Exatecan immunoconjugate

Abstract

The immunoconjugate of formula (I), or a pharmaceutically acceptable salt thereof, includes a linking group for linking the antibody targeting ligand (Ab) to a drug (D). Such immunoconjugate embodiments are useful for delivering drugs to selected cells or tissues, for example, to treat, inhibit, or alleviate cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 481,567, filed January 25, 2023, the contents of which are incorporated by reference herein in their entirety.

[0002] Array Statements This application contains a Sequence Listing which has been filed electronically and is incorporated herein by reference in its entirety. The Sequence Listing was created on January 23, 2023, is named ZENO_161PR, and is 46kb in size. [Background technology]

[0003] Field This application relates to conjugates that include a linking group for linking an antibody targeting ligand to a cell-lethal moiety (such as a drug), methods of making such conjugates, and methods of using such conjugates to deliver a cell-lethal moiety to selected cells or tissues, for example, to treat or inhibit cancer.

[0004] explanation Many 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 the ADC functions as a targeting agent to deliver the drug to selected cells or tissues, such as cancer cells or tumors. In the United States, the Food and Drug Administration (FDA) has approved the use of inotuzumab ozogamicin (trade name BESPONSA), gemtuzumab ozogamicin (trade name MYLOTARG), brentuximab vedotin (trade name ADCETRIS), ado-trastuzumab emtansine (trade name KADCYLA), mirvetuximab soravtansine-gynx (Elahere™), tisotumab vedotin-tftv (Tivdak™), loncatuximab tesirine- There are several approved ADC formulations, including lpyl (Zynlonta®), sacituzumab govitecan (Trodelvy®), trastuzumab deruxtecan (Enhertu®), enfortumab vedotin (Padcev®), polatuzumab vedotin-piiq (Polivy®), moxetumomab pasudotox (Lumoxiti®), and inotuzumab ozogamicin (Besponsa®).

[0005] U.S. Patent No. 10,155,821 discloses an ADC in which an antitumor compound is conjugated to an anti-HER2 antibody via a linker. See also U.S. Patent Application Publication Nos. 2020 / 0385486 and 2019 / 0077880. Trastuzumab deruxtecan is an example of an ADC in which an anti-HER2 antibody (trastuzumab) is conjugated to an antitumor compound (Dxd) 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 prior anti-HER2-based regimens in the metastatic setting. Figure 1 shows how the linker may connect the antibody (mAb) to the drug moiety. Summary of the Invention [Problem to be solved by the invention]

[0006] Although FDA approval marks a milestone in the ongoing development of therapeutic ADCs, there remains a need for improved ADCs that help address the long-felt demand for additional options to treat cancer and / or deliver therapeutic payloads to selected cells or tissues. [Means for solving the problem]

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

[0008] One embodiment is a compound of formula (I) Ab-[SL 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I) or a pharmaceutically acceptable salt thereof. [wherein Ab is an antibody or antigen-binding fragment; L 1 teeth,

[0009] [ka] and L 2 does not exist or

[0010] [ka] and Z 1 and Z 2 are each independently hydrogen, halogen, —NO2, —O—(C1-C6 alkyl), or C1-C6 alkyl; L 3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-; n 1 are independently integers from 0 to 12; L 4 is a tetrapeptide residue; L 5 is absent or -[NH(CH2)n 2 ]n 3 - and; n 2 is an integer between 0 and 6; n 3 is an integer between 0 and 2; L 6 does not exist or

[0011] [ka] and L 7 does not exist or

[0012] [ka] and D is a drug moiety; and n is an integer from 1 to 10. to provide.

[0013] In one embodiment, D in formula (I) has the structure:

[0014] [ka] [In the formula, R 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, such that R 1 and R 2 come together to form a ring; R 3 and R 4 is R 3 and R 4 are each individually hydrogen, -OH, -N3, -NH2, -NH(C=O)-CH2-R, provided that at least one of them is not hydrogen. 3D , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, and [(CY2) p O(CY2) q ] t and when C1-C6 alkyl or C2-C6 alkenyl is substituted, C1-C6 alkyl or C2-C6 alkenyl is selected from -OH and -NR 3BR 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); or R 3 and R 4 one of which is a substituted or unsubstituted -(C1-C6 alkyl)-X 2 or substituted or unsubstituted -(C2-C6 alkenyl)-X 2 and -(C1-C6 alkyl)-X 2 or -(C1-C6 alkenyl)-X 2 is substituted, -(C1-C6 alkyl)-X 2 or -(C1-C6 alkenyl)-X 2 -OH and -NR 3B R 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); R 3D are H, -CH3, -OH and -CH2Y 1 is selected from, where Y 1 is a halogen; X 2 -OR 9 , -SR 9 , or -NHR 9 and; R 5 and R 6 are each independently substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring; n 4 and n 5 Provided that at least one of the following is not 0, 4 and n 5are each individually 0, 1 or 2; each Y is individually H or halogen; each m is individually 1 or 2; each p is individually 1, 2, 3, 4, 5, or 6; each q is individually 0, 1, 2, 3, 4, 5, or 6; Each t is individually 1, 2, 3, 4, 5, or 6; R 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 and; R 8 is a 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 and R 9 is R 7 and R 9 One of them is definitely L 4 , L 5 , L 6 , or L 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 and; each X 3 are individually -H, -OH, -SH, or -NH2] or a pharmaceutically acceptable salt thereof.

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

[0016] [ka] [In the formula, R 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, so that R 1 and R 2 come together to form a ring; R 3 and R 4 is R 3 and R 4 are each individually hydrogen, -OH, -N3, -NH2, -NH(C=O)-CH2-R, provided that at least one of them is not hydrogen. 3D , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, and [(CY2) p O(CY2) q ] t and when C1-C6 alkyl or C2-C6 alkenyl is substituted, C1-C6 alkyl or C2-C6 alkenyl are selected from -OH and -NR 3B R 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); R 3D are H, -CH3, -OH and -CH2Y 1is selected from, where Y 1 is a halogen; R 5 and R 6 are each independently substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring; n 4 and n 5 is n 4 and n 5 are each individually 0, 1 or 2, provided that at least one of them is not 0; each Y is individually H or halogen; each m is individually 1 or 2; each p is individually 1, 2, 3, 4, 5, or 6; each q is individually 0, 1, 2, 3, 4, 5, or 6; Each t is individually 1, 2, 3, 4, 5, or 6; R 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 and; R 8 is a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3] The present invention provides a compound of formula (IV) having the 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 cancer or a 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 tumors.

[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 is a compound of formula (III): Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III) or a pharmaceutically acceptable salt thereof. [In the formula, Mi is,

[0022] [ka] and L 2 does not exist or

[0023] [ka] and Z 1 and Z 2 are each independently hydrogen, halogen, —NO2, —O—(C1-C6 alkyl), or C1-C6 alkyl; L3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-; n 1 are independently integers from 0 to 12; L 4 is a tetrapeptide residue; L 5 is absent or -[NH(CH2)n 2 ]n 3 - and; n 2 is an integer between 0 and 6; n 3 is an integer between 0 and 2; L 6 does not exist or

[0024] [ka] and L 7 does not exist or

[0025] [ka] and D is a drug moiety. to provide.

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

[0027] In one embodiment, Ab is a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:3 a heavy chain comprising: b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8; VLCDR2 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of AAS; and VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10 light chain containing an antibody or antigen-binding fragment thereof comprising: Provided are immunoconjugates, pharmaceutical compositions, methods of treatment, inhibition, or amelioration, uses, or processes of production described herein, wherein the antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0028] In one embodiment, Ab is a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15; VHCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; and VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17 a heavy chain comprising: b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 22; VLCDR2 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of DAY; and VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 24 light chain containing an antibody or antigen-binding fragment thereof comprising Provided are immunoconjugates, pharmaceutical compositions, methods of treatment, inhibition, or amelioration, uses, or processes of production described herein, wherein the antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0029] In one embodiment, Ab is a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 31 a heavy chain comprising: b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 36; VLCDR2 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of DAS; and VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 38 light chain containing an antibody or antigen-binding fragment thereof comprising: Provided are immunoconjugates, pharmaceutical compositions, methods of treatment, inhibition, or amelioration, uses, or processes of production described herein, wherein 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 and other embodiments are described in more detail below. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows trastuzumab deruxtecan antibody-drug conjugates. [Figure 2]FIG. 1 shows a reaction scheme for making compounds of formula (IV) where n4=2 and n5=0. [Figure 3A] FIG. 1 shows a reaction scheme for making immunoconjugates of formula (I). [Figure 3B] FIG. 1 shows a reaction scheme for the conjugate of formula (III). [Figure 4] FIG. 1 shows a reaction scheme for preparing compounds 1-11a, 1-11b, 1-11c, and 1-11d. [Figure 5] FIG. 1 shows reaction schemes for preparing compounds 2-15a, 2-15b, 2-15c, and 2-15d. [Figure 6] FIG. 1 shows a reaction scheme for preparing compounds 3-21a and 3-21b. [Figure 7] FIG. 1 shows a reaction scheme for preparing compounds 4-27a and 4-27b. [Figure 8] FIG. 1 shows a reaction scheme for preparing compounds 5-34a and 5-34b. [Figure 9] FIG. 1 shows a reaction scheme for preparing compounds 6-40a and 6-40b. [Figure 10] FIG. 1 shows a reaction scheme for making compounds 7-42a, 7-42b, 7-42c, and 7-42d. [Figure 11] FIG. 1 shows a reaction scheme for making compounds 8-47a, 8-47b, 8-47c, and 8-47d. [Figure 12] FIG. 1 shows a reaction scheme for making compounds 9-52a, 9-52b, 9-52c, and 9-52d. [Figure 13] FIG. 1 shows a reaction scheme for making an exemplary conjugate of formula (III). [Figure 14] FIG. 1 shows a reaction scheme for making compounds 10-60, exemplary conjugates of formula (III). [Figure 15]FIG. 1 shows reaction schemes for making compounds 10-59, which are exemplary intermediates in the preparation of exemplary conjugates of Formula (III). [Figure 16] Figure 1 shows cell-binding saturation data for anti-ROR-1 antibodies generated by the method described herein. The ROR-1-positive cell line JeKo-1 was incubated in a titration series with the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890, compared with the positive control antibody UC961. Cells were washed and stained with secondary antibodies. Cell-binding saturation was detected by flow cytometry and reported as mean fluorescence intensity (MFI). [Figure 17] Figure 1 shows ROR-1 receptor internalization data for the anti-ROR-1 antibodies ATX875, ATX-P-885, and ATX-P-890. The ROR-1-positive cell lines JeKo-1 and MDA-MB-468 were incubated with the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890, as well as the positive control antibody UC961, under supersaturating conditions to bind all available ROR-1 receptors. Cells were incubated at 37°C for four different time points (30 min, 1 h, 2 h, and 4 h) before internalization was stopped by washing and placing the cells on ice. Receptor internalization was determined by flow cytometry and reported as the percentage of receptor internalization compared to time zero. [Figure 18A] Figure 18A shows cell binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. Cell binning assays were performed to assess whether ATX-P-875, ATX-P-885, and ATX-P-890 bind to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. Figure 18A shows the staining profiles of antibodies that bind to the same epitope. [Figure 18B]Figure 18B shows cell binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. A cell binning assay was performed to assess whether ATX-P-875, ATX-P-885, and ATX-P-890 bind to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. Figure 18B shows the staining profiles of antibodies binding to different epitopes. ATX-P-875, ATX-P-885, and ATX-P-890 were separately incubated with various amounts of ROR-1+MDA-MB-468. The anti-ROR-1 antibodies were then fluorescently labeled with a secondary antibody. Finally, MDA-MB-468 cells coated with anti-ROR-1 antibodies were incubated with saturating doses of fluorescently labeled UC961 (Figure 18C) or 4A5 (Figure 18D) and analyzed by flow cytometry, and the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals were compared with the UC961 or 4A5 signals. [Figure 18C] Cell binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. Cell binning assays were performed to assess whether ATX-P-875, ATX-P-885, and ATX-P-890 bind to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. ATX-P-875, ATX-P-885, and ATX-P-890 were separately incubated with various amounts of ROR-1+MDA-MB-468. The anti-ROR-1 antibodies were then fluorescently labeled with a secondary antibody. Finally, MDA-MB-468 cells coated with anti-ROR-1 antibodies were incubated with saturating doses of fluorescently labeled UC961 (Figure 18C) or 4A5 (Figure 18D) and analyzed by flow cytometry, and the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals were compared with the UC961 or 4A5 signals. [Figure 18D]Cell binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. Cell binning assays were performed to assess whether ATX-P-875, ATX-P-885, and ATX-P-890 bind to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. ATX-P-875, ATX-P-885, and ATX-P-890 were separately incubated with various amounts of ROR-1+MDA-MB-468. The anti-ROR-1 antibodies were then fluorescently labeled with a secondary antibody. Finally, MDA-MB-468 cells coated with anti-ROR-1 antibodies were incubated with saturating doses of fluorescently labeled UC961 (Figure 18C) or 4A5 (Figure 18D) and analyzed by flow cytometry, and the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals were compared with the UC961 or 4A5 signals. [Figure 19] Figure 1 shows AC-SINS data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. Antibody developability was assessed by performing AC-SINS assays to evaluate the ability for self-interaction. Rituximab and infliximab were used as controls and demonstrated low and high shifts, respectively. Assay results for ATX-P-875, ATX-P-885, and ATX-P-890 were within the range determined by the control antibodies. [Figure 20] Figure 1 shows SPR biochemical binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 compared to the control anti-ROR-1 antibodies UC961 (ATX-P-453) and 4a5. [Figure 21-1] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-2] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-3]FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-4] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-5] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-6] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-7] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-8] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. [Figure 21-9] FIG. 1 shows the nucleotide and amino acid sequences of anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890. DETAILED DESCRIPTION OF THE INVENTION

[0032] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, published applications and other articles referenced herein are incorporated by reference in their entirety unless otherwise stated. In the event that there are multiple definitions for terms herein, those in this section shall prevail unless otherwise stated.

[0033] As used herein, a "conjugate" is a compound comprising two or more substances (e.g., an antibody, a linker moiety, and / or a drug moiety) joined 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 comprising an immunological substance such as an antibody.

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

[0035] When 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 described as being "unsubstituted or substituted," if substituted, the substituent(s) may be selected from one or more of the specified substituents. If no substituents are specified, it means that the specified "optionally substituted" or "substituted" group can be substituted with one or more group(s) individually and independently selected from 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-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, amino, mono-substituted amine group, di-substituted amine group, mono-substituted amine(alkyl) and di-substituted amine(alkyl).

[0036] As used herein, "C" is a set of integers where "a" and "b" are integers. a ~C b " refers to the number of carbon atoms in the group. A specified group can contain "a" to "b" carbon atoms, inclusive. Thus, for example, a "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 range described by these definitions is assumed.

[0037] When two "R" groups are described as being "together," the R groups and the atoms to which they are attached may form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle. For example, but not limited to, an ortho R on a phenyl ring1 and R 2 The substituent is -O-(CR 5 R 6 ) m -O-, and therefore R 1 and R 2 are shown to "come together" to form a ring, -O-(CR 5 R 6 ) m -O- is R 1 and R 2 covalently attached to the phenyl ring at the position:

[0038] [ka] This means forming

[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 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. The alkyl group may have 1 to 30 carbon atoms (as found herein, numerical ranges such as "1 to 30" refer 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., up to and including 30 carbon atoms, although this definition also covers occurrences of the term "alkyl" where no numerical range is specified). The alkyl group may also be a medium-sized alkyl having 1 to 12 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may be substituted or unsubstituted. Alkyl groups are typically monovalent unless the context dictates otherwise. For example, one skilled in the art would recognize a C1-C6 alkyl group having the following formula: -(C1-C6 alkyl)-X 2 Recognize that it is bivalent in

[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. An alkylene group is:

[0041] [ka] , then the number of carbon atoms, then " * For example,

[0042] [ka] represents ethylene. An alkylene group can have 1 to 30 carbon atoms (as used herein, numerical ranges such as "1 to 30" refer to each integer within the given range; for example, "1 to 30 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, although this definition also covers occurrences of the term "alkylene" where no numerical range is specified). An alkylene group can also be a medium-sized alkyl having 1 to 12 carbon atoms. An alkylene group can also be a lower alkyl having 1 to 4 carbon atoms. An alkylene group can be substituted or unsubstituted. For example, a lower alkylene group can be formed by replacing one or more hydrogens in the lower alkylene group and / or by C 3~6 Monocyclic cycloalkyl groups (e.g.,

[0043] [ka] ) by replacing both hydrogens on the same carbon.

[0044] As used herein, the term "alkenyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms containing carbon double bond(s), including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. Alkenyl groups can be unsubstituted or substituted.

[0045] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms containing a carbon triple bond(s), including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl, etc. Alkynyl groups can be unsubstituted or substituted.

[0046] The terms "halogen atom" or "halogen," as used herein, mean any one of the radioactive stable atoms in column 7 of the periodic table of the elements, such as fluorine, chlorine, bromine, and iodine.

[0047] As used herein, "heteroalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by 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 can be substituted or unsubstituted.

[0048] As used herein, "haloalkenyl" refers to an alkenyl group in which one or more hydrogen atoms are replaced by halogen (eg, 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 are replaced by halogen (eg, 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 are replaced by 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. A haloalkoxy can be substituted or unsubstituted.

[0051] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, and up to 18-membered monocyclic, bicyclic, and tricyclic ring systems in which carbon atoms, together with one to five heteroatoms, comprise the ring system. A heterocyclyl may contain one or more unsaturated bonds positioned in such a way that a completely delocalized pi-electron system does not occur throughout all rings. The heteroatom(s) are elements other than carbon, including, but not limited to, oxygen, sulfur, and nitrogen. A heterocycle may further contain one or more carbonyl or thiocarbonyl functional groups, so as to include within the definition oxo- and thio-systems, such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be fused, bridged, or joined together in a spiro fashion. As used herein, the term "fused" refers to two rings having two atoms and one common bond. As used herein, the term "bridged heterocyclyl" or "bridged heteroalicyclyl" refers to a compound in which a heterocyclyl or heteroalicyclyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings having one common atom, and the two rings are not connected by a bridge. Heterocyclyl and heteroalicyclyl groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), or 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. Furthermore, any nitrogen in a heteroalicyclyl can be quaternized. The heterocyclyl or heteroalicyclyl group can be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include, but are not limited to, 1,3-dioxin, 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, methyltriazolyl ... and benzo-fused analogs thereof (e.g., benzimidazolidinone, tetrahydroquinoline, thiamorpholine, thiazoline sulfoxide, thiamorpholine sulfone, and / or benzo-fused analogs thereof (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] Where the number of substituents is not specified (e.g., haloalkyl, haloalkenyl, haloalkynyl), one or more substituents can be present. For example, "haloalkyl" can include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" can 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 that has a single, unpaired electron such that the species containing the radical can be covalently bonded to another species. Thus, in this context, a radical is not necessarily a free group. Rather, a radical refers to a specific portion of a larger molecule. The term "radical" may be used interchangeably with the term "group."

[0054] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with an inorganic acid, such as a hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid (e.g., 2,3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting a compound with an organic acid, such as an aliphatic or aromatic carboxylic or sulfonic acid, for example, 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. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt, for example, an ammonium salt, an alkali metal salt, for example, sodium, potassium, or lithium salt, an alkaline earth metal salt, for example, calcium or magnesium salt, a carbonate salt, a bicarbonate salt, a salt with an organic base, for example, dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and amino acids such as arginine and lysine. In compounds of formula (I), those skilled in the art will recognize that when a salt is formed by protonation of a nitrogen-based group (e.g., NH), the nitrogen-based group may be associated with a positive charge (e.g., when NH is converted to NH + It is understood that the positive charge can be balanced by a negative counterion (e.g., Cl).

[0055] In any compound described herein having one or more chiral centers, unless the absolute stereochemistry is explicitly indicated, it is understood that each center may independently be in the R or S configuration, or a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, enantiomerically enriched, racemic, diastereomerically pure, diastereomerically enriched, or stereoisomeric mixtures. Furthermore, in any compound described herein having one or more double bond(s) that produce geometric isomers that may be defined as E or Z, it is understood that each double bond may independently be E or Z, or a mixture thereof. Similarly, it is understood that all tautomeric forms are intended to be included in any compound described.

[0056] It is understood that the compounds disclosed herein have an incomplete outermost shell, where the outermost shell is filled with hydrogen or its isotopes, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0057] It is understood that the compounds described herein can be isotopically labeled. It is understood that substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from superior metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Each chemical element represented in a compound structure can include any isotope of that element. For example, in a compound structure, a hydrogen atom may be expressly disclosed or understood as being present in the compound. At any position in a compound where a hydrogen atom can be present, the hydrogen atom may be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, reference to a compound herein encompasses all possible isotopic forms, unless the context clearly dictates otherwise.

[0058] It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, which include different crystalline packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and can be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, and alcoholates are formed when the solvent is alcohol. Furthermore, the compounds provided herein can exist in unsolvated as well as solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the compounds and methods provided herein.

[0059] Where a range of values ​​is provided, it is understood that the upper and lower limits, and each intervening value between the upper and lower limits of the range, are encompassed within the embodiments.

[0060] Terms and phrases used in this application, and variations thereof, unless expressly stated otherwise, particularly in the appended claims, should be construed as open-ended as opposed to limiting. As in the foregoing examples, the term "including" should be read to mean "including, without limitation," "including but not limited to," and the like; the term "comprising," as used herein, is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude further, unstated elements or method steps; the term "having" should be interpreted as "having at least"; and the term "includes" should be interpreted as "includes but is not limited to." The term "example" is used to provide illustrative examples of the items under discussion, not an exhaustive or limiting list thereof; use of terms such as "preferably," "preferred," "desired," or "desirable," and words of similar import, imply that a particular feature is critical, essential, or even important to structure or function, but should not be understood as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Furthermore, the term "comprising" is intended to be synonymous with 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 stated features or components, but may also include additional features or components.

[0061] With regard to the use of virtually any plural and / or singular term herein, those skilled in the art may convert from plural to singular and / or from singular to plural where appropriate to the context and / or application. Various singular / plural permutations may be expressly stated herein for clarity. The indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope.

[0062] It should be understood that the compound labels herein include similar numbers but may have letters associated therewith, and the identified compounds may be different (and unrelated). For example, compounds 2-15a are different compounds from compounds 2-15, but have similar ring structures.

[0063] compound Various embodiments disclosed herein include a polymerizable compound having the structure:

[0064] [ka] or a pharmaceutically acceptable salt thereof.

[0065] In various embodiments, R in formula (IV) 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 ) m-O-, such that R 1 and R 2 are taken together to form a ring. In one embodiment, R 1 and R 2 In one embodiment, at least one of R 1 and R 2 At least one of R 1 and R 2 In one embodiment, at least one of R 1 and R 2 In one embodiment, at least one of R 1 and R 2 At least one of the following is -OR 5 and R 5 is a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 1 and R 2 At least one of the groups is methoxy.

[0066] In one embodiment, R of formula (IV) 1 and R 2 At least one of the groups is -NR 5 R 6 and R 5 and R 6 are each independently substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring.

[0067] In one embodiment, R of formula (IV) 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 alkyl. 1 and R 2 At least one of R is C1-C3 alkyl. For example, in one embodiment, R 1 and R 2and at least one of R 1 and R 2 At least one of R is C1-C3 alkyl and the other is halogen. For example, in one embodiment, R 1 and R 2 At least one of is methyl and the other is fluoro.

[0068] In one embodiment, R of formula (IV) 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 haloalkyl. For example, in one embodiment, R 1 and R 2 and at least one of R is difluoromethyl. 1 and R 2 At least one of R is substituted or unsubstituted —O—(C1-C6 alkyl). For example, in one embodiment, R 1 and R 2 and at least one of R is methoxy. 1 and R 2 At least one of -[(CY2) p O(CY2) q ] t CY3. In one embodiment, R 1 and R 2 is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, and therefore R 1 and R 2 together to form a ring, -O-(CR 5 R 6 ) m The end of -O- is R 1 and R 2 The phenyl ring is covalently attached to the phenyl ring at position 1 to form a heterocyclyl ring.

[0069] In one embodiment, R of formula (IV) 1 and R 2 one of which is hydrogen and R 1and R 2 The other of R is halogen. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are hydrogen. 1 and R 2 None of these is hydrogen.

[0070] In one embodiment, R of formula (IV) 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently halogen. 1 and R 2 None of the above is a halogen.

[0071] In one embodiment, R of formula (IV) 1 and R 2 one of which is substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 alkyl. 1 and R 2 None of the is a substituted or unsubstituted C1-C6 alkyl.

[0072] In one embodiment, R of formula (IV) 1 and R 2 one of which is a substituted or unsubstituted C1-C6 haloalkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 Any of these is a substituted or unsubstituted C1-C6 haloalkyl.

[0073] In one embodiment, R of formula (IV) 1 and R 2 In one embodiment, one of R 1 and R 2 and R are independently substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, and therefore R 1 and R 2 are taken together to form a ring. In various embodiments, R 1 and R 2 are each individually selected from the group consisting of hydrogen, fluoro, methoxy, methyl, difluoromethyl, and —O—(CH)—O—, such that R 1 and R 2 come together to form a ring.

[0074] In various embodiments, R in formula (IV) 3 is R 3 and R 4 is hydrogen, -OH, -N3, -NH2, -NH(C=O)CH3, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or [(CY2) p O(CY2) q ] t OH. In one embodiment, R 3 is —OH. In one embodiment, R 3 is -N3. In one embodiment, R 3 is —NH. In one embodiment, R 3 is -NH(C=O)CH2-R 3D and R 3D are H, -CH3, -OH, and -CH2Y 1 and Y 1 is a halogen. For example, R 3 is -NH(C=O)-CH3, -NH(C=O)-CH2CH3, -NH(C=O)-CH2OH, -NH(C=O)-CH2CH2-Y 1 In some embodiments, Y 1 can be F or Cl. In one embodiment, R 3 is unsubstituted C1-C6 alkyl. In one embodiment, R 3is a substituted C1-C6 alkyl. Examples of C1-C6 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched). In one embodiment, R 3 is a substituted C2-C6 alkenyl. 3 is unsubstituted C2-C6 alkenyl. 3 is a substituted C1-C6 alkyl or a substituted C2-C6 alkenyl, the C1-C6 alkyl and / or C2-C6 alkenyl may each independently be -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 3 -OH and -NR 3B R 3C For example, in one embodiment, R 3 is methyl, -CHOH, -CHCHOH, -CHCH(OH)CHOH, -CH(NH))(CHOH), -CH(NH)(CHCHOH), -CH(NH(CH))(CHOH), -CH(NH(CH))(CHCHOH), -CH(N(CH))(CHOH), -CH(N(CH))(CHCHOH), -CH(NH(isopropyl))(CHOH), -CH(NH(isopropyl))(CHCHOH), -CHCH=CH, -CH(NH-(C(=O)CH))(CHOH), -CH(NH-(C(=O)CH))(CHCHOH), and -CH(NH-(C(=O)CH))(CHCH=CH). In some embodiments, R 3 -OH and -NR 3B R 3C In some embodiments, R3 is —C(═O) (unsubstituted C1-C6 alkyl) substituted with a substituted C1-C6 alkyl. Examples of suitable C1-C6 alkyls are described herein. In some embodiments, R 3 is a substituted C1-C6 alkyl substituted with one or more OH groups (e.g., 1, 2, 3, or 4 OH groups). 3 is -[(CY2) p O(CY2) q ] t OH. R 3 An exemplary -[(CY2) p O(CY2) q ] t OH groups include -CH2OCH2CH2OH.

[0075] In various embodiments, R in formula (IV) 4 is R 3 and R 4 is hydrogen, -OH, -N3, -NH2, -NH(C=O)CH3, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or [(CY2) p O(CY2) q ] t OH. In one embodiment, R 4 is hydrogen. In one embodiment, R 4 is —OH. In one embodiment, R 4 is -N3. In one embodiment, R 4 is —NH. In one embodiment, R 4 is -NH(C=O)CH2-R 3D and R 3D are H, -CH3, -OH, and -CH2Y 1 and Y 1 is a halogen. For example, R 4 is -NH(C=O)-CH3, -NH(C=O)-CH2CH3, -NH(C=O)-CH2OH, -NH(C=O)-CH2CH2-Y 1 In some embodiments, Y 1 can be F or Cl. In one embodiment, R4 is unsubstituted C1-C6 alkyl. In one embodiment, R 4 is a substituted C1-C6 alkyl. Examples of C1-C6 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched). In one embodiment, R 4 is a substituted C2-C6 alkenyl. 4 is unsubstituted C2-C6 alkenyl. 4 is a substituted C1-C6 alkyl or a substituted C2-C6 alkenyl, the C1-C6 alkyl and / or C2-C6 alkenyl may each independently be -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 4 -OH and -NR 3B R 3C For example, in one embodiment, R 3 is methyl, -CHOH, -CHCHOH, -CHCH(OH)CHOH, -CH(NH))(CHOH), -CH(NH)(CHCHOH), -CH(NH(CH))(CHOH), -CH(NH(CH))(CHCHOH), -CH(N(CH))(CHOH), -CH(N(CH))(CHCHOH), -CH(NH(isopropyl))(CHOH), -CH(NH(isopropyl))(CHCHOH), -CHCH=CH, -CH(NH-(C(=O)CH))(CHOH), -CH(NH-(C(=O)CH))(CHCHOH), and -CH(NH-(C(=O)CH))(CHCH=CH). In some embodiments, R 4 -OH and -NR 3B R3C In some embodiments, R 4 is —C(═O) (unsubstituted C1-C6 alkyl) substituted with a substituted C1-C6 alkyl. Examples of suitable C1-C6 alkyls are described herein. In some embodiments, R 4 is a substituted C1-C6 alkyl substituted with one or more OH groups (e.g., 1, 2, 3, or 4 OH groups). 4 is [(CY2) p O(CY2) q ] t OH. R 4 An exemplary -[(CY2) p O(CY2) q ] t OH groups include -CH2OCH2CH2OH.

[0076] In some embodiments, R of formula (IV) 3 and R 4 is hydrogen, and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 is hydrogen, and R 3 and R 4 and the other of R is a substituted C1-C6 alkyl. 3 and R 4 is hydrogen, and R 3 and R 4 The other of the groups is -OH and -NR 3B R 3C is a substituted C1-C6 alkyl substituted by 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). In some embodiments, R 3 and R 4 is hydrogen, and R 3 and R4 and the other of R is a substituted or unsubstituted C alkenyl. 3 and R 4 one of which is -N3 and R 3 and R 4 and the other of R is a substituted or unsubstituted C alkenyl. 3 and R 4 one of R is -OH; 3 and R 4 In still yet other embodiments, the other of R in formula (IV) is substituted or unsubstituted C alkenyl. 3 and R 4 one of which is -NH2 and R 3 and R 4 The other of R is a substituted or unsubstituted C alkenyl. For example, R in formula (IV) 3 and R 4 one of which is -OH, -N3, -NH2, or -NH(C=O)-CH2-R 3D and R 3 and R 4 The other of the R groups in formula (IV) is -CHCH=CH. 3 and R 4 is hydrogen, and R 3 and R 4 and the other of R is -CH(NH))(CHOH), -CH(NH)(CHCHOH), -CH(NH(CH))(CHOH), -CH(NH(CH))(CHCHOH), -CH(N(CH))(CHOH), -CH(N(CH))(CHCHOH), -CH(NH(isopropyl))(CHOH), -CH(NH(isopropyl))(CHCHOH), -CH(NH-(C(=O)CH))(CHOH), -CH(NH-(C(=O)CH))(CHCHOH), or -CH(NH-(C(=O)CH))(CHCH=CH). In some embodiments, R of formula (IV) 3 and R 4 one of R is -OH; 3and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 one of which is -NH2 and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 One of the groups is -NH(C=O)-CH2-R 3D and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 one of R is -OH; 3 and R 4 The other of R in formula (IV) is a hydroxy-substituted C1-C6 alkyl, such as -CH2OH, -CH2CH2OH, and -CH2CH(OH)CH2OH. 3 and R 4 one of which is -NH2 and R 3 and R 4 The other of R is a hydroxy-substituted C1-C6 alkyl, such as -CH2OH, -CH2CH2OH, and -CH2CH(OH)CH2OH. As provided herein, in some embodiments, one or more hydroxy groups can be present on the hydroxy-substituted C1-C6 alkyl, such as 1, 2, 3, or 4 OH groups. In some embodiments, R 3 is -OH, R 4 is the unsubstituted C 1~6 Alkyl, e.g., not methyl; R 4 is -OH, R 3 is the unsubstituted C 1~6 In some embodiments, R 3 is -NH2, R 4 is the unsubstituted C 1~6 alkyl, e.g., methyl, but not R 3 If is NH2, R 4 is the unsubstituted C1~6 In some embodiments, R 3 and R 4 R, provided that at least one of them is not -CH3. 3 and R 4 In some embodiments, one of R 3 and R 4 are not each unsubstituted C1-C6 alkyl, e.g., CH3. In some embodiments of this paragraph, R 1 may be substituted or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched); R 2 In some embodiments of this paragraph, R 1 may be unsubstituted C1-C6 alkyl; R 2 may be halogen (e.g., F or Cl). In some embodiments of this paragraph, R 7 can be H.

