 70% ethyl acetate in petroleum ether to afford N,N'-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide (4-33) (7.5 g, 51% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 11.81 (s, 1H), 8.20 - 8.37 (m, 2H), 2.94 - 3.03 (m, 1H), 2.77 - 2.88 (m, 1H), 2.65 (m, 1H), 2.06 - 2.18 (m, 6H), 1.85 (m, 1H), 1.79 (s, 3H), 1.32 (s, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -105.03.

[0333] N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4-34): To a stirred mixture of N,N'-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (4-33) (7.50 g, 24.4 mmol) in methanol (105 mL) was added HCl / MeOH (105 mL, 4 M). After stirring at 25 °C for 2 h, the reaction mixture was concentrated, diluted with dichloromethane (300 mL), extracted with saturated aqueous NaHCO3 (2 × 200 mL), washed with brine, dried over Na2SO4, filtered, concentrated, and dried under vacuum to give N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (4-34) (5.50 g, 89% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.91 (s, 1H), 7.39 (s, 2H), 6.36 (d, J = 12.4 Hz, 1H), 2.78 - 2.89 (m, 1H), 2.67 - 2.75 (m, 2H), 1.97 (d, J = 1.20 Hz, 3H), 1.83 - 1.88 (m, 1H), 1.80 (s, 3H), 1.27 (s, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -108.38. LCMS (ESI+) m / z: C 14 H 18 FN2O2 + calculated for [MH] + : 265.1, found: 265.1.

[0334] N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4-35): To a stirred mixture of N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (500 mg, 1.89 mmol) (4-34) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (547 mg, 2.08 mmol) in toluene (25 mL) at 120 °C were added pyridine 4-methylbenzenesulfonate (71.3 mg, 0.283 mmol) and o-cresol (1.44 mL, 13.8 mmol) under argon. After removing the water formed by stirring at 130 °C for 13 h using a Dean-Stark trap, the mixture was concentrated and the residue was purified by silica gel column chromatography eluting with 7% methanol in dichloromethane to give N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4-35) (362 mg, 39% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.78 (d, J = 11.2 Hz, 1H), 7.30 (s, 1H), 6.53 (d, J = 10.0 Hz, 1H), 5.25 - 5.54 (m, 4H), 4.81 - 4.90 (m, 1H), 3.24 - 3.29 (m, 1H), 2.86 - 3.11 (m, 3H), 2.39 (s, 3H), 1.95 (d, J = 3.6 Hz, 3H), 1.84 - 1.89 (m, 2H), 1.50 (d, J = 4.8 Hz, 3H), 0.87 (d, J = 5.2 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.94. LCMS (ESI+) m / z: C 27 H 27 FN3O5 + calculated for [MH] + : 492.1, found: 492.2.

[0335] (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (4-36) and (1R,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (4-37): A stirred mixture of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4-35) (600 mg, 1.22 mmol) in dioxane (6 mL) and concentrated hydrochloric acid (6 mL, 12 M) was heated at 100 °C for 24 h in a sealed tube under argon. The reaction mixture was concentrated and purified by preparative HPLC to give (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (4-36) (61.0 mg, yield 10%) (Compound 4-36 may be the opposite stereoisomer of that shown) and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (4-37) (85.0 mg, yield 14%) (Compound 4-37 may be the opposite stereoisomer of that shown). Note: The stereochemistry at the F-ring is arbitrarily assigned.

[0336] 4-36: 11H NMR (400 MHz, DMSO-D6) δ ppm 8.97 (s, 3H), 7.89 (d, J = 10.8 Hz, 1H), 7.36 (s, 1H), 5.73 - 5.81 (m, 1H), 5.56 - 5.65 (m, 1H), 5.41 - 5.49 (m, 2H), 3.22 (d, J = 4.4 Hz, 2H), 2.34 - 2.44 (m, 5H), 1.82 - 1.94 (m, 5H), 0.88 (t, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.24. LCMS (ESI+) m / z: C 25 H 25 FN3O4 + Calculated [M+H] for + : 450.1, found: 450.1

[0337] 4 - 37: 1 1H NMR (400 MHz, DMSO-D6) δ ppm 8.98 (s, 3H), 7.89 (d, J = 10.8 Hz, 1H), 7.36 (s, 1H), 5.71 - 5.79 (m, 1H), 5.57 - 5.64 (m, 1H), 5.46 (s, 2H), 3.18 - 3.26 (m, 2H), 2.36 - 2.44 (m, 5H), 1.81 - 1.94 (m, 5H), 0.87 (t, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.22. LCMS (ESI+) m / z: C 25 H 25 FN3O4 + Calculated [M+H] for + : 450.1, found: 450.1

[0338] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-38) and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-39): To a stirred mixture of (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (4-36) (50.0 mg, 0.102 mmol) in acetonitrile (10 mL) was added diisopropylethylamine (0.286 mL, 1.65 mmol), and the mixture was stirred at 20 °C for 0.5 h, then 1,4-dibromobutan-2-ol (0.0954 mL, 0.823 mmol) and sodium iodide (123 mg, 0.823 mmol) were added. After stirring at 125 °C for 24 h under microwave, the reaction mixture was concentrated, and the residue was purified by silica gel chromatography eluting with 0% - 10% methanol in ethyl acetate, followed by chiral SFC (equipment: Waters SFC80 preparative SFC, column: DAICEL CHIRALPAK IG (250 mm *Purified by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is IPA, gradient: B% = 60% isocratic elution mode, flow rate: 80 g / min, wavelength: 220 nm, column temperature: 40 °C, system backpressure: 100 bar), (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-38) (1.80 mg, yield 3.3%) (Compound 4-38 may be the opposite stereoisomer of that represented) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxypyrrolidin-1-yl)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (4-39) (1.70 mg, yield 3.1%) (Compound 4-39 may be the opposite stereoisomer of that represented) were obtained. Note: The stereochemistry in the F-ring and pyrrolidine ring is arbitrarily assigned.

[0339] 4-38: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.75 (d, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.49 (s, 1H), 5.34 - 5.50 (m, 3H), 5.23 (d, J = 20.0 Hz, 1H), 4.74 (d, J = 3.6 Hz, 1H), 4.22 (brs, 1H), 3.38 (brs, 1H), 2.88 - 2.98 (m, 1H), 2.66 - 2.82 (m, 3H), 2.48 (brs, 1H), 2.39 (s, 3H), 2.07 - 2.17 (m, 2H), 1.92 - 2.02 (m, 1H), 1.78 - 1.92 (m, 2H), 1.58 - 1.68 (m, 1H), 1.48 (s, 3H), 0.88 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -112.14. LCMS (ESI+) m / z: C 29 H 31 FN3O5 + calculated for [MH] +: 520.2, measured value: 520.3.

[0340] 4 - 39: 1 H NMR (400 MHz, DMSO - D6) δ ppm 7.75 (d, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.49 (s, 1H), 5.56 - 5.65 (m, 1H), 5.34 - 5.45 (m, 3H), 4.73 (d, J = 3.2 Hz, 1H), 4.24 (brs, 1H), 3.38 (brs, 1H), 2.85 - 3.01 (m, 3H), 2.43 - 2.49 (m, 1H), 2.31 - 2.41 (m, 4H), 2.07 - 2.22 (m, 2H), 1.92 - 1.98 (m, 1H), 1.77 - 1.92 (m, 2H), 1.62 - 1.67 (m, 1H), 1.49 (s, 3H), 0.88 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, DMSO - D6) δ ppm - 112.15. LCMS (ESI+) m / z: C 29 H 31 FN3O5 + Calculated [MH] for + : 520.2, measured value: 520.2.

[0341] Example 5 (1R,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - ((R) - 3 - hydroxypyrrolidin - 1 - yl) - 1,4 - dimethyl - 2,3,12,15 - tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13(1H,9H) - dione (5 - 40) and (1R,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - ((S) - 3 - hydroxypyrrolidin - 1 - yl) - 1,4 - dimethyl - 2,3,12,15 - tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13(1H,9H) - dione (5 - 41) (Figure 12)

[0342]

Chemical Structure

[0343] 5-40 and 5-41 were prepared in the same manner as 4-38 and 4-39, using 4-37 instead of 4-36. Compounds 5-40 and 5-41 may be the opposite stereoisomers of those represented. Note: The stereochemistry in the F-ring and pyrrolidine ring is arbitrarily assigned.

[0344] 5-40: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.71 (d, J = 10.8 Hz, 1H), 7.28 (s, 1H), 6.49 (s, 1H), 5.54 - 5.64 (m, 1H), 5.32 - 5.44 (m, 3H), 4.74 (d, J = 3.2 Hz, 1H), 4.24 (brs, 1H), 3.37 (brs, 1H), 2.83 - 2.99 (m, 3H), 2.41 - 2.49 (m, 1H), 2.31 - 2.40 (m, 4H), 2.07 - 2.20 (m, 2H), 1.93 - 2.03 (m, 1H), 1.82 - 1.87 (m, 2H), 1.61 - 1.70 (m, 1H), 1.48 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -112.15. LCMS (ESI+) m / z: C 29 H 31 FN3O5 + calculated for [MH] + : 520.2, found: 520.2.

[0345] 5-41: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.71 (d, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.49 (s, 1H), 5.38 - 5.47 (m, 3H), 5.22 (d, J = 20.0 Hz, 1H), 4.75 (d, J = 3.6 Hz, 1H), 4.19 - 4.27 (m, 1H), 3.37 (brs, 1H), 2.86 - 2.98 (m, 1H), 2.64 - 2.83 (m, 3H), 2.50 - 2.52 (m, 1H), 2.38 (s, 3H), 2.05 - 2.22 (m, 2H), 1.92 - 2.02 (m, 1H), 1.79 - 1.92 (m, 2H), 1.57 - 1.70 (m, 1H), 1.47 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H). 1919F NMR (376 MHz, DMSO-d6) δ ppm -112.13. LCMS (ESI+) m / z: C 29 H 31 FN3O5 + Calculated for [MH] + : 520.2, found: 520.2.

[0346] Example 6 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)(methyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (6-44) (Figure 13)

[0347]

Chemical Structure

[0348] (1S,9S)-1-((2-((tert-Butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-42): To a suspension of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (200 mg, 0.411 mmol) in dichloroethane (8 mL) was added triethylamine (57.2 mL, 0.411 mmol), and the mixture was stirred at 15 °C for 0.5 h. 2-((tert-Butyldimethylsilyl)oxy)acetaldehyde (156 mL, 0.823 mmol) was added. After stirring at 15 °C for 15 h, sodium triacetoxyborohydride (95.9 mg, 0.457 mmol) was added, and the mixture was further stirred at 15 °C for 3 h. Then the reaction mixture was quenched with water (20 mL) and extracted with dichloroethane (3 × 20 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel chromatography eluting with 35% ethyl acetate in petroleum ether to give (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6 - 42) (60.0 mg, yield 21%). 1 H NMR(400MHz,CDCl3)δppm7.69(d,J=10.5Hz,1H),7.60(s,1H),5.77(d,J=16.3Hz,1H),5.42-5.66(m,2H),5.31(d,J=16.3Hz,1H),3.65-3.93(m,3H),3.26-3.38(m,1H),2.95-3.11(m,1H),2.89(m,1H),2.30-2.50(m,5H),2.03-2.14(m,1H),1.77-2.00(m,3H),1.49(s,3H),1.05(t,J=7.3Hz,3H),0.89(s,9H),0.08(s,3H),0.10(s,3H). 19 F NMR(376MHz,CDCl3)δppm-111.20。LCMS(ESI+)m / z:C 33 H 43FN3O5Si + Calculated [MH] for + : 608.3, measured value: 608.2.

[0349] (1S,9S)-1-((2-((tert-Butyldimethylsilyl)oxy)ethyl)(methyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-43): To a stirred solution of (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-42) (50.0 mg, 0.0822 mmol) in methanol (2.0 mL) were added acetic acid (0.00941 mL, 0.164 mmol) and paraformaldehyde (12.3 mg, 0.409 mmol), and the mixture was stirred at 25 °C for 1 h, then sodium cyanoborohydride (5.16 mg, 0.0822 mmol) was added. After stirring at 40 °C for 15 h, another batch of paraformaldehyde (12.3 mg, 0.409 mmol) was added, and the mixture was stirred at 40 °C for 1 h, then sodium cyanoborohydride (20.6 mg, 0.329 mmol) was added. After stirring at 40 °C for an additional 15 h, the reaction mixture was quenched with water (1.0 mL), cooled to 0 °C, the pH was adjusted to 8 with aqueous NaHCO3, and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel chromatography eluting with 10% ethyl acetate in dichloromethane to give (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-43) (19.0 mg, 37% yield). 11H NMR (400 MHz, CDCl3) δ ppm 7.70 (d, J = 10.5 Hz, 1H), 7.61 (s, 1H), 5.77 (d, J = 16.3 Hz, 1H), 5.50 - 5.62 (m, 1H), 5.36 - 5.47 (m, 1H), 5.31 (d, J = 16.3 Hz, 1H), 3.77 - 3.91 (m, 2H), 3.70 (s, 1H), 3.36 (brd, J = 15.9 Hz, 1H), 2.82 - 3.07 (m, 2H), 2.48 - 2.62 (m, 1H), 2.39 - 2.46 (m, 3H), 2.30 (brs, 4H), 2.05 - 2.17 (m, 1H), 1.79 - 2.01 (m, 2H), 1.53 (s, 3H), 1.06 (t, J = 7.3 Hz, 3H), 0.87 (s, 9H), 0.06 (s, 3H), 0.10 (s, 3H). 19 19F NMR (376 MHz, CDCl3) δ ppm -111.00. LCMS (ESI+) m / z: C 34 H 45 FN3O5Si + calculated for [MH] + : 622.3, found: 622.2.