[0077] In some embodiments, the compound of formula (IV) or a pharmaceutically acceptable salt thereof has the structure of formula (IV-a), or a pharmaceutically acceptable salt thereof:

[0078] [ka] [In the formula, R 1 , R 2 , R 3A and R 7 is as provided herein in formula (IV); R 3 is -OH, -CH3, -NH2 or -NH(C=O)CH3; b is individually 1, 2, or 3] may have.

[0079] In some embodiments, each R 3Amay independently be OH. In some embodiments, each R 3A can independently be H. In some embodiments, each R 3A are independently -NR 3B R 3C where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). For example, —NR 3B R 3C can be -NH, -NHAc, -NHCH(CH), or -N(CH). In some embodiments of this paragraph, b can be 1. In other embodiments of this paragraph, b can be 2. In still other embodiments of this paragraph, b can be 3.

[0080] In some embodiments, the compound of formula (IV) is R 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, so that R 1 and R 2 together form a ring; R 3 and R 4 is R 3 and R 4 are each individually hydrogen, -OH, -N3, -NH2, -NH(C=O)-CH2-R, provided that at least one of them is not hydrogen. 3D , substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, and [(CY2) p O(CY2) q ]t and when C1-C6 alkyl or C2-C6 alkenyl is substituted, C1-C6 alkyl or C2-C6 alkenyl is selected from -OH and -NR 3B R 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); R 3D are H, -CH3, -OH and -CH2Y 1 is selected from, where Y 1 is a halogen; R 5 and R 6 are each independently substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring; n 4 and n 5 Provided that at least one of the following is not 0, 4 and n 5 are each independently 0, 1, or 2; each Y is independently H or halogen; each m is independently 1 or 2; each p is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 1, 2, 3, 4, 5, or 6; R 7 H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 and;R 8 is a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t It's CY3.

[0081] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt thereof, is:

[0082] [ka]

[0083] [ka] [In the formula, R 1 , R 2 , R 3B , R 3C and R 7 In some embodiments of this paragraph, R 3B and R 3C and may each be hydrogen. In other embodiments of this paragraph, R 3B and R 3C may be hydrogen, and R 3B and R 3C and the other of R may be a substituted C1-C6 alkyl. 3B and R 3C may be hydrogen, and R 3B and R 3C The other of R may be an unsubstituted C1-C6 alkyl. 3B and R 3C may be hydrogen, and R 3B and R 3C The other of R may be -C(=O) (unsubstituted C1-C6 alkyl). 3B and R 3C one of which may be unsubstituted C1-C6 alkyl, and R 3B and R 3C The other of the may be -C(=O) (unsubstituted C1-C6 alkyl). Suitable C1-C6 alkyls include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (straight chain or branched), and hexyl (straight chain or branched).

[0084] In various embodiments, R in formula (IV) 7 H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 and R 8 is as described elsewhere herein. In one embodiment, R 7 is H. In one embodiment, R 7 -COR 8 In one embodiment, R 7 -CO2R 8 In one embodiment, R 7 is -(CO)-NHR 8 is.

[0085] In various embodiments, R in formula (IV) 8 is a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3, and the variables p, q, t, and Y are as described elsewhere herein. 8 is a substituted or unsubstituted C1-C6 alkyl. In one embodiment, R 8 is a substituted or unsubstituted C1-C6 haloalkyl. In one embodiment, R 8 is -[(CY2) p O(CY2) q ] t It's CY3.

[0086] In various embodiments, m in formula (IV) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

[0087] In various embodiments, n in formula (IV) 4 and n 5 is n 4 and n 5 are each individually 0, 1, or 2, provided that at least one of is not 0. In one embodiment, n 4 and n 5 and n are both 1. In one embodiment, 4is 0, and n 5 is 1. In one embodiment, n 4 is 0, and n 5 is 2. In one embodiment, n 4 is 1 and n 5 is 0. In one embodiment, n 4 is 2 and n 5 is 0.

[0088] In various embodiments, each Y in formula (IV) is individually H or halogen. In one embodiment, each Y is hydrogen. In one embodiment, -CY2 is -CH2. In one embodiment, -CY3 is -CH3. In one embodiment, -CY3 is -CHF2. In one embodiment, -CY3 is -CH2F. In one embodiment, -CY3 is CF3.

[0089] In various embodiments, each p in formula (IV) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, p is 1. In one embodiment, p is 2.

[0090] In various embodiments, each q in formula (IV) is independently 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q is 1. In one embodiment, q is 2.

[0091] In various embodiments, each t in formula (IV) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, t is 1. In one embodiment, p is t.

[0092] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0093] [ka] It can be expressed by:

[0094] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0095] [ka] It can be expressed by:

[0096] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0097] [ka]

[0098] [ka] It can be expressed by:

[0099] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0100] [ka] It can be expressed by:

[0101] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt thereof, is

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] [ka] Not selected from.

[0108] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt thereof, is

[0109] [ka] Not selected from.

[0110] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt thereof, is

[0111] [ka]

[0112] [ka]

[0113] [ka]

[0114] [ka] Not selected from.

[0115] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt thereof, is

[0116] [ka]

[0117] [ka] Not selected from.

[0118] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0119] [ka] It can be expressed by:

[0120] In various embodiments, the compound of formula (IV) has a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0121] [ka] It can be expressed by:

[0122] In various embodiments, the compound of formula (IV) has the structure selected from the following compounds in Table A, or a pharmaceutically acceptable salt thereof:

[0123] [Table 1-1]

[0124] [Table 1-2]

[0125] [Table 1-3]

[0126] [Table 1-4]

[0127] [Table 1-5]

[0128] [Table 1-6] It can be expressed by:

[0129] Conjugates Various embodiments disclosed herein include a polymerizable compound having the structure: Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III) or a pharmaceutically acceptable salt thereof.

[0130] In various embodiments, Mi in formula (III) is

[0131] [ka] and D is a drug moiety; -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - is a linker connecting Mi to D.

[0132] In various embodiments, L of formula (III) 2 does not exist or

[0133] [ka] ,or

[0134] [ka] and Z 1 and Z 2 is each independently hydrogen, halogen, —NO2, —O—(C1-C6 alkyl), or C1-C6 alkyl. In one embodiment, L in formula (III) 2 In one embodiment, L in formula (III) is absent. 2 teeth,

[0135] [ka] In one embodiment, L of formula (III) 2 teeth,

[0136] [ka] is.

[0137] In various embodiments, Z in formula (III) 1 and Z 2 are each independently hydrogen, halogen, —NO, —O—(C1-C6 alkyl), or C1-C6 alkyl. 1 and Z 2 At least one of Z is hydrogen. 1 and Z 2 At least one of Z is halogen. 1 and Z 2 At least one of Z is -NO. 1 and Z 2 At least one of Z is -O-(C1-C6 alkyl). For example, in one embodiment, Z 1 and Z 2 and at least one of Z is methoxy. 1 and Z 2 At least one of Z is C1-C6 alkyl. 1 and Z 2 At least one of is methyl.

[0138] In various embodiments, L of formula (III) 3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-, and n 1 are independently integers from 0 to 12. In one embodiment, L 3 is -(CH2)n 1 For example, in one embodiment, L 3 is —C(═O)—. In one embodiment, L 3 is -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-. For example, in one embodiment, L 3 is —CHC(═O)—. In one embodiment, n 1 is an integer of 1 to 12, for example, 1 to 6 or 1 to 3.

[0139] In various embodiments, L of formula (III) 4 is a tetrapeptide residue. For example, in one embodiment, L 4 is a tetrapeptide residue selected from GGFG (gly-gly-phe-gly), EGGF (glu-gly-gly-phe), SGGF (ser-gly-gly-phe), and KGGF (lys-gly-gly-phe).

[0140] In various embodiments, L of formula (III) 5 is absent or -[NH(CH2)n 2 ]n 3 - and n 2 is an integer from 0 to 6, and n 2 is an integer from 0 to 2. In one embodiment, L 5 is absent. 5 is -[NH(CH2)n 2 ]n 3 For example, in one embodiment, L 5 is -NH-. In another embodiment, L 5 is -NHCH2-.

[0141] In various embodiments, L of formula (III) 6 does not exist or

[0142] [ka] In one embodiment, L 6 is absent. 6 teeth,

[0143] [ka] is.

[0144] In various embodiments, L of formula (III) 7 does not exist or

[0145] [ka] In one embodiment, L 7 is absent. 7 teeth,

[0146] [ka] In one embodiment, L 7 teeth,

[0147] [ka] In one embodiment, L 7 teeth,

[0148] [ka] In one embodiment, L 7 teeth,

[0149] [ka] In various embodiments, D of the conjugate of formula (III) is a drug moiety described herein (e.g., in the "Drug Moiety" section below). In one embodiment, D is a cytotoxic anticancer drug moiety.

[0150] In various embodiments, the conjugate of formula (III) is:

[0151] [ka]

[0152] [ka] or a pharmaceutically acceptable salt of any of the foregoing.

[0153] In various embodiments, the conjugate of formula (III) is:

[0154] [ka]

[0155] [ka] or a pharmaceutically acceptable salt of any of the foregoing.

[0156] In some embodiments, the conjugate of Formula (III) or a pharmaceutically acceptable salt thereof is

[0157] [ka]

[0158] [ka] Not selected from.

[0159] In some embodiments, the conjugate of Formula (III) or a pharmaceutically acceptable salt thereof is

[0160] [ka]

[0161] [ka]

[0162] [ka]

[0163] [ka]

[0164] [ka] [In the formula, Z 1 and Z 2 are each independently selected from hydrogen, fluoro, chloro, -NO2, and -OCH3. Not selected from.

[0165] In some embodiments, the conjugate of formula (III) is

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] [ka]

[0171] [ka]

[0172] [ka]

[0173] [ka]

[0174] [ka]

[0175] [ka]

[0176] [ka]

[0177] [ka]

[0178] [ka] Or, it is not selected from a pharmaceutically acceptable salt of any of the foregoing.

[0179] Drug portion In various embodiments, D of the immunoconjugate of Formula (I) or the conjugate of Formula (III) is a drug moiety. The drug moiety is linked to a linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - can be any compound of formula (IV) described herein (e.g., above in the "Compounds" section) with appropriate modifications so that - is attached to D. For example, in various embodiments, the drug moiety D has the structure:

[0180] [ka] or a pharmaceutically acceptable salt thereof.

[0181] Those skilled in the art will appreciate that compounds of formula (II) may be prepared by reacting R 3 Or R 4 (X 2 and R 9 (if defined to include) or R 7 via the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - will understand that it will bind to

[0182] In various embodiments, R in formula (II) 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 )m -O-, such that R 1 and R 2 are taken together to form a ring. In one embodiment, R 1 and R 2 In one embodiment, at least one of R 1 and R 2 At least one of R 1 and R 2 In one embodiment, at least one of R 1 and R 2 In one embodiment, at least one of R 1 and R 2 At least one of the following is -OR 5 and R 5 is hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3. For example, in one embodiment, R 1 and R 2 At least one of the groups is methoxy.

[0183] In one embodiment, R of formula (II) 1 and R 2 At least one of the groups is -NR 5 R 6 and R 5 and R 6 are each independently substituted or unsubstituted C1-C6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring.

[0184] In one embodiment, R 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 alkyl. 1 and R 2At least one of R is C1-C3 alkyl. For example, in one embodiment, R 1 and R 2 and at least one of R 1 and R 2 At least one of R is C1-C3 alkyl and the other is halogen. For example, in one embodiment, R 1 and R 2 At least one of is methyl and the other is fluoro.

[0185] In one embodiment, R 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 haloalkyl. For example, in one embodiment, R 1 and R 2 and at least one of R is difluoromethyl. 1 and R 2 At least one of R is substituted or unsubstituted —O—(C1-C6 alkyl). For example, in one embodiment, R 1 and R 2 and at least one of R is methoxy. 1 and R 2 At least one of -[(CY2) p O(CY2) q ] t CY3. In one embodiment, R 1 and R 2 is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, and therefore R 1 and R 2 together to form a ring, -O-(CR 5 R 6 ) m The end of -O- is R 1 and R 2 The phenyl ring is covalently attached to the phenyl ring at position 1 to form a heterocyclyl ring.

[0186] In one embodiment, R of formula (II) 1 and R 2 one of which is hydrogen and R 1 and R 2 The other of R is halogen. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is hydrogen and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are hydrogen. 1 and R 2 None of these is hydrogen.

[0187] In one embodiment, R of formula (II) 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is halogen, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently halogen.1 and R 2 None of the above is a halogen.

[0188] In one embodiment, R of formula (II) 1 and R 2 one of which is substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 one of which is substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 alkyl. 1 and R 2 None of the is a substituted or unsubstituted C1-C6 alkyl.

[0189] In one embodiment, R of formula (II) 1 and R 2 one of which is a substituted or unsubstituted C1-C6 haloalkyl, and R 1 and R 2 and the other of R is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 is not a substituted or unsubstituted C1-C6 haloalkyl.

[0190] In one embodiment, R of formula (II) 1 and R 2 In one embodiment, one of R 1 and R 2 and R are independently substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R2 is not substituted or unsubstituted —O—(C1-C6 alkyl). In one embodiment, R 1 and R 2 is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, and therefore R 1 and R 2 are taken together to form a ring. In various embodiments, R 1 and R 2 are each individually selected from the group consisting of hydrogen, fluoro, methoxy, methyl, difluoromethyl, and —O—(CH)—O—, such that R 1 and R 2 come together to form a ring.

[0191] In various embodiments, R in formula (II) 3 is R 3 and R 4 is hydrogen, -OH, -N3, -NH2, -NH(C=O)CH3, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or [(CY2) p O(CY2) q ] t OH. In one embodiment, R 3 is —OH. In one embodiment, R 3 is -N3. In one embodiment, R 3 is —NH. In one embodiment, R 3 is -NH(C=O)CH2-R 3D and R 3D are H, -CH3, -OH, and -CH2Y 1 and Y 1 is a halogen. For example, R 3 is -NH(C=O)-CH3, -NH(C=O)-CH2CH3, -NH(C=O)-CH2OH, -NH(C=O)-CH2CH2-Y 1 In some embodiments, Y 1 can be F or Cl. In one embodiment, R 3is unsubstituted C1-C6 alkyl. In one embodiment, R 3 is a substituted C1-C6 alkyl. Examples of C1-C6 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched). In one embodiment, R 3 is a substituted C2-C6 alkenyl. 3 is unsubstituted C2-C6 alkenyl. 3 is a substituted C1-C6 alkyl or a substituted C2-C6 alkenyl, the C1-C6 alkyl and / or C2-C6 alkenyl may each independently be -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 3 -OH and -NR 3B R 3C For example, in one embodiment, R 3 is methyl, -CHOH, -CHCHOH, -CHCH(OH)CHOH, -CH(NH))(CHOH), -CH(NH)(CHCHOH), -CH(NH(CH))(CHOH), -CH(NH(CH))(CHCHOH), -CH(N(CH))(CHOH), -CH(N(CH))(CHCHOH), -CH(NH(isopropyl))(CHOH), -CH(NH(isopropyl))(CHCHOH), -CHCH=CH, -CH(NH-(C(=O)CH))(CHOH), -CH(NH-(C(=O)CH))(CHCHOH), and -CH(NH-(C(=O)CH))(CHCH=CH). In some embodiments, R 3 -OH and -NR 3B R 3CIn some embodiments, R 3 is —C(═O) (unsubstituted C1-C6 alkyl) substituted with a substituted C1-C6 alkyl. Examples of suitable C1-C6 alkyls are described herein. In some embodiments, R 3 is a substituted C1-C6 alkyl substituted with one or more OH groups (e.g., 1, 2, 3, or 4 OH groups). 3 is -[(CY2) p O(CY2) q ] t OH. R 3 An exemplary -[(CY2) p O(CY2) q ] t OH groups include -CH2OCH2CH2OH.

[0192] In various embodiments, R in formula (II) 4 is R 3 and R 4 is hydrogen, -OH, -N3, -NH2, -NH(C=O)CH3, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, or [(CY2) p O(CY2) q ] t OH. In one embodiment, R 4 is hydrogen. In one embodiment, R 4 is —OH. In one embodiment, R 4 is -N3. In one embodiment, R 4 is —NH. In one embodiment, R 4 is -NH(C=O)CH2-R 3D and R 3D are H, -CH3, -OH, and -CH2Y 1 and Y 1 is a halogen. For example, R 4 is -NH(C=O)-CH3, -NH(C=O)-CH2CH3, -NH(C=O)-CH2OH, -NH(C=O)-CH2CH2-Y 1In some embodiments, Y 1 can be F or Cl. In one embodiment, R 4 is unsubstituted C1-C6 alkyl. In one embodiment, R 4 is a substituted C1-C6 alkyl. Examples of C1-C6 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched). In one embodiment, R 4 is a substituted C2-C6 alkenyl. 4 is unsubstituted C2-C6 alkenyl. 4 is a substituted C1-C6 alkyl or a substituted C2-C6 alkenyl, the C1-C6 alkyl and / or C2-C6 alkenyl may each independently be -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 4 -OH and -NR 3B R 3C For example, in one embodiment, R 3is methyl, -CHOH, -CHCHOH, -CHCH(OH)CHOH, -CH(NH))(CHOH), -CH(NH)(CHCHOH), -CH(NH(CH))(CHOH), -CH(NH(CH))(CHCHOH), -CH(N(CH))(CHOH), -CH(N(CH))(CHCHOH), -CH(NH(isopropyl))(CHOH), -CH(NH(isopropyl))(CHCHOH), -CHCH=CH, -CH(NH-(C(=O)CH))(CHOH), -CH(NH-(C(=O)CH))(CHCHOH), and -CH(NH-(C(=O)CH))(CHCH=CH). In some embodiments, R 4 -OH and -NR 3B R 3C In some embodiments, R 4 is —C(═O) (unsubstituted C1-C6 alkyl) substituted with a substituted C1-C6 alkyl. Examples of suitable C1-C6 alkyls are described herein. In some embodiments, R 4 is a substituted C1-C6 alkyl substituted with one or more OH groups (e.g., 1, 2, 3, or 4 OH groups). 4 is -[(CY2) p O(CY2) q ] t OH. R 4 An exemplary -[(CY2) p O(CY2) q ] t OH groups include -CH2OCH2CH2OH.

[0193] In some embodiments, R of formula (II) 3 and R 4 is hydrogen, and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 is hydrogen, and R 3 and R4 and the other of R is a substituted C1-C6 alkyl. 3 and R 4 is hydrogen, and R 3 and R 4 The other of the groups is -OH and -NR 3B R 3C is a substituted C1-C6 alkyl substituted by 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). In some embodiments, R 3 and R 4 is hydrogen, and R 3 and R 4 The other of these is a substituted or unsubstituted C 2~6 In another embodiment, R of formula (II) is alkenyl. 3 and R 4 one of which is -N3 and R 3 and R 4 The other of these is a substituted or unsubstituted C 2~6 In yet another embodiment, R of formula (II) is alkenyl. 3 and R 4 one of R is -OH; 3 and R 4 The other of these is a substituted or unsubstituted C 2~6 In still yet other embodiments, R of formula (II) is alkenyl. 3 and R 4 one of which is -NH2 and R 3 and R 4 The other of these is a substituted or unsubstituted C 2~6 For example, R in formula (II) is alkenyl. 3 and R 4 one of which is -OH, -N3, -NH2, or -NH(C=O)CH2-R 3D and R 3 and R 4 The other of the R groups is -CHCH=CH. In some embodiments, the R groups of formula (II) 3 and R4 is hydrogen, and R 3 and R 4 and the other of R is —CH(NH))(CHOH), —CH(NH)(CHCHOH), —CH(NH(CH))(CHOH), —CH(NH(CH))(CHCHOH), —CH(N(CH))(CHOH), —CH(N(CH))(CHCHOH), —CH(NH(isopropyl))(CHOH), —CH(NH(isopropyl))(CHCHOH), —CH(NH—(C(═O)CH))(CHOH), —CH(NH—(C(═O)CH))(CHCHOH), or —CH(NH—(C(═O)CH))(CHCH═CH). In some embodiments, R of Formula (II) 3 and R 4 one of R is -OH; 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 one of which is -NH2 and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 One of the groups is -NH(C=O)-CH2-R 3D and R 3 and R 4 and the other of R is a substituted or unsubstituted C1-C6 alkyl. 3 and R 4 one of R is -OH; 3 and R 4 The other of R is a hydroxy-substituted C-C alkyl, such as -CHOH, -CHCHOH, and -CHCH(OH)CHOH. 3 and R 4 one of which is -NH2 and R 3 and R 4The other of R is a hydroxy-substituted C1-C6 alkyl, such as -CH2OH, -CH2CH2OH, and -CH2CH(OH)CH2OH. As provided herein, in some embodiments, one or more hydroxy groups can be present on the hydroxy-substituted C1-C6 alkyl, such as 1, 2, 3, or 4 OH groups. In some embodiments, R 3 is -OH, R 4 is the unsubstituted C 1~6 Alkyl, e.g., not methyl; R 4 is -OH, R 3 is the unsubstituted C 1~6 In some embodiments, R 3 is -NH2, R 4 is the unsubstituted C 1~6 alkyl, e.g., methyl, but not R 3 is -NH2, R 4 is the unsubstituted C 1~6 In some embodiments, R 3 and R 4 R, provided that at least one of them is not -CH3. 3 and R 4 In some embodiments, one of R 3 and R 4 are unsubstituted C 1~6 In some embodiments, R is not an alkyl, e.g., CH. 3 and / or R 4 is not selected from -CHOH, -CHCHOH and -CHCHCHOH. In some embodiments of this paragraph, R 1 may be substituted or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (linear or branched), and hexyl (linear or branched)); R 2 In some embodiments of this paragraph, R 1 may be unsubstituted C1-C6 alkyl; R 2may be halogen (e.g., F or Cl). In some embodiments of this paragraph, R 7 may be H.

[0194] In some embodiments, R 3 and R 4 one of which is substituted or unsubstituted -(C1-C6 alkyl)-X 2 and -(C1-C6 alkyl)-X 2 is substituted, -(C1-C6 alkyl)-X 2 -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 3 and R 4 one of which is substituted or unsubstituted -(C2-C6 alkenyl)-X 2 and -(C2-C6 alkenyl)-X 2 When substituted, -(C2-C6 alkenyl)-X 2 -OH and -NR 3B R 3C One or more R selected from 3A groups (e.g., 1, 2, or 3 R 3A group), where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl). 3B R 3C may be NH. In other embodiments, -NR 3B R 3C may be -NH(substituted C1-C6 alkyl). In yet other embodiments, -NR 3B R 3Cmay be N(unsubstituted C1-C6 alkyl). In some embodiments, -NR 3B R 3C may be NH(substituted C1-C6 alkyl). In other embodiments, -NR 3B R 3C may be N(substituted C1-C6 alkyl). In some embodiments, -NR 3B R 3C may be -NH(-C(=O)(unsubstituted C1-C6 alkyl)). In other embodiments, -NR 3B R 3C may be N(unsubstituted C1-C6 alkyl) (-C(=O)(unsubstituted C1-C6 alkyl)). In some embodiments, R 3 and R 4 one of which is a substituted or unsubstituted -(C1-C6 alkyl)-X substituted by one or more (e.g., 1, 2, or 3) hydroxy groups; 2 In some embodiments, R 3 and R 4 One of the groups is -NR 3B R 3C substituted or unsubstituted -(C1-C6 alkyl)-X 2 In some embodiments, R 3 and R 4 One of the groups is -NR 3B R 3C and substituted or unsubstituted -(C1-C6 alkyl)-X substituted by one or more (e.g., 1, 2, or 3) hydroxy groups. 2 In some embodiments, R 3 and R 4 one of which is a substituted or unsubstituted -(C2-C6 alkenyl)-X substituted by one or more (e.g., 1, 2, or 3) hydroxy groups; 2 In some embodiments, R 3 and R 4 One of the groups is -NR 3B R 3C substituted or unsubstituted -(C2-C6 alkenyl)-X 2In some embodiments, R 3 and R 4 One of the groups is -NR 3B R 3C and substituted or unsubstituted -(C2-C6 alkenyl)-X substituted by one or more (e.g., 1, 2, or 3) hydroxy groups. 2 For example, R 3 and R 4 one of which is -(CH2)-CH(OH)-(CH2)-X 2 In various embodiments, X 2 -OR 9 , -SR 9 , or -NHR 9 and R 9 is R 7 and R 9 One of them is definitely L 4 , L 5 , L 6 , or L 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 In such embodiments, the compound of formula (II) is R 3 or R 4 are X 2 Contains R 9 But, L 4 , L 5 , L 6 , or L 7 If R 3 or R 4 via the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -Bind to.

[0195] In some embodiments, the compound of Formula (II) or a pharmaceutically acceptable salt thereof has the structure of Formula (II-a), or a pharmaceutically acceptable salt thereof:

[0196] [ka] [In the formula, R 1 , R 2 , R 3A and R 7 is as provided herein in formula (II); R 3 is -OH, -CH3, -NH2 or -NH(C=O)CH2R 3D and; b is individually 1, 2, or 3] may have.

[0197] In some embodiments, each R 3A can be OH. In some embodiments of this paragraph, b can be 1. In other embodiments of this paragraph, b can be 2. In still other embodiments of this paragraph, b can be 3.

[0198] R 3 or R 4 is X 2 In each such embodiment, R 9 L 4 , L 5 , L 6 , or L 7 and thus, whereby the compound of formula (II) is 3 or R 4 via linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - Provides the option to combine

[0199] In various embodiments, R in formula (II) 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L6 , or L 7 and each R 8 are individually 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 In one embodiment, R 7 is H. In one embodiment, R 7 -COR 8 In one embodiment, R 7 -CO2R 8 In one embodiment, R 7 is -(CO)-NHR 8 Those skilled in the art will recognize that R 7 But, H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 If the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 The bond of the compound of formula (II) to R 3 or R 4 (Thus, R 9 ) to understand that.

[0200] In various embodiments, each R of formula (II) 8 are individually 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 and X 3 is —H, —OH, —SH, or —NH. In one embodiment, each R 8 are individually substituted or unsubstituted C1-C6 alkyl-X 3 In one embodiment, each R 8are individually substituted or unsubstituted C1-C6 haloalkyl-X 3 In one embodiment, each R 8 are individually -[(CY2) p O(CY2) q ] t CY2-X 3 is.

[0201] In various embodiments, X in formula (II) 2 -OR 9 , -SR 9 , or -NHR 9 and R 9 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 In one embodiment, X 2 -OR 9 In one embodiment, X 2 -SR 9 In one embodiment, X 2 -NHR 9 is.

[0202] In various embodiments, R in formula (II) 9 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 and R 8 is a 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 In one embodiment, R 9 is H. In one embodiment, R 9 -COR 8 In one embodiment, R9 -CO2R 8 In one embodiment, R 9 is -(CO)-NHR 8 Those skilled in the art will recognize that R 9 But, H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 If the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 The bond of the compound of formula (II) to R 7 Understand that this is done through

[0203] In various embodiments, R in formula (II) 9 L 4 , L 5 , L 6 , or L 7 In one embodiment, R 9 L 4 In one embodiment, R 9 L 5 In one embodiment, R 9 L 6 In one embodiment, R 9 L 7 Those skilled in the art will recognize that R 9 But, L 4 , L 5 , L 6 , or L 7 If the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 The bond of the compound of formula (II) to R 3 or R 4 In one embodiment, R 7 and R 9 One of them is definitely L 4 , L 5 , L 6 , or L 7where a covalent bond connects the drug D to the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - and thereby connects to Mi.

[0204] In various embodiments, each X in formula (II) 3 are individually -H, -OH, -SH, or NH2. In one embodiment, X 3 is H. In one embodiment, X 3 is —OH. In one embodiment, X 3 is -SH. In one embodiment, X 3 is -NH2.

[0205] In various embodiments, m in formula (II) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

[0206] In various embodiments, n 4 and n 5 n in formula (II) is not 0, 4 and n 5 are each independently 0, 1, or 2. In one embodiment, n 4 and n 5 and n are both 1. In one embodiment, 4 is 0, and n 5 is 1. In one embodiment, n 4 is 0, and n 5 is 2. In one embodiment, n 4 is 1 and n 5 is 0. In one embodiment, n 4 is 2 and n 5 is 0.

[0207] In various embodiments, each Y in formula (II) is individually H or halogen. In one embodiment, each Y is hydrogen. In one embodiment, -CY2 is -CH2. In one embodiment, -CY3 is -CH3. In one embodiment, -CY3 is -CHF2. In one embodiment, -CY3 is -CH2F. In one embodiment, -CY3 is -CF3.

[0208] In various embodiments, each p in formula (II) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, p is 1. In one embodiment, p is 2.

[0209] In various embodiments, each q in Formula (II) is independently 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q is 1. In one embodiment, q is 2.

[0210] In various embodiments, each t in formula (II) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, t is 1. In one embodiment, p is t.

[0211] In some embodiments, the compound of formula (II) is R 1 and R 2 are 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, or substituted or unsubstituted -O-(CR 5 R 6 ) m -O-, so that R 1 and R 2 together form a ring; R 3 and R 4 is R 3 and R 4are each individually hydrogen, -OH, -N3, -NH2, -NH(C=O)-CH2-R, provided that at least one of them is not hydrogen. 3D , substituted or unsubstituted C2-C6 alkenyl and [(CY2) p O(CY2) q ] t and when C1-C6 alkyl or C2-C6 alkenyl is substituted, C1-C6 alkyl or C2-C6 alkenyl is selected from -OH and -NR 3B R 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); or R 3 and R 4 one of which is a substituted or unsubstituted -(C1-C6 alkyl)-X 2 or substituted or unsubstituted -(C2-C6 alkenyl)-X 2 and -(C1-C6 alkyl)-X 2 or -(C1-C6 alkenyl)-X 2 is substituted, -(C1-C6 alkyl)-X 2 or -(C1-C6 alkenyl)-X 2 -OH and -NR 3B R 3C One or more R selected from 3A substituted by a group, where R 3B and R 3C are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, or —C(═O)(unsubstituted C1-C6 alkyl); R 3D are H, -CH3, -OH and -CH2Y 1 is selected from, where Y 1 is a halogen; X 2 -OR 9 , -SR 9 , or -NHR 9 and;R 5 and R 6are each independently substituted or unsubstituted C1-C6 alkyl; or R 5 and R 6 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring; n 4 and n 5 is n 4 and n 5 are each independently 0, 1, or 2, provided that at least one of is not 0; each Y is independently H or halogen; each m is independently 1 or 2; each p is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 1, 2, 3, 4, 5, or 6; R 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 and;R 8 is a 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 and R 9 is R 7 and R 9 One of them is definitely L 4 , L 5 , L 6 , or L 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 , or L 7 and each X 3 are individually -H, -OH, -SH, or -NH2.

[0212] Immunoconjugates Various embodiments disclosed herein provide a compound having the structure: Ab-[SL 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I) or a pharmaceutically acceptable salt thereof.

[0213] In various embodiments, L of formula (III) 1 teeth,

[0214] [ka] is.

[0215] In various embodiments, L of formula (III) 2 does not exist or

[0216] [ka] ,or

[0217] [ka] and Z 1 and Z 2 is each independently hydrogen, halogen, —NO2, —O—(C1-C6 alkyl), or C1-C6 alkyl. In one embodiment, L in formula (III) 2 In one embodiment, L in formula (III) is absent. 2 teeth,

[0218] [ka] In one embodiment, L of formula (III) 2 teeth,

[0219] [ka] is.

[0220] In various embodiments, Z in formula (III) 1 and Z 2 are each independently hydrogen, halogen, —NO, —O—(C1-C6 alkyl), or C1-C6 alkyl. 1 and Z 2 At least one of Z is hydrogen. 1 and Z 2 At least one of Z is halogen. 1 and Z 2 At least one of Z is -NO. 1 and Z 2 At least one of Z is -O-(C1-C6 alkyl). For example, in one embodiment, Z 1 and Z 2 and at least one of Z is methoxy. 1 and Z 2 At least one of Z is C1-C6 alkyl. 1 and Z 2 At least one of is methyl.

[0221] In various embodiments, L of formula (III) 3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-, and n 1 are independently integers from 0 to 12. In one embodiment, L 3 is -(CH2)n 1 For example, in one embodiment, L 3 is —C(═O)—. In one embodiment, L 3 is -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-. For example, in one embodiment, L3 is —CHC(═O)—. In one embodiment, n 1 is an integer of 1 to 12, for example, 1 to 6 or 1 to 3.

[0222] In various embodiments, L of formula (III) 4 is a tetrapeptide residue. For example, in one embodiment, L 4 is a tetrapeptide residue selected from GGFG (gly-gly-phe-gly), EGGF (glu-gly-gly-phe), SGGF (ser-gly-gly-phe), and KGGF (lys-gly-gly-phe).

[0223] In various embodiments, L of formula (III) 5 is absent or -[NH(CH2)n 2 ]n 3 - and n 2 is an integer from 0 to 6, and n 3 is an integer from 0 to 2. In one embodiment, L 5 is absent. 5 is -[NH(CH2)n 2 ]n 3 For example, in one embodiment, L 5 is -NH-. In another embodiment, L 5 is -NHCH2-.

[0224] In various embodiments, L of formula (III) 6 does not exist or

[0225] [ka] In one embodiment, L 6 is absent. 6 teeth,

[0226] [ka] is.

[0227] In various embodiments, L of formula (III) 7 does not exist or

[0228] [ka] In one embodiment, L 7 is absent. 7 teeth,

[0229] [ka] In one embodiment, L 7 teeth,

[0230] [ka] In one embodiment, L 7 teeth,

[0231] [ka] In one embodiment, L 7 teeth,

[0232] [ka] is.

[0233] In various embodiments, D of the immunoconjugate of Formula (I) is a drug moiety described herein (e.g., in the "Drug Moiety" section above). 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] nThe "S" in D (shown in bold) may be a sulfur present in a cysteine ​​(e.g., a cysteine ​​that may be present in the antibody itself, a fragment of the antibody, an antigen-binding fragment thereof, a portion of the antigen-binding fragment, and / or a linker attached to the antibody or antigen-binding fragment). In one embodiment, D is a cytotoxic anti-cancer drug moiety. In one embodiment, the drug moiety is exatecan.

[0234] In various embodiments, the Ab of formula (III) is an antibody or antigen-binding fragment. In one embodiment, the Ab specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1), Her2, TROP2, Her3, B7-H3, GPR20, or CEACAM5. In one embodiment, the Ab binds to the surface of a cancer cell. In one embodiment, the Ab is an anti-HER2 antibody.

[0235] In various embodiments, the immunoconjugate compound of formula (I) is

[0236] [ka]

[0237] [ka] or a pharmaceutically acceptable salt of any of the foregoing. can be selected from:

[0238] In various embodiments, the immunoconjugate compound of formula (I) is

[0239] [ka]

[0240] [ka] Alternatively, it may be selected from a pharmaceutically acceptable salt of any of the foregoing.

[0241] In various embodiments, the immunoconjugate compound of formula (I) is

[0242] [ka]

[0243] [ka]

[0244] [ka]

[0245] [ka]

[0246] [ka]

[0247] [ka]

[0248] [ka]

[0249] [ka]

[0250] [ka]

[0251] [ka]

[0252] [ka]

[0253] [ka]

[0254] [ka] Or, it is not selected from a pharmaceutically acceptable salt of any of the foregoing.

[0255] In various embodiments, the immunoconjugate compound of formula (I) is

[0256] [ka]

[0257] [ka] Or, it is not selected from pharmaceutically acceptable salts thereof.

[0258] In various embodiments, the immunoconjugate compound of Formula (I), or a pharmaceutically acceptable salt thereof, is

[0259] [ka]

[0260] [ka]

[0261] [ka]

[0262] [ka] [In the formula, Z 1 and Z 2 are each independently selected from hydrogen, fluoro, chloro, -NO2, and -OCH3. Not selected from.

[0263] Pharmaceutical Composition Some embodiments described herein relate to pharmaceutical compositions, which may 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 a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0264] The term "pharmaceutical composition" refers to a mixture of one or more compounds and / or salts disclosed herein with other chemical components, such as diluents or carriers. Pharmaceutical compositions facilitate the administration of a compound to an organism. Pharmaceutical compositions can also be obtained by reacting a compound with an inorganic or organic acid, 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 specific intended route of administration.

[0265] The term "physiologically acceptable" defines a carrier, diluent or excipient that does not abrogate the biological activity and properties of the compound and does not cause obvious damage or injury to the animal to which the composition is intended to be delivered.

[0266] As used herein, "carrier" refers to a compound that facilitates the uptake of a compound into cells or tissues.For example, but not limited to, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the uptake of many organic compounds into target cells or tissues.

[0267] As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that lacks significant pharmacological activity but is pharmaceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose amount is too small for production and / or administration. It can also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is, for example, but not limited to, a buffered aqueous solution such as phosphate-buffered saline, which mimics the pH and isotonicity of human blood.

[0268] As used herein, "excipient" refers to an essentially inert substance added to a pharmaceutical composition to provide the composition with, but not limited to, bulk, uniformity, stability, binding capacity, lubrication, dissolvability, etc. For example, stabilizers such as antioxidants and metal chelators are excipients. In one embodiment, the pharmaceutical composition includes an antioxidant and / or a metal chelator. A "diluent" is a type of excipient.

[0269] The pharmaceutical compositions described herein can be administered to a human patient by themselves or in pharmaceutical compositions mixed with other active ingredients, or carriers, diluents, excipients, or combinations thereof, as in combination therapy. The appropriate formulation will depend on the chosen route of administration. Techniques for the formulation and administration of the compounds described herein are known to those skilled in the art.

[0270] The pharmaceutical compositions disclosed herein can be prepared by known methods, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, encapsulating, or tableting processes. In certain embodiments, the compositions are lyophilized and then reconstituted, for example, in buffered saline, at the time of administration. Furthermore, 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.

[0271] Multiple techniques of administering 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.

[0272] For example, compounds, salts and / or compositions can be administered locally rather than systemically, for example, by injecting or implanting compounds directly into the affected area, often in slow-release or sustained-release formulations.Furthermore, compounds can be administered in targeted drug delivery systems, for example, in liposomes coated with targeting ligands for specific cell or tissue types.Liposomes will be selectively targeted and taken up by target cells or tissues.

[0273] The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as 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 government agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting the agency's approval of the drug form for human or animal administration. Such notice may, for example, be labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions that may include the compounds and / or salts described herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a designated condition.

[0274] Methods of Use and Treatment Some embodiments described herein relate to methods of treating, inhibiting, or alleviating cancer or tumors, which may include administering to a subject having cancer or tumor an effective amount of a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof). Other embodiments described herein relate to the use of an effective amount of a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for treating, inhibiting, or alleviating a cancer or tumor described herein. Still other embodiments described herein relate to an effective amount of a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) for treating, inhibiting, or ameliorating a cancer or tumor described herein.

[0275] Examples of cancers and tumors that may be considered for response to one or more treatments described herein 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 tumors, 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.

[0276] As used herein, "subject" refers to an animal that is the object of treatment or therapy, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and specifically mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and specifically humans. In some embodiments, the subject may be a human. In some embodiments, the subject may be a child and / or infant, such as a child or infant with a fever. In other embodiments, the subject may be an adult.

[0277] As used herein, the terms "treat," "treating," "treatment," "therapeutic," and "therapy" do not necessarily mean a cure or eradication of a disease or condition. Any alleviation of any undesirable signs or symptoms of a disease or condition, to any extent, can be considered treatment and / or cure. Additionally, treatment can include effects that may worsen a subject's overall sense of health or condition.

[0278] The terms "therapeutically effective amount" and "effective amount" are used to refer to the amount of an active compound or pharmaceutical agent that elicits a specified biological or medical response. For example, a therapeutically effective amount of a compound, salt, or composition can be the amount necessary to prevent, alleviate, or ameliorate the symptoms of a disease or condition, or to prolong the survival of the subject being treated. This response occurs in a tissue, system, animal, or human, and includes alleviating the signs or symptoms of the disease or condition being treated. Determining an effective amount is well within the capabilities of one of ordinary skill in the art in light of the disclosure provided herein. The therapeutically effective amount of a compound disclosed herein required as a dose depends on the route of administration, the type of animal, including humans, being treated, and the physical characteristics of the particular animal under consideration. The dose can be adapted to achieve the desired effect, but will depend on body weight, diet, concurrent medication, and other factors as will be recognized by those skilled in the pharmaceutical arts.

[0279] For example, an effective amount of a compound is an amount that results in (a) a reduction, alleviation, or elimination of one or more symptoms caused by cancer, (b) a reduction in tumor size, (c) tumor elimination, and / or (d) long-term disease stability (growth arrest) of the tumor. In the treatment of lung cancer (e.g., non-small cell lung cancer), a therapeutically effective amount is an amount that reduces or eliminates cough, shortness of breath, and / or pain.

[0280] The amount of immunoconjugate compound of Formula (I), drug compound of Formula (IV), or pharmaceutically acceptable salt thereof required for therapeutic use will vary not only according to the particular compound or salt selected, but also according to the route of administration, the nature and / or symptoms of the disease or condition being treated, and the age and condition of the patient, and ultimately, the discretion of the nurse or physician. In the case of administration of a pharmaceutically acceptable salt, the dosage may be calculated as the free base. As will be understood by those skilled in the art, in certain circumstances, it may be necessary to administer the compounds disclosed herein in amounts above or even above the dosage ranges set forth herein in order to effectively and aggressively treat a particular ongoing disease or condition.

[0281] In general, however, suitable doses are often in the range of about 0.5 mg / kg to about 10 mg / kg. For example, suitable doses may range from about 1.0 mg / kg to about 7.5 mg / kg of body weight every three weeks or every week, e.g., about 1.5 mg / kg to about 5.0 mg / kg of the recipient's body weight every three weeks or every week, about 2.0 mg / kg to about 4.0 mg / kg of the recipient's body weight every three weeks or every week, or any amount therebetween. The compound may be administered in unit dosage forms, for example, containing 1-500 mg, 10-100 mg, 5-50 mg, or any amount therebetween of active ingredient per unit dosage form.

[0282] The desired dose may conveniently be presented in a single dose or in divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, for example, into a number of discrete loosely spaced administrations.

[0283] As is readily apparent to those skilled in the art, the useful in vivo dosage and specific mode of administration will vary depending on the age, weight, severity of the affliction, the mammalian species being treated, the specific compound used, and the specific use for which these compounds are used.Determination of effective dosage levels, which are the dosage levels required to achieve the desired results, can be achieved by those skilled in the art using conventional methods, such as human clinical trials, in vivo tests, and in vitro tests.For example, the useful dosage of the immunoconjugate compound of formula (I), the drug compound of formula (IV), or its pharmaceutically acceptable salt can be determined by comparing their in vitro activity and in vivo activity in animal models.Such comparisons can be made by comparison with established drugs, such as cisplatin and / or gemcitabine.

[0284] Dosage amount and interval can be individually adjusted to provide plasma levels of the active moiety sufficient to maintain a modulating effect, or minimum effective concentration (MEC). The MEC varies for each compound but can be estimated from in vivo and / or in vitro data. The dosage necessary to achieve the MEC depends on individual characteristics and the route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using the MEC value. Compositions should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably 30-90% and more preferably 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0285] Of note, nurses know how and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunction. Conversely, nurses also know to adjust treatment to higher levels if the clinical response is insufficient (precluding toxicity). The magnitude of the dose in the management of the disorder of interest will vary depending on the severity of the disease or condition being treated and the route of administration. The severity of the disease or condition may be assessed, for example, in part, by standard predictive evaluation methods. Furthermore, administration, and perhaps administration frequency, may also vary depending on the age, weight, and response of the individual patient. Programs comparable to those discussed above may be used in veterinary medicine.

[0286] The compounds, salts, and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound or a subset of compounds sharing a particular chemical moiety can be established by determining the in vitro toxicity of a cell line, such as a mammalian, preferably a human cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, 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 several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, those skilled in the art can be guided by the state of the art in selecting the appropriate model, dose, route of administration, and / or regimen.

[0287] synthesis The drug compounds of formula (IV), or pharmaceutically acceptable salts thereof, can be prepared in a variety of ways by those skilled in the art using known techniques as guided by the detailed teachings provided herein. For example, in one embodiment, the drug compounds of formula (IV) are prepared according to the general schemes shown in Figures 2 and 4-12.

[0288] Conjugates of formula (III) can be made in a variety of ways by those skilled in the art using known techniques as guided by the detailed teachings provided herein. For example, in one embodiment, conjugates of formula (II) are prepared according to the general schemes shown in Figures 13 and 14. Although illustrated with specific linkers and payloads, one skilled in the art will understand that other linkers and / or payloads can be used in a similar manner.

[0289] Immunoconjugates of Formula (I) can be made in a variety of ways by those skilled in the art using known techniques as guided by the detailed teachings provided herein. For example, in one embodiment, immunoconjugates of Formula (I) are prepared according to the general scheme shown in Figure 3. Examples of conjugates of Formula (III) and intermediates that can be used to prepare immunoconjugates of Formula (I) are provided in Figure 15. In one embodiment, the process for producing an immunoconjugate described herein comprises reacting the conjugate with an effective amount of a thiol-functionalized antibody or antigen-binding fragment, as described herein, under reaction conditions effective to form the immunoconjugate. In one embodiment, the process further comprises reducing the antibody or antigen-binding fragment under reducing conditions effective to form the thiol-functionalized antibody or antigen-binding fragment. [Example]

[0290] Further embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0291] The following abbreviations are used herein and have the specified definitions: Ac is acetyl (-C(=O)CH), AcOH is acetic acid, AcO is acetic anhydride, DCM is dichloromethane, AgOAc is silver acetate, DMAP is 4-dimethylaminopyridine, DMF is N,N-dimethylformamide, DMFDMA is N,N-dimethylformamide dimethyl acetal, DMSO is dimethyl sulfoxide, ESI is electrospray ionization, EtOAc is ethyl acetate, h is hour, HCHO is formaldehyde, HPLC is high performance liquid chromatography, KHMDS is potassium bis(trimethylsilyl)amide, LCMS is liquid chromatography / mass spectrometry, MeOH is methanol, and MsOH is p-toluene. is sulfonic acid, NaBH(OAc)3 is sodium triacetoxyborohydride, NMO is N-methylmorpholine N-oxide, NMR is nuclear magnetic resonance, OTBS is tert-butyldimethylsilyl ether, PhMe is toluene, PIDA is (diacetoxyiodo)benzene, P(OEt)3 is triethyl phosphite, PPh3 is triphenylphosphine, PyH is pyridinium, PPTS is pyridinium p-toluenesulfonate, SFC is supercritical fluid chromatography, t-BuOOH is tert-butyl hydroperoxide, TEA is triethylamine, TLC is thin layer chromatography, THF is tetrahydrofuran, and TsOH is p-toluenesulfonic acid.

[0292] Example 1 (1S,9S)-1-((S)-2,3-dihydroxypropyl)-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 (1-7A), (1S,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-8A), (1R Synthesis of (1R,9S)-1-((S)-2,3-dihydroxypropyl)-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 (1-9A) and (1R,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-10A)

[0293] [ka]

[0294] N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-2): To a stirred mixture of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-1) (7.50 g, 31.9 mmol, 1.0 equiv.) in toluene (150 mL) at -70 °C was added potassium bis(trimethylsilyl)amide (1 M, 63.8 mL, 2.0 equiv.). After stirring at -70 °C for 1 h, a solution of 3-iodoprop-1-ene (5.36 g, 31.9 mmol, 2.91 mL, 1.0 equiv.) in toluene (75.0 mL) was added, and the mixture was stirred at -70 °C for 1 h. Another reaction on the same scale was carried out in parallel. The two reactions were combined for further investigation. After quenching at -70 °C by the dropwise addition of HO (200 mL) and slowly warming to 25 °C, the mixture was extracted with ethyl acetate (3 × 200 mL). The combined organic phases were washed with brine (500 mL), dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-2) (9.70 g, 55% yield). 1 H NMR(400MHz,DMSO-d6) δ ppm 12.09(br s,1H) 8.29(d,J=13.13Hz,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(376MHz,DMSO-D6) δ ppm -104.42. LCMS(ESI+) m / z:[MH] + C 16 H 19 FNO2 + Calculated value: 276.1, Measured value: 276.1.

[0295] N-(7-(2,3-dihydroxypropyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-3A): To a stirred mixture of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-2) (1.0 g, 3.63 mmol, 1.0 equiv.) in acetone (20 mL) and water (1 mL) was added potassium osmate(VI) dihydrate (535 mg, 1.45 mmol, 0.4 equiv.) and 4-methylmorpholine N-oxide monohydrate (850 mg, 7.26 mmol, 766 μL, 2.0 equiv.). After stirring at 25° C. for 2 h, the reaction mixture was quenched by the addition of water (200 mL) and saturated sodium thiosulfate solution (20 mL) at 25° C. and extracted with ethyl acetate (3×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(7-(2,3-dihydroxypropyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-3A) (700 mg, 62% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.97-12.22(m,1H),8.28(d,J=13.18Hz,1H),4.45-4.60(m,2H),3.53-3.66(m,1H),3.23-3.30(m,1H),2.93 -3.05(m,1H),2.81-2.91(m,1H),2.71-2.80(m,1H),2.08-2.25(m,8H),1.67-1.91(m,2H),1.40-1.50(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.74. LCMS(ESI+) m / z:[M + Na] + C 16 H 20 FNO4Na + Calculated value: 332.1, Measured value: 332.1.

[0296] 8-amino-2-(2,3-dihydroxypropyl)-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-4A): To a stirred mixture of N-(7-(2,3-dihydroxypropyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-3A) (700 mg, 2.25 mmol, 1.0 equiv) in methanol (28 mL) was added hydrochloric acid (2 M, 28 mL). After stirring at 60 °C for 3 h, the mixture was quenched at 25 °C by the addition of saturated sodium bicarbonate solution (56 mL), the pH was adjusted to 7, and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give 8-amino-2-(2,3-dihydroxypropyl)-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-4A) (270 mg, 65% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.22-7.54(m,2H),6.28-6.46(m,1H),4.37-4.60(m,2H),3.55-3.73(m,1H),3.17-3.29(m,2H),2.79-2.9 1(m,1H),2.63-2.76(m,2H),2.08-2.16(m,1H),1.93-2.04(m,4H),1.68-1.89(m,1H),1.33-1.59(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.74. LCMS(ESI+) m / z:[MH] + C 14 H 19 FNO3 + Calculated value: 268.1, Measured value: 268.1.

[0297] (9S)-1-(2,3-dihydroxypropyl)-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 (1-6A): A mixture of 8-amino-2-(2,3-dihydroxypropyl)-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-4A) (400 mg, 1.20 mmol, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (346 mg, 1.32 mmol, 1.1 equiv), o-cresol (945 mg, 8.74 mmol, 90 μL, 7.3 equiv), and pyridinium 4-toluenesulfonate (45.13 mg, 179 μm, 0.15 equiv) in toluene (20 mL) was degassed, then purged with argon three times, and stirred at 120 °C under an argon atmosphere for 16 h. The mixture was then quenched at 25 °C by the addition of water (15 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / methanol) to give (9S)-1-(2,3-dihydroxypropyl)-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 (1-6A) (300 mg, 28% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.74(br d,J=11.37Hz,1H),7.30(s,1H),6.51(s,1H),5.28-5.52(m,3H),4.23-5.01(m,2H),3.53-3.74(m,2 H),2.90-3.25(m,4H),2.28-2.42(m,4H),1.57-2.06(m,5H),1.29-1.49(m,1H),0.75-0.96(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.99,-111.98,-111.84. LCMS(ESI+) m / z:[MH] + C 27 H 28 FN2O6 +Calculated value: 495.2, Found value: 495.2. SFC (retention times = 0.720 min, 0.827 min, 1.749 min, 2.134 min).

[0298] (1S,9S)-1-((S)-2,3-dihydroxypropyl)-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 (1-7A), (1S,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-8A), (1R,9S)-1-((S)-2,3-dihydroxypropyl)-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 (1-9A), (1R,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-10A): (9S)-1-(2,3-dihydroxypropyl)-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 (1-6A) (300 mg, 606 μmol) was first separated by chiral SFC to give (1S,9S)-1-((S)-2,3-dihydroxypropyl)-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. (1S,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-7A) and (1S,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-7B). Hydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-8A), (1R,9S)-1-((S)-2,3-dihydroxypropyl)-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- A mixture of 10,13-dione (1-9A) (45.4 mg, 15% yield), (1R,9S)-1-((R)-2,3-dihydroxypropyl)-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 (1-10A) (45.6 mg, 15% yield) was obtained. Note: The stereochemistry of the four products was arbitrarily assigned. SFC separation method: Apparatus: Waters SFC80Q preparative SFC; Column: DAICEL CHIRALCEL OD (250 mm * 30 mm, 10 μm); mobile phase: A is CO2 and B is methanol; gradient: B% = 38% isocratic elution mode; flow rate: 65 g / min; wavelength: 220 nm; column temperature: 40 °C; system back pressure: 100 bar.

[0299] The mixture of 1-7A and 1-8A (70.2 mg) was again separated by chiral SFC to give 1-7A (23.29 mg, 7.6% yield) and 1-8A as a mixture with 1-7A. SFC separation method: Apparatus: Waters SFC80 preparative SFC; Column: REGIS(s,s)WHELK-O1 (250 mm * 30 mm, 10 μm); mobile phase: A is CO2 and B is IPA; gradient: B% = 55% isocratic elution mode; flow rate: 60 g / min; wavelength: 220 nm; column temperature: 40 °C; system back pressure: 100 bar.

[0300] The above mixture of 1-8A and 1-7A was then further separated by SFC to give 1-8A (9.5 mg, 3.1% yield). SFC separation method: Apparatus: Waters SFC150AP preparative SFC; Column: REGIS(s,s)WHELK-O1 (250 mm * 30 mm, 10 μm); mobile phase: A is CO2 and B is MeOH; gradient: B% = 50% isocratic elution mode; flow rate: 70 g / min; wavelength: 220 nm; column temperature: 35 °C; system back pressure: 120 bar. 1-7A spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.74(br d,J=10.88Hz,1H),7.30(s,1H),6.51(s,1H),5.18-5.53(m,4H),4.65-4.75(m,1H),4.43(br t,J=5.13Hz,1H),3.58(br s,2H),3.16-3.28(m,2H),3.07-3.15(m,2H),2.27-2.41(m,4H),1.95-2.1 1(m,1H),1.80-1.94(m,2H),1.69-1.78(m,1H),1.53-1.66(m,1H),0.87(br t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.996. LCMS(ESI+) m / z:[MH] + C 27 H 28 O6N2F +Calculated value: 495.2, measured value: 495.3. SFC (holding time = 1.654 minutes). 1-8Aのスペクトル: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.74(br d,J=11.13Hz,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.28(s,2H),4 .88(d,J=5.75Hz,1H),4.54(t,J=5.63Hz,1H),3.67-3.76(m,1H),3.59(br d,J=11.76Hz,1H),3.36-3.41(m,1H),3.22(dt,J=11.07,5.72Hz,1H),3.10-3.18(m,1H),2. 92-3.08(m,1H),2.29-2.42(m,4H),1.70-1.98(m,4H),1.33-1.49(m,1H),0.83-0.92(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.849. LCMS(ESI+) m / z:[MH] + C 27 H 28 O6N2F + Calculated value: 495.2, measured value: 495.3. SFC (holding time = 2.032 minutes). 1-9Aのスペクトル: 1 H NMR (400MHz, DMSO-D6) δ ppm 7.74(d,J=11.13Hz,1H),7.30(s,1H),6.52(s,1H),5.24-5.52(m,4H),4.73(br s,1H),4.45(br s,1H),3.64-3.95(m,1H),3.57(br s,1H),3.18-3.24(m,1H),3.04-3.14(m,2H),2.38(s,3H),2.31(br d,J=12.96Hz,1H),1.95-2.10(m,1H),1.81-1.92(m,2H),1.69-1.80(m,1H),1.54-1.66(m,1H),1.11-1.27(m,1H),0.87(br t,J=7.21Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.988. LCMS(ESI+) m / z:[MH] + C27 H 28 O6N2F + Calculated value: 495.2, Found value: 495.3. SFC (retention time = 1.538 min). 1-10A spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.73(d,J=11.13Hz,1H),7.29(s,1H),6.51(s,1H),5.43(s,2H),5.28(s,2H),4.87(br d,J=5.50Hz,1H),4.54(br s,1H),3.67-3.77(m,1H),3.54-3.64(m,1H),3.36-3.42(m,1H),3.19-3.27(m,1H),3.09-3.18(m,1H) ,2.93-3.07(m,1H),2.27-2.43(m,4H),1.70-1.98(m,4H),1.34-1.47(m,1H),0.87(t,J=7.34Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.849. LCMS(ESI+) m / z:[MH] + C 27 H 28 O6N2F + Calculated: 495.2, Found: 495.3. SFC (retention time = 1.679 min). Alternative and / or additional synthetic routes to the compounds described in Example 1 can be found in FIG.

[0301] Example 2 Synthesis of (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-15) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-16)

[0302] [ka]

[0303] 8-Amino-6-fluoro-2-hydroxy-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-13A): To a stirred mixture of N-(3-fluoro-7-hydroxy-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-12A) (150 mg, 177 μmol, 1.0 equiv.) in methanol (6.00 mL) was added hydrochloric acid (2 M, 6.00 mL). After stirring at 60 °C for 3 h, the mixture was cooled to room temperature, the pH was adjusted to approximately 4 by adding saturated aqueous sodium bicarbonate, and extracted with ethyl acetate (4 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give 8-amino-6-fluoro-2-hydroxy-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-13A) (95 mg, 74% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.41(br s,2H) 6.37(d,J=12.51Hz,1H) 4.93(s,1H) 4.66(t,J=5.94Hz,1H) 3.60(dd,J=11.01,6.00Hz,1H) 3.35(dd,J=11.01,5.88Hz,1H) 2.70-2.86(m,2H) 2.16(dt,J=13.35,5.58Hz,1H) 1.97(d,J=1.13Hz,3H) 1.79-2.02(m,1H). LCMS(ESI+) m / z:[MH] + C 12 H 15 FNO3 + Calculated value: 240.1, measured value: 240.3.

[0304] (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (2-14A): To a stirred mixture of 8-amino-6-fluoro-2-hydroxy-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-13A) (80 mg, 334 μmol, 1.0 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (176 mg, 668 μmol, 2.0 equiv.) in toluene (4 mL) was added toluene-4-sulfonic acid (23.0 mg, 133 μmol, 0.4 equiv.). After stirring at 120° C. for 16 h, the mixture was cooled to room temperature, quenched by the addition of HO (10 mL) at 20° C., and extracted with dichloromethane (3×20 mL). The combined organic phases were washed with brine (50 mL), dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / methanol) to give (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-14A) (80 mg, 46% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.76(d,J=10.88Hz,1H) 7.31(s,1H) 6.50(d,J=2.13Hz,1H) 5.73(d,J=2.50Hz,1H) 5.44(br d,J=7.63Hz,4H) 4.88-5.02(m,1H) 3.48-3.65(m,2H) 3.14-3.24(m,1H) 2.94-3.10(m,1H) 2.44-2.49(m,1H) 2.36(s,3H) 1.94-2.03(m,1H) 1.82-1.91(m,2H) 0.84-0.90(m,3H). 19F NMR(376MHz,DMSO-D6) δ ppm -112.28. LCMS(ESI+) m / z:[MH] + C 25 H 24 FN2O6 + Calculated value: 467.1, Measured value: 467.2. SFC (RT = 2.284 min, 2.571 min).

[0305] (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-15), and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-16). (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (2-14A) (80 mg, 171 μmol) was purified by SFC (apparatus: Waters SFC150AP preparative SFC; column: IH (250 mm *30 mm, 10 μm); mobile phase: A is CO2 and B is methanol; gradient: B% = 35% isocratic elution mode; flow rate: 70 g / min; wavelength: 220 nm; column temperature: 35 °C; system back pressure: 120 bar) to separate (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-15) (compound 2-15 may be the opposite enantiomer of that shown) (17.2 mg) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (2-16) (compound 2-16 may be the opposite enantiomer of that shown) (23.5 mg) were obtained. Note: The stereochemistry of the two products has been arbitrarily assigned. Spectrum of 2-15: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.76(d,J=10.88Hz,1H) 7.31(s,1H) 6.50(s,1H) 5.74(s,1H) 5.44(br d,J=9.01Hz,4H) 4.96(t,J=6.19Hz,1H) 3.45-3.67(m,2H) 3.14-3.24(m,1H) 2.95-3.10(m,1H) 2.39-2.46(m,1H) 2.36(s,3H) 1.98(td,J=13.13,5.50Hz,1H) 1.79-1.92(m,2H) 0.87(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.25. LCMS(ESI+) m / z:[MH] + C 25 H 24 FN2O6 + Calculated value: 467.1, Measured value: 467.3. SFC (RT=2.292 min). Spectrum of 2-16: 1H NMR(400MHz,DMSO-D6) δ ppm 7.77(d,J=10.92Hz,1H) 7.31(s,1H) 6.51(s,1H) 5.74(s,1H) 5.44(d,J=7.91Hz,4H) 4.91-5.03(m,1H) 3.50-3.66(m,2H) 3.13-3.26(m,1H) 2.96-3.11(m,1H) 2.39-2.46(m,1H) 2.36(s,3H) 1.98(td,J=13.02,5.83Hz,1H) 1.80-1.92(m,2H) 0.88(t,J=7.28Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.27. LCMS(ESI+) m / z:[MH] + C 26 H 26 FN2O5 + Calculated value: 467.1, Measured value: 467.3. SFC (RT = 2.580 min).

[0306] Example 3 Synthesis of (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-24) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-25)

[0307] [ka]

[0308] N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-17A): A stirred mixture of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-1) (40.0 g, 170 mmol, 1.0 equiv) in 1,1-dimethoxy-N,N-dimethylmethanamine (400 mL) was heated at 110° C. for 5 h, then cooled to 15° C. and concentrated to give N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-17A) (46.6 g, 94% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6) δ ppm 13.07(s,1H),8.20(d,J=13.26Hz,1H),7.73(s,1H),3.15(s,6H),2.68-2.81(m,4H),2.12(d,J=1.75Hz,3H),2.07(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -108.358. LCMS(ESI+) m / z:[MH] + C 16 H 20 FN2O2 + Calculated value: 291.1, measured value: 291.2.