[0350] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)(methyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (6-44): To a stirred solution of (1S,9S)-1-((2-((tert-Butyldimethylsilyl)oxy)ethyl)(methyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-43) (19.0 mg, 0.0305 mmol) in dioxane (0.45 mL) was added HCl / dioxane (0.45 mL, 4 M). After stirring at 15 °C for 1 h, the reaction mixture was filtered and the filter cake was washed with tert-butyl methyl ether (3 × 1 mL). The resulting solid was dissolved in water (0.5 mL) and lyophilized to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)(methyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)dione hydrochloride (6-44) (10.2 mg, 61% yield) (Compound 6-44 may be the opposite stereoisomer of that represented). Note: The stereochemistry in the F-ring is arbitrarily assigned. 1 H NMR (400 MHz, D2O) δ ppm 7.03 - 7.37 (m, 2H), 5.30 - 5.57 (m, 4H), 3.67 - 3.94 (m, 2H), 3.44 - 3.64 (m, 1H), 3.12 - 3.35 (m, 3H), 2.87 - 2.98 (m, 1H), 2.42 - 2.79 (m, 4H), 2.12 - 2.33 (m, 6H), 1.89 (q, J = 7.3 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). 19 F NMR (376 MHz, D2O) δ ppm -108.73. LCMS (ESI+) m / z: C 28 H 31 FN3O5 + Calculated for [MH] + : 508.2, found: 508.1.

[0351] Example 7 (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)(methyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (7-45) (Figure 14)

[0352]

Chem.

[0353] 7-45 was prepared in the same manner as 6-44 using 4-37 instead of 4-36. Compound 7-45 may be the opposite stereoisomer of that represented. Note: The stereochemistry in the F ring is arbitrarily assigned.

[0354] 7-45: 1 H NMR (400 MHz, CD3OD) δ ppm 7.51 - 7.93 (m, 2H), 5.33 - 5.73 (m, 4H), 3.79 - 3.83 (m, 2H), 3.40 - 3.44 (m, 1H), 3.38 - 3.50 (m, 2H), 2.79 - 3.19 (m, 2H), 2.28 - 2.55 (m, 6H), 2.06 - 2.27 (m, 2H), 1.91 - 2.03 (m, 2H), 1.41 - 1.72 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). 19 F NMR (400 MHz, CD3OD) δ ppm -113.03. LCMS (ESI+) m / z: C 28 H 31 FN3O5 + calculated for [MH] + : 508.2, found: 508.2.

[0355] Example 8 Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-N-(2-hydroxyethyl)acetamide (8-47) (Figure 15)

[0356] [Chem.]

[0357] N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (8-46): Acetic anhydride (100 mg, 0.987 mmol) was added to a stirred solution of (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-10,13(1H,9H)-dione (6-42) (60.0 mg, 0.0987 mmol) in pyridine (0.6 mL). After stirring at 15 °C for 12 h, the reaction mixture was purified directly by silica gel chromatography eluting with 30% ethyl acetate in dichloromethane to afford N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (8-46) (26.0 mg, 31% yield). 11H NMR (400 MHz, DMSO-d6) δ ppm 7.74 (d, J = 10.8 Hz, 1H), 6.98 (s, 1H), 5.40 - 5.59 (m, 4H), 3.70 (q, J = 5.2 Hz, 2H), 3.29 (brs, 1H), 2.95 - 3.09 (m, 1H), 2.74 - 2.85 (m, 1H), 2.38 (s, 4H), 2.20 (s, 3H), 2.10 - 2.17 (m, 3H), 1.95 (brd, J = 1.6 Hz, 1H), 1.41 (s, 3H), 0.90 (brt, J = 7.2 Hz, 3H), 0.81 (s, 9H), 0.02 (s, 6H). 19 19F NMR (376 MHz, DMSO-d6) δ ppm -112.09. LCMS (ESI+) m / z: C 35 H 45 FN3O6Si + calculated for [MH]+ + : 650.3, found: 650.2.

[0358] N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-N-(2-hydroxyethyl)acetamide (8-47): To a stirred solution of N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (8-46) (28.0 mg, 0.0431 mmol) in dioxane (1.12 mL) was added HCl / dioxane (215 mL, 2 M). After stirring at 15 °C for 1 h, the reaction mixture was concentrated and the residue was triturated with tert-butyl methyl ether (5 mL) at 20 °C for 5 min, filtered, and the resulting solid was purified by preparative TLC (ethyl acetate, R fBy further purification with (yield = 0.5), N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-N-(2-hydroxyethyl)acetamide (8-47) (10.0 mg, yield 45%) was obtained (Compound 8-47 may be the opposite stereoisomer of the one shown). Note: The stereochemistry in the F ring is arbitrarily assigned. 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.70 (d, J = 10.8 Hz, 1H), 7.08 (s, 1H), 5.33 - 5.63 (m, 4H), 3.52 (td, J = 5.6, 2.0 Hz, 2H), 3.29 (brd, J = 17.2 Hz, 1H), 2.99 (brt, J = 13.6 Hz, 1H), 2.70 - 2.79 (m, 1H), 2.36 (s, 3H), 2.25 - 2.33 (m, 1H), 2.14 - 2.18 (m, 4H), 2.04 - 2.12 (m, 2H), 1.92 - 2.00 (m, 1H), 1.37 (s, 3H), 0.89 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.54. LCMS (ESI+) m / z: C 29 H 31 FN3O6 + calculated for [MH] + : 536.2, found: 536.2.

[0359] Example 9 N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-N-(2-hydroxyethyl)acetamide hydrochloride (9-48) (Figure 16)

[0360]

Chemical Structure

[0361] 9-48 was prepared in the same manner as 8-47, using 4-37 instead of 4-36. Compound 9-48 may be the opposite stereoisomer of that represented. Note: The stereochemistry in the F-ring is arbitrarily assigned.

[0362] 9-48: 1 H NMR (400 MHz, DMSO-D6) δ ppm 9.50 - 9.72 (m, 1H), 8.81 - 9.03 (m, 1H), 7.84 - 7.91 (m, 1H), 7.06 (s, 1H), 5.77 - 5.88 (m, 1H), 5.45 - 5.60 (m, 3H), 5.13 - 5.40 (m, 1H), 3.64 (brd, J = 4.0 Hz, 2H), 3.20 (brd, J = 5.2 Hz, 2H), 3.08 (brs, 2H), 2.62 - 2.66 (m, 1H), 2.41 (s, 3H), 2.26 - 2.31 (m, 1H), 2.22 (s, 3H), 2.14 (dt, J = 10.4, 7.2 Hz, 2H), 1.96 (brs, 3H), 0.90 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.19. LCMS (ESI+) m / z: C 29 H 31 FN3O6 + calculated for + [MH]: 536.2, found: 536.2.

[0363] Example 10 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxyazetidin-1-yl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione 2,2,2-trifluoroacetate (10-49) (Figure 17)

[0364]

Chemical Structure

[0365] To a stirred mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (100 mg, 0.188 mmol) and diisopropylethylamine (131 mL, 0.752 mmol) in N,N-dimethylacetamide (2 mL) was added 2-(chloromethyl)oxirane (29.5 mL, 0.376 mmol) at 25 °C. After stirring at 120 °C for 10 h, the reaction mixture was concentrated and the residue was purified by silica gel chromatography eluting with 10% methanol in dichloromethane and further purified by preparative HPLC (acidic conditions under TFA buffer) to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxyazetidin-1-yl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione 2,2,2-trifluoroacetate (10-49) (9.25 mg, yield 8.1%) (Compound 10-49 may be the opposite stereoisomer of that represented). Note: The stereochemistry in the F-ring is arbitrarily assigned. 1 H NMR (400 MHz, CD3OD) δ ppm 7.78 (d, J = 10.5 Hz, 1H), 7.64 (s, 1H), 5.53 - 5.65 (m, 2H), 5.36 - 5.49 (m, 2H), 5.15 (brs, 1H), 4.58 (brs, 2H), 4.06 - 4.46 (m, 3H), 3.36 (brs, 1H), 2.96 - 3.09 (m, 1H), 2.73 (brd, J = 15.6 Hz, 1H), 2.46 (s, 3H), 2.28 - 2.40 (m, 1H), 1.88 - 2.03 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). 19 F NMR (376 MHz, CD3OD) δ ppm -77.05 (3F), -111.31 (1F). LCMS (ESI+) m / z: C 27 H 27 FN3O5 + calculated for [MH] + : 492.1, found: 492.1.

[0366] Example 11 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-(hydroxymethyl)azetidin-1-yl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione 2,2,2-trifluoroacetate (11-52) (Figure 18)

[0367] [Chemical Formula]

[0368] 2-((Benzyloxy)methyl)propane-1,3-diyl bis(trifluoromethanesulfonate (11-50): To a stirred mixture of 2-((benzyloxy)methyl)propane-1,3-diol (200 mg, 1.02 mmol) in dichloromethane (2 mL) were added diisopropylethylamine (0.390 mL, 2.24 mmol) and trifluoromethanesulfonic anhydride (0.369 mL, 2.24 mmol) at 0 °C. After stirring at 15 °C for 20 h, the reaction mixture was quenched with water (5 mL), extracted with dichloromethane (3 × 10 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel chromatography eluting with 2% ethyl acetate in petroleum ether to give 2-((benzyloxy)methyl)propane-1,3-diyl bis(trifluoromethanesulfonate) (11-50) (437 mg, 93% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 7.29 - 7.42 (m, 5H), 4.58 - 4.70 (m, 4H), 4.54 (s, 2H), 3.59 (d, J = 5.50 Hz, 2H), 2.64 (m, 1H). 19 F NMR (376 MHz, CDCl3) δ ppm -74.30.

[0369] (1S,9S)-1-(3-((Benzyloxy)methyl)azetidin-1-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (11-51): To a stirred mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (exatecan) (50.0 mg, 0.094 mmol) in acetonitrile (10 mL) was added diisopropylethylamine (81.9 mL, 0.470 mmol) at 15 °C, and the mixture was stirred at 15 °C for 0.5 h. A solution of 2-((benzyloxy)methyl)propane-1,3-diyl bis(trifluoromethanesulfonate) (11-50) (86.6 mg, 0.188 mmol) in acetonitrile (2.5 mL) was added. After stirring at 60 °C for 15 h, the reaction mixture was cooled to 15 °C, concentrated, and the residue was purified by silica gel chromatography eluting with 7% methanol in dichloromethane to give (1S,9S)-1-(3-((benzyloxy)methyl)azetidin-1-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (11-51) (32.0 mg, 57% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 7.55 - 7.70 (m, 2H), 7.29 - 7.39 (m, 5H), 5.76 (d, J = 16.4 Hz, 1H), 5.60 (dd, J = 6.0, 1.6 Hz, 1H), 5.38 (s, 2H), 5.28 - 5.34 (m, 1H), 4.51 (s, 2H), 3.91 - 4.01 (m, 2H), 3.78 (brs, 1H), 3.55 (brd, J = 6.4 Hz, 2H), 3.03 - 3.31 (m, 4H), 2.75 (m, 1H), 2.40 (s, 3H), 1.87 - 1.98 (m, 4H), 1.05 (t, J = 7.2 Hz, 3H).19 19F NMR (376 MHz, CDCl3) δ ppm -110.71. LCMS (ESI+) m / z: C 35 H 35 FN3O5 + calculated for [MH] + : 596.2, found: 596.3.

[0370] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-(hydroxymethyl)azetidin-1-yl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (2,2,2-trifluoroacetic acid (11-52): To a stirred mixture of (1S,9S)-1-(3-((benzyloxy)methyl)azetidin-1-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (11-51) (32.0 mg, 0.0537 mmol) in dichloromethane (4 mL) was added methanesulfonic acid (0.8 mL) at 0 °C. After stirring at 15 °C for 1 h, the reaction mixture was concentrated and the residue was dissolved in dimethyl sulfoxide (3 mL) and purified by preparative HPLC (acidic conditions under TFA buffer) to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-(hydroxymethyl)azetidin-1-yl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate 2,2,2-trifluoroacetic acid (11-52) (12.5 mg, yield 37%). 11H NMR (400 MHz, D2O) δ ppm 7.26 - 7.36 (m, 2H), 5.40 - 5.52 (m, 2H), 5.27 - 5.38 (m, 2H), 5.20 (brs, 1H), 4.69 - 4.72 (m, 2H), 4.33 - 4.41 (m, 1H), 4.15 (brt, J = 8.8 Hz, 1H), 3.61 - 3.72 (m, 2H), 3.31 (brdd, J = 18.4, 6.0 Hz, 1H), 3.02 (m, 1H), 2.75 - 2.97 (m, 2H), 2.40 - 2.54 (m, 1H), 2.28 (s, 3H), 1.90 (q, J = 7.2 Hz, 2H), 0.88 (t, J = 7.2 Hz, 3H). 19 19F NMR (376 MHz, D2O) δ ppm -75.60, -108.10. LCMS (ESI+) m / z: C 28 H 29 FN3O5 + calculated for [MH] + : 506.2, found: 506.1.

[0371] Example 12 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-58) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-59) (Figure 19)

[0372]

Chem.