[0309] N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-18A): To a stirred mixture of N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-17A) (46.0 g, 158 mmol, 1.0 equiv) in dichloromethane (920 mL) was added 1 N hydrochloric acid solution (920 mL) at 15 °C. After stirring for 15 h at 15 °C, the mixture was extracted with dichloromethane (3 × 600 mL), and the combined organic phases were washed with brine, dried over NaSO, filtered, and concentrated to give N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-18A) (41.0 g, 98% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6) δ ppm 12.55(br s,1H),11.45(br s,1H),8.26(d,J=13.26Hz,1H),7.87(s,1H),2.80(br t,J=6.69Hz,2H),2.55(br t,J=6.50Hz,2H),2.10-2.16(m,6H). 19 F NMR(376MHz,DMSO-D6) δ ppm -105.54. LCMS(ESI+) m / z:[MH] + C 14 H 15 FNO3 + Calculated value: 264.1, measured value: 264.5.

[0310] N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-19A): To a stirred mixture of N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-18A) (41.0 g, 155 mmol, 1.0 equiv) in dichloromethane (420 mL) and acetic acid (4.2 mL) was added sodium triacetoxyborohydride (39.6 g, 93.4 mmol, 1.2 equiv) in portions at 20° C. After stirring at 30° C. for 15 hours under a nitrogen atmosphere, the mixture was quenched by the addition of water (250 mL) and extracted with dichloromethane (3×350 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-19A) (34.0 g, 82% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 12.13(s,1H),8.29(d,J=13.26Hz,1H),4.68(t,J=5.44Hz,1H),3.62-3.82(m,2H),3.03(dt,J=17.51,4.63Hz,1H) ,2.76-2.88(m,1H),2.63-2.73(m,1H),2.17-2.24(m,1H),2.15(s,3H),2.13(d,J=1.50Hz,3H),1.82-1.93(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.25. LCMS(ESI+) m / z:[MH] + C 14 H 17 FNO3 + Calculated value: 266.1, measured value: 266.2.

[0311] N-(7-((2-((Tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-20A): To a stirred mixture of N-[7-fluoro-3-(hydroxymethyl)-8-methyl-4-oxo-tetralin-5-yl]acetamide (3-19A) (2.5 g, 9.42 mmol, 1 equiv.) in dichloromethane (25 mL) was added AgO (21.8 g, 94.2 mmol, 10 equiv.) and tert-butyl-(2-iodoethoxy)-dimethyl-silane (53.95 g, 188.48 mmol, 20 equiv.). After stirring at 60 °C under a nitrogen atmosphere for 36 h, the mixture was cooled to room temperature and filtered to remove the AgO. It was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-20A) (3.35 g, 83% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 12.10(s,1H),8.29(d,J=13.13Hz,1H),3.66-3.79(m,4H),3.43-3.51(m,2H),3.03(dt,J=17.45,4.22Hz,1H),2. 76-2.94(m,2H),2.17-2.26(m,1H),2.11-2.16(m,6H),1.88(qd,J=12.09,4.38Hz,1H),0.84(s,9H),0.01(s,6H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.08. LCMS(ESI+) m / z:[MH] + C 22 H 35 FNO4Si + Calculated value: 424.2, measured value: 424.3.

[0312] N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-21A): To a stirred mixture of N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-20A) (1.1 g, 2.60 mmol, 1 equiv.) and cesium carbonate (169 mg, 519 μmol, 0.2 equiv.) in DMSO (22 mL) was added triethyl phosphite (862 mg, 5.19 mmol, 890 μL, 2.0 equiv.). The reaction mixture was degassed under vacuum, then purged with O three times and stirred for 3 h at 25 °C under O (15 psi). It was then quenched by the addition of water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-21A) (250 mg, 21% yield). 1 H NMR(400MHz,CDCl3) δ ppm 11.85(br s,1H),8.46(d,J=12.8Hz,1H),4.09(s,1H),3.64-3.77(m,4H),3.51-3.62(m,2H),2.95-3.05(m,1H),2. 80-2.92(m,1H),2.38-2.51(m,1H),2.24(s,3H),2.07-2.18(m,4H),0.87(s,9H),0.03(d,J=8.9Hz,6H). 19 F NMR(376MHz,CDCl3) δ ppm -100.715. LCMS(ESI+) m / z:[MH] + C 22 H 35 FNO5Si + Calculated value: 440.2, Measured value: 440.2.

[0313] 8-Amino-6-fluoro-2-hydroxy-2-((2-hydroxyethoxy)methyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (3-22A): A mixture of N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-21A) (450 mg, 1.02 mmol, 1 equiv.) in HCl (2 M, 18 mL) and methanol (18 mL) was stirred at 60 °C for 2.5 h, then cooled to room temperature, concentrated to remove methanol, quenched by the addition of saturated sodium bicarbonate solution at 25 °C, adjusted to pH 7, and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were dried over NaSO, filtered, and concentrated to give 8-amino-6-fluoro-2-hydroxy-2-(2-hydroxyethoxymethyl)-5-methyl-tetralin-1-one (3-22A) (200 mg, 68% yield), which was used directly in the next step without further purification. LCMS(ESI+)m / z:[M−H] + C 14 H 19 Calculated value of FNO4: 384.1, measured value: 384.1.

[0314] (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 N-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-23A): To a stirred mixture of 8-amino-6-fluoro-2-hydroxy-2-(2-hydroxyethoxymethyl)-5-methyl-tetralin-1-one (3-22A) (200 mg, 705 μmol, 1.0 equiv.) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione (1-4) (371 mg, 1.41 mmol, 2.0 equiv.) in toluene (10 mL) was added 4-methylbenzenesulfonic acid (48.6 mg, 282 μmol, 0.4 equiv.). The mixture was degassed and purged with argon three times, stirred under argon at 120 °C for 3 hours, cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / methanol) to give (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-23A) (100 mg, 27% yield). LCMS (ESI+) m / z: [MH] + C 27 H 28 FN2O7 + Calculated value: 511.2, Found value: 511.2. SFC (retention times = 1.845 min, 2.002 min).

[0315] (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-24) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-25). (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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 (3-23A) (100 mg) was purified by chiral HPLC (column: DAICEL CHIRALCEL OD (250 mm) * 30 mm, 10 μm); mobile phase: [CO2-methanol]; B%: 35%, isocratic elution mode) gradient: B% = 50%, isocratic elution mode; flow rate: 70 g / min; wavelength: 220 nm; column temperature: 35 °C; system back pressure: 120 bar) to separate (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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-di 3-24) (28.0 mg) (compound 3-24 may be the opposite enantiomer of that shown) and ((1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1-((2-hydroxyethoxy)methyl)-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) (3-25) (20.8 mg) (compound 3-25 may be the opposite enantiomer of that shown). Note: The stereochemistry of the two products has been arbitrarily assigned. 3-24 spectrum: 1H NMR(400MHz,CD3OD):δ ppm 7.57(s,1H),7.51(d,J=10.8Hz,1H),5.49-5.62(m,3H),5.37(d,J=16.3H z,1H),3.54-3.83(m,6H),3.20-3.29(m,1H),3.04-3.18(m,1H),2.69(br dd,J=12.8,3.3Hz,1H),2.39(s,3H),2.10(td,J=13.2,5.6Hz,1H),1.97(qd,J=7.2,2.3Hz,2H),1.02(t,J=7.3Hz,3H). 19 F NMR(376MHz,CD3OD) δ ppm -113.051. LCMS(ESI+) m / z:[MH] + C 27 H 28 FN2O7 + Calculated value: 511.2, Found value: 511.3. SFC (retention time = 2.005 min). 3-25 spectrum: 1 H NMR(400MHz,CD3OD):7.59(s,1H),7.53(br d,J=10.6Hz,1H),5.45-5.61(m,3H),5.35(d,J=16.3Hz,1H),3.47-3.75(m,6H),3.26(br d,J=4.5Hz,1H),3.00-3.17(m,1H),2.69(br dd,J=12.9,3.3Hz,1H),2.40(s,3H),2.18(td,J=13.2,5.6Hz,1H),1.88-2.02(m,2H),0.99(t,J=7.4Hz,3H). 19 F NMR(376MHz,CD3OD) δ ppm -112.862. LCMS(ESI+) m / z:[MH] + C 27 H 28 FN2O7 + Calculated value: 511.2, Found value: 511.3. SFC (retention time = 1.847 min). Alternative and / or additional synthetic routes to the compounds described in Example 3 can be found in FIG.

[0316] Example 4 Synthesis of (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (4-29) and (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (4-30)

[0317] [ka]

[0318] N-(7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-fluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4-26A): To a mixture of N-(3-fluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-11A) (4.00 g, 15.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (40 mL) and tetrahydrofuran (40 mL) was added sodium hydride (2.55 g, 63.6 mmol, 60 wt%, 4.0 equiv.) at 0° C., stirred for 0.5 h at 0° C., and 2-[tert-butyl(dimethyl)silyl]oxyethyl 4-methylbenzenesulfonate (10.5 g, 31.8 mmol, 2.0 equiv.) was added at 0° C., and stirred for 2.5 h at 0° C. The reaction mixture was then quenched by the addition of saturated ammonium chloride (70 mL) and citric acid (0.1 mol / L, 30 mL) and extracted with ethyl acetate (4 × 100 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-[3-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl]acetamide (4-26A) (1.50 g, 13% yield). 1 H NMR(DMSO-D6,400MHz):δ ppm 11.88(s,1H),8.29(d,J=13.0Hz,1H),5.35(s,1H),3.44-3.48(m,2H),2.53-2.57(m,2H),2.29(t,J=4.9Hz,1H),2.20(br d,J=5.4Hz,1H),2.15(s,3H),2.11(d,J=1.3Hz,3H),1.74-1.85(m,2H),0.80(s,9H),-0.02(s,6H) LCMS(ESI+) m / z:[M−H] + C 21 H 33 FNO4Si + Calculated value: 410.2, measured value: 410.3.

[0319] 8-Amino-6-fluoro-2-hydroxy-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (4-27A): To a stirred mixture of N-[3-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl]acetamide (4-26A) (1.50 g, 3.66 mmol, 1.0 equiv) in methanol (60 mL) was added HCl (2 M, 60 mL) at 60° C. After stirring at 60° C. for 2.5 hours, the mixture was cooled to room temperature, adjusted to pH 4 by addition of saturated sodium bicarbonate, and extracted with ethyl acetate (4×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give 8-amino-6-fluoro-2-hydroxy-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (4-27A) (310 mg, 33% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.43(br s,2H) 6.38(d,J=12.51Hz,1H) 4.96(s,1H) 4.30-4.39(m,1H) 3.54-3.63(m,1H) 3.37-3.46(m,1H) 2.70-2.88(m,2H) 2.04-2.14(m,1H) 1.97(s,3H) 1.82-1.93(m,1H) 1.60-1.77(m,2H). LCMS(ESI+) m / z:[MH-H2O] + C 13 H 15 FNO2 + Calculated value: 254.1, measured value: 236.2.

[0320] (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (4-28A): A stirred mixture of 8-amino-6-fluoro-2-hydroxy-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (4-27A) (310 mg, 1.22 mmol, 1.0 equiv.), (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione (1-4) (644 mg, 2.45 mmol, 2.0 equiv.), and 4-methylbenzenesulfonic acid (84.3 mg, 489 μm, 0.4 equiv.) in toluene (15.5 mL) was degassed, purged with argon three times, and stirred for 16 hours at 120° C. The mixture was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (3×60 mL). The combined organic phases were dried over Na2SO4, filtered, concentrated, and the residue was purified by preparative HPLC (column: Phenomenex luna C18 (250 * Purification by chromatography (70 mm, 15 μm); mobile phase: [HO (0.2% formic acid)-acetonitrile]; gradient 25% to 55% B 20.0 min) gave (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (4-28A) (40 mg, 6% yield). LCMS (ESI+) m / z: [MH] + C 26 H 26 FN2O6 + Calculated value: 481.1, measured value: 481.4.

[0321] (1S,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (4-29) and (1R,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (4-30). (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-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 (4-28A) (40.0 mg, 83.2 μmol) was purified by SFC (instrument: not defined; column: REGIS(s,s) WHELK-O1 (250 mm * 30 mm, 5 μm); (Mobile phase: A is CO2 and B is methanol; Gradient: B% = 50.00% isocratic elution mode; Flow rate: 70.00 g / min; Monitor wavelength: 220 nm; Column temperature: 40 °C; System back pressure: 100 bar) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (4-29) (compound 4-29 may be the opposite enantiomer of that shown) (10.2 mg) and (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (4-30) (compound 4-30 may be the opposite enantiomer of that shown) (5.00 mg) were obtained. Note: The stereochemistry of the two products was arbitrarily assigned. Spectrum of 4-29: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.76(d,J=10.8Hz,1H),7.31(s,1H),6.50(s,1H),5.81(s,1H),5.28-5.52(m,4H),4.47(t,J=4.9Hz ,1H),3.70-3.81(m,1H),3.57-3.67(m,1H),3.05-3.26(m,2H),2.34-2.46(m,1H),2.37(s,3H),1.95 -2.03(m,1H),1.77-1.90(m,4H),0.88(br t,J=7.3Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.26. LCMS(ESI+) m / z:[MH] + C 26 H 26 FN2O6 + Calculated: 481.1, Found: 481.2. SFC (retention time = 1.879 min). 4-30 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.76(d,J=10.8Hz,1H),7.31(s,1H),6.50(s,1H),5.81(s,1H),5.28-5.52(m,4H),4.47(t,J=4.9Hz,1H) ,3.70-3.81(m,1H),3.57-3.67(m,1H),3.05-3.26(m,2H),2.34-2.46(m,4H),1.77-2.08(m,5H),0.88(br t,J=7.3Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.27. LCMS(ESI+) m / z:[MH] + C 26 H 26 FN2O6 + Calculated: 481.1, Found: 481.3. SFC (retention time = 2.091 min). Alternative and / or additional synthetic routes to the compounds described in Example 4 can be found in FIG.

[0322] Example 5 Synthesis of (1S,9S)-1-azido-9-ethyl-5-fluoro-9-dihydroxy-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 (5-38) and (1R,9S)-1-azido-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 (5-39)

[0323] [ka]

[0324] N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-31A): To a stirred mixture of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-2) (5.0 g, 18.1 mmol, 1.0 equiv.) in water (33.7 mL) and 1,4-dioxane (101 mL), 2,6-dimethylpyridine (3.89 g, 36.3 mmol, 4.23 mL, 2.0 equiv.), osmium tetroxide (92.3 mg, 363 μmol, 18.8 μL, 0.02 equiv.), and sodium periodate (15.5 g, 72.6 mmol, 4.03 mL, 4.0 equiv.) were added. Two more vials were set up as above, and all three reaction mixtures were combined. After stirring for 3 h at 25 °C, the mixture was quenched by the addition of water (50 mL) and extracted with dichloromethane (4 × 200 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-31A) (8.0 g, 69% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.95(s,1H) 9.74(d,J=1.00Hz,1H) 8.28(d,J=13.26Hz,1H) 3.15-3.26(m,1H) 2.97-3.06(m,1H) 2.82-2.93(m,2H) 2.58(dd,J=17.39,5.00Hz,1H) 2.11-2.15(m,7H) 1.80-1.92(m,1H). LCMS(ESI+) m / z:[MH] + C 15 H 17 FNO3 + Calculated value: 278.1, measured value: 278.3.

[0325] N-(3-fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-32A): To a stirred mixture of N-(3-fluoro-4-methyl-8-oxo-7-(2-oxoethyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-31A) (7.6 g, 23.8 mmol, 1.0 equiv) in tetrahydrofuran (152 mL) and water (76 mL) was added sodium borohydride (180 mg, 4.76 mmol, 0.2 equiv) at 0° C. After stirring for 0.5 h at 0° C., the mixture was extracted with ethyl acetate (4×200 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(3-fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-32A) (4.83 g, 72% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 12.08(s,1H) 8.26(d,J=13.26Hz,1H) 4.49(t,J=5.19Hz,1H) 3.47-3.59(m,2H) 2.96(dt,J=17.60,4.64Hz,1H) 2.75-2.88(m,1H) 2.61-2.72(m,1H) 2.15(s,3H) 2.10(s,3H) 1.98-2.06(m,1H) 1.68-1.80(m,1H) 1.44-1.56(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.67. LCMS(ESI+) m / z:[M + Na] + C 15 H 18 FNO3Na + Calculated value: 302.1, Measured value: 302.1.

[0326] 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)ethyl acetate (5-33A): To a stirred mixture of N-(3-fluoro-7-(2-hydroxyethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-32A) (4.0 g, 14.3 mmol, 1.0 equiv) in dichloromethane (80 mL) was added pyridine (27.2 g, 343 mmol, 27.7 mL, 24 equiv), N,N-dimethylpyridin-4-amine (174 mg, 1.43 mmol, 0.1 equiv), and AcO (23.3 g, 229 mmol, 21.5 mL, 16 equiv) at 0° C. After stirring for 3 hours at 25° C., the reaction mixture was quenched by the addition of ice water (60 mL) at 0° C., adjusted to pH 7 by the addition of aqueous HCl (1 M) at 0° C., and extracted with dichloromethane (4 × 100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give ethyl 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-33A) (3.2 g, 69% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 12.01(s,1H) 8.26(d,J=13.26Hz,1H) 4.06-4.21(m,2H) 2.93-3.05(m,1H) 2.78-2.91(m,1H) 2.63-2.74(m,1H) 2.13-2.22(m,5H) 2.11(s,3H) 1.99(s,3H) 1.64-1.86(m,2H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.47. LCMS(ESI+) m / z:[MH] + C 17 H 21 FNO4 + Calculated value: 322.1, measured value: 322.2.

[0327] 2-(8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)ethyl acetate (5-34A): To a stirred mixture of ethyl 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-33A) (3.2 g, 10.0 mmol, 1.0 equiv.) in acetic acid (64 mL) was added pyridinium tribromide (3.52 g, 11.0 mmol, 1.1 equiv.). After stirring for 12 hours at 50° C., the mixture was quenched by the addition of ice water (60 mL), the pH was adjusted to 7 by the addition of saturated sodium bicarbonate solution at 0° C., and extracted with dichloromethane (3×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give ethyl 2-(8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-34A) (2.4 g, 58% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.81(s,1H) 8.33-8.40(m,1H) 4.16-4.37(m,2H) 3.03-3.13(m,1H) 2.84-2.96(m,1H) 2.52-2.69(m,3H) 2.29-2.40(m,1H) 2.19(s,3H) 2.14-2.18(m,3H) 1.99(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -104.47. LCMS(ESI+) m / z:[MH] + C 17 H 20 BrFNO4 + Calculated values: 400.0, 402.0 Measured values: 400.1, 402.1.

[0328] 2-(8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)ethyl acetate (5-35A): To a stirred mixture of ethyl 2-(8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-34A) (500 mg, 1.25 mmol, 1.0 equiv.) in dimethyl sulfoxide (10 mL) was added sodium azide (162 mg, 2.50 mmol, 2 equiv.) After stirring at 20°C for 4 hours under an argon atmosphere, the mixture was quenched with ice water (40 mL), adjusted to pH 8 by the addition of saturated sodium bicarbonate solution at 0°C, and extracted with ethyl acetate (3 x 50 mL). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give ethyl 2-(8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-35A) (184 mg, 38% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.54(s,1H) 8.29(d,J=13.01Hz,1H) 4.14-4.20(m,2H) 2.94-3.00(m,2H) 2.21(br d,J=5.13Hz,2H) 2.19(s,3H) 2.14-2.18(m,2H) 2.12(d,J=1.38Hz,3H) 1.93(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -102.81. LCMS(ESI+) m / z:[MH] + C 17 H 20 FN4O4 + Calculated value: 363.1, Measured value: 363.1.

[0329] 8-Amino-2-azido-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (5-36A): To a stirred mixture of ethyl 2-(8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (5-35A) (180 mg, 496 μmol, 1 equiv.) in methanol (7.2 mL) was added hydrochloric acid (2 M, 7.20 mL, 28.9 equiv.). After stirring at 60° C. for 2.5 hours and cooling to room temperature, the mixture was quenched by the addition of saturated sodium bicarbonate solution (approximately 12 mL), adjusted to pH 7, and extracted with ethyl acetate (3×30.0 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated to give 8-amino-2-azido-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (5-36A) (130 mg, 94% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6) δ ppm 7.36-7.63(m,2H) 6.42(d,J=12.51Hz,1H) 4.64(t,J=5.19Hz,1H) 3.48-3.65(m,2H) 2.79(br t,J=5.94Hz,2H) 2.10-2.21(m,2H) 1.97(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -106.57. LCMS(ESI+) m / z:[M + Na] + C 13 H 15 FN4O2Na + Calculated value: 301.1, Measured value: 301.1.

[0330] (9S)-1-Azido-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 (5-37A): A stirred mixture of 8-amino-2-azido-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (5-36A) (130 mg, 467 μmol, 1.0 equiv.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (245 mg, 934 μmol, 2.0 equiv.), and 4-methylbenzenesulfonic acid (32.1 mg, 186 μmol, 0.4 equiv.) in toluene (6.5 mL) was degassed, then purged with argon three times and stirred at 120° C. under an argon atmosphere for 16 hours. The mixture was cooled to room temperature, quenched by the addition of water (20 mL), and extracted with ethyl acetate (5×30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give (9S)-1-azido-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 (5-37A) (150 mg, 27% yield). LCMS (ESI+) m / z: [MH] + C 26 H 25 FN5O5 + Calculated value: 506.1, measured value: 506.3.

[0331] (1S,9S)-1-Azido-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 (5-38) and (1R,9S)-1-Azido-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 (5-39). (9S)-1-Azido-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 (5-37A) (150 mg, crude) was purified by HPLC (column: Phenomenex Luna C18 100 * 30mm * 5 μm; mobile phase: [HO (0.2% formic acid)-acetonitrile]; gradient: 30% to 50% B 12.0 min) and purified to give (1S,9S)-1-azido-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 (5-38). (Compound 5-38 may be the opposite enantiomer of the one shown.) (11. 4 mg) and (1R,9S)-1-azido-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 (5-39) (compound 5-39 may be the opposite enantiomer of that shown) (19.8 mg) were obtained. Note: The stereochemistry of the two products was assigned arbitrarily. Spectrum of 5-38: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.83(d,J=10.63Hz,1H) 7.32(s,1H) 6.53(s,1H) 5.37-5.47(m,4H) 4.66(t,J=5.00Hz,1H) 3.53-3.71(m,2H) 3.33-3.39(m,1H) 3.14-3.28(m,1H) 2.71-2.80(m,1H) 2.40(s,3H) 2.24-2.30(m,1H) 1.98(br t,J=6.25Hz,2H) 1.79-1.92(m,2H) 0.83-0.91(m,3H). 19F NMR(376MHz,DMSO-D6) δ ppm -111.66. LCMS(ESI+) m / z:[MH] + C 26 H 25 FN5O5 + Calculated value: 506.1, measured value: 506.3. SFC (holding time = 1.500 minutes). 5-39のスペクトル: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.83(d,J=10.76Hz,1H) 7.32(s,1H) 6.48-6.58(m,1H) 5.43(d,J=7.88Hz,4H) 4.66(t,J=5.07Hz,1H) 3.53-3.72(m,2H) 3.35(br s,1H) 3.14-3.27(m,1H) 2.72-2.80(m,1H) 2.40(s,3H) 2.27(td,J=12.66,5.07Hz,1H) 1.99(br t,J=6.13Hz,2H) 1.79-1.93(m,2H) 0.84-0.92 (m, 3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.66. LCMS(ESI+) m / z:[MH] + C 26 H 25 FN5O5 + Calculated value: 506.1, measured value: 506.3. SFC (holding time = 1.903 minutes).

[0332] Example 6 N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-45), N-((R)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-46), N- Synthesis of ((S)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-47) and N-((R)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-48)

[0333] [ka]

[0334] 2-Allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (6-40A): To a stirred mixture of N-(7-allyl-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-2) (1.0 g, 3.63 mmol, 1.0 equiv.) in methanol (30 mL) was added sulfuric acid (2.0 mL). Five additional vials were set up as above, and all six reaction mixtures were combined for further analysis. After stirring at 60 °C for 18 h under an argon atmosphere and cooling to room temperature, the reaction mixture was quenched with water (150 mL), adjusted to pH 7 by the addition of saturated sodium bicarbonate solution at 0 °C, and extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give 2-allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (6-40A) (3.2 g, 62% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.42(br s,2H),6.35(d,J=12.7Hz,1H),5.59-5.99(m,1H),4.93-5.23(m,2H),2.87(dt,J=17.3,4.5Hz,1H),2.52-2.74(m,2H),2.48(br s,1H),1.93-2.21(m,5H),1.54-1.70(m,1H).LCMS(ESI+) m / z:[MH] + C 14 H 17 FNO + Calculated value: 234.1, measured value: 234.4.

[0335] (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 (6-41A): To a suspension of 2-allyl-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (6-40A) (200 mg, 857 μmol, 1.0 equiv) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (451 mg, 1.71 mmol, 2.0 equiv) in toluene (10 mL) was added 4-methylbenzenesulfonic acid (59.1 mg, 342 μmol, 0.4 equiv) at 140 °C. Two more vials were set up as above, and all three reaction mixtures were combined for further analysis. After stirring at 140 °C for 16 h in a Dean-Stark reaction, the mixture was cooled to room temperature and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) 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 (6-41A) (300 mg, 21% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.74(br d,J=10.9Hz,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(br d,J=3.3Hz,1H),3.08(br s,2H),2.34-2.47(m,4H),2.18-2.33(m,2H),1.83-1.95(m,3H),0.87(br t,J=7.0Hz,3H). LCMS(ESI+) m / z:[MH] + C 27 H 26 FN2O4 + Calculated value: 461.1, Found value: 461.4. SFC (retention times = 1.020 min, 2.034 min).

[0336] (9S)-1-Allyl-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-42A): To a stirred mixture of (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 (6-41A) (15.1 g, 32.8 mmol, 1 equiv) in dichloromethane (300 mL) at 0 °C was added N,N-dimethylpyridin-4-amine (400 mg, 3.28 mmol, 0.1 equiv), pyridine (62.2 g, 787 mmol, 63.5 mL, 24 equiv), and AcO (53.5 g, 525 mmol, 49.3 mL, 16 equiv). After stirring for 2 h at 0 °C, the mixture was quenched by the addition of HO (500 mL) and extracted with dichloromethane (3 × 300 mL). The combined organic phases were washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give (9S)-1-allyl-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-42A) (9.3 g, 56% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.72(dd,J=11.07,3.44Hz,1H) 7.01(s,1H) 5.89-6.05(m,1H) 5.49(s,2H) 5.22-5.40(m,2H) 5.07-5.20(m,2H) 3.44(br d,J=4.88Hz,1H) 2.93-3.17(m,2H) 2.39-2.46(m,1H) 2.36(s,3H) 2.21-2.31(m,5H) 2.07-2.19(m,2H) 1.91-1.99(m,1H) 0.91(t,J=7.38Hz,3H). 19F NMR(376MHz,DMSO-D6) δ ppm -111.53. LCMS(ESI+) m / z:[MH] + C 29 H 28 FN2O5 + Calculated value: 503.1, measured value: 503.4.

[0337] (9S)-1-(1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-43A): (9S)-1-Allyl-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl in dichloromethane (100 mL) A stirred mixture of acetic acid (6-42A) (9.30 g, 18.5 mmol, 1.0 equiv), 3-methyl-1,4,2-dioxazol-5-one (18.7 g, 185 mmol, 10 equiv), dichloroiridium-1,2,3,4,5-pentamethylcyclopentane (4.48 g, 5.55 mmol, 0.3 equiv), AgSbF (5.09 g, 14.8 mmol, 0.8 equiv), and AgOAc (5.56 g, 33.3 mmol, 1.71 mL, 1.8 equiv) was degassed, then purged with nitrogen three times, and stirred at 40 °C under a nitrogen atmosphere for 12 h. After cooling to room temperature, the mixture was diluted with dichloromethane (200 mL), filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give (9S)-1-(1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-43A) (8.3 g, 68% yield). 1H NMR(400MHz,DMSO-D6) δ ppm 8.16-8.29(m,1H) 7.66-7.86(m,1H) 6.91-7.09(m,1H) 5.62-5.95(m,1H) 5.43-5.55(m,2H) 4.74-5.40(m,3H) 4.47-4.62(m,1H) 4.12(br s,1H) 3.50-3.69(m,1H) 2.90-3.17(m,2H) 2.29-2.42(m,4H) 2.22(d,J=4.77Hz,3H) 2.02-2.17(m,3H) 1.70-1.78(m,3H) 0.83-0.99(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.12. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6 + Calculated value: 560.3, measured value: 560.2.

[0338] N-(1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-44A): To a stirred mixture of (9S)-1-(1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-43A) (700 mg, 1.25 mmol, 1.0 equiv.) in methanol (14 mL) was added HCl (2 M, 28 mL). After stirring at 80 °C for 12 h, the mixture was cooled to 20 °C and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with brine, dried over NaSO, filtered, concentrated, and the residue was purified by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system, column: Phenomenex Luna C18 100). * 30mm *Purification by 5 μm column chromatography (mobile phase: A: HO (0.2% formic acid); B: acetonitrile, gradient: 20.00% to 50.00% B in 8.00 min, flow rate: 60.00 ml / min, monitor wavelength: 220 & 254 nm) afforded N-(1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-44A) (120 mg, 18% yield). LCMS (ESI+) m / z: [MH] + C 29 H 29 FN3O5 + Calculated value: 518.2, measured value: 518.3.

[0339] N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-45), N-((R)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-46), N -((S)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-47), and N-((R)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-48). N-(1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-44A) (120 mg, 0.23 mmol) was purified by HPLC (instrument: Gilson 281 semi-preparative HPLC system, column: Phenomenex Luna C18 100). * 30mm *Separation was performed using a 5 μm column, mobile phase: A: H2O (0.2% formic acid); B: acetonitrile, gradient: 25.00% to 45.00% B in 12.00 min, flow rate: 60.00 ml / min, monitor wavelength: 220 & 254 nm) and N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide. amide (6-45) (compound 6-45 may be the opposite enantiomer of that shown) (9.2 mg), N-((R)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-46) (compound 6-46 is shown) (10.3 mg), N-((S)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-47) (compound 6-47 is the opposite enantiomer of that shown) (3.6 mg), and N-((R)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)allyl)acetamide (6-48) (compound 6-48 may be the opposite enantiomer of that shown) (3.6 mg). Note: The stereochemistry of the four products was assigned arbitrarily. 6-45 spectrum: 1H NMR(400MHz,DMSO-D6) δ ppm 8.22(d,J=9.26Hz,1H) 7.75(d,J=11.01Hz,1H) 7.30(s,1H) 6.52(br s,1H) 5.70(ddd,J=16.85,10.54,6.00Hz,1H) 5.42(s,2H) 5.33(d,J=18.64Hz,1H) 5.11(d,J=18.64Hz,1H) 4.78-4.94(m,2H) 4.48-4.62(m,1H) 3.31-3.40(m,1H) 3.11-3.25(m,1H) 2.97-3.10(m,1H) 2.36(s,3H) 2.32(br s,1H) 1.78-1.98(m,6H) 0.89(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.76. LCMS(ESI+) m / z:[MH] + C 29 H 29 FN3O5 + Calculated value: 518.2, measured value: 518.3. SFC (holding time = 1.368 minutes). 6-46のスペクトル: 1 H NMR(400MHz, DMSO-D6) δ ppm 8.22(br d,J=9.13Hz,1H) 7.76(br d,J=10.88Hz,1H) 7.30(s,1H) 6.53(s,1H) 5.71(ddd,J=16.76,10.26,6.25Hz,1H) 5.43(s,2H) 5.33(br d,J=18.64Hz,1H) 5.12(br d,J=18.64Hz,1H) 4.74-4.93(m,2H) 4.49-4.60(m,1H) 3.31-3.40(m,1H) 3.09-3.20(m,1H) 2.97-3.08(m,1H) 2.36(br s,3H) 2.32(br s,1H) 1.77-1.99(m,6H) 0.87(br t,J=7.13Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.76. LCMS(ESI+) m / z:[MH] + C 29 H 29 FN3O5 +Calculated value: 518.2, measured value: 518.3. SFC (holding time = 2.727 minutes). 6-47のスペクトル: 1 H NMR(400MHz,DMSO-D6) δ ppm 8.28(d,J=9.26Hz,1H) 7.74(d,J=11.01Hz,1H) 7.31(s,1H) 6.53(s,1H) 5.92(ddd,J=17.42,9.91,7.94Hz,1H) 5.31-5.54(m,4H) 5.28(br d,J=17.14Hz,1H) 5.16(d,J=10.38Hz,1H) 4.60(q,J=8.88Hz,1H) 3.36-3.42(m,1H) 3.02-3.18(m,2H) 2.39(s,3H) 2.29-2.36(m,1H) 1.83-2.00(m,3H) 1.52(s,3H) 0.89(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.29. LCMS(ESI+) m / z:[MH] + C 29 H 29 FN3O5 + Calculated value: 518.2, measured value: 518.3. SFC (holding time = 1.859 minutes). 6-48のスペクトル: 1 H NMR(400MHz, DMSO-D6) δ ppm 8.25(d,J=9.26Hz,1H) 7.73(d,J=11.01Hz,1H) 7.30(s,1H) 6.52(s,1H) 5.85-5.99(m,1H) 5.30-5.50(m,4H) 5.27(d,J=17.13Hz,1H) 5.15(d,J=10.51Hz,1H) 4.60(q,J=8.80Hz,1H) 3.34-3.36(m,1H) 3.00-3.18(m,2H) 2.38(s,3H) 2.27-2.35(m,1H) 1.81-2.01(m,3H) 1.49(s,3H) 0.86(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.28. LCMS(ESI+) m / z:[MH] + C 29 H 29 FN3O5+ Calculated value: 518.2, Found value: 518.2. SFC (retention time = 2.897 min).

[0340] Example 7 Synthesis of (1S,9S)-1-amino-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 (7-50) and (1R,9S)-1-amino-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 (7-51)

[0341] [ka]

[0342] (9S)-1-amino-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 hydrochloride (7-49): To a stirred mixture of (9S)-1-azido-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 (5-37A) (450 mg, 489 μmol, 1.0 equiv.) in tetrahydrofuran (9 mL) and water (2.25 mL) was added PPh3 (256 mg, 979 μmol, 2.0 equiv.). After stirring at 50°C for 4 hours, the mixture was cooled to room temperature, quenched by the addition of water (20 mL), and adjusted to pH 2 by the addition of 1 N hydrochloric acid solution at 0°C. After separation, the aqueous phase was washed with ethyl acetate (2 × 20 mL) and concentrated to give (9S)-1-amino-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 hydrochloride (7-49) (230 mg, 86% yield). LCMS (ESI+) m / z: [MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, measured value: 480.3.

[0343] (1S,9S)-1-Amino-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 hydrochloride (7-50) and (1R,9S)-1-Amino-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 hydrochloride (7-51) (9S)-1-Amino-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 hydrochloride (7-49) (200 mg, 417 μmol) was purified by HPLC (column: Phenomenex Luna C18 100 * 30mm * 5 μm; Mobile phase: [HO (0.2% formic acid)-acetonitrile]; Gradient: B The separation was carried out in 8.0 min with a 1% to 30% yield to give (1S,9S)-1-amino-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 hydrochloride (7-50) and (1R,9S)-1-amino-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 hydrochloride (7-51). Each product was suspended in water (0.04% hydrochloric acid, 2 mL) and lyophilized to give (7-50) (compound 7-50 may be the opposite enantiomer of that shown) (5.0 mg) and (7-51) (compound 7-51 may be the opposite enantiomer of that shown) (10.3 mg). 7-50 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.74-7.82(m,1H) 7.29-7.35(m,1H) 6.49-6.54(m,1H) 5.69(br d,J=19.76Hz,1H) 5.40-5.53(m,3H) 3.59(br t,J=6.19Hz,2H) 3.08-3.20(m,2H) 2.30-2.41(m,4H) 2.00-2.12(m,1H) 1.79-1.98(m,4H) 0.88(t,J=7.32Hz,3H). 19F NMR(376MHz,DMSO-D6) δ ppm -112.45. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.3. SFC (retention time = 3.114 min). Spectrum of 7-51: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.84(d,J=10.63Hz,1H) 7.31-7.36(m,1H) 6.53(s,1H) 5.68(d,J=19.2Hz,1H) 5.53(d,J=18.8Hz,1H) 5.45(s,2H) 3.60-3.67(m,1H) 3.51-3.60(m,1H) 3.15-3.20(m,2H) 2.50 -2.54(m,1H) 2.39(s,3H) 1.99-2.24(m,3H) 2.04-2.09(m,1H) 1.91-2.03(m,2H) 1.80-1.91(m,2H) 0.88(t,J =7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.75. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.3 SFC (retention time = 1.822 min). Alternative and / or additional synthetic routes to the compounds described in Example 7 can be found in FIG.

[0344] Example 8 Synthesis of (1R,9S)-1-amino-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 hydrochloride (8-57) and (1S,9S)-1-amino-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 hydrochloride (8-58)

[0345] [ka]

[0346] N-(7-azido-3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-53A): To a stirred mixture of N-(7-azido-3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-52A) (900 mg, 3.26 mmol, 1.0 equiv) in formaldehyde (25.6 g, 316 mmol, 37 wt%, 97 equiv) was added triethylamine (329 mg, 3.26 mmol, 1.0 equiv) at 0° C. After stirring for 12 hours at 20° C., the mixture was quenched by the addition of water (20 mL) and extracted with dichloromethane (3×30 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give N-(7-azido-3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-53A) (700 mg, 21% yield). 1H NMR(400MHz,DMSO-D6) δ ppm 11.63(s,1H),8.30(d,J=13.01Hz,1H),5.47(t,J=5.88Hz,1H),3.81(d,J=5.88Hz,2H),2.93(br d,J=6.13Hz,2H),2.12-2.20(m,4H),2.11(d,J=1.38Hz,3H),1.98-2.08(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -102.781. LCMS(ESI+) m / z:[MH] + C 14 H 16 FN4O3 + Calculated value: 307.1, measured value: 307.5.

[0347] 8-Amino-2-azido-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (8-54A): To a stirred mixture of N-(7-azido-3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-53A) (700 mg, 2.29 mmol, 1.0 equiv) in methanol (28 mL) was added hydrochloric acid (2 M, 28 mL). After stirring at 60 °C for 3 h, the mixture was cooled to room temperature, adjusted to pH 8 with saturated sodium bicarbonate solution, and extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give 8-amino-2-azido-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (8-54A) (500 mg, 41% yield). 1 H NMR(400MHz,CDCl3) δ ppm 6.31-6.85(m,2H),6.23(d,J=11.51Hz,1H),3.96(d,J=1.63Hz,2H),2.76-2.99(m,3H),2.08-2.18(m,1H),2.07(s,3H),1.85-2.01(m,1H). 19F NMR(376MHz,CDCl3) δ ppm -103.729. LCMS(ESI+) m / z:[MH] + C 12 H 14 FN4O2 + Calculated value: 265.1, measured value: 265.5.

[0348] (9S)-1-Azido-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 (8-55A): To a stirred mixture of 8-amino-2-azido-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (8-54A) (500 mg, 1.89 mmol, 1.0 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (966 mg, 3.78 mmol, 2.0 equiv.) in toluene (25 mL) was added 4-methylbenzenesulfonic acid (130 mg, 756 μmol, 0.4 equiv.). After stirring at 120° C. for 12 hours, the mixture was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (3×100 mL). The combined organic phases were dried over NaSO, filtered, and concentrated. The residue was purified by silica gel chromatography (ethyl acetate / methanol) to give (9S)-1-azido-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 (8-55A) (280 mg, 12% yield). 1H NMR(400MHz,DMSO-D6) δ ppm 7.81(d,J=10.8Hz,1H),7.31(d,J=2.1Hz,1H),6.52(s,1H),5.71(t,J=5.8Hz,1H),5.41-5.50(m,3H),4.13(t,J=6.7Hz,1H),3 .66-3.85(m,2H),3.03-3.18(m,1H),2.80-2.94(m,2H),2.39(s,3H),2.12-2.26(m,1H),1.80-1.89(m,2H),0.83-0.91(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.5. LCMS(ESI+) m / z:[MH] + C 25 H 23 FN5O5 + Calculated value: 492.1, Measured value: 492.4

[0349] (9S)-1-amino-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 hydrochloride (8-56A): To a stirred mixture of (9S)-1-azido-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 (8-55A) (230 mg, 467 μmol, 1.0 equiv.) in tetrahydrofuran (4.6 mL) and water (1.1 mL) was added PPh3 (245 mg, 935 μmol, 2.0 equiv.). After stirring for 12 hours at 50°C, the mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were dried over Na2SO4, filtered, concentrated, and the residue was purified by preparative HPLC (column: Phenomenex Luna C18 75 * 30mm *Purification by column chromatography (3 μm; mobile phase: [HO (0.04% HCl)-acetonitrile]; gradient 10% to 35% B 8.0 min) gave (9S)-1-amino-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 hydrochloride (8-56A) (100 mg, 45% yield). 1 H NMR(400MHz,CD3OD) δ ppm 7.75(dd,J=10.32,7.07Hz,1H),7.65(d,J=4.13Hz,1H),5.56-5.69(m,2H),5.36-5.50(m,2H),3 .95-4.10(m,2H),3.34-3.44(m,1H),3.17-3.29(m,1H),2.75(dt,J=13.07,4.53Hz,1H),2.46(br s,3H) 2.29-2.42(m,1H),1.91-2.03(m,2H),1.01(td,J=7.32,2.25Hz,3H). 19 F NMR(376MHz,CD3OD) δ ppm -111.89. LCMS(ESI+) m / z:[MH] + C 25 H 25 FN3O5 + Calculated value: 466.1, Measured value: 466.1

[0350] (1R,9S)-1-amino-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 hydrochloride (8-57) and (1S,9S)-1-amino-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 hydrochloride (8-58). (9S)-1-amino-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 hydrochloride (8-56A) (47 mg) was dissolved in methanol and separated by preparative HPLC to give (1R,9S)-1-amino-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 hydrochloride (8-56A) (47 mg). This afforded 11 mg of (1S,9S)-1-amino-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 hydrochloride (8-57) (compound 8-57 may be the opposite enantiomer of that shown) and 15 mg of (1S,9S)-1-amino-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 hydrochloride (8-58) (compound 8-58 may be the opposite enantiomer of that shown). Note: The stereochemistry at this carbon was arbitrarily assigned. Spectrum of 8-57: 1 H NMR(400MHz,DMSO-D6) δ ppm 9.08(br s,3H),7.88(d,J=10.51Hz,1H),7.36(s,1H),6.56(br s,1H),5.85(br s,1H),5.60(d,J=6.88Hz,2H),5.45(s,2H),3.80-3.92(m,2H),3.14-3.25(m,2H),2.56-2.65( m,1H),2.40(s,3H),2.17-2.27(m,1H),1.88(dt,J=14.10,6.89Hz,2H),0.88(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.32. LCMS(ESI+) m / z:[MH] + C 25 H 25 FN3O5 +Calculated value: 466.1, Measured value: 466.3. SFC (RT = 2.594 min). Spectrum of 8-58: 1 H NMR(400MHz,DMSO-D6) δ ppm 9.06(br s,3H),7.88(d,J=10.51Hz,1H),7.36(s,1H),6.48(br s,1H),5.87(br s,1H),5.59(s,2H),5.46(s,2H),3.80-3.90(m,2H),3.15-3.27(m,2H),2.61(dt,J=13.07 ,4.66Hz,1H),2.40(s,3H),2.16-2.26(m,1H),1.80-1.94(m,2H),0.87(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.28. LCMS(ESI+) m / z:[MH] + C 25 H 25 FN3O5 + Calculated value: 466.1, Measured value: 466.3. SFC (RT = 2.374 min). Alternative and / or additional synthetic routes to the compounds described in Example 8 can be found in FIG.

[0351] Example 9 Synthesis of (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-64) and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-65)

[0352] [ka]

[0353] 2-((8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)ethyl acetate (9-59): To a stirred mixture of N-(7-((2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3-20A) (6.50 g, 12.7 mmol, 1.1 equiv.) in acetic acid (130 mL) was added pyridinium tribromide (4.48 g, 14.01 mmol, 1 equiv.). After stirring at 50 °C for 18 h under a nitrogen atmosphere, the reaction mixture was cooled to room temperature, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give ethyl 2-((8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)acetate (9-59) (4.20 g, 76% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.85(s,1H),8.37(d,J=13.13Hz,1H),4.21(d,J=10.13Hz,1H),4.11-4.16(m,2H),3.94(d,J=10.13Hz,1H),3.71-3.78(m,2H),3. 12(dt,J=17.92,3.55Hz,1H),2.83-2.95(m,1H),2.42(dd,J=8.25,3.63Hz,2H),2.19(s,3H),2.16(d,J=1.38Hz,3H),1.98(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -101.87. LCMS(ESI+) m / z:[MH] + C 18 H 22 FNO5Br + Calculated values: 430.1, 432.1 Measured values: 430.2, 432.2.

[0354] 2-((8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)ethyl acetate (9-60): To a stirred mixture of ethyl 2-((8-acetamido-2-bromo-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)acetate (9-59) (4.20 g, 9.76 mmol, 1 equiv.) in DMSO (84 mL) was added sodium azide (1.27 g, 19.52 mmol, 2 equiv.) at 20° C. After stirring at 20° C. for 4 hours under a nitrogen atmosphere, the mixture was quenched by the addition of water (100 mL) at 0° C., adjusted to pH 8 by the addition of saturated sodium bicarbonate solution at 0° C., and extracted with dichloromethane (3×150 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give ethyl 2-((8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)acetate (9-60) (1.40 g, 36% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 11.55(s,1H),8.30(d,J=13.05Hz,1H),4.08-4.14(m,2H),3.87(s,2H),3.66(t,J=4.58Hz,2H), 2.90-2.98(m,2H),2.20-2.26(m,1H),2.19(s,3H),2.12(s,3H),2.05-2.11(m,1H),1.98(s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -102.54. LCMS(ESI+) m / z:[MH] + C 18 H 22 FN4O5 + Calculated value: 393.1, measured value: 393.2.

[0355] 8-Amino-2-azido-6-fluoro-2-((2-hydroxyethoxy)methyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (9-61): To a stirred mixture of ethyl 2-((8-acetamido-2-azido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methoxy)acetate (9-60) (1.40 g, 3.56 mmol) in methanol (28 mL) was added hydrochloric acid (2 M, 28.00 mL). After stirring at 60° C. for 3 hours, the mixture was cooled to room temperature, quenched by the addition of water (30 mL) at 20° C., adjusted to pH 7 by the addition of saturated sodium bicarbonate solution at 0° C., and extracted with ethyl acetate (3×40 mL). The combined organic phases were dried over NaSO, filtered, and concentrated to give 8-amino-2-azido-6-fluoro-2-((2-hydroxyethoxy)methyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (9-61) (1.00 g, 90% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6) δ ppm 7.48-7.58(m,1H),6.42(d,J=12.51Hz,1H),4.57-4.64(m,1H),3.72-3.87(m,2H),3.45-3.55(m,4H),2.78(br t,J=5.75Hz,2H),2.13-2.24(m,1H),1.97(s,3H),1.90-1.96(m,1H). 19 F NMR(376MHz,DMSO-D6) δ ppm -106.16. LCMS(ESI+) m / z:[MH] + C 14 H 18 FN4O3 + Calculated value: 309.2, Measured value: 309.2.

[0356] (9S)-1-Azido-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 (9-62): A stirred mixture of 8-amino-2-azido-6-fluoro-2-((2-hydroxyethoxy)methyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (9-61) (250 mg, 810 μmol, 1 equiv.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-4) (426 mg, 1.62 mmol, 2 equiv.), and 4-methylbenzenesulfonic acid (55.8 mg, 324 μmol, 0.4 equiv.) in toluene (11.5 mL) was degassed, then purged with argon three times, and stirred at 120° C. under an argon atmosphere for 18 hours. The mixture was then cooled to room temperature, quenched by the addition of water (20 mL) at 20 °C, and extracted with dichloromethane (3 × 20 mL). The combined organic phases were dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to give (9S)-1-azido-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 (9-62) (149 mg, 34% yield). LCMS (ESI+) m / z: [MH] + C 27 H 27 O6N5F + Calculated value: 536.1, measured value: 536.4.

[0357] (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-63): To a stirred mixture of (9S)-1-azido-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 (9-62) (149 mg, 278 μmol, 1 equiv.) in tetrahydrofuran (3 mL) and water (0.75 mL) was added PPh3 (145 mg, 556 μmol, 2 equiv.). After stirring for 12 h at 50 °C, the mixture was cooled to room temperature, quenched by the addition of aqueous HCl (0.5 M, 20 mL), and extracted with dichloromethane (3 × 20 mL). The aqueous phase was lyophilized to give (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-63) (80.0 mg, 45% yield), which was used directly in the next step without further purification. LCMS (ESI+) m / z: [MH] + C 27 H 29 O6N3F + Calculated value: 510.2, Found value: 510.3. SFC (retention times = 1.609 min, 1.941 min).

[0358] (1R,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-64) and (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-65) A mixture of (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-63) (100 mg, 157 μmol) in DMSO (5 mL) was separated by preparative HPLC to give (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-64) (compound 9-64 may be the opposite enantiomer of that shown) (5.20 mg) and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-65) (compound 9-65 may be the opposite enantiomer of that shown) (4.80 mg) were obtained. Note: * The stereochemistry at this carbon was arbitrarily assigned. Preparative HPLC separation method: Apparatus: Gilson 281 semi-preparative HPLC system, Column: Phenomenex Luna C18 100 * 30mm * 3 μm, Mobile phase: A: HO (0.04% HCl); B: Acetonitrile, Gradient: 10.00% to 45.00% B in 8.00 min, Flow rate: 25.00 ml / min, Monitor wavelength: 220 & 254 nm, Prep HPLC (9-64, retention time = 4.883 min) and SFC (9-65, retention time = 5.136 min). Spectrum of 9-64: 1H NMR (400MHz, DMSO-D6) δ ppm 9.21(br s,3H),7.88(d,J=10.51Hz,1H),7.36(s,1H),6.58(br s,1H),5.59(s,2H) 5.45(s,2H),3.88(s,2H),3.40 -3.50(m,4H),3.20 -3.30(m,1H),3.04 -3.18(m,1H),2.62-2.67(m,1H),2.40(s,3H),2.24(td,J=12.41,5.32Hz,1H),1.88(dt,J=14.23,6.96Hz,2H),0.88(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.24. LCMS(ESI+) m / z:[MH] + C 27 H 29 O6N3F + Calculated value: 510.2, measured value: 510.3. SFC (holding time = 1.929 minutes). 9-65のスペクトル: 1 H NMR (400MHz, DMSO-D6) δ ppm 9.18(br s,3H),7.89(d,J=10.51Hz,1H),7.36(s,1H),6.55(br s,1H),5.59(s,2H),5.46(s,2H),4.55-4.67(m,1H),3.86(s,2H),3.44-3.54(m,4H),3.26(br d,J=3.38Hz,1H),3.08-3.20(m,1H),2.67(br dd,J=8.25,4.75Hz,1H),2.40(s,3H),2.17-2.29(m,1H),1.80-1.94(m,2H),0.88(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.21. LCMS(ESI+) m / z:[MH] + C 27 H 29 O6N3F + Calculated value: 510.2, measured value: 510.3. SFC (holding time = 1.602 minutes).

[0359] Example 10 N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-68) and Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-69)

[0360] [ka]

[0361] 2-(((9S)-1-acetamido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methoxy)ethyl acetate (10-66): To a stirred mixture of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-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 hydrochloride (9-63) (120 mg, 220 μmol, 1.0 equiv) in dichloromethane (2.4 mL) was added triethylamine (222 mg, 2.20 mmol, 304 μL, 10 equiv) and stirred for 15 min at 20 °C, followed by the dropwise addition of AcO (112.3 mg, 1.10 mmol, 5.0 equiv) at 0 °C. After stirring for 12 h at 20 °C, the mixture was quenched by the addition of water (10 mL) and extracted with dichloromethane (3 × 10 mL). The combined organic phases were dried over NaSO, filtered, and concentrated to give ethyl 2-(((9S)-1-acetamido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methoxy)acetate (10-66) (110 mg, 52% yield), which was used directly in the next step without further purification. LCMS (ESI+) m / z: [MH] + C 31 H 33 FN3O8 + Calculated value: 594.2, measured value: 594.3.

[0362] N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-67): To a stirred mixture of 2-(((9S)-1-acetamido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methoxy)ethyl acetate (10-66) (110 mg, 185 μmol, 1 equiv., 67% purity) in methanol (4.4 mL) was added hydrochloric acid (2 M, 4.4 mL). After stirring at 60° C. for 1 hour, the mixture was cooled to room temperature, quenched by the addition of water (8.8 mL) at 20° C., and extracted with dichloromethane (3×10 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated, and the residue was purified by preparative TLC (ethyl acetate / methanol = 4 / 1) to give N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-67) (28 mg, 40% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 8.57(d,J=6.13Hz,1H),7.79(d,J=10.88Hz,1H),7.30(s,1H),6.51(d,J=11.51Hz, 1H),5.39-5.52(m,3H),4.85(dd,J=19.14,3.13Hz,1H),4.57-4.67(m,1H),3.69(br d,J=7.00Hz,2H),3.36-3.52(m,4H),3.17-3.27(m,1H),2.95-3.09(m,1H),2.80-2.88(m,1H), 2.38(s,3H),2.18-2.26(m,1H),1.99(d,J=3.13Hz,3H),1.77-1.92(m,2H),0.81-0.93(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.99. LCMS(ESI+) m / z:[MH] + C 29 H 31 FN3O7 +Calculated value: 552.2, Found value: 552.3. SFC (retention times = 1.193 min, 1.344 min).

[0363] N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-68) and N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-69) N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-67) (38 mg, 68 μmol) was purified by SFC (column: DAICEL CHIRALCEL OD (250 mm) *30 mm, 10 μm; mobile phase: [CO2-methanol]; B%: 15%, isocratic elution mode, flow rate: 70.00 g / min; monitor wavelength: 220 & 254 nm, column temperature: 40 °C), and N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1 10-68) (Compound 10-68 may be the opposite enantiomer of that shown) (4.01 mg) and N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethoxy)methyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (10-69) (Compound 10-69 may be the opposite enantiomer of that shown) (2.11 mg) were obtained. Note: Stereochemistry was assigned arbitrarily. Spectrum of 10-68: 1 H NMR(400MHz,DMSO-D6) δ ppm 8.58(s,1H),7.79(d,J=10.63Hz,1H),7.31(s,1H),6.51(s,1H),5.49(d,J=19.14Hz,1H ),5.42(s,2H),4.86(d,J=19.14Hz,1H),4.58-4.67(m,1H),3.66-3.74(m,2H),3.45-3. 50(m,3H),3.38-3.43(m,1H),3.08-3.10(m,1H),2.97-3.06(m,1H),2.81-2.91(m,1H), 2.38(s,3H),2.18-2.25(m,1H),1.98(s,3H),1.79-1.92(m,2H),0.87(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.98. LCMS(ESI+) m / z:[MH] + C 29 H 31 O7N3F +Calculated value: 552.2, measured value: 552.4. SFC (holding time = 1.189 minutes). 10-69のスペクトル: 1 H NMR (400MHz, DMSO-D6) δ ppm 8.56(s,1H),7.73-7.85(m,1H),7.30(s,1H),6.53(br s,1H),5.43(d,J=19.2Hz,1H),5.42(s,1H),4.85(d,J=19.2Hz,1H),4.59-4.69(m,1H),3.68(s,2H),3.43-3.49(m,4H),3.13-3. 20(m,1H),2.95-3.11(m,1H),2.82-2.93(m,1H),2.35-2.40(m,3H),2.18-2.27(m,1H),1.99(s,3H),1.78-1.93(m,2H),0.86(br t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.95. LCMS(ESI+) m / z:[MH] + C 29 H 31 O7N3F + Calculated value: 552.2, measured value: 552.4. SFC (holding time = 1.327 minutes).

[0364] Example 11 (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-76), (1S,9S)-1-((R)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-78), (1 Synthesis of (R,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-80) and (1R,9S)-1-((R)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-82)

[0365] [ka]

[0366] (1S,9S)-1-((S)-1-Acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-70), (1S,9S)-1-((R)-1-Acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (11-71), (1R,9S)-1-((S)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (11-72), (1R,9S)-1-((R)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (11-73), and (1R,9S)-1-((E)-3-acetamidoprop-1-en-1-yl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetate (11-74). (9S)-1-(1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (6-43A) (5.0 g) was subjected to SFC (apparatus: REGIS(s,s) WHEELK-01 (250 mm column) *The column was separated using a 50 mm column, 10 μm column, mobile phase: A is CO2 and B is methanol:acetonitrile = 1:1, gradient: B% = 60.00%, isocratic elution mode, flow rate: 200.00 g / min; monitor wavelength: 220 & 254 nm, column temperature: 40 °C, system back pressure: 100 bar) and (1S,9S)-1-((S)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl Acetic acid (11-70) (compound 11-70 may be the opposite enantiomer of that shown) (300 mg), (1S,9S)-1-((R)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (11-71) (compound 11-71 may be the opposite enantiomer of that shown) (230 mg), a mixture of 11-72 and 11-73, and (1R,9S)-1-((E)-3-acetamidoprop-1-en-1-yl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-74) (1.0 g) (compound 11-74 may be the opposite enantiomer of that shown) were obtained. The mixture of 11-72 and 11-73 was subjected to SFC (apparatus: Waters SFC80 preparative SFC system, column: DAICEL CHIRALCEL OD (250 mm) *30 mm, 10 μm); mobile phase: A is CO2 and B is methanol, gradient: B% = 38.00%, isocratic elution mode, flow rate: 64.00 g / min; monitor wavelength: 220 nm; column temperature: 40 °C, system back pressure: 100 bar) to further separate (1R,9S)-1-((S)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (11-72) (140 mg) (compound 11-72 may be the opposite enantiomer of that shown) and (1R,9S)-1-((R)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-73) (380 mg) (compound 11-73 may be the opposite enantiomer of that shown) were obtained. Note: The stereochemistry at these carbons was assigned arbitrarily. 11-70 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 8.21(d,J=9.13Hz,1H) 7.75(d,J=11.01Hz,1H) 7.01(s,1H) 5.70(ddd,J=16.85,10.47,6.07Hz,1H) 5.48(s,2H) 5.36(d,J=18.64Hz,1H) 5.12(d,J=18.64Hz,1H) 4.76-4.93(m,2H) 4.49-4.62(m,1H) 3.34-3.41(m,1H) 3.12-3.17(m,1H) 2.98-3.09(m,1H) 2.30-2.39(m,4H) 2.21(s,3H) 2.09-2.17(m,2H) 1.81-1.94(m,4H) 0.93(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.55. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6+ Calculated value: 560.2, measured value: 560.4. SFC (holding time = 0.771 minutes). 11-71のスペクトル: 1 H NMR(400MHz,DMSO-D6) δ ppm 8.26(d,J=9.13Hz,1H) 7.71(d,J=11.01Hz,1H) 7.00(s,1H) 5.91(ddd,J=17.39,10.01,7.88Hz,1H) 5.30-5.59(m,4H) 5.07-5.30(m,2H) 4.52-4.65(m,1H) 2.95-3.20(m,3H) 2.27-2.40(m,4H) 2.04-2.25(m,5H) 1.89-2.03(m,1H) 1.52(s,3H) 0.92(t,J=7.44Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.05. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6 + Calculated value: 560.2, measured value: 560.4. SFC (holding time = 1.050 minutes). 11-72のスペクトル: 1 H NMR(400MHz,DMSO-D6) δ ppm 8.24(d,J=9.16Hz,1H) 7.72(d,J=11.04Hz,1H) 7.00(s,1H) 5.91(ddd,J=17.44,9.91,7.91Hz,1H) 5.31(s,4H) 5.25(d,J=17.07Hz,1H) 5.13(d,J=10.67Hz,1H) 4.55-4.64(m,1H) 3.31-3.40(m,1H) 2.98-3.15(m,2H) 2.38(s,3H) 2.26-2.31(m,1H) 2.22(s,3H) 1.82-2.01(m,2H) 1.51(s,3H) 0.90(t,J=7.34Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.98. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6 + Calculated value: 560.2, measured value: 560.3. 11-73のスペクトル: 1 H NMR(400MHz, DMSO-D6) δ ppm 8.22(d,J=9.29Hz,1H) 7.75(d,J=10.92Hz,1H) 7.01(s,1H) 5.70(ddd,J=16.88,10.48,6.15Hz,1H) 5.49(d,J=1.63Hz,2H) 5.06-5.40(m,2H) 4.74-4.94(m,2H) 4.45-4.61(m,1H) 3.35-3.39(m,1H) 3.12-3.25(m,1H) 2.99-3.08(m,1H) 2.31-2.40(m,4H) 2.22(s,3H) 2.06-2.18(m,2H) 1.87(s,4H) 0.90(t,J=7.34Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.48. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6 + Calculated value: 560.2, measured value: 560.3. 11-74のスペクトル: 1 H NMR (400MHz, DMSO-D6) δ ppm 7.70-7.84(m,2H) 7.02(s,1H) 5.78(dd,J=15.45,6.57Hz,1H) 5.42-5.54(m,2H) 5.35(d,J=18.89Hz,1H) 5.18(dt,J=15.45,5.66Hz,1H) 5.04(d,J=18.76Hz,1H) 4.14(br s,1H) 3.59(br s,1H) 3.08-3.21(m,1H) 2.88-3.01(m,1H) 2.38(s,3H) 2.12-2.24(m,7H) 1.74 (s, 6H) 0.91(t, J = 7.38 Hz, 3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.12. LCMS(ESI+) m / z:[MH] + C 31 H 31 FN3O6 + Calculated value: 560.2, measured value: 560.4. SFC (holding time = 3.398 minutes).

[0367] (1S,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-75): (1S,9S)-1-((S)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl in dichloromethane (6 mL) To a stirred mixture of acetic acid (11-70) (300 mg, 536 μmol, 1.0 equiv) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (411 mg, 3.22 mmol, 466 μL, 6.0 equiv), a solution of iridium chloride; (1Z,5Z)-cycloocta-1,5-diene (36.0 mg, 53.6 μmol, 0.1 equiv) and 2-diphenylphosphanylethyl(diphenyl)phosphane (42.7 mg, 107 μmol, 0.2 equiv) in dichloromethane (1 mL) was added. After stirring for 3 h at 20° C., a mixture of 3-oxidodioxaborirane sodium tetrahydrate (412 mg, 2.68 mmol, 515 μL, 5.0 equiv) in H 2 O (3 mL) was added and stirred for 12 h at 20° C. The reaction mixture was extracted with dichloromethane (3 × 10 mL), and the combined organic phases were washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / methanol) to give (1S,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-75) (50 mg, 16% yield). 1H NMR(400MHz,DMSO-D6) δ ppm 7.81(br d,J=9.3Hz,1H) 7.73(br d,J=11.0Hz,1H) 7.02(s,1H) 5.49(s,2H) 5.43(br d,J=18.5Hz,1H) 5.18(d,J=18.6Hz,1H) 4.07-4.19(m,2H) 3.21-3.29(m,2H) 3.10-3.17(m,1H) 2.95-3.06(m,1H) 2.31-2.41(m,5H) 2.12-2.21(m,5H) 1.80-1.94(m,1H) 1.73(s,3H) 1.57-1.65(m,1H) 1.33-1.43(m,1H) 0.92(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.68. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O7 + Calculated value: 578.2, measured value: 578.4.