[0373] N-(7-Allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-53): A solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7) (7.50 g, 31.9 mmol, 1.0 eq) in PhMe (150 mL) was added with potassium bis(trimethylsilyl)amide (1.0 M, 63.8 mL, 2.0 eq) at -70 °C, stirred at -70 °C for 1 h, and then a solution of 3-iodoprop-1-ene (5.36 g, 31.9 mmol, 2.91 mL, 1.0 eq) in PhMe (75 mL) was added. After stirring at -70 °C for 1 h, this was quenched with H2O (100 mL) at -70 °C and warmed gradually to 25 °C. Another reaction on the same scale was set up and worked up in the same manner as described above. The combined mixture was extracted with ethyl acetate (3 × 200 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash chromatography (petroleum ether / ethyl acetate = 100 / 1 - 94 / 6) to afford N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-53) (9.7 g, yield 55%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.09 (brs, 1H) 8.29 (d, J = 13.13 Hz, 1H) 5.69 - 5.97 (m, 1H) 4.98 - 5.21 (m, 2H) 2.93 - 3.07 (m, 1H) 2.77 - 2.90 (m, 1H) 2.64 - 2.74 (m, 1H) 2.54 - 2.63 (m, 1H) 2.05 - 2.26 (m, 8H) 1.65 - 1.78 (m, 1H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -104.42. LCMS (ESI+) m / z: C 16 H 19 FNO2 + calculated for [MH] + : 276.1, found: 276.1.

[0374] N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-54): A solution of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-53) (1.50 g, 5.45 mmol, 1.0 equiv) in dichloromethane (30 mL) and methanol (15 mL) was cooled to -70 °C, and then ozone (generation rate: 30 g / h) was bubbled into the mixture at -70 °C for 10 minutes to obtain a pale blue solution. Then, O2 was bubbled into the reaction mixture at -70 °C for 15 minutes to remove the excess ozone, Me2S (846 mg, 13.6 mmol, 1.00 mL, 2.5 equiv) was added, the mixture was warmed to 0 °C and stirred at 0 °C for 1 hour. This was quenched with H2O (50 mL), extracted with ethyl acetate (3 × 50.0 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-54) as a crude product (1.60 g), which was used directly without further purification. LCMS (ESI+) m / z: C 15 H 16 FNO3 + calculated for [MH] + : 278.1, found: 278.1.

[0375] N-(3-Fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-55): A solution of crude N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-54) (1.60 g, crude) in THF (36 mL) and H2O (18 mL) was added NaBH4 (65.5 mg, 1.73 mmol) at 25 °C. After stirring at 0 °C for 0.5 h, it was quenched with H2O (50 mL), extracted with ethyl acetate (3 × 100 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 7 / 3) to give N-(3-fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-55) (600 mg, 39% yield over two steps). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 12.09 (s, 1H) 8.29 (d, J = 13.26 Hz, 1H) 4.50 (t, J = 5.19 Hz, 1H) 3.47 - 3.58 (m, 2H) 2.93 - 3.04 (m, 1H) 2.78 - 2.90 (m, 1H) 2.62 - 2.75 (m, 1H) 2.10 - 2.22 (m, 7H) 1.98 - 2.06 (m, 1H) 1.68 - 1.83 (m, 1H) 1.42 - 1.59 (m, 1H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -104.70. LCMS (ESI+) m / z: C 15 H 16 FNO + calculated for + [MH - H2O]: 262.1, found: 262.1.

[0376] 8-Amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (12-56): To a solution of N-(3-fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-55) (600 mg, 2.15 mmol, 1.0 eq) in methanol (24.0 mL) was added HCl (2 M, 24.0 mL, 22 eq). After stirring at 60 °C for 3 h, the reaction mixture was adjusted to pH 7 at 0 °C with saturated aqueous NaHCO3 and extracted with ethyl acetate (3 × 30.0 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 4 / 1) to give 8-amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (12-56) (360 mg, 70% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.40 (brs, 2H) 6.35 (d, J = 12.63 Hz, 1H) 4.46 (t, J = 5.25 Hz, 1H) 3.44 - 3.56 (m, 2H) 2.85 (dt, J = 17.32, 4.85 Hz, 1H) 2.60 - 2.74 (m, 1H) 2.44 - 2.50 (m, 1H) 1.89 - 2.15 (m, 5H) 1.59 - 1.74 (m, 1H) 1.36 - 1.51 (m, 1H) 19 F NMR (376 MHz, DMSO-D6) δ ppm -108.49. LCMS (ESI+) m / z: C 13 H 17 FNO2 + calculated for + [MH]: 238.1, found: 238.1.

[0377] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-57): A mixture of 8-amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (12-56) (180 mg, 0.759 mmol, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (220 mg, 0.834 mmol, 1.1 equiv), and 4-methylbenzenesulfonic acid (52.3 mg, 0.303 mmol, 0.4 equiv) in PhMe (10 mL) was degassed and purged with argon three times, and then stirred at 120 °C for 16 h under an argon atmosphere. Another reaction was set up on the same scale. The two combined reaction mixtures were quenched with H2O (10 mL) at 25 °C, extracted with ethyl acetate (3 × 10.0 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-57) (200 mg, yield 28%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.71 (brd, J = 10.79 Hz, 1H) 7.29 (d, J = 1.13 Hz, 1H) 6.50 (d, J = 1.63 Hz, 1H) 5.43 (s, 2H) 5.27 (s, 2H) 4.73 (t, J = 4.89 Hz, 1H) 3.58 - 3.67 (m, 1H) 3.53 (brd, J = 3.64 Hz, 2H) 2.95 - 3.15 (m, 2H) 2.35 (s, 3H) 2.28 (brd, J = 13.18 Hz, 1H) 1.81 - 1.98 (m, 3H) 1.71 (brd, J = 4.52 Hz, 2H) 0.88 (brt, J = 7.28 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.93. LCMS (ESI+) m / z: C 26 H 26 FN2O5 + calculated for [MH] +: 465.1, Measured value: 465.3. SFC (Retention time = 1.656 min, 1.970 min).

[0378] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-58) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-59): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-57) (200 mg, 0.430 mmol, 1.0 equivalent) was used in SFC (Equipment: Waters SFC150AP preparative SFC, Column: DAICEL CHIRALPAK AD (250 mm *Separated by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is ethyl alcohol, gradient: B% = 50% isocratic elution mode, flow rate: 70 g / min, wavelength: 220 nm, column temperature: 35 °C, system backpressure: 120 bar), (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-58) (Compound 12-58 may be the opposite enantiomer of the one represented) (60 mg, yield 30%) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-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 (12-59) (Compound 12-59 may be the opposite enantiomer of the one represented) (55 mg, yield 28%) were obtained. Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0379] 12-48: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.73 (d, J = 11.13 Hz, 1H) 7.30 (s, 1H) 6.51 (s, 1H) 5.43 (s, 2H) 5.29 (s, 2H) 4.74 (t, J = 5.07 Hz, 1H) 3.58 - 3.67 (m, 1H) 3.49 - 3.57 (m, 2H) 2.98 - 3.17 (m, 2H) 2.37 (s, 3H) 2.29 (brd, J = 13.38 Hz, 1H) 1.80 - 1.99 (m, 3H) 1.66 - 1.79 (m, 2H) 0.87 (t, J = 7.32 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.91. LCMS (ESI+) m / z: C 26 H 26 Calculated for FN2O5 [MH] + : 465.1, found: 465.3. SFC (retention time = 1.659 min).

[0380] 12-49: 11H NMR (400 MHz, DMSO-D6) δ ppm 7.73 (d, J = 11.01 Hz, 1H) 7.30 (s, 1H) 6.50 (s, 1H) 5.43 (s, 2H) 5.29 (s, 2H) 4.74 (t, J = 5.13 Hz, 1H) 3.58 - 3.66 (m, 1H) 3.53 (dt, J = 10.60, 5.27 Hz, 2H) 2.98 - 3.17 (m, 2H) 2.37 (s, 3H) 2.25 - 2.33 (m, 1H) 1.80 - 2.00 (m, 3H) 1.66 - 1.78 (m, 2H) 0.88 (t, J = 7.32 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.91. LCMS (ESI+) m / z: C 26 H 26 FN2O5 + calculated for [MH] + : 465.1, found: 465.3. SFC (retention time = 1.976 min).

[0381] Example 13 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-63) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-64) (Figure 20)

[0382]

Chem.

[0383] N-(3-Fluoro-7-(3-hydroxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (13-60): A solution of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-53) (700 mg, 2.54 mmol, 1.0 eq) in dichloromethane (30 mL) at room temperature was added with chloro(1Z,5Z)-cycloocta-1,5-dieneiridium(III) (85.3 mg, 0.127 mmol, 0.05 eq) and 1,2-bis-(diphenylphosphino)ethane (101 mg, 0.254 mmol, 0.1 eq), stirred for 10 minutes, and then 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (389 mg, 3.05 mmol, 0.442 mL, 1.2 eq) was added. After stirring for 25 minutes, it was cooled to 0 °C, and 2M NaOH (12 mL) and 30% aqueous H2O2 solution (27.5 g, 242 mmol, 23 mL, 30%, 95 eq) were then added under vigorous stirring. Then, it was stirred at 0 °C for 1.5 hours, quenched with saturated aqueous Na2S2O3 solution (50 mL), extracted with dichloromethane (3 × 100 mL), the combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 7 / 3) to obtain N-(3-fluoro-7-(3-hydroxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (13-60) (340 mg, yield 45%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.12 (s, 1H) 8.29 (d, J = 13.13 Hz, 1H) 4.41 (t, J = 5.19 Hz, 1H) 3.42 (q, J = 6.09 Hz, 2H) 2.94 - 3.04 (m, 1H) 2.79 - 2.91 (m, 1H) 2.53 - 2.64 (m, 1H) 2.06 - 2.22 (m, 7H) 1.69 - 1.91 (m, 2H) 1.36 - 1.60 (m, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -104.66. LCMS (ESI+) m / z: C 16 H 19 FNO2 + calculated for + [MH-H2O]: 276.1, found: 276.1.

[0384] 8-Amino-6-fluoro-2-(3-hydroxypropyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (13-61): To a solution of N-(3-fluoro-7-(3-hydroxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (13-60) (340 mg, 1.16 mmol, 1.0 eq) in methanol (15 mL) was added HCl (2 M, 13 mL, 23 eq). After stirring at 60 °C for 3 h, the reaction mixture was adjusted to pH = 7 at 0 °C with saturated aqueous NaHCO3, extracted with ethyl acetate (3 × 30 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 7 / 3) to give 8-amino-6-fluoro-2-(2-hydroxypropyl)-5-methyl-3,4-dihydronaphthalen-1(3H)-one (13-61) (200 mg, yield 68%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.42 (brs, 2H) 6.35 (d, J = 12.64 Hz, 1H) 4.39 (t, J = 5.19 Hz, 1H) 3.37 - 3.43 (m, 2H) 2.85 (dt, J = 17.52, 5.13 Hz, 1H) 2.62 - 2.76 (m, 1H) 2.31 - 2.44 (m, 1H) 2.01 - 2.14 (m, 1H) 1.97 (d, J = 1.19 Hz, 3H) 1.62 - 1.85 (m, 2H) 1.30 - 1.58 (m, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -108.52. LCMS (ESI+) m / z: C 14 H 19 FNO2 + calculated for + [MH]: 252.1, found: 252.1.

[0385] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-62): A mixture of 8-amino-6-fluoro-2-(3-hydroxypropyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (13-61) (200 mg, 0.795 mmol, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (230 mg, 0.872 mmol, 1.1 equiv), and toluene-4-sulfonic acid (54.7 mg, 0.316 mmol, 0.4 equiv) in PhMe (3.5 mL) was degassed and purged with argon three times, and then stirred at 120 °C for 16 h under an argon atmosphere. It was cooled to 25 °C, quenched with H2O (10 mL), extracted with ethyl acetate (3 × 10 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-62) (200 mg, 52% yield). 11H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (d, J = 11.13 Hz, 1H) 7.30 (d, J = 1.22 Hz, 1H) 6.52 (d, J = 2.08 Hz, 1H) 5.44 (s, 2H) 5.31 - 5.40 (m, 1H) 5.25 (brd, J = 3.42 Hz, 1H) 4.39 - 4.47 (m, 1H) 3.40 - 3.51 (m, 2H) 3.35 - 3.39 (m, 1H) 3.10 (brd, J = 3.30 Hz, 2H) 2.38 (s, 3H) 2.30 (brd, J = 13.20 Hz, 1H) 1.81 - 1.99 (m, 3H) 1.51 - 1.73 (m, 4H) 0.88 (t, J = 7.34 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -111.87. LCMS (ESI+) m / z: C 27 H 28 F N2O5 + calculated for [MH] + : 479.2, found: 479.4. SFC (retention times = 0.842 min, 1.830 min).

[0386] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-63) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-64): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-62) (200 mg, 0.42 mmol) was separated by chiral SFC (equipment: Waters SFC150AP preparative SFC, column: DAICEL CHIRALPAK IG (250 mm *Separated by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is ethyl alcohol (0.1% NH3·H2O), gradient: B% = 37% isocratic elution mode, flow rate: 70 g / min, wavelength: 220 nm, column temperature: 35 °C, system back pressure: 120 bar), (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-63) (Compound 13-63 may be the opposite enantiomer of the one represented) (36.8 mg, yield 18%) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-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 (13-64) (Compound 13-64 may be the opposite enantiomer of the one represented) (76.2 mg, yield 38%) were obtained. Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0387] 13-63: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (d, J = 11.13 Hz, 1H) 7.30 (s, 1H) 6.51 (s, 1H) 5.43 (s, 2H) 5.32 - 5.39 (m, 1H) 5.18 - 5.26 (m, 1H) 4.42 (t, J = 5.13 Hz, 1H) 3.43 - 3.49 (m, 2H) 3.35 (brs, 1H) 2.97 - 3.17 (m, 2H) 2.37 (s, 3H) 2.29 (brd, J = 12.63 Hz, 1H) 1.79 - 2.00 (m, 3H) 1.51 - 1.77 (m, 4H) 0.87 (t, J = 7.25 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -111.87. LCMS (ESI+) m / z: C 27 H 28 FN2O5 + calculated for [MH] + : 479.2, found: 479.5. Chiral SFC (RT = 0.837 min).

[0388] 13 - 64: 1 1H NMR (400 MHz, DMSO - D6) δ ppm 7.73 (d, J = 11.04 Hz, 1H) 7.30 (s, 1H) 6.52 (s, 1H) 5.43 (s, 2H) 5.31 - 5.40 (m, 1H) 5.18 - 5.28 (m, 1H) 4.43 (t, J = 5.14 Hz, 1H) 3.42 - 3.51 (m, 2H) 3.33 - 3.39 (m, 1H) 2.98 - 3.16 (m, 2H) 2.37 (s, 3H) 2.24 - 2.34 (m, 1H) 1.80 - 1.99 (m, 3H) 1.51 - 1.77 (m, 4H) 0.87 (t, J = 7.34 Hz, 3H). 19 19F NMR (376 MHz, DMSO - D6) δ ppm - 111.87. LCMS (ESI+) m / z: C 27 H 28 F N2O5 + calculated for [MH] + : 479.2, found: 479.5. Chiral SFC (RT = 1.803 min).

[0389] Example 14 (1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - (2 - hydroxyethyl) - 1,4 - dimethyl - 1,2,3,9,12,15 - hexahydro - 10H,13H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13 - dione (14 - 70) and (1R,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - (2 - hydroxyethyl) - 1,4 - dimethyl - 1,2,3,9,12,15 - hexahydro - 10H,13H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13 - dione (14 - 71) (Figure 21)

[0390]

Chemical Structure

[0391] N-(7-Allyl-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-65): To a solution of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-23) (12.0 g, 48.1 mmol, 1.0 equiv) in PhMe (240 mL) was added potassium bis(trimethylsilyl)amide (1.0 M, 96.2 mL, 96.2 mmol, 2.0 equiv) dropwise at -70 °C. The mixture was stirred at -70 °C for 1 h, and 3-iodoprop-1-ene (8.09 g, 48.1 mmol, 4.39 mL, 1.0 equiv) was added. After stirring at -70 °C for 1 h, the reaction was quenched with H2O (200 mL) at 25 °C, and the mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 96 / 4) to give N-(7-allyl-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-65) (10.3 g, 74% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.12 (s, 1H), 8.31 (d, J = 13.26 Hz, 1H), 5.69 - 5.85 (m, 1H), 5.04 - 5.15 (m, 2H), 2.80 - 3.00 (m, 2H), 2.30 - 2.40 (m, 1H), 2.10 - 2.25 (m, 7H), 1.92 - 2.02 (m, 1H), 1.77 - 1.91 (m, 1H), 1.11 (s, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -104.43. LCMS (ESI+) m / z: C 17 H 21 FNO2 + Calculated for + [MH]: 290.1, found: 290.1.

[0392] N-(3-Fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-66): A solution of N-(7-allyl-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-65) (1.50 g, 5.18 mmol, 1.0 eq) in dichloromethane (30 mL) was cooled to -70 °C, and then ozone (generation rate: 30 g / h) was bubbled into the mixture for 10 minutes to obtain a pale blue solution. Next, O2 was bubbled into the mixture at -70 °C for 15 minutes to remove the excess ozone, and then Me2S (805 mg, 12.9 mmol, 0.951 mL, 2.5 eq) was added. The mixture was warmed to 25 °C and stirred at 25 °C for 0.5 hour, then quenched with H2O (15 mL), extracted with ethyl acetate (3 × 40 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to obtain N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-66) (1.40 g, crude). LCMS (ESI+) m / z: C 16 H 19 FNO3 + Calculated for [MH] + : 292.1, found: 292.1.

[0393] N-(3-Fluoro-7-(2-hydroxyethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-67): A solution of N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-66) (1.40 g, crude) in THF (28 mL) and water (14 mL) was added with NaBH4 (36.3 mg, 0.961 mmol, 0.5 eq.). After stirring at 0 °C for 0.5 h, this was quenched with H2O (7 mL), extracted with ethyl acetate (3 × 10 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 7 / 3) to obtain N-(3-fluoro-7-(2-hydroxyethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-67) (330 mg, yield 21%). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 11.99 (s, 1H), 8.30 (d, J = 13.13 Hz, 1H), 4.39 (t, J = 5.07 Hz, 1H), 3.35 - 3.53 (m, 2H), 2.80 - 3.00 (m, 2H), 2.10 - 2.17 (m, 6H), 1.97 - 2.08 (m, 1H), 1.81 - 1.91 (m, 1H), 1.70 - 1.80 (m, 1H), 1.60 - 1.69 (m, 1H), 1.13 (s, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -105.12. LCMS (ESI+) m / z: C 16 H 19 FNO2 + calculated for + [M - H2O]+: 276.1, found: 276.1.

[0394] 8-Amino-6-fluoro-2-(2-hydroxyethyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-68): A solution of N-(3-fluoro-7-(2-hydroxyethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-67) (330 mg, 1.13 mmol, 1.0 eq) in MeOH (13.2 mL) was treated with HCl (2 M, 13.2 mL, 23.4 eq). After stirring at 60 °C for 3 h, the reaction mixture was cooled to 25 °C, quenched with saturated NaHCO3 to adjust the pH to 7, extracted with ethyl acetate (3 × 30.0 mL), and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 7 / 3) to give 8-amino-6-fluoro-2-(2-hydroxyethyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-68) (270 mg, 95% yield). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 7.40 (brs, 2H), 6.36 (d, J = 12.63 Hz, 1H), 4.34 (t, J = 5.19 Hz, 1H), 3.37 - 3.51 (m, 2H), 2.64 - 2.89 (m, 2H), 1.89 - 2.02 (m, 4H), 1.66 - 1.84 (m, 2H), 1.52 - 1.65 (m, 1H), 1.08 (s, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -108.69. LCMS (ESI+) m / z: C 14 H 17 FNO + Calculated for + [M H - H2O]: 234.1, found: 234.1.

[0395] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-69): A mixture of 8-amino-6-fluoro-2-(2-hydroxyethyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-68) (270 mg, 1.07 mmol, 1.0 eq), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (311 mg, 1.18 mmol, 1.1 eq), and 4-methylbenzenesulfonic acid (74.0 mg, 0.429 mmol, 0.4 eq) in PhMe (13.5 mL) was degassed and purged with argon three times, and then stirred at 120 °C for 16 h under an argon atmosphere. The reaction mixture was then diluted with H2O (10 mL), extracted with ethyl acetate (3 × 10 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (ethyl acetate / methanol = 100 / 1 to 98 / 2) to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolone-10,13-dione (KP-6363-5) as a pale red solid (180 mg, yield 35%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (d, J = 10.67 Hz, 1H), 7.31 (s, 1H), 6.51 (d, J = 2.01 Hz, 1H), 5.32 - 5.54 (m, 4H), 4.45 - 4.51 (m, 1H), 3.39 - 3.62 (m, 2H), 2.98 - 3.23 (m, 2H), 2.37 (s, 3H), 2.08 - 2.24 (m, 1H), 1.80 - 1.98 (m, 5H), 1.53 (d, J = 4.14 Hz, 3H), 0.83 - 0.92 (m, 3H). 19 F NMR (376 MHz, DMSO) δ ppm -112.52. LCMS (ESI+) m / z: C 27 H 28 FN2O5 + calculated for [MH] + : 479.2, found: 479.3.

[0396] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-70) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-71): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-69) (180 mg, 0.37 mmol) was subjected to SFC (equipment: Waters SFC150AP preparative SFC, column: REGIS (s,s) WHELK-O1 (250 mm *Separated by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is ethyl alcohol, gradient: B% = 50% isocratic elution mode, flow rate: 70 g / min, wavelength: 220 nm, column temperature: 35 °C, system back pressure: 120 bar), (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-70) (Compound 14-70 may be the opposite enantiomer of the one represented) (40.3 mg, yield 22.4%) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (14-71) (Compound 14-71 may be the opposite enantiomer of the one represented) (60.5 mg, yield 33.6%) were obtained. Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0397] 14-70: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.75 (d, J = 10.76 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.31 - 5.54 (m, 4H), 4.48 (t, J = 5.07 Hz, 1H), 3.40 - 3.59 (m, 2H), 3.00 - 3.21 (m, 2H), 2.37 (s, 3H), 2.08 - 2.22 (m, 1H), 1.77 - 2.02 (m, 5H), 1.54 (s, 3H), 0.88 (t, J = 7.32 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -112.53. LCMS (ESI+) m / z: C 27 H 28 FN2O5 + calculated for [MH] + : 479.2, found: 479.4. SFC (retention time = 0.801 min).

[0398] 14-71: 11H NMR (400 MHz, DMSO-D6) δ ppm 7.75 (d, J = 10.76 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.32 - 5.54 (m, 4H), 4.49 (t, J = 5.00 Hz, 1H), 3.39 - 3.62 (m, 2H), 2.97 - 3.21 (m, 2H), 2.38 (s, 3H), 2.15 (dt, J = 12.73, 6.21 Hz, 1H), 1.79 - 2.01 (m, 5H), 1.53 (s, 3H), 0.87 (t, J = 7.32 Hz, 3H). 19 19F NMR (376 MHz, DMSO-D6) δ ppm -112.51. LCMS (ESI+) m / z: C 27 H 28 F N2O5 + calculated for [MH] + : 479.2, found: 479.3. SFC (retention time = 1.223 min).

[0399] Example 15 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-75) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-76) (Figure 22)

[0400]

Chemical Structure

[0401] N-(3-Fluoro-7-(3-hydroxypropyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (15-72): A solution of N-(7-allyl-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-65) (800 mg, 2.76 mmol, 1.0 equiv) in dichloromethane (40 mL) at room temperature was added with chloro(1,5-cyclooctadiene)iridium(I) dimer (92.9 mg, 0.138 mmol, 0.05 equiv) and 1,2-bis(diphenylphosphino)ethane (110 mg, 0.276 mmol, 0.1 equiv), stirred for 10 minutes, and then 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (424 mg, 3.32 mmol, 0.481 mL, 1.2 equiv) was added. After stirring at room temperature for 25 minutes, it was cooled to 0 °C, and NaOH (2 M, 8 mL) and H2O2 (8 mL, 30 wt%) were successively added and stirred for 1.5 hours. The reaction mixture was quenched with saturated Na2S2O3 (16 mL) at 0 °C, extracted with ethyl acetate (3 × 20 mL), the combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 65 / 35) to obtain N-(3-fluoro-7-(3-hydroxypropyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (15-72) (520 mg, yield 61%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.16 (s, 1H), 8.32 (d, J = 13.20 Hz, 1H), 4.38 (t, J = 5.26 Hz, 1H), 3.33 - 3.38 (m, 2H), 2.81 - 2.97 (m, 2H), 2.16 (s, 3H), 2.12 (d, J = 1.59 Hz, 3H), 1.94 - 2.03 (m, 1H), 1.83 - 1.92 (m, 1H), 1.32 - 1.62 (m, 4H), 1.11 (s, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -104.67. LCMS (ESI+) m / z: C 17 H 21 FNO2 + calculated for + [MH-H2O]: 290.1, found: 290.1.

[0402] 8-Amino-6-fluoro-2-(3-hydroxypropyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (15-73): To a solution of N-(3-fluoro-7-(3-hydroxypropyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (15-72) (520 mg, 1.69 mmol, 1.0 eq) in MeOH (26 mL) was added HCl (2 M, 26 mL, 24 eq). After stirring at 60 °C for 3 h, the reaction mixture was cooled to 25 °C, quenched with saturated NaHCO3 to adjust the pH to 7, extracted with ethyl acetate (3 × 30 mL), the combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 65 / 35) to afford 8-amino-6-fluoro-2-(3-hydroxypropyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (15-73) (310 mg, yield 69%). 1 1H NMR (400 MHz, DMSO-D6) δ ppm 7.41 (brd, J = 1.75 Hz, 2H), 6.35 (d, J = 12.63 Hz, 1H), 4.36 (t, J = 5.19 Hz, 1H), 3.35 (brs, 1H), 3.31 (brs, 1H), 2.64 - 2.84 (m, 2H), 1.95 - 2.05 (m, 3H), 1.86 - 1.94 (m, 1H), 1.72 - 1.82 (m, 1H), 1.28 - 1.55 (m, 4H), 1.07 (s, 3H). 19 19F NMR (376 MHz, DMSO) δ ppm -108.72. LCMS (ESI+) m / z: C 15 H 19 NFO + calculated for + [MH - H2O]: 248.1, found: 248.1.

[0403] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-74): A mixture of 8-amino-6-fluoro-2-(3-hydroxypropyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (15-73) (310 mg, 1.17 mmol, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (338 mg, 1.28 mmol, 1.1 equiv), and p-toluenesulfonic acid (80.6 mg, 0.468 mmol, 0.4 equiv) in PhMe (15 mL) was degassed and purged with argon three times, and then stirred at 120 °C for 16 h under an argon atmosphere. This was cooled to 25 °C, diluted with water (8 mL), extracted with ethyl acetate (3 × 10 mL), the combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 15 / 85) to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-74) (220 mg, yield 38%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (brd, J = 10.63 Hz, 1H), 7.30 (s, 1H), 6.51 (d, J = 2.13 Hz, 1H), 5.32 - 5.57 (m, 4H), 4.40 (q, J = 4.79 Hz, 1H), 3.34 - 3.45 (m, 2H), 3.08 (brs, 2H), 2.38 (s, 3H), 2.06 - 2.20 (m, 1H), 1.75 - 2.02 (m, 4H), 1.27 - 1.73 (m, 6H), 0.87 (t, J = 7.21 Hz, 3H). 1919F NMR (376 MHz, DMSO-d6) δ ppm -112.45. LCMS (ESI+) m / z: C 28 H 30 N2O5F + calculated for [MH] + : 493.2, found: 493.4.