[0368] (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-76): To a stirred mixture of (1S,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-75) (50 mg, 86.5 μmol, 1.0 equiv.) in methanol (1.0 mL) was added HCl (2 M, 2 mL). After stirring at 60 °C for 20 h, the mixture was cooled to room temperature, filtered, and the filtrate was purified by preparative HPLC (instrument: Gilson 281 semi-preparative HPLC system, column: Phenomenex Luna C18 100). * 40mm *Purification by HPLC using a 5 μm column, mobile phase: A: HO (0.04% HCl); B: acetonitrile, gradient: 10.00% to 40.00% B in 8.00 min, flow rate: 60.00 ml / min, monitor wavelength: 220 & 254 nm) gave (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-76) (10.2 mg, 23% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 8.04(br d,J=0.88Hz,3H) 7.80(d,J=10.88Hz,1H) 7.32(s,1H) 6.36-6.68(m,1H) 5.44(s,2H) 5.39(br d,J=18.89Hz,1H) 5.17-5.26(m,1H) 3.54-3.63(m,2H) 3.27-3.34(m,2H) 3.22(br d,J=13.76Hz,1H) 3.01-3.14(m,1H) 2.58(br s,1H) 2.39(s,3H) 1.94-2.06(m,1H) 1.87(tt,J=13.68,6.96Hz,2H) 1.63-1.79(m,1H) 1.22-1.38(m,1H) 0.89(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.64. LCMS(ESI+) m / z:[MH] + C 27 H 29 FN3O5 + Calculated: 494.2, Found: 494.4. SFC (retention time = 3.178 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0369] (1S,9S)-1-((R)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-77): 11-77 was synthesized in the same manner as 11-75. Spectrum of 11-77: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.93(d,J=8.88Hz,1H) 7.71(d,J=11.01Hz,1H) 7.00(s,1H) 5.28-5.59(m,4H) 4.34(t,J=5.00Hz,1H) 4.16(qd,J=8.98,3.44Hz,1H) 3.36-3.45(m,1H) 3.32-3.35(m,1H) 3.21-3.28(m,1H) 3.02-3.09(m,2H) 2.31-2.40(m,4H) 2.21(s,3H) 2.11-2.18(m,2H) 1.92-2.02(m,1H) 1.68(br d,J=4.13Hz,2H) 1.49(s,3H) 0.92(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.14. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O7 + Calculated value: 578.2, Measured value: 578.4

[0370] (1S,9S)-1-((R)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-78): 11-78 was synthesized in the same manner as 11-76. Spectrum of 11-78: 1H NMR(400MHz,DMSO-D6) δ ppm 7.73-8.02(m,4H) 7.34(s,1H) 6.14-6.89(m,1H) 5.37-5.50(m,3H) 5.25-5.34(m,1H) 3.42-3.65(m,4H) 3.08(br d,J=6.88Hz,2H) 2.51-2.56(m,1H) 2.38(s,3H) 1.97-2.11(m,1H) 1.79-1.91(m,3H) 1.65-1.77(m,1H) 0.88(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.91. LCMS(ESI+) m / z:[MH] + C 27 H 29 FN3O5 + Calculated: 494.2, Found: 494.3. SFC (retention time = 2.531 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0371] (1R,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-79): 11-79 was synthesized in the same manner as 11-75. Spectrum of 11-79: 1H NMR(400MHz,DMSO-D6) δ ppm 7.90(br d,J=8.88Hz,1H) 7.71(d,J=11.13Hz,1H) 7.00(s,1H) 5.19-5.61(m,4H) 4.34(t,J=5.00Hz,1H) 4.06-4.25(m,1H) 3.38-3.42(m,1H) 3.27-3.29(m,2H) 3.10(br d,J=7.38Hz,2H) 2.30-2.39(m,4H) 2.22(s,3H) 2.09-2.13(m,2H) 1.86-2.02(m,1H) 1.59-1.75(m,2H) 1.38-1.53(m,3H) 0.90(br t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.07. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O7 + Calculated value: 578.2, measured value: 578.4.

[0372] (1R,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-80): 11-80 was synthesized in the same manner as 11-76. 11-80 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.79-7.87(m,3H) 7.34(s,1H) 6.56(s,1H) 5.37-5.50(m,3H) 5.26-5.34(m,1H) 4.88(br s,1H) 3.45-3.73(m,4H) 3.08(br d,J=7.38Hz,2H) 2.50-2.56(m,1H) 2.38(s,3H) 1.96-2.08(m,1H) 1.79-1.95(m,3H) 1.65-1.78(m,1H) 0.87(t,J=7.32Hz,3H). 19F NMR(376MHz,DMSO-D6) δ ppm -111.91. LCMS(ESI+) m / z:[MH] + C 27 H 29 FN3O5 + Calculated: 494.2, Found: 494.4. SFC (retention time = 4.328 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0373] (1R,9S)-1-((R)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-81): 11-81 was synthesized in the same manner as 11-75. Spectrum of 11-81: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.69-7.85(m,2H) 7.02(s,1H) 5.35-5.58(m,3H) 5.21(d,J=18.64Hz,1H) 4.07-4.17(m,2H) 3.35-3.38(m,1H) 3.22-3.30(m,2H) 3.17(d,J=5.25Hz,1H) 2.95-3.05(m,1H) 2.32-2.39(m,4H) 2.22(s,3H) 2.08-2.19(m,2H) 1.80-1.94(m,1H) 1.67-1.75(m,3H) 1.51-1.65(m,1H) 1.31-1.44(m,1H) 0.90(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.65. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O7 + Calculated value: 578.2, measured value: 578.5.

[0374] (1R,9S)-1-((R)-1-amino-3-hydroxypropyl)-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 hydrochloride (11-82): 11-82 was synthesized in the same manner as 11-76. Spectrum of 11-82: 1H NMR(400MHz,DMSO-D6) δ ppm 8.17(br s,3H) 7.79(d,J=10.88Hz,1H) 7.32(s,1H) 6.20-6.80(m,1H) 5.44(s,2H) 5.38(d,J=18.89Hz,1H) 5.13-5.29(m,1H) 3.46-3.57(m,2H) 3.38-3.44(m,1H) 3.32(dt,J=11.16,5.74Hz,1H) 3.23(br d,J=12.88Hz,1H) 2.99-3.14(m,1H) 2.60(br 1H NMR(400MHz,DMSO-D6+ D2O) δ ppm 7.75(d,J=10.88Hz,1H) 7.35(s,1H) 5.30-5.49(m,3H) 5.19(d,J=18.76Hz,1H) 3.49-3.61(m,2H) 3.35-3.44(m,1H) 3.26-3.35(m,1H) 3.12-3.25(m,1H) 2.98-3.11(m,1H) 2.47(br s,1H) 2.36(s,3H) 1.95-2.09(m,1H) 1.85(tt,J=14.45,7.13Hz,2H) 1.73(br dd,J=13.57,6.57Hz,1H) 1.25-1.42(m,1H) 0.85(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.67. LCMS(ESI+) m / z:[MH] + C 27 H 29 FN3O5+ Calculated: 494.2, Found: 494.4. SFC (retention time = 6.619 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0375] Example 12 N-((2R,3R)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,3-dihydroxypropyl)acetamide (12-86) and Synthesis of N-((2S,3S)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,3-dihydroxypropyl)acetamide (12-87)

[0376] [ka]

[0377] (1S,9S)-1-((1R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-83): (1R,9S)-1-((E)-3-acetamidoprop-1-en-1-yl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl in acetone (17.5 mL), HO (2.5 mL), and tert-butyl alcohol (5 mL) To a stirred mixture of acetic acid 11-74 (500 mg, 893 μmol, 1.0 equiv.), 4-methylmorpholine 4-oxide (439 mg, 3.75 mmol, 396 μL, 4.2 equiv.), osmium tetroxide (45.4 mg, 178 μmol, 9.27 μL, 0.2 equiv.), and 2-hydroperoxy-2-methyl-propane (8.05 mg, 89.3 μmol, 8.57 μL, 0.1 equiv.) was added. After stirring for 12 h at 20 °C, the mixture was quenched by the addition of HO (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated to give (1S,9S)-1-((1R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-83) (160 mg, 30% yield), which was used directly in the next step without purification. LCMS (ESI+) m / z: [MH] + C 31 H 33 FN3O8 + Calculated: 594.2, Found: 594.3. SFC (retention times = 1.847 min, 2.465 min).

[0378] (1S,9S)-1-((1R,2R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-84) and (1S,9S)-1-((1S,2S)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (12-85): (1S,9S)-1-((1R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-83) (190 mg, 320 μmol) was purified by SFC (apparatus: Waters SFC80Q preparative SFC system; column: REGIS(S,S)WHELK-O1 (250 mm *25 mm, 10 μm); mobile phase: A is CO2 and B is ethyl alcohol; gradient: B% = 56.00% isocratic elution mode; flow rate: 80.00 g / min; monitor wavelength: 220 nm; column temperature: 40 °C; system back pressure: 100 bar) and (1S,9S)-1-((1R,2R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl Acetic acid (12-84) (34.0 mg) and (1S,9S)-1-((1S,2S)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-85) (41.0 mg) were obtained. Spectrum of 12-84: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.84(t,J=5.69Hz,1H) 7.74(d,J=11.01Hz,1H) 7.01(s,1H) 5.41-5.57(m,3H) 5.29(d,J=19.01Hz,1H) 5.01(d,J=7.13Hz,1H) 4.60(d,J=5.88Hz,1H) 3.84(q,J=6.88Hz,1H) 3.48-3.62(m,2H) 3.17(br t,J=6.00Hz,2H) 3.03-3.11(m,1H) 2.85-3.02(m,1H) 2.26-2.36(m,4H) 2.21(s,3H) 2.14(dt,J=9.47,7.08Hz,2H) 1.86-1.97(m,1H) 1.80(s,3H) 0.90(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.17. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O8 +Calculated: 594.2, Found: 594.4. SFC (retention time = 1.212 min). 12-85 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.72(d,J=10.88Hz,1H) 7.57(br t,J=5.32Hz,1H) 7.01(s,1H) 5.45-5.61(m,3H) 5.27(d,J=19.39Hz,1H) 4.94(d,J=7.50Hz,1H) 4.77(d,J=7.25Hz,1H) 3.51-3.57(m,1H) 3.43-3.50(m,1H) 3.13-3.24(m,2H) 2.98-3.04(m,2H) 2.92(dt,J=13.32,4.72Hz,1H) 2.58-2.65(m,1H) 2.36(s,3H) 2.22(s,3H) 2.14(dt,J=10.69,7.16Hz,2H) 1.80-1.92(m,1H) 1.65(s,3H) 0.90(t,J=7.38Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.92. LCMS(ESI+) m / z:[MH] + C 31 H 33 FN3O8 + Calculated: 594.2, Found: 594.2. SFC (retention time = 1.605 min).

[0379] N-((2R,3R)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,3-dihydroxypropyl)acetamide (12-86): A mixture of (1S,9S)-1-((1R,2R)-3-acetamido-1,2-dihydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (12-84) (63.0 mg, 106 μmol) in methanesulfonic acid (1.2 mL) was stirred for 6 h at 25 °C, then filtered, and the residue was purified by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system; column: Phenomenex Luna C18 100 * 30mm * Purify by HPLC (3 μm; mobile phase: A: HO (0.2% formic acid); B: acetonitrile, gradient: B 10.00% to 50.00% in 8.00 min; flow rate: 25.00 mL / min, monitor wavelength: 220 & 254 nm) to obtain N-((2R,3R)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,3-dihydroxypropyl)acetamide (12-86) (15.2 mg, 25% yield). Note: * The stereochemistry at these carbons was arbitrarily assigned. 1 H NMR(400MHz,DMSO-D6) δ ppm 7.86(br t,J=5.57Hz,1H) 7.76(d,J=11.13Hz,1H) 7.31(s,1H) 6.47-6.56(m,1H) 5.36-5.54(m,3H) 5.29(d,J=19.01Hz,1H) 5.03(br d,J=6.88Hz,1H) 4.67(d,J=5.75Hz,1H) 3.84(q,J=6.50Hz,1H) 3.48-3.61(m,2H) 3.05-3.22(m,3H) 2.84-3.01(m,1H) 2.29-2.42(m,4H) 1.76-1.97(m,6H) 0.87(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.44. LCMS(ESI+) m / z:[MH]+ C 29 H 31 FN3O7 + Calculated: 552.2, Found: 552.2. SFC (retention time = 2.308 min).

[0380] N-((2S,3S)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,3-dihydroxypropyl)acetamide (12-87): 12-87 was synthesized in the same manner as 12-86. Spectrum of 12-87: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.73(d,J=10.88Hz,1H) 7.62(br t,J=5.44Hz,1H) 7.31(s,1H) 6.51(s,1H) 5.52(d,J=19.51Hz,1H) 5.43(s,2H) 5.27(d,J=19.39Hz,1H) 4.96(br d,J=7.00Hz,1H) 4.82(d,J=7.25Hz,1H) 3.44-3.58(m,2H) 3.12-3.24(m,2H) 2.92-3.08(m,3H) 2.59-2.66(m,1H) 2.36(s,3H) 1.78-1.94(m,3H) 1.65(s,3H) 0.87(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.19.LCMS(ESI+) m / z:[MH] + C 29 H 31 FN3O7 + Calculated value: 552.2, Found value: 552.2. SFC (retention time = 2.999 min).

[0381] Example 13 N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-88), N -((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-89), N- ((S)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-90), and Synthesis of N-((R)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-91)

[0382] [ka]

[0383] N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-88): A mixture of (1S,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-75) (120 mg, 208 μmol) and methanesulfonic acid (1.2 mL) was stirred at 60 °C for 1 h, cooled to room temperature, diluted with methanol (0.5 mL), filtered, and the filtrate was purified by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system, column: Phenomenex Luna C18 100 * 30mm * Purification by HPLC using a 3 μm column, mobile phase: A: HO (0.2% formic acid); B: acetonitrile, gradient: 10.00% to 50.00% B in 8.00 min, flow rate: 25.00 ml / min, monitor wavelength: 220 & 254 nm) gave N-((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-88) (12.3 mg, 11% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.81(d,J=9.26Hz,1H) 7.75(d,J=11.01Hz,1H) 7.31(s,1H) 6.52(s,1H) 5.36-5.49(m,3H) 5.19(d,J=18.64Hz,1H) 4.06-4.20(m,2H) 3.36(br s,1H) 3.29(br s,2H) 3.16(br dd,J=11.44,4.57Hz,1H) 3.00(br dd,J=17.70,4.57Hz,1H) 2.29-2.39(m,4H) 1.79-1.96(m,3H) 1.73(s,3H) 1.54-1.68(m,1H) 1.31-1.43(m,1H) 0.89(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.89. LCMS(ESI+) m / z:[MH]+ C 29 H 31 FN3O6 + Calculated value: 536.2, Found value: 536.2. SFC (retention time) = 1.146 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0384] N-((R)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-89): 13-89 was synthesized in the same manner as 13-88. Spectrum of 13-89: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.93(d,J=8.82Hz,1H) 7.72(d,J=10.97Hz,1H) 7.30(s,1H) 6.51(s,1H) 5.39-5.51(m,3H) 5.28-5.36(m,1H) 4.34(t,J=5.01Hz,1H) 4.11-4.23(m,1H) 3.37-3.45(m,1H) 3.27(br d,J =7.63Hz,2H) 3.09(br d,J=6.68Hz,2H) 2.35-2.44(m,4H) 1.81-2.03(m,3H) 1.59-1.79(m,2H) 1.48(s,3H) 0.88(t,J=7.33Hz,3H) 19 F NMR(376MHz,DMSO-D6) δ ppm -112.37. LCMS(ESI+) m / z:[MH] + C 29 H 31 FN3O6 + Calculated: 536.2, Found: 536.2. SFC (retention time) = 1.589 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0385] N-((S)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-90): 13-90 was synthesized in the same manner as 13-88. 13-90 spectrum: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.90(br d,J=8.94Hz,1H) 7.72(d,J=10.97Hz,1H) 7.30(s,1H) 6.50(s,1H) 5.26-5.53(m,4H) 4.36(t,J=4.95Hz,1H) 4.10-4.24(m,1H) 3.35-3.46(m,2H) 3.21-3.27(m,1H) 3.02-3.15(m,2H) 2.35-2.44(m,4H) 1.92-2.03(m,1H) 1.82-1.89(m,2H) 1.61-1.79(m,2H) 1.45(s,3H) 0.87(t,J=7.27Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.38. LCMS(ESI+) m / z:[MH] + C 29 H 31 FN3O6 + Calculated value: 536.2, Found value: 536.2. SFC (retention time) = 2.448 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0386] N-((R)-1-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropyl)acetamide (13-91): 13-91 was synthesized in the same manner as 13-88. Spectrum of 13-91: 1H NMR(400MHz,DMSO-D6) δ ppm 7.80(d,J=9.13Hz,1H) 7.74(d,J=10.88Hz,1H) 7.31(s,1H) 6.53(s,1H) 5.35-5.49(m,3H) 5.20(d,J=18.64Hz,1H) 4.05-4.20(m,2H) 3.36(br d,J=8.88Hz,1H) 3.29(br d,J=4.13Hz,2H) 3.12-3.22(m,1H) 3.00(br dd,J=17.76,4.13Hz,1H) 2.28-2.41(m,4H) 1.78-1.96(m,3H) 1.72(s,3H) 1.55-1.67(m,1H) 1.30-1.45(m,1H) 0.87(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.92. LCMS(ESI+) m / z:[MH] + C 29 H 31 FN3O6 + Calculated: 536.2, Found: 536.2. SFC (retention time) = 2.064 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0387] Example 14 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-92), (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-93), (1 Synthesis of (R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-94) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-95)

[0388] [ka]

[0389] (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 (11-76): To a stirred mixture of (1S,9S)-1-((S)-1-acetamido-3-hydroxypropyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl acetic acid (11-75) (290 mg, 502 μmol, 1.0 equiv.) in methanol (2.9 mL) was added methanesulfonic acid (434 mg, 4.52 mmol, 323 μL, 9.0 equiv.). After stirring at 50 °C for 12 h, the mixture was cooled to room temperature and adjusted to pH 7 by the addition of N,N-diisopropylethylamine. The resulting mixture was used directly in the next step without further purification. LCMS(ESI+)m / z:[M−H] + C 27 H 29 FN3O5 + Calculated value: 494.2, measured value: 494.3.

[0390] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-92): The above mixture of (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 (11-76) was diluted with methanol (3 mL), acetone (353 mg, 6.08 mmol, 446 μL, 20 equiv.) was added, and the mixture was stirred for 1 hour at 40° C. Sodium cyanoborohydride (95.5 mg, 1.51 mmol, 5.0 equiv.) was then added. After stirring for 13 hours at 25°C, the mixture was filtered, and the filtrate was subjected to preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system; column: Phenomenex Gemini-NX 150 * 30mm *Purify by HPLC using a 5 µm column; mobile phase: A: HO (0.2% formic acid); B: acetonitrile; gradient: B 15.00% to 45.00% in 20.00 min; flow rate: 25.00 mL / min, monitor wavelength: 220–254 nm) to obtain (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-92) (15.8 mg, 12% yield over two steps). 1 H NMR(400MHz,DMSO-D6) δ ppm 7.73(br d,J=10.88Hz,1H) 7.31(s,1H) 6.50(s,1H) 5.29-5.53(m,4H) 4.20-4.67(m,1H) 3.32-3.37(m,3H) 3.23-3.28(m,1H) 3.02-3.17(m,2H) 2.45(br d,J=3.63Hz,1H) 2.36(s,3H) 1.70-2.05(m,4H) 1.36-1.52(m,3H) 0.89(br t,J=7.25Hz,6H) 0.58(br s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.25. LCMS(ESI+) m / z:[MH] + C 30 H 35 FN3O5 + Calculated value: 536.2, Found value: 535.8. SFC (retention time) = 1.913 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0391] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-93): 14-93 was synthesized in the same manner as 14-92. Spectrum of 14-93: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.71(d,J=11.01Hz,1H) 7.31(s,1H) 6.53(br s,1H) 5.61(br d,J=18.64Hz,1H) 5.32-2.43(m,3H) 3.25-3.32(m,3H) 2.95-3.08(m,2H) 2.91(br t,J=7.88Hz,1H) 2.32-2.40(m,4H) 2.10-2.21(m,1H) 1.74-2.02(m,3H) 1.65-1.72(m,1H),1.52-1.65(m,1H) 0.87(br t,J=7.19Hz,3H) 0.76(d,J=6.13Hz,3H) 0.19(br d,J=6.00Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.22. LCMS(ESI+) m / z:[MH] + C 30 H 35 FN3O5 + Calculated: 536.2, Found: 536.3. SFC (retention time = 2.167 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0392] (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-94): 14-94 was synthesized in the same manner as 14-92. Spectrum of 14-94: 1H NMR(400MHz,DMSO-D6) δ ppm 7.73(d,J=11.01Hz,1H) 7.31(s,1H) 6.48(s,1H) 5.65(br d,J=18.76Hz,1H) 5.43(s,2H) 5.35(br d,J=18.8Hz,1H) 3.44-3.62(m,2H) 3.31-3.40(m,1H),2.99-3.14(m,2H) 2.85-2.92(m,1H) 2.34-2.42(m,4H) 2.04-2.19(m,1H) 1.75-1.96(m,3H) 1.65-1.74(m,1H) 1.53-1.64(m,1H) 0.87(t,J=7.32Hz,3H) 0.76(br s,3H) 0.12(br s,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.34. LCMS(ESI+) m / z:[MH] + C 30 H 35 FN3O5 + Calculated value: 536.2, Found value: 536.2. SFC (retention time) = 1.372 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0393] (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxy-1-(isopropylamino)propyl)-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 (14-95): 14-95 was synthesized in the same manner as 14-92. 14-95 spectrum: 1H NMR(400MHz,DMSO-D6) δ ppm 7.73(br d,J=11.13Hz,1H) 7.31(s,1H) 6.43-6.55(m,1H) 5.31-5.48(m,4H) 4.22-4.66(m,1H) 3.33-3.47(m,3H) 3.25-3.30(m,1H) 3.02-3.18(m,2H) 2.42-2.47(m,1H) 2.36(s,3H) 1.66-2.03(m,4H) 1.43(br s,3H) 0.87(br t,J=7.32Hz,6H) 0.34-0.71(m,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.25. LCMS(ESI+) m / z:[MH] + C 30 H 35 FN3O5 + Calculated value: 536.2, Found value: 535.8. SFC (retention time) = 1.541 min. Note: * The stereochemistry at these carbons was arbitrarily assigned. Alternative and / or additional synthetic routes to the compounds described in Example 14 can be found in FIG.

[0394] Example 15 (1S,9S)-1-((S)-1(dimethylamino)-3-hydroxypropyl)-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 (15-96), (1S,9S)-1-((R)-1(dimethylamino)-3-hydroxypropyl)-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 (15-97), (1 Synthesis of (S,9S)-1-((R)-1(dimethylamino)-3-hydroxypropyl)-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 (15-98) and (1R,9S)-1-((R)-1(dimethylamino)-3-hydroxypropyl)-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 (15-99)

[0395] [ka]

[0396] (1S,9S)-1-((S)-1-(dimethylamino)-3-hydroxypropyl)-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 (15-96): A mixture of (1S,9S)-1-((S)-1-amino-3-hydroxypropyl)-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 (11-76) (150 mg, 303 μmol, 1.0 equiv.) and paraformaldehyde (155 mg, 4.56 mmol, 15 equiv.) in methanol (3 mL) was stirred at 40 °C for 1 h, followed by the addition of sodium cyanoborohydride (57.3 mg, 912 μmol, 3.0 equiv.). The mixture was then stirred for 13 hours at 25°C, filtered, and the filtrate was analyzed by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system; column: Phenomenex Gemini-NX 150 * 30mm * 5 μm; mobile phase: A: HO (0.2% formic acid); B: acetonitrile; gradient: B 15.00% to 45.00% in 20.00 min; flow rate: 25.00 mL / min, monitor wavelength: 220–254 nm) to give (1S,9S)-1-((S)-1(dimethylamino)-3-hydroxypropyl)-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 (15-96) (20.5 mg, 12% yield). 1 H NMR (400MHz, methanol-D4) δ ppm 7.54-7.66(m,2H) 5.59(d,J=16.26Hz,1H) 5.24-5.46(m,3H) 3.45(br d,J=9.76Hz,1H) 3.17-3.28(m,1H) 3.03-3.16(m,2H) 2.92-3.03(m,2H) 2.68(br d,J=11.51Hz,1H) 2.53(s,6H) 2.41(s,3H) 1.82-2.06(m,4H) 1.12-1.23(m,1H) 1.01(t,J=7.38Hz,3H). 19 F NMR (376 MHz, methanol-D4) δ ppm -112.83. LCMS (ESI+) m / z: [MH] +C 29 H 33 FN3O5 + Calculated: 522.2, Found: 522.2. Chiral HPLC (retention time) = 6.542 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0397] (1S,9S)-1-((R)-1(dimethylamino)-3-hydroxypropyl)-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 (15-97): 15-97 was synthesized in the same manner as 15-96. Spectrum of 15-97: 1 H NMR (400MHz, methanol-D4) δ ppm 7.53-7.67(m,2H),5.58(d,J=16.26Hz,1H),5.38(d,J=16.26Hz,1H),5.32(s,2H) ),3.60-3.73(m,1H),3.53(dt,J=10.38,7.00Hz,2H),3.07-3.24(m,2H),2.99(br t,J=7.75Hz,1H),2.30-2.56(m,10H),1.74-2.22(m,5H),1.02(t,J=7.38Hz,3H). 19 F NMR (376 MHz, methanol-D4) δ ppm -113.304. LCMS (ESI+) m / z: [MH] + C 29 H 33 FN3O5 + Calculated: 522.2, Found: 522.2. SFC (retention time = 6.693 min). Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0398] (1S,9S)-1-((S)-1(dimethylamino)-3-hydroxypropyl)-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 (15-98): 15-98 was synthesized in the same manner as 15-96. Spectrum of 15-98: 1H NMR (400 MHz, methanol-D4) δ ppm 7.61-7.70 (m, 2H), 5.61 (d, J = 16.26 Hz, 1H), 5.28-5.47 (m, 3H), 3.69 (dt, J = 11.57, 5.85 Hz, 1H), 3.46-3.60 (m, 2H), 3.07-3.24 (m, 2H), 2.94-3.05 (m, 1H), 2.31-2.48 (m, 10H), 2.02-2.15 (m, 2H), 1.93-2.02 (m, 2H), 1.81-1.93 (m, 1H), 1.01 (t, J = 7.38 Hz, 3H). 19 F NMR (376 MHz, methanol-D4) δ ppm -113.37. LCMS (ESI+) m / z: [MH] + C 29 H 33 FN3O5 + Calculated: 522.2, Found: 522.2. Chiral HPLC (retention time) = 8.066 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0399] (1R,9S)-1-((R)-1(dimethylamino)-3-hydroxypropyl)-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 (15-99): 15-99 was synthesized in the same manner as 15-96. 15-99 spectrum: 1H NMR (400MHz, methanol-D4) δ ppm 7.58-7.71(m,2H) 5.62(d,J=16.26Hz,1H) 5.29-5.48(m,3H) 3.48(br d,J=9.76Hz,1H) 3.24(br dd,J=13.45,4.44Hz,1H) 3.09-3.21(m,2H) 3.05(dt,J=10.63,6.82Hz,2H) 2.66-2.74(m,1H) 2.57(s,6H) 2.45(s,3H) 1.87-2.06(m,4H) 1.24(dq,J=11.73,6.89Hz,1H) 1.03(t,J=7.38Hz,3H). 19 F NMR (376 MHz, methanol-D4) δ ppm -112.89. LCMS (ESI+) m / z: [MH] + C 29 H 33 FN3O5 + Calculated: 522.2, Found: 522.2. Chiral HPLC (retention time) = 7.606 min. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0400] Example 16 (1S,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-102), (1S,9S)-1-((S)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-105), (1 Synthesis of (R,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-108) and (1R,9S)-1-((S)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-111)

[0401] [ka]

[0402] (1S,9S)-1-((R)-1-acetamido-2-oxoethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-100): (1S,9S)-1-((S)-1-acetamidoallyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl in dioxane (3.0 mL) and water (1.0 mL) To a stirred mixture of acetic acid (11-70) (100 mg, 179 μmol, 1.0 equiv.), osmium(VIII) oxide (4.54 mg, 17.9 μmol, 0.1 equiv.), 2,6-dimethylpyridine (38.3 mg, 357 μmol, 41.6 μL, 2.0 equiv.), and sodium periodate (153 mg, 715 μmol, 39.6 μL, 4.0 equiv.) was added. After stirring for 3 h at 25 °C, the mixture was quenched by the addition of saturated sodium sulfate solution (5 mL) and extracted with dichloromethane (3 × 5 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered, and concentrated to give (1S,9S)-1-((R)-1-acetamido-2-oxoethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-100), which was used directly without further purification. LCMS (ESI+) m / z: [MH] + C 30 H 29 FN3O7 + Calculated value: 562.2, measured value: 562.2.

[0403] (1S,9S)-1-((R)-1-acetamido-2-hydroxyethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-101): To a stirred mixture of (1S,9S)-1-((R)-1-acetamido-2-oxoethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-100) (100 mg, 142 μmol, 1.0 equiv) in tetrahydrofuran (2.0 mL) and water (1.0 mL) was added NaBH (2.69 mg, 71.2 μmol, 0.5 equiv). After stirring for 1 h at 20 °C, it was extracted with dichloromethane (3 × 5 mL), and the combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and concentrated to give (1S,9S)-1-((R)-1-acetamido-2-hydroxyethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-101) (60 mg, 59% yield over two steps), which was used directly in the next step. 1 H NMR(400MHz,DMSO-D6) δ ppm 8.45(s,1H) 8.01(d,J=9.41Hz,1H) 7.69(d,J=11.00Hz,1H) 7.00(s,1H) 5.42-5.59(m,3H) 5.27-5.38(m,1H) 4.91-5.06(m,1H) 4.10(tt,J=8.85,4.42Hz,1H) 3.74(br dd,J=11.07,4.83Hz,1H) 3.44(br d,J=11.62Hz,2H) 3.04-3.19(m,2H) 2.37(s,4H) 2.20(s,3H) 2.10-2.18(m,2H) 1.92-1.98(m,1H) 1.39(s,3H) 0.92(t,J=7.34Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.30. LCMS(ESI+) m / z:[MH] + C 30 H 31 FN3O7 + Calculated value: 564.2, measured value: 564.3.