[0404] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-75) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-76): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-74) (220 mg, 0.447 mmol) was subjected to SFC (equipment: Waters SFC80Q preparative SFC, column: REGIS (s,s) WHELK-O1 (250 mm *Separated by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is ethyl alcohol, gradient: B% = 54% isocratic elution mode, flow rate: 75 g / min, wavelength: 220 nm, column temperature: 40 °C, system back pressure: 100 bar), (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-75) (Compound 15-75 may be the opposite enantiomer of the one represented) (65.2 mg, yield 30%) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (15-76) (Compound 15-76 may be the opposite enantiomer of the one represented) (90.5 mg, yield 41%) were obtained. Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0405] 15-75: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (brd, J = 10.76 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.29 - 5.59 (m, 4H), 4.40 (t, J = 5.14 Hz, 1H), 3.32 - 3.40 (m, 2H), 3.08 (brs, 2H), 2.37 (s, 3H), 2.06 - 2.19 (m, 1H), 1.72 - 1.95 (m, 4H), 1.58 - 1.69 (m, 1H), 1.50 (s, 3H), 1.34 - 1.48 (m, 2H), 0.87 (brt, J = 7.21 Hz, 3H). 19 F NMR (376 MHz, DMSO-D6) δ ppm -112.45. LCMS (ESI+) m / z: C 28 H 30 FN2O5 + calculated for [MH] + : 493.2, found: 493.5. SFC (retention time = 0.761 min).

[0406] 15 - 76: 1 1H NMR (400 MHz, DMSO - D6) δ ppm 7.75 (d, J = 10.76 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.34 - 5.52 (m, 4H), 4.42 (t, J = 5.14 Hz, 1H), 3.32 - 3.40 (m, 2H), 3.08 (brs, 2H), 2.37 (s, 3H), 2.07 - 2.21 (m, 1H), 1.76 - 1.96 (m, 4H), 1.57 - 1.70 (m, 1H), 1.49 (s, 3H), 1.32 - 1.45 (m, 2H), 0.87 (brt, J = 7.21 Hz, 3H). 19 19F NMR (376 MHz, DMSO - D6) δ ppm - 112.44. LCMS (ESI+) m / z: C 28 H 30 FN2O5 + calculated [MH]+ for + : 493.2, found: 493.4. SFC (retention time = 1.165 min).

[0407] Example 16 (1R,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - ((2 - hydroxyethoxy)methyl) - 1,4 - dimethyl - 1,2,3,9,12,15 - hexahydro - 10H,13H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13 - dione (16 - 82) and (1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 1 - ((2 - hydroxyethoxy)methyl) - 1,4 - dimethyl - 1,2,3,9,12,15 - hexahydro - 10H,13H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinoline - 10,13 - dione (16 - 83) (Figure 23)

[0408]

Chemical Structure

[0409] N - (7 - ((allyloxy)methyl) - 3 - fluoro - 4,7 - dimethyl - 8 - oxo - 5,6,7,8 - tetrahydronaphthalen - 1 - yl)acetamide (16 - 77): To a solution of N-(3-fluoro-7-(hydroxymethyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-24) (5.00 g, 17.9 mmol, 1.0 eq) in CH2Cl2 (100 mL) were added Ag2O (41.4 g, 179 mmol, 10 eq) and 3-iodoprop-1-ene (60.1 g, 358 mmol, 20 eq). After stirring at 50 °C for 60 h under an argon atmosphere, the reaction mixture was cooled to room temperature, filtered through a pad of celite, the filter cake was washed with CH2Cl2, the combined filtrates were washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was subjected to silica gel flash column chromatography eluting with 20% ethyl acetate in petroleum ether to give N-(7-((allyloxy)methyl)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-77) (3.5 g, 61% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 12.09 (brd, J = 3.4 Hz, 1H), 8.30 (d, J = 13.2 Hz, 1H), 5.73 - 5.96 (m, 1H), 5.02 - 5.29 (m, 2H), 3.90 - 3.99 (m, 2H), 3.62 - 3.82 (m, 1H), 3.34 (s, 1H), 2.82 - 3.03 (m, 2H), 2.00 - 2.30 (m, 7H), 1.81 - 1.94 (m, 1H), 1.04 - 1.17 (m, 3H). LCMS (ESI+) m / z: C 18 H 23 FNO3 + Calculated for + [MH]: 320.1, found: 320.3.

[0410] N-(3-Fluoro-4,7-dimethyl-8-oxo-7-((2-oxoethoxy)methyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-78): A solution of N-(7-((allyloxy)methyl)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-77) (3.50 g, 10.9 mmol, 1.0 eq) in CH2Cl2 (68 mL) and MeOH (34 mL) was stirred at -78 °C, and then ozone was bubbled into the mixture for 15 minutes to obtain a pale blue mixture. Me2S (1.69 g, 27.3 mmol, 1.95 mL, 2.5 eq) was added at -78 °C. This was warmed to 25 °C, stirred at 25 °C for 1 hour, quenched with water (100 mL), extracted with dichloromethane (3 × 200 mL), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to obtain N-(3-fluoro-4,7-dimethyl-8-oxo-7-((2-oxoethoxy)methyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-78) (1.50 g, crude), which was used directly in the next step without further purification.

[0411] N-(3-Fluoro-7-((2-hydroxyethoxy)methyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-79): To a solution of N-(3-fluoro-4,7-dimethyl-8-oxo-7-((2-oxoethoxy)methyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-78) (1.50 g, 3.73 mmol, 1.0 eq) in THF (30 mL) and H2O (15 mL), NaBH4 (45.0 mg, 1.19 mmol, 0.32 eq) was added portionwise at 0 °C. After stirring at 0 °C for 30 minutes, it was quenched with H2O (50 mL), extracted with ethyl acetate (3 × 100 mL), the combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography eluted with 40% ethyl acetate in petroleum ether to obtain N-(3-fluoro-7-((2-hydroxyethoxy)methyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-79) (800 mg, 23% yield over 2 steps).1 1H NMR (400 MHz, DMSO-D6) δ ppm 12.09 (brd, J = 8.75 Hz, 1H), 8.30 (brd, J = 13.13 Hz, 1H), 4.60 (brd, J = 2.38 Hz, 1H), 3.66 - 3.80 (m, 1H), 3.33 - 3.52 (m, 4H), 3.14 - 3.24 (m, 1H), 2.79 - 3.03 (m, 2H), 2.06 - 2.25 (m, 7H), 1.86 (dt, J = 8.04, 5.36 Hz, 1H), 1.11 (brd, J = 9.63 Hz, 3H). LCMS (ESI+) m / z: C 17 H 23 FNO4 + Calculated for [MH] + : 324.1, found: 324.3.

[0412] 8-Amino-6-fluoro-2-((2-hydroxyethoxy)methyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (16-80): To a solution of N-(3-fluoro-7-((2-hydroxyethoxy)methyl)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (16-79) (800 mg, 2.47 mmol, 1.0 equiv) in MeOH (32 mL) was added 2N hydrochloric acid solution (32 mL) at 20 °C. After stirring at 60 °C for 1 h under argon protection, this was cooled to 0 °C, the pH was adjusted to 8 with saturated aqueous NaHCO3, and it was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography eluting with 35% ethyl acetate in petroleum ether to give 8-amino-6-fluoro-2-((2-hydroxyethoxy)methyl)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (16-80) (350 mg, 50% yield). 11H NMR (400 MHz, DMSO-D6) δ ppm 7.45 (brs, 2H), 6.35 (d, J = 12.59 Hz, 1H), 4.50 (t, J = 5.38 Hz, 1H), 3.70 (d, J = 9.05 Hz, 1H), 3.36 - 3.47 (m, 4H), 3.25 (d, J = 9.05 Hz, 1H), 2.69 - 2.88 (m, 2H), 2.10 - 2.16 (dt, J = 8.99, 4.55 Hz, 1H), 1.99 (s, 3H), 1.75 (dt, J = 13.60, 5.55 Hz, 1H), 1.04 (s, 3H). LCMS (ESI+) m / z: C 15 H 21 FNO3 + calculated for [MH] + : 282.1, found: 282.2.

[0413] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-81): A mixture of 8-amino-6-fluoro-2-((2-hydroxyethoxy)methyl)-2,5-dimethyl-3,4-dihydro-naphthalen-1(2H)-one (16-80) (30.0 mg, 0.106 mmol, 1.0 equiv) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (83.8 mg, 0.319 mmol, 3.0 equiv) in PhMe (1.5 mL) was stirred at 120 °C until all solids dissolved, and then p-toluenesulfonic acid (11.0 mg, 0.064 mmol, 0.6 equiv) was added in one portion at 120 °C under argon protection. A further 10 reactions were set up as above, and after stirring at 120 °C for 2 h, all 11 reaction mixtures were combined. The combined mixture was concentrated, and the residue was subjected to silica gel flash column chromatography eluting with ethyl acetate to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-81) (85 mg, 14% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.76 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.28 - 5.46 (m, 4H), 3.35 - 3.44 (m, 6H), 3.08 - 3.19 (m, 1H), 2.19 - 2.29 (m, 5H), 2.18 (t, J = 6.2 Hz, 2H), 1.22 (s, 3H), 0.79 - 0.90 (m, 3H). LCMS (ESI+) m / z: C 28 H 30 FN2O6 + calculated for [MH] + : 509.2, found: 509.2.

[0414] (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-82) and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-83): (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-81) (60.0 mg, 0.117 mmol) was dissolved in MeOH, and chiral SFC (equipment: Waters SFC80 preparative SFC, column: DAICEL CHIRALPAK AD (250 mm *Separated by (column dimensions: 30 mm, 10 μm), mobile phase: A is CO2 and B is ethyl alcohol, gradient: B% = 50% isocratic elution mode, flow rate: 80 g / min, wavelength: 220 nm, column temperature: 40 °C, system backpressure: 100 bar), (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-82) (Compound 16-82 may be the opposite enantiomer of the one shown) (5.0 mg, yield 8.3%) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-83) (Compound 16-83 may be the opposite enantiomer of the one shown) (9.0 mg, yield 15%) were obtained. Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0415] 16-82: 1 H NMR (400 MHz, CDCl3) δ ppm 7.67 (brd, J = 10.4 Hz, 1H), 7.61 (s, 1H), 5.75 (brd, J = 16.3 Hz, 1H), 5.47 (brs, 2H), 5.30 (brd, J = 15.8 Hz, 1H), 3.50 - 4.02 (m, 7H), 3.12 (brt, J = 6.0 Hz, 2H), 2.41 (brs, 4H), 1.85 - 2.08 (m, 4H), 1.53 (s, 3H), 1.05 (brt, J = 7.1 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ ppm -111.40. LCMS (ESI+) m / z: C 28 H 30 FN2O6 + calculated for [MH] + : 509.2, found: 509.5. SFC (RT = 1.842 min).

[0416] 16-83:1 1H NMR (400 MHz, CDCl3) δ ppm 7.67 (broad d, J = 10.5 Hz, 1H), 7.61 (s, 1H), 5.75 (broad d, J = 16.4 Hz, 1H), 5.47 (broad s, 2H), 5.32 (s, 1H), 3.47 - 4.06 (m, 7H), 3.12 (broad t, J = 5.9 Hz, 2H), 2.30 - 2.50 (m, 4H), 1.82 - 2.04 (m, 4H), 1.53 (s, 3H), 0.98 - 1.13 (m, 3H). 19 19F NMR (376 MHz, CDCl3) δ ppm -111.40. LCMS (ESI+) m / z: C 28 H 30 FN2O6 + Calculated for [MH]+ + : 509.2, found: 509.5. SFC (RT = 2.103 min).

[0417] Example 17 (1S,9S)-1-Allyl-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 (17-86) and (1R,9S-1-allyl-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 (17-87) (Figure 24)

[0418]

Chemical formula

[0419] 2-Allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (17-84): A solution of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-53) (1.0 g, 3.63 mmol, 1.0 equiv) in MeOH (30 mL) was added with sulfuric acid (2.0 mL) and stirred at 60 °C for 18 h under an argon atmosphere. Five more reactions were set up as above, and after cooling to room temperature, all six reaction mixtures were combined. These were quenched with water (150 mL), the pH was adjusted to 7 at 0 °C with saturated aqueous NaHCO3, extracted with EtOAc (3 × 300 mL), the combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was subjected to silica gel flash column chromatography eluting with 10% ethyl acetate in petroleum ether to give 2-allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (17-84) (3.2 g, 63% yield). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.42 (brs, 2H), 6.35 (d, J = 12.7 Hz, 1H), 5.59 - 5.99 (m, 1H), 4.93 - 5.23 (m, 2H), 2.87 (dt, J = 17.3, 4.5 Hz, 1H), 2.52 - 2.74 (m, 2H), 2.48 (brs, 1H), 1.93 - 2.21 (m, 5H), 1.54 - 1.70 (m, 1H). LCMS (ESI+) m / z: C 14 H 17 FNO + calculated for [MH] + : 234.1, found: 234.4.

[0420] (9S)-1-allyl-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 (17-85): A suspension of 2-allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (17-84) (200 mg, 0.857 mmol, 1.0 eq) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (10) (451 mg, 1.71 mmol, 2.0 eq) in PhMe (10 mL) was added with 4-methylbenzenesulfonic acid (59.1 mg, 0.342 mmol, 0.4 eq) at 140 °C, and stirred for 16 h with a Dean-Stark trap to remove the water generated during the reaction. Another two reactions were set up as above. The three reaction mixtures were combined, concentrated, and the residue was subjected to silica gel flash chromatography eluted with 50% ethyl acetate in petroleum ether to give (9S)-1-allyl-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 (17-85) (300 mg, yield 25%). 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74 (brd, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.88 - 6.12 (m, 1H), 5.27 - 5.51 (m, 4H), 5.02 - 5.25 (m, 2H), 3.45 (brd, J = 3.3 Hz, 1H), 3.08 (brs, 2H), 2.34 - 2.47 (m, 4H), 2.18 - 2.33 (m, 2H), 1.83 - 1.95 (m, 3H), 0.87 (brt, J = 7.0 Hz, 3H). LCMS (ESI+) m / z: C 27 H 26 FN2O4 + calculated for [MH] + : 461.1, found: 461.4.

[0421] (1S,9S)-1-Allyl-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 (17-86) and (1R,9S)-1-allyl-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 (17-87): (9S)-1-Allyl-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 (17-85) (150 mg, 0.326 mmol) was separated by SFC (equipment: Waters SFC80 preparative SFC system, column: REGIS (s,s) WHELK-O1 (250 mm * × 25 mm, 10 μm), mobile phase: A was CO2 and B was methanol, gradient: B% = 60.00% isocratic elution mode, flow rate: 75.00 g / min, monitoring wavelength: 220 & 254 nm, column temperature: 40 °C, system back pressure: 100 bar) to obtain (1S,9S)-1-allyl-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 (17-86) (compound 17-86 may be the opposite enantiomer of that shown) (25.2 mg, yield 17%) and (1R,9S)-1-allyl-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 (17-87) (compound 17-87 may be the opposite enantiomer of that shown) (11.9 mg, yield 8%). Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0422] 17 - 86: 1 1H NMR (400 MHz, DMSO - D6) δ ppm 7.76 (d, J = 11.1 Hz, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.91 - 6.05 (m, 1H), 5.24 - 5.47 (m, 4H), 5.09 - 5.20 (m, 2H), 3.40 - 3.50 (m, 1H), 3.03 - 3.15 (m, 2H), 2.36 - 2.47 (m, 4H), 2.21 - 2.31 (m, 2H), 1.79 - 1.99 (m, 3H), 0.88 (t, J = 7.3 Hz, 3H). 19 19F NMR (376 MHz, DMSO - D6) δ ppm - 111.77. LCMS (ESI+) m / z: C 27 H 26 FN2O4 + Calculated [M + H] for + : 461.1, found: 461.3. SFC (retention time = 1.020 min).

[0423] 17 - 87: 1 1H NMR (400 MHz, DMSO - D6) δ ppm 7.74 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.87 - 6.06 (m, 1H), 5.24 - 5.51 (m, 4H), 5.03 - 5.21 (m, 2H), 3.40 - 3.52 (m, 1H), 3.01 - 3.16 (m, 2H), 2.36 - 2.48 (m, 4H), 2.20 - 2.34 (m, 2H), 1.79 - 2.02 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H). 19 19F NMR (376 MHz, DMSO - D6) δ ppm - 111.78. LCMS (ESI+) m / z: C 27 H 26 FN2O4 + Calculated [M + H] for + : 461.1, found: 461.2. SFC (retention time = 2.047 min).

[0424] Example 18 (S)-2-(2-(2-(3-(4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamido)acetamido)-N-(2-(((2-(((1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-94) (Figure 25)

[0425]

Chem.

[0426] (3-(4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoyl)glycylglycyl-L-phenylalanine (18-90): A mixture of (S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropanoic acid (18-89) and N-ethyl-N,N-diisopropylamine was added dropwise to a mixture of 2,5-dioxopyrrolidin-1-yl 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-88) in acetonitrile. The reaction mixture was stirred at 25 °C for 5 hours, filtered, and the filtrate was purified by preparative HPLC to obtain (3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoyl)glycylglycyl-L-phenylalanine (18-90).

[0427] (9H-Fluoren-9-yl)methyl (2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (18-92): To a mixture of (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-10,13-dione (16-82) in dichloromethane at 25 °C, scandium(III) trifluoromethanesulfonate and [[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate (18-91) are added in three portions over 1.5 hours. The reaction mixture is stirred at 30 °C for 36 hours, quenched with water, and extracted with dichloromethane. The combined organic phases are washed with brine, dried over Na2SO4, filtered, concentrated, and the residue is purified by silica gel flash column chromatography eluting with 10% to 100% dichloromethane in ethyl acetate to give (9H-fluoren-9-yl)methyl (2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (18-92).

[0428] 2-Amino-N-((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (18-93): Piperidine was added to a mixture of (9H-fluoren-9-yl)methyl (9H-fluoren-9-yl)methyl (2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (18-92) in N,N-dimethylformamide. The reaction mixture was stirred at 0 °C for 1 hour, filtered, and the filtrate was purified by preparative HPLC to give 2-amino-N-((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (18-93).

[0429] (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-94): (2S)-2-[[2-[[2-[3-[4-(2,5-Dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoic acid (18-90), 2-amino-N-((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (18-93), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate, and 4-methylmorpholine in N,N-dimethylformamide were stirred at 25 °C for 1 hour, filtered, and the filtrate was purified by preparative HPLC to obtain (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamide)-N-(2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-94) (Compound 18-94 may be the opposite enantiomer of that represented). Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0430] Example 19 (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (19-97) (Figure 26)

[0431]

Chemical formula

[0432] (9H-Fluoren-9-yl)methyl (2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (19-95): To a mixture of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (16-83) in dichloromethane at 25 °C, scandium(III) trifluoromethanesulfonate and [[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate (18-91) are added in three portions over 1.5 hours. The reaction mixture is stirred at 30 °C for 36 hours, quenched with water, and extracted with dichloromethane. The combined organic phases are washed with brine, dried over Na2SO4, filtered, concentrated, and the residue is purified by silica gel flash column chromatography eluting with 10% to 100% dichloromethane in ethyl acetate to give (9H-fluoren-9-yl)methyl (9H-fluoren-9-yl)methyl (2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (19-95).

[0433] 2-Amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (19-96): Piperidine was added to a mixture of (9H-fluoren-9-yl)methyl(9H-fluoren-9-yl)methyl(2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (19-95) in N,N-dimethylformamide. The reaction mixture was stirred at 0 °C for 1 hour, filtered, and the filtrate was purified by preparative HPLC to give 2-amino-N-((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (19-96).

[0434] (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (19-97): (2S)-2-[[2-[[2-[3-[4-(2,5-Dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoic acid (18-90) (see Example 18) in N,N-dimethylformamide, 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)acetamide (19-96), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate, and 4-methylmorpholine were stirred at 25 °C for 1 hour, filtered, and the filtrate was purified by preparative HPLC to obtain (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamide)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)methoxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (19-97) (Compound 19-97 may be the opposite enantiomer of that represented). Note: The stereochemistry at the F-ring carbon is arbitrarily assigned.

[0435] Example 20 CTG assay of payloads (Jeko-1 and MDA-MB-468) The CTG assay is a method for determining the number of viable cells in a culture based on the quantification of the ATP present, which is an indicator of metabolically active cells. The cell assay requires the addition of a single reagent, Cell Titer Glo, in which case the cells are lysed and a luminescent signal is generated. The luminescent signal is proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. In this assay, it was ensured that the cells were in the logarithmic phase for either Jeko-1 or MDA-MB-468. The cells were transferred to 96-well plates and treated with a 3-fold serial dilution (10-point dilution) of the compound starting from 1 mM down to 0.0000508 mM for 72 hours. Cell viability was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. The percentage of viable cells at each compound concentration was determined by normalizing to the luminescence of the vehicle control and plotted as a percentage of the viability vs. dose response curve by non-linear fitting in GraphPad Prism software. The compound IC 50 was calculated as the concentration of the compound that killed 50% of the cells. Representative assay results are summarized in Tables 1 and 2.

[0436] Example 21 Human hepatocyte clearance (HHEP CL) Human hepatocytes in Williams E medium (from 10 male and female human donors, final concentration 0.5×10 6A suspension (cells / mL) was incubated for 90 minutes with a test compound (0.90% acetonitrile and 0.10% DMSO, final concentration 1 mM) and a positive control (7-ethoxycoumarin, 7-hydroxycoumarin, 0.90% acetonitrile, and 0.10% DMSO, final concentration 3 mM) while constantly shaking at 37 °C and approximately 600 rpm in an incubator with 5% CO2 and 95% humidity. The total volume of the incubation was 200 μl. Samples (25 μL) were taken out at T0, 15, 30, 60, and 90 minutes and added to ice-cold stop solution (acetonitrile containing 200 ng / mL of tobutamide and labetalol as internal standards) (125 μl) in the middle, vortexed at 500 rpm for 10 minutes, and centrifuged at 3220×g at 4 °C for 20 minutes. The analysis plate was sealed and stored at 4 °C until LCMS analysis. The hepatocyte viability at pre-incubation was 84.5%. Representative assay results are summarized in Tables 1 and 2.

[0437] Example 22 Human liver microsome clearance (HLM CL) The working solution was prepared by adding 5 μL of the compound and the control stock solution (10 mM in dimethyl sulfoxide, DMSO) to 495 μL of acetonitrile (ACN) (intermediate solution concentration: 100 μM, 99% CAN and 1% DMSO). The microsome working solution at the appropriate concentration was prepared in 100 mM potassium phosphate buffer. The reaction plate containing the mixture of the compound and microsomes was pre-incubated at 37 °C for 10 minutes, and then 98 mL of a solution of 2 mM NADPH and 2 mM MgCl2 was added to initiate the reaction. The final concentrations of the incubation medium were as follows. Microsomes - 0.5 mg protein / mL, test compound / control compound - 1 mM, NADPH - 1 mM, MgCl2 - 1 mM, acetonitrile 0.99%, DMSO 0.01%. Incubation was carried out at 37 °C for 60 minutes. Samples were taken at T0, T5, T15, T30, T45, and T60 and added to ice-cold stop solution (acetonitrile containing 200 ng / mL of tobutamide and labetalol as internal standards) (125 μl) in the middle, shaken for 10 minutes, and centrifuged at 4000 rpm at 4 °C for 20 minutes. The analytical plate was analyzed by LCMS. Representative assay results are summarized in Tables 1 and 2.

[0438] The human liver microsome clearance assay evaluates metabolism by the cytochrome P450 system (phase I enzymes). These enzymes oxidize the substrate by incorporating an oxygen atom into the hydrocarbon, thus causing the introduction of a hydroxyl group, or N-, O-, and S-dealkylation of the substrate, forming more polar products that are more easily removed. The human hepatocyte clearance assay more broadly measures the overall cellular metabolism (phase I and phase II enzyme pathways) of the test compound. Phase II enzymes catalyze conjugation reactions of metabolites of foreign substances with charged species, such as glutathione, sulfate, glycine, or glucuronic acid, to form more polar compounds for easier clearance.

[0439] Payloads with higher intrinsic clearance may provide a better therapeutic index due to their potentially lower systemic plasma exposure. (Maderna, A.; Doroski, M; Subramanyam, C.; Porte, A.; Leverett, C. A.; Vetelino, B. C.; Chen, Z.; Risley, H.; Parris, K.; Pandit, J.; Varghese, A. H.; Shanker, S.; Song, C.; Sukuru, S. C. K.; Farley, K. A.; Wagenaar, M. M.; Shapiro, M. J.; Musto, S.; Lam, M-H.; Loganzo, F.; O’Donnell, C. J. “Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications” Journal Medicinal Chemistry, 2014, 57, 10527-10543). In Table 1, payloads with higher intrinsic clearance are likely to have an improved safety profile because payloads that are potentially toxic to healthy cells are rapidly removed from the plasma, reducing the opportunity for interaction with healthy cells.

[0440] Example 23 PAMPA (Parallel Artificial Membrane Permeability Assay) PAMPA is a method for determining the permeability of substances from a donor compartment, through a lipid-infused artificial membrane, into an acceptor compartment. See Ottaviani, G.; Martel, S.; Carrupt, P-A. “Parallel Artificial Membrane Permeability Assay: A New Membrane for the Fast Prediction of Passive Human Skin Permeability”, Journal of Medicinal Chemistry, 2006, 49 (13), 3948-3954. A multiwell microtiter plate is used as the donor, with the membrane / acceptor compartment placed on top, and the whole assembly is commonly referred to as a “sandwich”. At the start of the test, the drug is added to the donor compartment and the acceptor compartment is drug-free. After an incubation period that may include agitation, the sandwich is separated and the amount of drug is measured in each compartment. Mass balance enables calculation of the drug remaining in the membrane.

[0441] PAMPA was performed by Pion Inc. using GIT-0 lipid and a 5 μM donor solution in PRISMA buffer (containing 0.05% DMSO) at pH 5.0 and pH 7.4. Higher PAMPA data have been associated with better bystander killing. (Ogitani Y.; Hagihara K.; Oitate, M.; Naito, H.; Agatsuma T. “Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity” Cancer Science, 2016, 107 (7), 1039-1046).

[0442] Representative assay results are summarized in Tables 1 and 2. Higher permeability is important because it means a greater likelihood of "bystander killing." That is, when the payload neutralizes a tumor cell, a more permeable payload is more likely to escape the neutralized tumor cell and then become embedded in adjacent tumor cells. Once there, it can neutralize the tumor cell, escape, and become embedded in another adjacent tumor cell, repeating this process.

[0443] [Table 1]

[0444] [Table 2]

[0445] Example 24 Development of Anti-ROR-1 Specific Monoclonal Antibodies By using an antibody development campaign that employed three strategies, novel and diverse anti-ROR-1 specific monoclonal antibodies that bind to multiple regions of the ROR-1 extracellular domain (ECD) were developed: (1) Mice in cohort 1 were immunized with full-length ROR-1 ECD, (2) Mice in cohorts 2 and 3 were immunized with the ROR-1 IgG-like domain, and (3) Mice in cohort 4 were immunized with a short region of the human IgG-like sequence of ROR-1. After immunizing the mice, monoclonal antibodies were generated using conventional approaches. Briefly, unique variable heavy and light chain pairs from hybridoma and phage display campaigns were cloned into a vector designed to express full-length antibodies as IgG in HEK293 cells under the control of the CMV promoter. The antibody expression vector was complexed with polyethyleneimine and transfected into HEK293 cultures. After shaking at 37°C for 5 days in 293 cell culture medium, the antibody was captured on an agarose-based protein A resin. After several stringent washes, the antibody was eluted in glycine solution (pH 3), neutralized with Hepes (pH 9), and the buffer was exchanged to PBS.