[0404] (1S,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-102): To a stirred mixture of (1S,9S)-1-((R)-1-acetamido-2-hydroxyethyl)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-ylacetic acid (16-101) (60 mg, 106 μmol, 1.0 equiv.) in methanol (1.2 mL) was added methanesulfonic acid (0.6 mL). After stirring at 50 °C for 12 h, the mixture was cooled to room temperature, filtered, and the filtrate was purified by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system, column: Phenomenex Luna C18 75). * 30mm * Purification by HPLC using a 3 μm column, mobile phase: A: water (0.04% HCl); B: acetonitrile, gradient: 20.00% to 53.00% B in 8.00 min, flow rate: 25.00 ml / min, monitor wavelength: 220 & 254 nm) gave (1S,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-102) (23.1 mg, 45% yield). 1H NMR(400MHz,DMSO-D6) δ ppm 8.29(br d,J=1.67Hz,3H) 7.79(d,J=10.85Hz,1H) 7.31(s,1H) 6.53(br s,1H) 5.19-5.61(m,5H) 3.66(br d,J=10.13Hz,1H) 3.21-3.38(m,3H) 3.10-3.17(m,1H) 3.04(br dd,J=17.76,3.93Hz,1H) 2.44-2.48(m,1H) 2.38(s,3H) 1.96-2.10(m,1H) 1.87(tt,J=14.01,7.03Hz,2H) 0.89(t,J=7.27Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.92. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.2. Chiral HPLC (retention times = 6.297 min / 12.278 min) showed only two peaks, and a P1 / P2 ratio of 1.70 / 98.30, with 96.6% dehydrogenase. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0405] (1S,9S)-1-((S)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-105): 16-105 was synthesized in the same manner as 16-102. Spectrum of 16-105: 1H NMR(400MHz,DMSO-D6) δ ppm 7.79-7.87(m,3H) 7.34(s,1H) 6.55(br s,1H) 5.23-5.61(m,5H) 3.82-3.92(m,1H) 3.74(br d,J=10.76Hz,1H) 3.57(br d,J=10.01Hz,1H) 3.39-3.44(m,1H) 3.02-3.17(m,2H) 2.46(br s,1H) 2.37(s,3H) 1.95-2.08(m,1H) 1.88(dt,J=13.60,6.89Hz,2H) 0.88(t,J=7.32Hz,3H). 1 H NMR (400 MHz, DMSO-D 6, D2O) δ ppm 7.76(d,J=10.88Hz,1H) 7.37(s,1H) 5.36-5.51(m,3H) 5.19-5.32(m,1H) 3.74-3.79(m,1H) 3.67-3.74(m,1H) 3.53(br d,J=9.88Hz,1H) 3.21-3.32(m,1H) 2.98-3.10(m,2H) 2.42(br d,J=15.38Hz,1H) 2.34(s,3H) 1.95-2.08(m,1H) 1.85(tt,J=13.73,7.10Hz,2H) 0.85(t,J=7.32Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.13. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.2. Chiral HPLC (retention times = 5.525 min / 11.031 min) showed only two peaks, and a P1 / P2 ratio of 0.93 / 99.07, 98.14% dextromethorphan. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0406] (1R,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-108): 16-108 was synthesized in the same manner as 16-102. Spectrum of 16-108: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.45-8.20(m,3H) 7.23-7.41(m,1H) 6.25-6.79(m,1H) 4.88-5.83(m,4H) 3.82-3.93(m,1H) 3.75(br d,J=10.76Hz,1H) 3.58(br d,J=9.76Hz,1H) 3.05-3.16(m,3H) 2.46(br d,J=2.00Hz,1H) 2.38(s,3H) 1.95-2.03(m,1H) 1.81-1.94(m,2H) 0.88(br t,J=7.25Hz,3H). 1 H NMR(400MHz,DMSO-D6,D2O) δ ppm 7.79(d,J=10.76Hz,1H) 7.37(s,1H) 5.20-5.55(m,4H) 3.81-3.87(m,1H) 3.73(br s,1H) 3.55(br d,J=10.01Hz,1H) 3.32(dt,J=8.72,4.33Hz,1H) 2.96-3.18(m,2H) 2.43(br d,J=12.63Hz,1H) 2.36(s,3H) 1.95-2.08(m,1H) 1.87(tt,J=14.63,7.25Hz,2H) 0.86(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.12. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.2. Chiral HPLC (retention times = 6.277 min / 12.288 min) showed only two peaks, and a P1 / P2 ratio of 98.96 / 1.04, 97.92% dextromethorphan. Note:* The stereochemistry at these carbons was arbitrarily assigned.

[0407] (1R,9S)-1-((S)-1-amino-2-hydroxyethyl)-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 hydrochloride (16-111): 16-111 was synthesized in the same manner as 16-102. Spectrum of 16-111: 1 H NMR(400MHz,DMSO-D6) δ ppm 7.60-8.21(m,2H) 7.33(s,1H) 6.54(s,1H) 5.22-5.58(m,5H) 3.60-3.65(m,1H) 3.40-3.42(m,1H) 3.18-3.28(m,2H) 3.11-3.17(m,1H) 3.02-3.10(m,1H) 2.46(br s,1H) 2.39(s,3H) 1.95-2.06(m,1H) 1.81-1.94(m,2H) 0.87(t,J=7.25Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -111.91. LCMS(ESI+) m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.1, Found value: 480.2. Chiral HPLC (retention times = 5.521 min / 11.027 min) showed only two peaks, and a P1 / P2 ratio of 99.43 / 0.57, 98.86% dehydrogenase. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0408] Example 17 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-112), (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((S)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-114), (1 Synthesis of (R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-113) and (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-115)

[0409] [ka]

[0410] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-112): To a stirred mixture of (1S,9S)-1-((R)-1-amino-2-hydroxyethyl)-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 (16-102) (100 mg, 209 μmol, 1.0 equiv) in methanol (2.0 mL) was added acetone (242 mg, 4.18 mmol, 20 equiv) and heated at 40 °C for 2 h, followed by the addition of sodium cyanoborohydride (65.5 mg, 1.05 mmol, 5.0 equiv). After stirring for 14 hours at 25°C, the mixture was filtered, and the filtrate was analyzed by preparative HPLC (apparatus: Gilson 281 semi-preparative HPLC system, column: Phenomenex Gemini C18 75 * 40mm * Purification by HPLC (3 μm column, mobile phase: A: water (0.2% formic acid); B: acetonitrile, gradient: 15.00% to 40.00% B in 20.00 min, flow rate: 25.00 mL / min, monitor wavelength: 220 & 254 nm) gave (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-112) (9.1 mg, 8% yield). 1 H NMR(400MHz,DMSO-D6) δ ppm 8.39(s,0.63H) 7.72(d,J=11.01Hz,1H) 7.30(s,1H) 6.50(br s,1H) 5.59(d,J=19.14Hz,1H) 5.42(d,J=2.00Hz,2H) 5.24(d,J=19.26Hz,1H) 4.63-5.03(m,1H) 3.20(br s,2H) 2.99-3.05(m,1H) 2.86(br dd,J=11.19,2.56Hz,1H) 2.63-2.77(m,4H) 2.31-2.41(m,4H) 1.79-1.93(m,3H) 0.94(d,J=6.13Hz,3H) 0.89(t,J=7.32Hz,3H) 0.77(d,J=6.00Hz,3H).1 H NMR (400 MHz, DMSO-D 6, D2O) δ ppm 8.33(s,0.63H) 7.69(d,J=10.88Hz,1H) 7.32(s,1H) 5.56(d,J=19.14Hz,1H) 5.33-5.46(m,2H) 5.22(d,J=19.14Hz,1H) 3.41(br dd,J=11.32,3.94Hz,1H) 3.34(br s,1H) 3.10-3.24(m,1H) 2.95-3.07(m,1H) 2.83(br dd,J=11.26,2.75Hz,1H) 2.73-2.78(m,1H) 2.59-2.71(m,3H) 2.33(s,3H) 1.78-1.93(m,3H) 0.93(d,J=6.00Hz,3H) 0.87(t,J=7.32Hz,3H) 0.76(d,J=6.13Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.34. LCMS(ESI+) m / z:[MH] + C 29 H 33 FN3O5 + Calculated: 522.2, Found: 522.2. SFC (retention time = 1.021 min) showed only one peak, 100% de. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0411] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-113): 17-113 was synthesized in the same manner as 17-112. Spectrum of 17-113: 1H NMR(400MHz,DMSO-D6) δ ppm 8.41(s,0.50H) 7.72(d,J =11.01Hz,1H) 7.30(s,1H) 6.50(br s,1H) 5.70(d,J=18.64Hz,1H) 5.37-5.50(m,2H) 5.32(d,J=18.64Hz,1H) 4.72-4.97(m,1H) 3.56-3.73(m,2H) 3.07(br d,J=7.25Hz,2H) 2.62(br d,J=9.76Hz,1H) 2.37(s,4H) 2.10-2.19(m,1H) 1.88(dt,J=13.91,6.86Hz,3H) 0.89(t,J=7.32Hz,3H) 0.70(d,J=6.13Hz,3H) -0.02(d,J=6.00Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.56. LCMS(ESI+) m / z:[MH] + C 29 H 33 FN3O5 + Calculated value: 522.2, Found value: 522.2. SFC (retention time = 0.839 min) showed only one peak, 100% de. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0412] (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((R)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-114): 17-114 was synthesized in the same manner as 17-112. Spectrum of 17-114: 1H NMR(400MHz,DMSO-D6) δ ppm 8.26(s,0.54H) 7.72(d,J=11.01Hz,1H) 7.31(s,1H) 6.47(br s,1H) 5.71(d,J=18.76Hz,1H) 5.43(d,J=1.63Hz,2H) 5.32(d,J=18.76Hz,1H) 4.58-5.09(m,1H) 3.67-3.73(m,1H) 3.63(br d,J=2.38Hz,1H) 3.28-3.31(m,3H) 3.07(br d,J=7.25Hz,2H) 2.60(br d,J =10.01Hz,1H) 2.31-2.41(m,4H) 2.10-2.37(m,1H) 1.76-1.98(m,3H) 0.87(t,J=7.38Hz,3H) 0.69(d,J=6.13Hz,3H) -0.08(d,J=6.00Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.56. LCMS(ESI+) m / z:[MH] + C 29 H 33 FN3O5 + Calculated value: 522.2, Found value: 522.2. SFC (retention time = 1.031 min / 1.779 min) showed only two peaks, and a P1 / P2 ratio of 1.94 / 98.06, 96.12% dextromethorphan. Note: * The stereochemistry at these carbons was arbitrarily assigned.

[0413] (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((S)-2-hydroxy-1-(isopropylamino)ethyl)-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 (17-115): 17-115 was synthesized in the same manner as 17-112. Spectrum of 17-115: 1H NMR(400MHz, DMSO-D6) δ ppm 8.35(s,0.35H) 7.79(d,J=11.01Hz,1H) 7.38(s,1H) 6.45-6.69(m,1H) 5.66(d,J=19.39Hz,1H) 5.50(s,2H) 5.32(d,J=19.26Hz,1H) 4.92(br s,1H) 3.25(br d,J=12.76Hz,2H) 3.09(br dd,J=17.26,4.38Hz,2H) 2.92-2.97(m,1H) 2.72-2.83(m,4H) 2.43(s,3H) 1.84-2.02(m,3H) 1.01(d,J=6.13Hz,3H) 0.94(br t,J=7.25Hz,3H) 0.82(d,J=6.13Hz,3H). 1 H NMR(400MHz,DMSO-D6,D2O) δ ppm 8.34(s,0.35H) 7.69(d,J=10.88Hz,1H) 7.33(s,1H) 5.55(br d,J=19.26Hz,1H) 5.33-5.47(m,2H) 5.16-5.29(m,1H) 3.41(br dd,J=11.07,3.94Hz,1H) 3.35(br d,J=2.25Hz,1H) 3.12-3.20(m,1H) 3.00(br dd,J=17.39,3.75Hz,1H) 2.84(br dd,J=11.13,3.00Hz,1H) 2.73-2.77(m,1H) 2.67(br s,1H) 2.64-2.69(m,2H) 2.33(s,3H) 1.86(dq,J=14.43,7.07Hz,3H) 0.92(d,J=6.13Hz,3H) 0.83-0.88(m,3H) 0.74(d,J=6.00Hz,3H). 19 F NMR(376MHz,DMSO-D6) δ ppm -112.32. LCMS(ESI+) m / z:[MH] + C 29 H 33 FN3O5 + Calculated value: 522.2, measured value: 522.2.SFC (holding time = 0.839 minutes), 3つのピーク, P1 / P2 / P3=1.03 / 2.21 / 96.76, 93.52% deformation. Note: *The stereochemistry at these carbons was arbitrarily assigned. Alternative and / or additional synthetic routes to the compounds described in Example 17 can be found in FIG.

[0414] Example A CTG assay of payloads (Jeko-1 and MDA-MB-468) The CTG assay is a method for determining the number of viable cells in culture based on quantification of ATP abundance, an indicator of metabolically active cells. The cell assay requires the addition of a single reagent, Cell Titer Glo, which results in cell lysis and the generation of a luminescent signal. The luminescent signal is proportional to the amount of ATP abundance. The amount of ATP is directly proportional to the number of cells present in culture. In our assay, cells are confirmed to be in the logarithmic phase of Jeko-1 or MDA-MB-468. Cells were transferred to 96-well plates and treated with compounds in 3-fold serial dilutions (10-point dilutions) starting from 1 mM 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 by the luminescence of the vehicle control and plotted into a percentage viability vs. dose-response curve by a nonlinear fit in GraphPad Prism software. Compound IC 50 was calculated as the concentration of compound that killed 50% of the cells. The results for Jeko-1 are summarized in Table 7.

[0415] Example B Human Hepatocyte Clearance (HHEP CL) Suspension of human hepatocytes (from 10 mixed-sex donors, final concentration 0.5 × 10) in Williams' medium 6Cells (cells / mL) were incubated with test compounds (0.90% acetonitrile and 0.10% DMSO, final concentration 1 mM) and positive controls (7-ethoxycoumarin, 7-hydroxycoumarin, 0.90% acetonitrile and 0.10% DMSO, final concentration 3 mM) at 37°C for 90 min with constant shaking at approximately 600 rpm in a 5% CO2 and 95% humidity incubator. The total incubation volume was 200 μL. Samples (25 mL) were taken at TO, 15, 30, 60, and 90 min and added midway to 125 μL of ice-cold stop solution (acetonitrile containing 200 ng / mL tobutamide and labetalol as internal standards), vortexed at 500 rpm for 10 min, and centrifuged at 3220 × g for 20 min at 4°C. The analysis plates were sealed and stored at 4°C until LCMS analysis. Hepatocyte viability upon preincubation was determined to be 84.5%. HHEPCL results are summarized in Table 7.

[0416] Example C Human Liver Microsomal Clearance (HLM CL) Working solutions were prepared by adding 5 μL of compound and control stock solutions (10 mM in dimethyl sulfoxide, DMSO) to 495 μL of acetonitrile (ACN) (intermediate solution concentration: 100 μM, 99% ACN and 1% DMSO). Microsome working solutions of appropriate concentrations were prepared in 100 mM potassium phosphate buffer. After preincubating the reaction plate containing the compound and microsome mixture for 10 min at 37 °C, 98 mL of 2 mM NADPH and 2 mM MgCl2 solution 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 for 60 min at 37 °C. Samples were taken at T0, T5, T15, T30, T45, and T60, added midway to ice-cold stop solution (acetonitrile containing 200 ng / mL tobutamide and labetalol as internal standards) (125 μl), shaken for 10 min, and centrifuged at 4000 rpm for 20 min at 4° C. Analysis plates were analyzed by LCMS.

[0417] The human liver microsome clearance assay evaluates metabolism via the cytochrome P450 system (phase I enzymes). These enzymes oxidize substrates by incorporating oxygen atoms into hydrocarbons, thus introducing hydroxyl groups or N-, O-, and S-dealkylating the substrate to form more polar products that are more susceptible to clearance. The human hepatocyte clearance assay measures the broader, global cellular metabolism of the test compound (phase I and phase II enzyme pathways). Phase II enzymes catalyze the conjugation of xenobiotic metabolites and charged species, such as glutathione, sulfur, glycine, or glucuronic acid, to form even more polar compounds that facilitate clearance.

[0418] Payloads that exhibit high intrinsic clearance may offer better therapeutic indices due to their potentially lower systemic plasma exposure. (Maderna, A.; Doroski, M; Subramanyam, C.; Porte, A.; Leverett, CA; Vetelino, BC; Chen, Z.; Risley, H.; Parris, K.; Pandit, J.; Varghese, AH; Shanker, S.; Song, C.; Sukuru, SCK; Farley, KA; Wagenaar, MM; Shapiro, MJ; Musto, S.; Lam, MH.; Loganzo, F.; O'Donnell, CJ "Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications" Journal of Medicinal Chemistry, 2014, 57, 10527-10543). Payloads that exhibit high intrinsic clearance are likely to have an improved safety profile, as potentially toxic payloads are quickly cleared from plasma, reducing the chance of interaction with healthy cells. HLM CL results are summarized in Table 7.

[0419] Example D PAMPA (Parallel Artificial Membrane Permeability Assay) PAMPA is a method for determining the permeability of substances from a donor compartment to an acceptor compartment through a lipid-infused artificial membrane. (See Ottaviani, G.; Martel, S.; Carrupt, PA. "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 for the donor, and the membrane / acceptor compartment is placed on top. The entire assembly is commonly called a "sandwich." At the start of the test, the drug is added to the donor compartment, while the acceptor compartment is drug-free. After an incubation period, which may include agitation, the sandwich is separated, and the amount of drug is measured in each compartment. A mass balance allows for the calculation of the drug remaining in the membrane.

[0420] PAMPA was performed by Pion Inc. using GIT-0 lipid and a 5 mM donor solution at pH 5.0 and pH 7.4 PRISMA buffer (containing 0.05% DMSO). Higher PAMPA data correlated 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.)

[0421] High permeability is important because it suggests a superior ability for "bystander killing." That is, as the payload neutralizes a tumor cell, the more permeable the payload is, the more likely it is to detach from the neutralized tumor cell and then attach to a neighboring tumor cell. Once it neutralizes a tumor cell, detaches, and attaches to another neighboring tumor cell, the process repeats. The PAMPA results are summarized in Table 7.

[0422] Example E Development of anti-ROR-1 specific monoclonal antibody Novel and diversified anti-ROR-1-specific monoclonal antibodies were developed to bind to multiple regions of the ROR-1 extracellular domain (ECD) by employing an antibody development campaign employing three strategies: (1) immunizing mice in cohort 1 with the full-length ROR-1 ECD; (2) immunizing mice in cohorts 2 and 3 with the ROR-1 IgG-like domain; and (3) immunizing mice in cohort 4 with a short region of the human IgG-like sequence of ROR-1. After immunization of mice, monoclonal antibodies were generated using a conventional approach. Briefly, unique variable heavy and light chain pairs from the hybridoma and phage display campaigns were cloned into vectors designed to express full-length antibodies as IgG in HEK293 cells under the control of a CMV promoter. The antibody expression vectors were complexed with polyethyleneimine and transferred to HEK293 cultures. After 5 days of shaking in 293 cell culture medium at 37°C, the antibody was captured onto an agarose-based Protein A resin. After several rigorous washes, the antibody was eluted in a glycine solution, pH 3, neutralized with Hepes, pH 9, and buffer exchanged into PBS.

[0423] Several monoclonal antibodies were developed using these approaches, and the resulting antibodies underwent further screening to evaluate specific antibody characteristics. To fully characterize the novel antibodies, several assays were performed. First, antibody binding to the ROR-1 epitope was confirmed biochemically and in ROR-1-positive cell lines. Binding specificity was assessed biochemically 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 self-association assessment by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS).

[0424] Example F Assays to evaluate saturating concentrations and human ROR-1 binding affinities of anti-ROR-1 specific monoclonal antibodies A cell-binding saturation assay was developed to evaluate the extent to which the anti-ROR-1 antibodies developed in Example 16 bind to endogenously expressed extracellular ROR-1 protein on cell lines. More specifically, the anti-ROR-1 monoclonal antibodies developed in Example 16, e.g., ATX-P-875, ATX-P-885, and ATX-P-890, were analyzed in the 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 flow cytometric detection. The mean fluorescence intensity (MFI) was determined by analysis using the cytometer software. The binding of ATX-P-875, ATX-P-885, and ATX-P-890 was compared with the cell-binding saturation data of the monoclonal anti-ROR-1 antibody UC961 (see Figure 16). As shown in Figure 16, the cell binding saturation of antibodies ATX-P-875, ATX-P-885, and ATX-P-890 was comparable to that of UC961, but higher concentrations of ATX-P-875 were required to achieve saturation compared to UC961. ATX-P-890 and ATX-P-885 each had similar or improved binding saturation concentrations compared to UC961. The comparable saturation to UC961 demonstrated that the anti-ROR-1 antibodies, ATX-P-875, ATX-P-885, and ATX-P-890, have similar affinities to the clinically approved antibody UC961 for the human ROR-1 target.

[0425] Example G Assay to evaluate the ability of anti-ROR-1 specific monoclonal antibodies to internalize ROR-1 on human ROR-1 positive tumor cells After determining a saturating concentration (74 nM) in a binding assay, the anti-ROR-1 antibodies developed herein (ATX-P-875, P-885, and P-890) were evaluated for their ability to internalize ROR-1 receptors on human ROR-1-positive cells (Jeko-1 and MDA-MB-468). Briefly, ROR-1-positive cell lines were incubated with the antibodies under supersaturating conditions to ensure binding to all available ROR-1 receptors. Excess antibody was washed away, and cells were incubated at 37°C for the indicated times over a 4-hour time course. At the end of each time point, internalization was stopped by placing an aliquot of cells on ice. Remaining surface antibody was detected using a labeled secondary antibody and flow cytometry. Percent internalization was calculated based on time zero, which assumed 100% available receptors were present on the cell surface. The results in Figure 17 demonstrated that all antibodies internalized the ROR-1 receptor on Jeko-1 and MDA-MB-468 cells by at least 75% reduction over 4 hours. Unexpectedly, in MDA-MB-468, internalization of two anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) was improved over the clinically used UC961 anti-ROR-1 antibody, providing evidence that the ATX-P-875 and ATX-P-890 antibodies have improved ability to internalize the ROR-1 receptor from the surface of solid tumors.

[0426] Example H 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 described anti-ROR-1 monoclonal antibodies UC961 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 separately incubated with various amounts of ROR-1-expressing cells (MDA-MB-468). In step 2, the samples were incubated with a fluorescently labeled secondary antibody that recognizes the novel antibody. Finally, in step 3, ROR-1-expressing cells coated with ATX-P-875, ATX-P-885, and ATX-P-890 were incubated with saturating doses of labeled UC961 (Dy650-UC961) or 4A5 antibody (PE 4A5) and analyzed by flow cytometry. The UC961 and 4A5 staining signals were then compared with 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 18A below shows staining profiles predicting whether the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bind to the same epitope as the UC961 and 4A5 antibodies. Figure 18B shows profiles predicting whether the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bind to distinct epitopes on ROR-1 than the UC961 or 4A5 antibodies. Briefly, when binding to the same epitope, increasing concentrations of the novel antibody block binding of the pre-labeled competing antibody, thereby reducing the signal of the high-concentration competing antibody. When antibodies bind to separate epitopes, increasing doses of each antibody, both novel and competing, result in increased staining due to lack of competition for receptor binding. Cell binning data obtained with MDA-MB-468 cells showed that ATX-P-885 clearly binds to the same epitope as UC961, and both ATX-P-875 and ATX-P-890 clearly bind to the same epitope as 4A5 (see Figures 18C, 18D, and 19).The ability of the antibodies developed herein to bind to different ROR-1 epitopes provides the opportunity to regulate targets in a variety of ways.

[0427] Example I Biochemical binning study of anti-ROR-1 specific monoclonal antibodies Biochemical binning by SPR was also evaluated for anti-ROR-1 antibodies (ATX-P-875, P-885, and P-890) compared to the control anti-ROR-1 antibodies UC961 and 4a5. In these experiments, 10 μg / ml of purified cloned Hu / Cy / Rh ROR-1-His protein was covalently coupled to an HC30M chip. Individual dilutions of each antibody at 10 μg / mL were injected onto the chip, and binding was assessed by Carterra SPR. Unexpectedly, the data demonstrated three distinct binding epitopes between ATX-P-875, ATX-P-885, and ATX-P-890, with ATX-885 being the only antibody that conferred partial blocking to the UC961 antibody (see Figure 20). Cell binning only assessed the ability of the antibodies to block either of two clinically used ROR-1 antibodies, UC961 or 4a5. Biochemical SPR assessment 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 still failed to block UC961.

[0428] Example J Antibody characteristics The antibody characteristics of ATX-P-875, ATX-P-885, and ATX-P-890 compared to UC961 are summarized in Figure 19 and Tables 1-6. An initial evaluation of antibody developability was performed by AC-SINS to assess the potential for self-interaction (Figure 19). The control antibody adalimumab showed the expected low shift, while infliximab showed the expected high shift. The anti-ROR-1 antibodies developed herein, ATX-P-875, ATX-P-885, and ATX-P-890, are consistent with the control antibody, which does not exhibit significant self-interaction, and do not appear to pose significant developability risks. Furthermore, the binding characteristics of monoclonal antibodies ATX-P-875, ATX-P-885, and ATX-P-890 were compared to those of UC961 in further experiments. Tables 1-4 provide antibody characterization data compared to the known ROR-1-binding antibody UC961, including tabulated results for biochemical binding to purified protein and by SPR (Tables 1-4), cellular binding (EC50) to the ROR-1-positive cell lines Jeko-1 and MDA-MB-468 (Table 5), and cellular internalization (% internalization) (Table 6). Of particular note, the decreased affinity (KD: 1.09E-08) of ATX-P-885 compared to UC961 and other anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) may provide unexpected therapeutic efficacy. Weaker binding to the ROR-1 epitope may allow the ATX-P-885 antibody to further infiltrate tumors and reach more distant cells expressing the ROR-1 target.

[0429] [Table 2]

[0430] [Table 3]

[0431] [Table 4]

[0432] [Table 5]

[0433] [Table 6]

[0434] [Table 7]

[0435] [Table 8]

[0436] Example K Preparation of antibody-drug conjugates Synthesis of immunoconjugates was accomplished as described in this Example. Antibodies were produced as described in Example E and suspended in PBS pH 7.2 with protein concentrations ranging between 10 and 20 mg / ml. A molecular weight of 150,000 Da was used for all antibodies for reduction and conjugation calculations.

[0437] Each antibody was prepared for reduction by adding 5% v / v of 500 mM Tris, 25 mM EDTA, pH 8.5, followed by the addition of TCEP (6 equivalents of TCEP in water, 10 mM stock), and the mixture was maintained for 2 hours at 20° C. This reduction step forms a cysteine ​​residue, Cys-SH, on the antibody that facilitates bioconjugation with a toxin linker, i.e., a compound of formula (III) described herein.

[0438] After DMA was added and gently mixed with the reduced protein solution to achieve a final 10% v / v concentration in the conjugation mixture, the toxin-linker stock solution (12 equiv. in DMA, 50 mM) was added and gently mixed. Bioconjugation proceeded overnight at 20 °C for approximately 16–20 h, and was complete within 2 h, including an extension time to allow for maleimide ring-opening.

[0439] The crude conjugate was buffer exchanged into PBS pH 7.4 using a gravity-fed NAP25 (small scale) or flow HiPrep G25 (large scale) with columns prepared and operated according to the manufacturer's instructions (Cytivia). To remove residual toxin, a 100 mg / ml slurry of activated charcoal (Sigma / C9157) in PBS pH 7.4 was prepared and added to achieve 1 mg of carbon per 1 mg of starting antibody weight. This was mixed gently for 2 hours, sufficient to maintain the carbon in suspension. The carbon was then removed by centrifugation at 4000 g. Polysorbate 20 (PS20) was 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 was finally filtered through a suitably sized 0.2 μm PES filter (Chromatography Direct / FIL-S-PES-022-13-100-S) under grade A laminar flow. The final product was analyzed as follows: monomer and [ADC] mg / ml by SEC HPLC, mean DAR by PLRP, residual toxin by RP-HPLC, and endotoxin by Endosafe kinetic chromogenic assay.

[0440] The analytical process was carried out on an Agilent 1100 or 1260 HPLC system.

[0441] Example L CTG assay of antibody-drug conjugates The novel ROR-1 antibody-drug conjugates (ADCs) were evaluated by CTG assay for payload screening in a manner similar to that described in Example A and Table 7. By way of example only and in no way limiting the scope of the antibody-drug conjugates of the present disclosure, a total of three unique antibodies, ATX-P-875, ATX-P-885, and ATX-P-890, are conjugated to novel linkers / payloads or compounds of formula (III), including, but not limited to, compounds 14-92, 14-93, 14-94, 14-95, 15-96, 15-97, 15-98, 15-99, and any exemplary compound of formula (III) described herein. Briefly, ROR-positive (JeKo-1 / MDA-MB-468) or ROR-negative (Ramos) cells were transferred to 96-well plates and treated with test ADCs at 3-fold serial dilutions (10-point dilutions) starting from 1 mM to 0.0000508 mM for 72 hours. Cell viability was analyzed by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. The percentage of viable cells at each ADC concentration was determined by normalizing with the luminescence of the vehicle control and plotted into a percentage viability vs. dose-response curve by a nonlinear fit in GraphPad Prism software. The IC of each test ADC was calculated. 50 was calculated as the concentration of compound that kills 50% of the cells and was evaluated against UC961. The ADCs described herein, including those prepared by conjugating antibodies ATX-P-875, ATX-P-885, and ATX-P-890 to compounds 14-92, 14-93, 14-94, 14-95, 15-96, 15-97, 15-98, 15-99, and any exemplary compound of formula (III), have an IC 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 a CTG assay with Jeko-1 or MDA-MB-468 cells. 50 It has a value.

[0442] Moreover, while the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, those skilled in the art will appreciate that numerous and various modifications can be made without departing from the spirit of the present disclosure. It is therefore to be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but will cover all modifications and variations that fall within the true scope and spirit of the present disclosure.

[0443] Sequence Listing SEQ ID NO: 1 ATX-P-875 VH CDR1(Kabat) GFTFSNAW SEQ ID NO: 2 ATX-P-875 VH CDR2(Kabat) IKSKTDGGTT SEQ ID NO: 3 ATX-P-875 VH CDR3(Kabat) TTGPDDLDY SEQ ID NO:4 ATX-P-875 VH nt GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACA ACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA SEQ ID NO:5 ATX-P-875 VH AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSS SEQ ID NO:6 ATX-P-875 HC IgG1-Fc nt SEQ ID NO:7 ATX-P-875 HC IgG1-Fc AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG SEQ ID NO:8 ATX-P-875 VL CDR1(Kabat) QSISSY ATX-P-875 VL CDR2(Kabat) AAS SEQ ID NO: 10 ATX-P-875 VL CDR3(Kabat) QQYDNLPIT SEQ ID NO: 11 ATX-P-875 VL nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA Sequence number 12 ATX-P-875 VL AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIK Sequence number 13 ATX-P-875 kappa LC nt GACATCCAGATGACCCAGTTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGAAAGCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCCGATCACCTTGGCCAAGGGACACGACTGGAGATTAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT sequence number 14 ATX-P-875 カムパ LC AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence number 15 ATX-P-885 VH CDR1(Kabat) GGSFSGYY SEQ ID NO: 16 ATX-P-885 VH CDR2(Kabat) INHSGST SEQ ID NO: 17 ATX-P-885 VH CDR3(Kabat) AREGVYEDY SEQ ID NO: 18 ATX-P-885 VH nt CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCA ACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 19 ATX-P-885 VH AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSS SEQ ID NO: 20 ATX-P-885 HC IgG1-Fc nt SEQ ID NO: 21 ATX-P-885 HC IgG1-Fc AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 22 ATX-P-885 VL CDR1(Kabat) QSVSNY ATX-P-885 VL CDR2(Kabat) DAY SEQ ID NO: 24 ATX-P-885 VL CDR3(Kabat) QQRSNWPLT SEQ ID NO: 25 ATX-P-885 VL nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAA Sequence number 26 ATX-P-885 VL AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIK Sequence number 27 ATX-P-885 kappa LC nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGAAAGAGCCACCCTCTCCTGCAGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTGGCCAAGGGACACGACTGGAGATTAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT sequence no. 28 ATX-P-885 カムパ LC AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence no. 29 ATX-P-890 VH CDR1(Kabat) GYTFTGYY SEQ ID NO: 30 ATX-P-890 VH CDR2(Kabat) INPNSGGT SEQ ID NO: 31 ATX-P-890 VH CDR3(Kabat) VRDQVQLERFDS SEQ ID NO: 32 ATX-P-890 VH nt CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGCACAAACT ATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 33 ATX-P-890 VH AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSS SEQ ID NO: 34 ATX-P-890 HC IgG1-Fc nt SEQ ID NO: 35 ATX-P-890 HC IgG1-Fc AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG SEQ ID NO: 36 ATX-P-890 VL CDR1(Kabat) QDISNY ATX-P-890 VL CDR2(Kabat) DAS SEQ ID NO: 38 ATX-P-890 VL CDR3(Kabat) QQYDNLPPT SEQ ID NO: 39 ATX-P-890 VL nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAA Sequence number 40 ATX-P-890 VL AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIK Sequence number 41 ATX-P-890 kappa LC nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAAACAGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT sequence no. 42 ATX-P-890 カムパ LC AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFFTISSLQPEDIATYYCQQYDNLPPTGFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. Formula (I) Ab-[S-L １ -L ２ -L ３ -L ４ -L ５ -L ６ -L ７ -D] ｎ (I) or a pharmaceutically acceptable salt thereof. wherein Ab is an antibody or antigen-binding fragment; L １ teeth, 【Chemistry 1】 and L ２ does not exist or 【Chemistry 2】 and Z １ and Z ２ are hydrogen, halogen, and -NO ２ , —O—(C １ ~C ６ alkyl), or C １ ~C ６ 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 are independently integers from 0 to 12, L ４ is a tetrapeptide residue, L ５ is absent or -[NH(CH ２ ) n ２ ]n ３ - and n ２ is an integer from 0 to 6, n ３ is an integer from 0 to 2, L ６ does not exist or 【Transformation 3】 and L ７ does not exist or 【Chemistry 4】 and D is a drug moiety, and and n is an integer from 1 to 10.