[0446] Using these approaches, several monoclonal antibodies were developed and the produced antibodies were subjected to further screening to evaluate the specific characteristics of the antibodies. To fully evaluate the characteristics of the novel antibodies, several assays were performed. First, the antibody binding to the ROR-1 epitope was confirmed both biochemically and in ROR-1 positive cell lines. The binding specificity was biochemically evaluated by screening for binding to human ROR-2 protein and rodent ROR-1 protein, as well as in cell-based assays. Further screening parameters included analysis of antibody internalization, epitope binning against known anti-ROR-1 antibodies (UC961 and 4a5), binding to the human ROR-1 Ig-like domain, thermal shift, and evaluation of self-interaction by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS).

[0447] Example 25 Assay for Evaluating the Saturation Concentration and Human ROR-1 Binding Affinity of Anti-ROR-1 Specific Monoclonal Antibodies A cell binding saturation assay was developed to evaluate how well the anti-ROR-1 antibodies developed in Example 22 bound to the extracellular ROR-1 protein endogenously expressed on the cell lines. More specifically, the anti-ROR-1 monoclonal antibodies developed in Example 22, such as ATX-P-875, ATX-P-885, and ATX-P-890, were analyzed in a cell binding assay. Briefly, two ROR-1 positive cell lines, JeKo-1 and MDA-MB-468, were incubated with titration series concentrations of each antibody construct. The cells were then washed and subjected to secondary antibody staining and detection by flow cytometry. The mean fluorescence intensity (MFI) was determined by analysis with cytometer software. The binding of ATX-P-875, ATX-P-885, and ATX-P-890 was compared to the cell binding saturation data of the monoclonal anti-ROR-1 antibody UC-961. (See Figures 27A - 27B). As shown in Figures 27A - 27B, the cell binding saturation for the antibodies ATX-P-875, ATX-P-885, and ATX-P-890 was equivalent to that of UC-961, although a higher concentration of ATX-P-875 was required compared to UC-961 to achieve saturation. ATX-P-890 and ATX-P-885 were either comparable or improved compared to UC-961 at the concentrations required to achieve binding saturation. The comparable saturation to UC-961 demonstrates that the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 have an affinity for the human ROR-1 target similar to that of the clinically approved antibody UC-961.

[0448] Example 26 Assay for evaluating the ability of anti-ROR-1 specific monoclonal antibodies to internalize ROR-1 on human ROR-1 positive tumor cells After determining the saturation concentration (74 nM) in a binding assay, the anti-ROR-1 antibodies (ATX-P-875, P-885, P-890) developed herein were evaluated for their ability to internalize the ROR-1 receptor on human ROR-1 positive cells (JeKo-1 and MDA-MB-468). Briefly, ROR-1 positive cell lines were incubated with the antibodies under supersaturated conditions to bind to all available ROR-1 receptors. Excess antibody was washed away, and the cells were incubated for the specified time over a 4-hour time course at 37°C. At the end of each time point, internalization was stopped by placing a fixed aliquot of the cells on ice. Antibody remaining on the surface was detected using a labeled secondary antibody and flow cytometry. The percent internalization was calculated based on time 0, which was assumed to have 100% of the available receptors on the cell surface. The results in Figures 28A - 28B demonstrate that all antibodies internalize the ROR-1 receptor on JeKo-1 and MDA-MB-468 cells by at least a 75% decrease over 4 hours. Unexpectedly, in MDA-MB-468, the internalization of two anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) was improved over the clinically used UC-961 anti-ROR-1 antibody, providing evidence that the ATX-P-875 and ATX-P-890 antibodies have an improved ability to internalize the ROR-1 receptor from the surface of solid tumors.

[0449] Example 27 Epitope Binding Studies of Anti-ROR-1 Specific Monoclonal Antibodies Cell binning was also used to determine whether the monoclonal antibodies ATX-P-875, ATX-P-885, and ATX-P-890 bind to the same epitope as the previously known anti-ROR-1 binding monoclonal antibodies UC-961 and 4A5 (control). In step 1 of the cell binning experiment, the ATX-P-875, ATX-P-885, and ATX-P-890 monoclonal antibodies were incubated separately with ROR-1 expressing cells (MDA-MB-468) in various amounts. In step 2, a fluorescently labeled secondary antibody that recognizes the novel antibody was incubated with the samples. And finally, in step 3, the ROR-1 expressing cells coated with ATX-P-875, ATX-P-885, and ATX-P-890 were incubated with a saturating dose of labeled UC-961 (Dy650-UC 961) or 4A5 antibody (PE 4A5) and analyzed by flow cytometry. The UC-961 and 4A5 staining signals were then compared to the novel antibody staining signals to determine whether the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bind to the same epitope as the known ROR-1 binding antibodies UC961 and 4A5. Figure 29A shows the staining profile expected if the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bind to the same epitope as the UC-961 and 4A5 antibodies. Figure 29B shows the expected profile if the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bind to an epitope on ROR-1 different from the UC-961 or 4A5 antibodies. Briefly, when binding to the same epitope, increasing the concentration of the novel antibody blocks the binding of the pre-labeled competing antibody, thereby reducing the signal of the competitor at higher concentrations. When the antibodies bind to separate epitopes, each antibody (novel antibody and competing antibody) does not compete for binding to the receptor, so staining increases with increasing dose. The cell binning data obtained with MDA-MB-468 cells showed that ATX-P-885 bound significantly to the same epitope as UC-961 and both ATX-P-875 and ATX-P-890 bound significantly to the same epitope as 4A5. (See Figures 29C-29H and Figure 30).The ability of the antibodies developed herein to bind to different ROR-1 epitopes provides opportunities to modulate the target in various ways.

[0450] Example 28 Biochemical binding studies of anti-ROR-1 specific monoclonal antibodies Biochemical binding by SPR was also evaluated for anti-ROR1 antibodies (ATX-P-875, P-885, P-890) compared to control anti-ROR-1 antibodies UC-961 and 4a5. In these experiments, 10 μg / ml of purified cloned protein of Hu / Cy / Rh ROR1-His was covalently bound to an HC30M chip. Individual dilutions of each antibody at 10 μg / mL were injected onto the chip and binding was evaluated by Carterra SPR. Unexpectedly, the data demonstrated that there were three different binding epitopes among ATX-P-875, ATX-P-885, and ATX-P-890, and ATX-885 was the only antibody that conferred partial blockade to the UC-961 antibody (see Figure 31). Cellular binding evaluated only the ability of anti-ROR-1 antibodies (ATX-P-875, P-885, P-890) to block either of the two clinically used ROR-1 antibodies, UC-961 or 4a5. Biochemical SPR evaluations also tested the ability of antibodies to block each other and found that ATX-P-875 was able to block the binding of 4A5 as well as ATX-P-885, but was still unable to block UC-961.

[0451] Example 29 Antibody characterization The antibody characterization of ATX-P-875, ATX-P-885, and ATX-P-890 compared to UC-961 is summarized in FIGS. 31 and Tables 3-6. The initial assessment of antibody developability was performed by AC-SINS to evaluate the potential for self-interaction (FIG. 28). The control antibody rituximab shows a low shift as expected, while the control antibody infliximab shows a high shift as expected. The anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 developed herein do not show significant self-interaction and thus are consistent with control antibodies that do not pose a significant development risk. Furthermore, the binding characteristics of the monoclonal antibodies ATX-P-875, ATX-P-885, and ATX-P-890 were compared with those of UC961 in further experiments. Tables 3 and 4 provide the characterization data of this antibody compared to the known ROR-1 binding antibody UC-961, including biochemical binding to purified protein and the results shown in the table measured by SPR (Tables 3-4), cell binding (EC50) to ROR-1 positive cell lines JeKo-1 and MDA-MB-468 (Table 5), and cellular internalization (% internalization) (Table 6). Of particular note, the affinity of ATX-P-875 (KD: 1.09E-08), which is reduced compared to UC-961 and other anti-ROR-1 antibodies (ATX-P-885 and ATX-P-890), may provide an unexpected therapeutic benefit. By not binding as strongly to the ROR-1 epitope, the ATX-P-885 antibody is thought to be able to penetrate further into the tumor and reach cells further away that express the ROR-1 target.

[0452]

Table 3

[0453]

Table 4

[0454]

Table 5

[0455]

Table 6

[0456] Example 30 Preparation of Antibody-Drug Conjugates The synthesis of the immunoconjugate is achieved as described in this example. Antibodies are produced as described in Example 24 and suspended in PBS pH 7.2 at a protein concentration in the range of 10 - 20 mg / ml. A molecular weight of 150,000 Da was used for all antibodies for reduction and conjugation calculations.

[0457] Each antibody is prepared for reduction by the addition of 5% v / v of 500 mM Tris, 25 mM EDTA, pH 8.5, followed by the addition of TCEP (6 equivalents, 10 mM stock of TCEP in water), and the mixture is maintained at 20 °C for 2 hours. This reduction step forms cysteine residues Cys-SH on the antibody, facilitating bioconjugation with the toxin-linker, i.e., the compound of formula (III) described herein.

[0458] DMA is added and gently mixed with the above reduction protein solution to achieve a final 10% v / v during conjugation, after which the toxin-linker stock solution (12 equivalents, 50 mM in DMA) is added and gently mixed. The bioconjugation is allowed to proceed at 20 °C overnight, about 16 - 20 hours. The ring-opening of the maleimide ring is completed within 2 hours with an extended time that allows it.

[0459] The crude conjugate is buffer-exchanged into PBS pH 7.4 using gravity-fed NAP25 (small scale) or Flow HiPrep G25 (large scale), and the column is prepared and operated according to the manufacturer's (Cytivia) instructions. To remove residual toxins, a 100 mg / ml slurry of activated carbon (Sigma / C9157) in PBS pH 7.4 is prepared and added to achieve 1 mg of carbon per 1 mg of starting antibody mass. This is gently mixed for 2 hours to keep the carbon well suspended in the suspension. The carbon is then removed by centrifugation at 4000 g. Polysorbate 20 (PS20) is added from a 10% w / v stock solution in PBS pH 7.4 to achieve a final 0.02% PS20 w / v in the product. The antibody-drug conjugate (ADC) product is finally filtered through a 0.2 μm PES filter of appropriate size (Chromatography Direct / FIL-S-PES-022-13-100-S) under grade A laminar flow. The final product is analyzed as follows. Monomer and [ADC] mg / ml (by SEC HPLC), average DAR (by PLRP), residual toxins (by RP-HPLC), and endotoxin (by Endosafe kinetic chromogenic).

[0460] The analytical process was performed on an HPLC instrument Agilent 1100 or 1260.

[0461] Example 31 CTG assay of antibody-drug conjugate The novel ROR-1 antibody-drug conjugate (ADC) is evaluated by the CTG assay in a manner similar to that described in Example 20 and Tables 1 and 2 for payload screening. Without limiting the scope of the antibody-drug conjugates of the present disclosure, but merely by way of example, a total of three unique antibodies, ATX-P-875, ATX-P-885, and ATX-P-890, are conjugated to a novel linker / payload or to any of the compounds of Formula (III) described throughout paragraphs

[0121] to

[0123] beginning with "In various embodiments, the conjugate of Formula (III) can be represented by a structure selected from...", including but not limited to the exemplary compounds of Formula (III). Briefly, ROR-positive (JeKo-1 / MDA-MB-468) or ROR-negative (Ramos) cells are transferred to 96-well plates and treated with the test ADCs in 3-fold serial dilutions starting from 1 mM down to 0.0000508 mM (10-point dilution) for 72 hours. According to the manufacturer's instructions, the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) is used to analyze cell viability. The percentage of viable cells at each ADC concentration is determined by normalizing to the luminescence of the vehicle control and plotted as a percentage of the survival versus dose response curve by non-linear fitting in GraphPad Prism software. The IC 50 is calculated as the concentration of the compound that kills 50% of the cells and benchmarked against UC-961. The ADCs described herein, including those prepared by conjugating the antibodies ATX-P-875, ATX-P-885, and ATX-P-890 to any of the compounds 18-94, 19-97, and the exemplary compounds of Formula (III), have an IC 50 value of less than 500 nM (e.g., less than 300 nM, less than 100 nM, less than 50 nM, or less than 30 nM) based on the CTG assay using Jeko-1 or MDA-MB-468 cells.

[0462] Furthermore, although the above text has been described in some detail as figures and examples for clarity and understanding, it will be understood by those skilled in the art that numerous various modifications can be made without departing from the spirit of the present disclosure. Therefore, the forms disclosed herein are merely illustrative and are not intended to limit the scope of the present disclosure. On the contrary, it should be clearly understood that all modifications and alternative forms along the true scope and spirit of the present invention are included.

[0463] Sequence Listing SEQ ID NO: 1 ATX-P-875 VH CDR1 (Kabat) GFTFSNAW

[0464] SEQ ID NO: 2 ATX-P-875 VH CDR2 (Kabat) IKSKTDGGTT

[0465] SEQ ID NO: 3 ATX-P-875 VH CDR3 (Kabat) TTGPDDLDY

[0466] SEQ ID NO: 4 ATX-P-875 VH nt GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA

[0467] Accession number: 5 ATX-P-875 VH AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSS

[0468] Accession number: 6 ATX-P-875 HC IgG1-Fc nt

[0469] Accession number: 7 ATX-P-875 HC IgG1-Fc AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0470] Accession number: 8 ATX-P-875 VL CDR1 (Kabat) QSISSY

[0471] ATX-P-875 VL CDR2 (Kabat) AAS

[0472] Accession number: 10 ATX-P-875 VL CDR3 (Kabat) QQYDNLPIT

[0473] Accession number: 11 ATX-P-875 VL nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0474] Accession number: 12 ATX-P-875 VL AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIK

[0475] Accession number: 13 ATX-P-875 Kappa LC nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0476] Sequence number: 14 ATX-P-875 Kappa LC AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0477] Sequence number: 15 ATX-P-885 VH CDR1 (Kabat) GGSFSGYY

[0478] Sequence number: 16 ATX-P-885 VH CDR2 (Kabat) INHSGST

[0479] Sequence number: 17 ATX-P-885 VH CDR3 (Kabat) AREGVYEDY

[0480] Sequence number: 18 ATX-P-885 VH nt CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0481] Sequence number: 19 ATX-P-885 VH AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSS

[0482] Sequence number: 20 ATX-P-885 HC IgG1-Fc nt

[0483] Accession number: 21 ATX-P-885 HC IgG1-Fc AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0484] Accession number: 22 ATX-P-885 VL CDR1 (Kabat) QSVSNY

[0485] ATX-P-885 VL CDR2 (Kabat) DAY

[0486] Accession number: 24 ATX-P-885 VL CDR3 (Kabat) QQRSNWPLT

[0487] Accession number: 25 ATX-P-885 VL nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0488] Sequence number: 26 ATX-P-885 VL AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIK

[0489] Sequence number: 27 ATX-P-885 Kappa LC nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0490] Sequence number: 28 ATX-P-885 Kappa LC AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0491] Sequence number: 29 ATX-P-890 VH CDR1 (Kabat) GYTFTGYY

[0492] Sequence number: 30 ATX-P-890 VH CDR2 (Kabat) INPNSGGT

[0493] Sequence number: 31 ATX-P-890 VH CDR3 (Kabat) VRDQVQLERFDS

[0494] Sequence number: 32 ATX-P-890 VH nt CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0495] Sequence number: 33 ATX-P-890 VH AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSS

[0496] Sequence number: 34 ATX-P-890 HC IgG1-Fc nt

[0497] Accession number: 35 ATX-P-890 HC IgG1-Fc AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0498] Accession number: 36 ATX-P-890 VL CDR1 (Kabat) QDISNY

[0499] ATX-P-890 VL CDR2 (Kabat) DAS

[0500] Accession number: 38 ATX-P-890 VL CDR3 (Kabat) QQYDNLPPT

[0501] Accession number: 39 ATX-P-890 VL nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAA

[0502] Accession number: 40 ATX-P-890 VL AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIK

[0503] Accession number: 41 ATX-P-890 Kappa LC nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0504] Accession No.: 42 ATX-P-890 Kappa LC AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An immunoconjugate having formula (I), A[S]- １ -L ２ -L ３ -L ４ -L ５ -L ６ -L ７ -D] ｎ (I) During the ceremony, Ab is an antibody or its antigen-binding fragment. L １ is 【Chemistry 1】 And, L ２ Does it not exist? 【Chemistry 2】 And, Z １ and Z ２ These are, individually, hydrogen, halogen, and NO. ２ , -O-(C １ -C ６ Alkyl), or C １ -C ６ It is alkyl, L ３ is, -(CH ２ )n １ -C (=O)- or -(CH ２ CH ２ O)n １ - (CH ２ )n １ C (= O) - n １ These are independent integers from 0 to 12. L ４ It is a tetrapeptide residue, L ５ It does not exist, or -[NH(CH ２ )n ２ ]n ３ - and n ２ These are integers from 0 to 6, n ３ is an integer between 0 and 2, L ６ It does not exist, or 【Transformation 3】 And, L ７ Does it not exist? 【Chemistry 4】 And, D is the drug portion, and n is an integer between 1 and 10. D is the drug portion of formula (II) having the following structure: 【Transformation 5】 During the ceremony, R1 and R2 are each individually selected from the group consisting of hydrogen, halogen, -CN, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted -O-(C1-C6 alkyl), substituted or unsubstituted -O-(C1-C6 haloalkyl), and -[(CY2)pO(CY2)q]tCY3, or substituted or unsubstituted -O-(CR5R6)m-O-, thereby R1 and R2 together form a ring. R3 is selected from the group consisting of hydrogen, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted 4-membered or 5-membered heterocyclyl, and [(CY2)pO(CY2)q]tCY3. R4 is selected from the group consisting of substituted or unsubstituted -(C1-C6 alkyl)-X2, substituted or unsubstituted -(C1-C6 haloalkyl)-X2, substituted or unsubstituted -(C1-C6 alkenyl)-X2, substituted or unsubstituted -(C1-C6 haloalkenyl)-X2, substituted or unsubstituted -(C1-C6 alkynyl)-X2, and substituted or unsubstituted -(C1-C6 haloalkynyl)-X2. X2 is -OR 9, -SR 9, or -NHR 9, R5 and R6 are each individually substituted or unsubstituted C1-C6 alkyl groups, or R5 and R6, together with the nitrogen atoms to which they are bonded, form substituted or unsubstituted four-membered or five-membered heterocyclines. n4 and n5 are each 0, 1, or 2, respectively, except that both n4 and n5 cannot be 0. Each Y is individually either H or a halogen. Each m is either 1 or 2. Each p is 1, 2, 3, 4, 5, or 6, Each q is 0, 1, 2, 3, 4, 5, or 6, Each t is 1, 2, 3, 4, 5, or 6, R7 is H, -COR8, -CO2R8, -(CO)-NHR8, L4, L5, L6, or L7. R8 is a substituted or unsubstituted C1-C6 alkyl-X3, a substituted or unsubstituted C1-C6 haloalkyl-X3, or -[(CY2)pO(CY2)q]tCY2-X3, R9 is H, -COR8, -CO2R8, -(CO)-NHR8, L4, L5, L6, or L7, provided that strictly one of R7 and R9 is L4, L5, L6, or L7, and Each X3 is an immunoconjugate that is individually -H, -OH, -SH, or -NH2.

2. The immunoconjugate according to claim 1, wherein L4 is gly-gly-phe-gly (GGFG).

3. The immunoconjugate according to claim 1, wherein R1 is a C1-C3 alkyl group and R2 is a halogen group.

4. The immunoconjugate according to claim 1, wherein R1 is methyl and R2 is F.

5. The immunoconjugate of formula (I) is 【Chemistry 6-1】 【Chemistry 6-2】 An immunoconjugate according to claim 1, selected from the group consisting of the following.

6. The immunoconjugate of formula (I) is 【Chemistry 7-1】 【Chemistry 7-2】 An immunoconjugate according to claim 1, selected from the group consisting of the following.

7. The immunoconjugate of formula (I) is 【Chemistry 8-1】 【Chemistry 8-2】 An immunoconjugate according to claim 1, selected from the group consisting of the following.

8. The immunoconjugate of formula (I) is 【Chemistry 9-1】 【Chemistry 9-2】 An immunoconjugate according to claim 1, selected from the group consisting of the following.

9. The immunoconjugate of formula (I) is 【Chemistry 10】 An immunoconjugate according to claim 1, selected from the group consisting of the following.

10. A compound of formula (IV) having the following structure or a pharmaceutically acceptable salt thereof, 【Chemistry 11】 During the ceremony, R1 and R2 are each individually selected from the group consisting of hydrogen, halogen, -CN, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted -O-(C1-C6 alkyl), substituted or unsubstituted -O-(C1-C6 haloalkyl), and -[(CY2)pO(CY2)q]tCY3, or substituted or unsubstituted -O-(CR5R6)m-O-, thereby R1 and R2 together form a ring. R3 is selected from the group consisting of hydrogen, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted 4- or 5-membered heterocyclyl, and -[(CY2)pO(CY2)q]tCY3. R4 is selected from the group consisting of substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, and substituted or unsubstituted C2-C6 alkynyl. Alternatively, R3 and R4, together with the carbon atoms to which they are bonded, may form an unsubstituted four-membered, five-membered, or six-membered heterocycline, or a four-membered, five-membered, or six-membered heterocycline substituted with an optionally substituted C1-C3 alkyl group. R5 and R6 are each individually substituted or unsubstituted C1-C6 alkyl groups, or R5 and R6, together with the nitrogen atoms to which they are bonded, form substituted or unsubstituted four-membered or five-membered heterocyclines. n4 and n5 are each 0, 1, or 2, respectively, except that both n4 and n5 cannot be 0. Each Y is individually either H or a halogen. Each m is either 1 or 2. Each p is 1, 2, 3, 4, 5, or 6, Each q is 0, 1, 2, 3, 4, 5, or 6, and Each t is 1, 2, 3, 4, 5, or 6, R7 is H, -COR8, -CO2R8, or -(CO)-NHR8, and R8 is a compound or a pharmaceutically acceptable salt thereof, which is a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2)pO(CY2)q]tCY3.

11. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein R1 is a C1-C3 alkyl group and R2 is a halogen.

12. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein R1 is methyl and R2 is F.

13. The compound of formula (IV) is 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 A compound according to claim 10 or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

14. The compound of formula (IV) is 【Chemistry 13】 A compound according to claim 10 or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

15. The compound of formula (IV) is 【Chemistry 14-1】 【Chemistry 14-2】 A compound according to claim 10 or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

16. The compound of formula (IV) is 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 A compound according to claim 10 or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

17. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 1 to 9, or a compound according to any one of claims 10 to 16 or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

18. A pharmaceutical composition for use in a method for treating cancer or tumor in an individual requiring treatment of cancer or tumor, comprising an effective amount of the immunoconjugate described in any one of claims 1 to 9, or the compound described in any one of claims 10 to 16 or a pharmaceutically active salt thereof.

19. The pharmaceutical composition according to claim 18, wherein the cancer or tumor is selected from lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

20. A conjugate having formula (III), Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III) During the ceremony, Mi is 【Chemistry 16】 And, Does L2 not exist? 【Chemistry 17】 And, Z1 and Z2 are, respectively, hydrogen, halogen, NO2, -O-(C1-C6 alkyl), or C1-C6 alkyl. L3 is -(CH2)n1-C(=O)- or -(CH2CH2O)n1-(CH2)n1C(=O)-, n 1 is an independent integer between 0 and 12. L4 is a tetrapeptide residue, L 5 is either nonexistent or -[NH(CH2)n2]n3-, n² is an integer between 0 and 6. n 3 is an integer between 0 and 2, L 6 does not exist, or [Chemistry 18] And, Does L 7 not exist? 【Chemistry 19】 and D is the drug portion of formula (II) having the following structure: 【Chemistry 20】 During the ceremony, R1 and R2 are each individually selected from the group consisting of hydrogen, halogen, -CN, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted -O-(C1-C6 alkyl), substituted or unsubstituted -O-(C1-C6 haloalkyl), and -[(CY2)pO(CY2)q]tCY3, or substituted or unsubstituted -O-(CR5R6)m-O-, thereby R1 and R2 together form a ring. R3 is selected from the group consisting of hydrogen, -OR5, -NR5R6, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted 4-membered or 5-membered heterocyclyl, and [(CY2)pO(CY2)q]tCY3. R4 is selected from the group consisting of substituted or unsubstituted -(C1-C6 alkyl)-X2, substituted or unsubstituted -(C1-C6 haloalkyl)-X2, substituted or unsubstituted -(C1-C6 alkenyl)-X2, substituted or unsubstituted -(C1-C6 haloalkenyl)-X2, substituted or unsubstituted -(C1-C6 alkynyl)-X2, and substituted or unsubstituted -(C1-C6 haloalkynyl)-X2. X2 is -OR 9, -SR 9, or -NHR 9, R5 and R6 are each individually substituted or unsubstituted C1-C6 alkyl groups, or R5 and R6, together with the nitrogen atoms to which they are bonded, form substituted or unsubstituted four-membered or five-membered heterocyclines. n4 and n5 are each 0, 1, or 2, respectively, except that both n4 and n5 cannot be 0. Each Y is individually either H or a halogen. Each m is either 1 or 2. Each p is 1, 2, 3, 4, 5, or 6, Each q is 0, 1, 2, 3, 4, 5, or 6, Each t is 1, 2, 3, 4, 5, or 6, R7 is H, -COR8, -CO2R8, -(CO)-NHR8, L4, L5, L6, or L7. R8 is a substituted or unsubstituted C1-C6 alkyl-X3, a substituted or unsubstituted C1-C6 haloalkyl-X3, or -[(CY2)pO(CY2)q]tCY2-X3, R9 is H, -COR8, -CO2R8, -(CO)-NHR8, L4, L5, L6, or L7, provided that strictly one of R7 and R9 is L4, L5, L6, or L7, and Each X³ is a conjugate that is individually -H, -OH, -SH, or -NH².

21. The conjugate according to claim 20, wherein L4 is gly-gly-phe-gly (GGFG).

22. The conjugate according to claim 20, wherein R1 is a C1-C3 alkyl group and R2 is a halogen.

23. The conjugate according to claim 20, wherein R1 is methyl and R2 is F.

24. The conjugate having formula (III) 【Chemistry 21-1】 【Chemistry 21-2】 A conjugate according to claim 20, selected from the group consisting of the following.

25. The conjugate having formula (III) 【Chemistry 22-1】 【Chemistry 22-2】 A conjugate according to claim 20, selected from the group consisting of the following.

26. The conjugate having formula (III) 【Chemistry 23-1】 【Chemistry 23-2】 A conjugate according to claim 20, selected from the group consisting of the following.

27. ​​The conjugate having formula (III) 【Chemistry 24-1】 【Chemistry 24-2】 A conjugate according to claim 20, selected from the group consisting of the following.

28. The conjugate having formula (III) 【Chemistry 25】 A conjugate according to claim 20, selected from the group consisting of the following.

Images