2. L ２ The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein:

3. L ２ but, 【Transformation 5】 2. The immunoconjugate of claim 1 , wherein:

4. L ２ but, 【Transformation 6】 2. The immunoconjugate of claim 1 , wherein:

5. Z １ and Z ２ 5. The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein at least one of:

6. Z １ and Z ２ 5. The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein at least one of:

7. Z １ and Z ２ At least one of the following is -NO ２ 5. The immunoconjugate of claim 1 , wherein:

8. Z １ and Z ２ At least one of the groups is —O—(C １ ~C ６ 5. The immunoconjugate of claim 1 , wherein R is 1 or 2; R is 2 or 3; R is 3 or 4; or a pharmaceutically acceptable salt thereof.

9. Z １ and Z ２ At least one of １ ~C ６ 5. The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

10. L ３ But -(CH ２ ) n １ 10. The immunoconjugate of any one of claims 1 to 9, wherein the group is -C(=O)-, or a pharmaceutically acceptable salt thereof.

11. L ３ But -(CH ２ CH ２ O)n １ - (CH ２ ) n １ 10. The immunoconjugate of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein C(=O)-.

12. L ４ 12. The immunoconjugate of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein is gly-gly-phe-gly (GGGFG).

13. L ５ 12. The immunoconjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

14. L ５ is -[NH(CH ２ ) n ２ ]n ３ 12. The immunoconjugate of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein:

15. L ６ 15. The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein:

16. L ６ but, 【Transformation 7】 15. The immunoconjugate of any one of claims 1 to 14, wherein:

17. L ７ 17. The immunoconjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein:

18. L ７ but, 【Transformation 8】 17. The immunoconjugate of any one of claims 1 to 16, wherein:

19. L ７ but, 【Chemistry 9】 17. The immunoconjugate of any one of claims 1 to 16, wherein:

20. L ７ but, 【Chemistry 10】 17. The immunoconjugate of any one of claims 1 to 16, wherein:

21. D is a compound having the structure: 【Chemistry 11】 [In the formula, R １ and R ２ are each independently hydrogen, halogen, -CN, -OR ５ , -NR ５ R ６ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C １ ~C ６ haloalkyl, substituted or unsubstituted —O—(C １ ~C ６ alkyl), substituted or unsubstituted —O—(C １ ~C ６ haloalkyl), -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ３ or substituted or unsubstituted —O—(CR ５ R ６ ) ｍ —O—, so that R １ and R ２ come together to form a ring, R ３ and R ４ is R ３ and R ４ are each independently hydrogen, —OH, —N, provided that at least one of them is not hydrogen. ３ , -NH ２ , -NH(C=O)-CH ２ -R ３Ｄ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C ２ ~C ６ Alkenyl and [(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ OH, C １ ~C ６ Alkyl or C ２ ~C ６ When alkenyl is substituted, C １ ~C ６ Alkyl or C ２ ~C ６ Alkenyl is an alkyl group selected from the group consisting of -OH and -NR ３Ｂ R ３Ｃ One or more R selected from ３Ａ group, where R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ alkyl, or —C(═O)(unsubstituted C １ ~C ６ alkyl), or R ３ and R ４ one of which is a substituted or unsubstituted -(C １ ~C ６ alkyl)-X ２ or substituted or unsubstituted -(C ２ ~C ６ alkenyl)-X ２ and -(C １ ~C ６ alkyl)-X ２ or -(C １ ~C ６ alkenyl)-X ２ is substituted, -(C １ ~C ６ alkyl)-X ２ or -(C １ ~C ６ alkenyl)-X ２ represents —OH and —NR ３Ｂ R ３Ｃ One or more R selected from ３Ａ substituted by a group, where R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or —C(═O)(unsubstituted C １ ~C ６ alkyl), R ３Ｄ is H, -CH ３ , —OH and —CH ２ Y １ wherein Y is selected from the group consisting of １ is a halogen, X ２ is -OR ９ , -SR ９ , or -NHR ９ and R ５ and R ６ are each individually substituted or unsubstituted C １ ~C ６ alkyl or R ５ and R ６ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring, n ４ and n ５ is n ４ and n ５ are each individually 0, 1, or 2, provided that at least one of them is not 0; each Y is individually H or halogen; each m is independently 1 or 2; each p is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 1, 2, 3, 4, 5, or 6; R ７ is H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ , or L ７ and R ８ is a substituted or unsubstituted C １ ~C ６ Alkyl-X ３ , substituted or unsubstituted C １ ~C ６ Haloalkyl-X ３ , or - [(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ２ -X ３ and R ９ is R ７ and R ９ One of them is definitely L ４ , L ５ , L ６ , or L ７ H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ , or L ７ and each X ３ are individually —H, —OH, —SH, or —NH ２ is] 21. The immunoconjugate of any one of claims 1 to 20, wherein the drug moiety has formula (II):

22. R １ But C １ ~C ３ alkyl, and R ２ 22. The immunoconjugate of claim 21 , or a pharmaceutically acceptable salt thereof, wherein is a halogen.

23. R １ is methyl, and R ２ is F, or a pharmaceutically acceptable salt thereof.

24. R ３ and R ４ 24. The immunoconjugate of any one of claims 21 to 23, or a pharmaceutically acceptable salt thereof, wherein one of: is hydrogen.

25. R ３ and R ４ or a pharmaceutically acceptable salt thereof.

26. R ３ and R ４ One of the groups is -N ３ 24. The immunoconjugate of any one of claims 21 to 23, wherein:

27. R ３ and R ４ One of the groups is -NH ２ 24. The immunoconjugate of any one of claims 21 to 23, wherein:

28. R ３ and R ４ one of which is —NH(C═O)CH ２ R ３Ｄ 24. The immunoconjugate of any one of claims 21 to 23, wherein:

29. R ３ and R ４ the other of which is substituted or unsubstituted C １ ~C ６ 29. The immunoconjugate of any one of claims 24 to 28, or a pharmaceutically acceptable salt thereof, wherein:

30. R ３ and R ４ The other of these is a substitution C １ ~C ６ 30. The immunoconjugate of claim 29, or a pharmaceutically acceptable salt thereof, wherein:

31. Substitution C １ ~C ６ The alkyl is selected from —OH and —NR ３Ｂ R ３Ｃ and R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or C(=O)(unsubstituted C １ ~C ６ 31. The immunoconjugate of claim 30, wherein R is 1 or 2; R is 2 or 3; R is 3 or 4; R is 4 or 5; R is 5 or 6; R is 6 or 7; R is 7 or 8; R is 8 or 9; R is 9 or 10; R is 10 or 11; R is 11 or 12; R is 13 or 14; R is 15 or 16; R is 16 or 17; R is 18 or 19; R is 19 or 20; R is 19 or 21; R is 19 or 22; R is 19 or 23; R is 19 or 24; R is 19 or 25; R is 19 or 26; R is 19 or 27; R is 19 or 28; R is 19 or 29; R is 20 or

32. R ３Ｂ and R ３Ｃ and n is 0 or 1. The immunoconjugate of claim 31 , or a pharmaceutically acceptable salt thereof, wherein each is hydrogen.

33. R ３Ｂ is hydrogen, and R ３Ｃ is a substituted or unsubstituted C １ ~C ６ 32. The immunoconjugate of claim 31 , or a pharmaceutically acceptable salt thereof, wherein:

34. R ３Ｂ is hydrogen, and R ３Ｃ is unsubstituted C １ ~C ６ 34. The immunoconjugate of claim 33, or a pharmaceutically acceptable salt thereof, wherein:

35. R ３Ｂ and R ３Ｃ are each substituted or unsubstituted C １ ~C ６ 32. The immunoconjugate of claim 31 , or a pharmaceutically acceptable salt thereof, wherein:

36. R ３Ｂ and R ３Ｃ are unsubstituted C １ ~C ６ 35. The immunoconjugate of claim 34, or a pharmaceutically acceptable salt thereof, wherein:

37. R ３Ｂ is hydrogen, and R ３Ｃ is -C(=O) (unsubstituted C １ ~C ６ 32. The immunoconjugate of claim 31 , or a pharmaceutically acceptable salt thereof, wherein:

38. Substitution C １ ~C ６ 38. The immunoconjugate of any one of claims 30 to 37, or a pharmaceutically acceptable salt thereof, wherein alkyl is substituted with one or more OH groups.

39. Substitution C １ ~C ６ The alkyl is —CH ２ OH, -CH ２ CH ２ OH or -CH ２ CH(OH)CH ２ OH, or a pharmaceutically acceptable salt thereof.

40. R ３ and R ４ The other of these is a substitution C ２ ~C ６ 29. The immunoconjugate of any one of claims 24 to 28, or a pharmaceutically acceptable salt thereof, which is alkenyl.

41. Substitution C ２ ~C ６ The alkenyl is —NR ３Ｂ R ３Ｃ and R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or —C(═O)(unsubstituted C １ ~C ６ 41. The compound of claim 40, or a pharmaceutically acceptable salt thereof, wherein:

42. R ３ and R ４ The other of these is unsubstituted C ２ ~C ６ 29. The immunoconjugate of any one of claims 24 to 28, or a pharmaceutically acceptable salt thereof, which is alkenyl.

43. R ３ and R ４ the other of which is substituted or unsubstituted -(C １ ~C ６ alkyl)-X ２ 29. The immunoconjugate of any one of claims 24 to 28, wherein:

44. R ３ and R ４ the other of which is substituted or unsubstituted -(C ２ ~C ６ alkenyl)-X ２ 29. The immunoconjugate of any one of claims 24 to 28, wherein:

45. n ４ 45. The immunoconjugate of any one of claims 21 to 44, or a pharmaceutically acceptable salt thereof, wherein

46. n ５ 46. ​​The immunoconjugate of any one of claims 21 to 45, or a pharmaceutically acceptable salt thereof, wherein

47. Formula (II) has the structure: 【Chemistry 12】 wherein b is 1, 2, or 3.

22. The immunoconjugate of claim 21 having the structure of formula (II-a) having the following formula:

48. Each R ３Ａ 48. The immunoconjugate of claim 47, or a pharmaceutically acceptable salt thereof, wherein is -OH.

49. 49. The immunoconjugate of claim 47 or 48, or a pharmaceutically acceptable salt thereof, wherein b is 1.

50. 49. The immunoconjugate of claim 47 or 48, or a pharmaceutically acceptable salt thereof, wherein b is 2.

51. 49. The immunoconjugate of claim 47 or 48, or a pharmaceutically acceptable salt thereof, wherein b is 3.

52. R ３ and R ４ The other of the two is -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ 29. The immunoconjugate of any one of claims 24 to 28, or a pharmaceutically acceptable salt thereof, wherein R is OH.

53. R ７ is H, or a pharmaceutically acceptable salt thereof.

54. 54. The immunoconjugate of any one of claims 1 to 53, or a pharmaceutically acceptable salt thereof, wherein the Ab specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1), Her2, TROP2, Her3, B7-H3, GPR20, or CEACAM5.

55. 55. The immunoconjugate of any one of claims 1 to 54, or a pharmaceutically acceptable salt thereof, wherein the Ab binds to the surface of a cancer cell.

56. Formula (I) 【Chemistry 13-1】 【Chemistry 13-2】 or a pharmaceutically acceptable salt thereof.

57. Formula (I) 【Chemistry 14-1】 【Chemistry 14-2】 or a pharmaceutically acceptable salt thereof.

58. Formula (I) 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】 【Chemistry 15-5】 【Chemistry 15-6】 【Chemistry 15-7】 【Chemistry 15-8】 【Chemistry 15-9】 【Chemistry 15-10】 【Chemistry 15-11】 [Chemistry 15-12] 【Chemistry 15-13】 or a pharmaceutically acceptable salt thereof.

59. structure: 【Chemistry 16】 [In the formula, R １ and R ２ are each independently hydrogen, halogen, -CN, -OR ５ , -NR ５ R ６ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C １ ~C ６ haloalkyl, substituted or unsubstituted —O—(C １ ~C ６ alkyl), substituted or unsubstituted —O—(C １ ~C ６ haloalkyl), -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ３ or substituted or unsubstituted —O—(CR ５ R ６ ) ｍ —O—, so that R １ and R ２ come together to form a ring, R ３ and R ４ is R ３ and R ４ are each independently hydrogen, —OH, —N, provided that at least one of them is not hydrogen. ３ , -NH ２ , -NH(C=O)-CH ２ -R ３Ｄ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C ２ ~C ６ Alkenyl and [(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ OH, C １ ~C ６ Alkyl or C ２ ~C ６ When alkenyl is substituted, C １ ~C ６ Alkyl or C ２ ~C ６ Alkenyl is an alkyl group selected from the group consisting of -OH and -NR ３Ｂ R ３Ｃ One or more R selected from the group consisting of ３Ａ group, where R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ alkyl, or —C(═O)(unsubstituted C １ ~C ６ alkyl), R ３Ｄ is H, -CH ３ , —OH and —CH ２ Y １ wherein Y is selected from the group consisting of １ is a halogen, R ５ and R ６ are each individually substituted or unsubstituted C １ ~C ６ alkyl or R ５ and R ６ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring, n ４ and n ５ is n ４ and n ５ are each individually 0, 1, or 2, provided that at least one of them is not 0; each Y is individually H or halogen; each m is independently 1 or 2; each p is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 1, 2, 3, 4, 5, or 6; R ７ is H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ and R ８ is a substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C １ ~C ６ haloalkyl, or -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ３ is] 1. A compound of formula (IV) having the formula:

60. R １ is C １ ~C ３ alkyl, and R ２ 60. The compound of claim 59, or a pharmaceutically acceptable salt thereof, wherein is halogen.

61. R １ is methyl, and R ２ is F, or a pharmaceutically acceptable salt thereof.

62. R ３ and R ４ 62. The compound of any one of claims 59 to 61, or a pharmaceutically acceptable salt thereof, wherein one of: is hydrogen.

63. R ３ and R ４ 62. The compound of any one of claims 59 to 61, or a pharmaceutically acceptable salt thereof, wherein one of:

64. R ３ and R ４ One of them is -N ３ 62. The compound of any one of claims 59 to 61, wherein:

65. R ３ and R ４ One of them is -NH ２ 62. The compound of any one of claims 59 to 61, wherein:

66. R ３ and R ４ One of the is -NH(C=O)CH ２ R ３Ｄ 62. The compound of any one of claims 59 to 61, wherein:

67. R ３ and R ４ the other of which is substituted or unsubstituted C １ ~C ６ 67. The compound of any one of claims 62 to 66, or a pharmaceutically acceptable salt thereof, wherein:

68. R ３ and R ４ The other of these is a substitution C １ ~C ６ 68. The compound of claim 67, or a pharmaceutically acceptable salt thereof, wherein:

69. Substitution C １ ~C ６ The alkyl is selected from —OH and —NR ３Ｂ R ３Ｃ and R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or —C(═O)(unsubstituted C １ ~C ６ 69. The compound of claim 68, or a pharmaceutically acceptable salt thereof, wherein:

70. R ３Ｂ and R ３Ｃ 70. The compound of claim 69, or a pharmaceutically acceptable salt thereof, wherein each is hydrogen.

71. R ３Ｂ is hydrogen, and R ３Ｃ is a substituted or unsubstituted C １ ~C ６ 70. The compound of claim 69, or a pharmaceutically acceptable salt thereof, wherein: R is alkyl;

72. R ３Ｂ is hydrogen, and R ３Ｃ is unsubstituted C １ ~C ６ 72. The compound of claim 71, or a pharmaceutically acceptable salt thereof, wherein:

73. R ３Ｂ and R ３Ｃ are each substituted or unsubstituted C １ ~C ６ 70. The compound of claim 69, or a pharmaceutically acceptable salt thereof, wherein: R is alkyl;

74. R ３Ｂ and R ３Ｃ are unsubstituted C １ ~C ６ 74. The compound of claim 73, or a pharmaceutically acceptable salt thereof, wherein:

75. R ３Ｂ is hydrogen, and R ３Ｃ is -C(=O) (unsubstituted C １ ~C ６ 70. The compound of claim 69, or a pharmaceutically acceptable salt thereof, wherein:

76. Substitution C １ ~C ６ 76. The compound of any one of claims 68 to 75, or a pharmaceutically acceptable salt thereof, wherein alkyl is substituted with one or more OH groups.

77. Substitution C １ ~C ６ The alkyl is —CH ２ OH, -CH ２ CH ２ OH or -CH ２ CH(OH)CH ２ 76. The compound of claim 75, or a pharmaceutically acceptable salt thereof, wherein:

78. R ３ and R ４ The other of these is a substitution C ２ ~C ６ 67. The compound of any one of claims 62 to 66, or a pharmaceutically acceptable salt thereof, which is alkenyl.

79. Substitution C ２ ~C ６ The alkenyl is —NR ３Ｂ R ３Ｃ and R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or —C(═O)(unsubstituted C １ ~C ６ 79. The compound of claim 78, or a pharmaceutically acceptable salt thereof, wherein:

80. R ３ and R ４ The other of these is unsubstituted C ２ ~C ６ 67. The compound of any one of claims 62 to 66, or a pharmaceutically acceptable salt thereof, which is alkenyl.

81. n ４ 81. The compound of any one of claims 59 to 80, or a pharmaceutically acceptable salt thereof, wherein

82. n ５ 82. The compound of any one of claims 59 to 81, or a pharmaceutically acceptable salt thereof, wherein

83. Formula (IV) has the structure: 【Chemistry 17】 wherein b is 1, 2, or 3.

60. The compound of claim 59, having the structure of formula (IV-a):

84. Each R ３Ａ is independently -OH, or a pharmaceutically acceptable salt thereof.

85. Each R ３Ａ is independently H, or a pharmaceutically acceptable salt thereof.

86. Each R ３Ａ Independently -NR ３Ｂ R ３Ｃ where R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ alkyl, or —C(═O)(unsubstituted C １ ~C ６ 84. The compound of claim 83, or a pharmaceutically acceptable salt thereof, wherein:

87. 85. The compound of claim 83 or 84, or a pharmaceutically acceptable salt thereof, wherein b is 1.

88. 85. The compound of claim 83 or 84, or a pharmaceutically acceptable salt thereof, wherein b is 2.

89. 85. The compound of claim 83 or 84, or a pharmaceutically acceptable salt thereof, wherein b is 3.

90. R ３ and R ４ The other of the two is -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ 62. The compound of any one of claims 59 to 61, or a pharmaceutically acceptable salt thereof, wherein R is OH.

91. R ７ 91. The compound of any one of claims 59 to 90, or a pharmaceutically acceptable salt thereof, wherein:

92. The compound of formula (IV) 【Chemistry 18-1】 【Chemistry 18-2】 【Chemistry 18-3】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

93. The compound of formula (IV) 【Chemistry 19】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

94. The compound of formula (IV) 【Chemistry 20-1】 【Chemistry 20-2】 【Chemistry 20-3】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

95. The compound of formula (IV) 【Chemistry 21-1】 【Chemistry 21-2】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

96. The compound of formula (IV) 【Chemistry 22-1】 【Chemistry 22-2】 【Chemistry 22-3】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

97. The compound of formula (IV) 【Chemistry 23】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

98. The compound of formula (IV) 【Chemistry 24】 60. The compound of claim 59, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

99. 98. A pharmaceutical composition comprising an immunoconjugate of any one of claims 1 to 58, a compound of any one of claims 59 to 98, or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

100. 99. A method for treating, inhibiting, or alleviating cancer or tumors, comprising administering to a subject having cancer or tumors an effective amount of an immunoconjugate of any one of claims 1 to 58, a compound of any one of claims 59 to 98, or a pharmaceutically active salt thereof, or a pharmaceutical composition of claim 99.

101. 101. The method of claim 100, wherein the cancer or tumor is selected from lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal tumors, 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.

102. Use of an effective amount of an immunoconjugate of any one of claims 1 to 58, a compound of any one of claims 59 to 98, or a pharmaceutically active salt thereof, or a pharmaceutical composition of claim 99 in the manufacture of a medicament for treating, inhibiting, or ameliorating cancer or tumors.

103. 103. The use of claim 102, wherein the cancer or tumor is selected from lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal tumors, 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.

104. Formula (III), Mi-L ２ -L ３ -L ４ -L ５ -L ６ -L ７ -D (III) or a pharmaceutically acceptable salt thereof. [In the formula, Mi is, 【Chemistry 25】 and L ２ does not exist or 【Chemistry 26】 and Z １ and Z ２ are hydrogen, halogen, and -NO ２ , —O—(C １ ~C ６ alkyl), or C １ ~C ６ 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 are independently integers from 0 to 12, L ４ is a tetrapeptide residue, L ５ is absent or -[NH(CH ２ ) n ２ ]n ３ - and n ２ is an integer from 0 to 6, n ３ is an integer from 0 to 2, L ６ does not exist or 【Chemistry 27】 and L ７ does not exist or 【Chemistry 28】 and D is a drug moiety].

105. L ２ The conjugate of claim 104, or a pharmaceutically acceptable salt thereof, wherein

106. L ２ but, 【Chemistry 29】 105. The conjugate of claim 104, wherein:

107. L ２ but, 【Transformation 30】 105. The conjugate of claim 104, wherein:

108. Z １ and Z ２ 108. The conjugate of any one of claims 104 to 107, or a pharmaceutically acceptable salt thereof, wherein at least one of:

109. Z １ and Z ２ 108. The conjugate of any one of claims 104 to 107, or a pharmaceutically acceptable salt thereof, wherein at least one of: is a halogen.

110. Z １ and Z ２ At least one of the following is -NO ２ 108. The conjugate of any one of claims 104 to 107, wherein:

111. Z １ and Z ２ At least one of the groups is —O—(C １ ~C ６ 108. The conjugate of any one of claims 104 to 107, or a pharmaceutically acceptable salt thereof, wherein

112. Z １ and Z ２ At least one of １ ~C ６ 108. The conjugate of any one of claims 104 to 107, or a pharmaceutically acceptable salt thereof, wherein:

113. L ３ But -(CH ２ ) n １ 113. The conjugate of any one of claims 104 to 112, or a pharmaceutically acceptable salt thereof, which is -C(=O)-.

114. L ３ But -(CH ２ CH ２ O)n １ - (CH ２ ) n １ 113. The conjugate of any one of claims 104 to 112, or a pharmaceutically acceptable salt thereof, wherein C(=O)-.

115. L ４ is gly-gly-phe-gly (GGGFG), or a pharmaceutically acceptable salt thereof.

116. L ５ 116. The conjugate of any one of claims 104 to 115, or a pharmaceutically acceptable salt thereof, wherein

117. L ５ is -[NH(CH ２ ) n ２ ]n ３ -, or a pharmaceutically acceptable salt thereof.

118. L ６ 118. The conjugate of any one of claims 104 to 117, or a pharmaceutically acceptable salt thereof, wherein

119. L ６ but, 【Chemistry 31】 118. The conjugate of any one of claims 104 to 117, wherein:

120. L ７ 120. The conjugate of any one of claims 104 to 119, or a pharmaceutically acceptable salt thereof, wherein

121. L ７ but, 【Chemistry 32】 120. The conjugate of any one of claims 104 to 119, wherein:

122. L ７ but, 【Transformation 33】 120. The conjugate of any one of claims 104 to 119, wherein:

123. L ７ but, 【Transformation 34】 120. The conjugate of any one of claims 104 to 119, wherein:

124. D is a compound having the structure: 【Chemistry 35】 [In the formula, R １ and R ２ are each independently hydrogen, halogen, -CN, -OR ５ , -NR ５ R ６ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C １ ~C ６ haloalkyl, substituted or unsubstituted —O—(C １ ~C ６ alkyl), substituted or unsubstituted —O—(C １ ~C ６ haloalkyl), -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ３ or substituted or unsubstituted —O—(CR ５ R ６ ) ｍ —O—, so that R １ and R ２ come together to form a ring, R ３ and R ４ is R ３ and R ４ are each independently hydrogen, —OH, —N, provided that at least one of them is not hydrogen. ３ , -NH ２ , -NH(C=O)-CH ２ R ３Ｄ , substituted or unsubstituted C １ ~C ６ Alkyl, substituted or unsubstituted C ２ ~C ６ Alkenyl and [(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ OH, C １ ~C ６ Alkyl or C ２ ~C ６ When alkenyl is substituted, C １ ~C ６ Alkyl or C ２ ~C ６ Alkenyl is an alkyl group selected from the group consisting of -OH and -NR ３Ｂ R ３Ｃ One or more R selected from the group consisting of ３Ａ group, where R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ alkyl, or —C(═O)(unsubstituted C １ ~C ６ alkyl), R ３Ｄ is H, -CH ３ , —OH and —CH ２ Y １ wherein Y is selected from the group consisting of １ is a halogen, X ２ is -OR ９ , -SR ９ , or -NHR ９ and R ５ and R ６ are each individually substituted or unsubstituted C １ ~C ６ alkyl or R ５ and R ６ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4- or 5-membered heterocyclyl ring, n ４ and n ５ is n ４ and n ５ are each individually 0, 1, or 2, provided that at least one of them is not 0; each Y is individually H or halogen; each m is independently 1 or 2; each p is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 1, 2, 3, 4, 5, or 6; R ７ is H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ , or L ７ and R ８ is a substituted or unsubstituted C １ ~C ６ Alkyl-X ３ , substituted or unsubstituted C １ ~C ６ Haloalkyl-X ３ , or - [(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ CY ２ -X ３ and R ９ is R ７ and R ９ One of them is definitely L ４ , L ５ , L ６ , or L ７ H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ , or L ７ and Each X ３ are individually —H, —OH, —SH, or —NH ２ is] 124. The conjugate of any one of claims 104 to 123, wherein the drug moiety has formula (II):

125. R １ But C １ ~C ３ alkyl, and R ２ 125. The conjugate of claim 124, or a pharmaceutically acceptable salt thereof, wherein is a halogen.

126. R １ is methyl, and R ２ is F, or a pharmaceutically acceptable salt thereof.

127. R ３ and R ４ 127. The conjugate of any one of claims 124 to 126, or a pharmaceutically acceptable salt thereof, wherein one of: is hydrogen.

128. R ３ and R ４ or a pharmaceutically acceptable salt thereof.

129. R ３ and R ４ One of the groups is -N ３ 127. The conjugate of any one of claims 124 to 126, wherein:

130. R ３ and R ４ One of the groups is -NH ２ 127. The conjugate of any one of claims 124 to 126, wherein:

131. R ３ and R ４ one of which is —NH(C═O)CH ２ R ３Ｄ 127. The conjugate of any one of claims 124 to 126, wherein:

132. R ３ and R ４ the other of which is substituted or unsubstituted C １ ~C ６ 127. The conjugate of any one of claims 124 to 126, or a pharmaceutically acceptable salt thereof, wherein:

133. R ３ and R ４ The other of these is a substitution C １ ~C ６ 133. The conjugate of claim 132, or a pharmaceutically acceptable salt thereof, wherein:

134. Substitution C １ ~C ６ The alkyl is selected from —OH and —NR ３Ｂ R ３Ｃ and R ３Ｂ and R ３Ｃ are each independently hydrogen, substituted or unsubstituted C １ ~C ６ Alkyl or C(=O)(unsubstituted C １ ~C ６ 134. The conjugate of claim 133, or a pharmaceutically acceptable salt thereof, wherein:

135. R ３Ｂ and R ３Ｃ and R are each hydrogen, or a pharmaceutically acceptable salt thereof.

136. R ３Ｂ is hydrogen, and R ３Ｃ is a substituted or unsubstituted C １ ~C ６ 135. The conjugate of claim 134, or a pharmaceutically acceptable salt thereof, wherein:

137. R ３Ｂ is hydrogen, and R ３Ｃ is unsubstituted C １ ~C ６ 137. The conjugate of claim 136, or a pharmaceutically acceptable salt thereof, wherein:

138. R ３Ｂ and R ３Ｃ are each substituted or unsubstituted C １ ~C ６ 135. The conjugate of claim 134, or a pharmaceutically acceptable salt thereof, wherein:

139. R ３Ｂ and R ３Ｃ are unsubstituted C １ ~C ６ 139. The conjugate of claim 138, or a pharmaceutically acceptable salt thereof, wherein:

140. R ３Ｂ is hydrogen, and R ３Ｃ is -C(=O) (unsubstituted C １ ~C ６ 135. The conjugate of claim 134, or a pharmaceutically acceptable salt thereof, wherein:

141. Substitution C １ ~C ６ 141. The conjugate of any one of claims 133 to 140, or a pharmaceutically acceptable salt thereof, wherein the alkyl is substituted with one or more -OH groups.

142. Substitution C １ ~C ６ The alkyl is —CH ２ OH, -CH ２ CH ２ OH or -CH ２ CH(OH)CH ２ 142. The conjugate of claim 141, or a pharmaceutically acceptable salt thereof, wherein:

143. R ３ and R ４ The other of these is a substitution C ２ ~C ６ 132. The conjugate of any one of claims 124 to 131, or a pharmaceutically acceptable salt thereof, which is alkenyl.

144. R ３ and R ４ The other of these is unsubstituted C ２ ~C ６ 132. The conjugate of any one of claims 124 to 131, or a pharmaceutically acceptable salt thereof, which is alkenyl.

145. Formula (II) has the structure: 【Transformation 36】 wherein b is 1, 2, or 3.

125. The conjugate of claim 124, having the structure of formula (II-a):

146. Each R ３Ａ is -OH, or a pharmaceutically acceptable salt thereof.

147. 147. The conjugate of claim 145 or 146, or a pharmaceutically acceptable salt thereof, wherein b is 1.

148. 147. The conjugate of claim 145 or 146, or a pharmaceutically acceptable salt thereof, wherein b is 2.

149. 147. The conjugate of claim 145 or 146, or a pharmaceutically acceptable salt thereof, wherein b is 3.

150. R ３ and R ４ The other of the two is -[(CY ２ ) ｐ O (CY ２ ) ｑ ] ｔ 132. The conjugate of any one of claims 124 to 131, or a pharmaceutically acceptable salt thereof, wherein:

151. R ７ is H, or a pharmaceutically acceptable salt thereof.

152. The conjugate having the formula (III) 【Chemistry 37-1】 【Chemistry 37-2】 or a pharmaceutically acceptable salt thereof.

153. The conjugate having the formula (III) 【Chemistry 38-1】 【Chemistry 38-2】 or a pharmaceutically acceptable salt thereof.

154. Formula (III) is 【Chemistry 39-1】 【Chemistry 39-2】 【Chemistry 39-3】 【Chemistry 39-4】 【Chemistry 39-5】 【Chemistry 39-6】 【Chemistry 39-7】 【Chemistry 39-8】 【Chemistry 39-9】 【Chemistry 39-10】 【Chemistry 39-11】 【Chemistry 39-12】 【Chemistry 39-13】 or a pharmaceutically acceptable salt thereof.

155. A process for producing an immunoconjugate, comprising reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with a conjugate described in any one of claims 104 to 154 under reaction conditions effective to form the immunoconjugate described in any one of claims 1 to 58.

156. 156. The process of claim 155, further comprising reducing the antibody or antigen-binding fragment under reducing conditions effective to form a thiol-functionalized antibody or antigen-binding fragment.

157. Ab is, a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8; A VLCDR2 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of AAS; and VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10 light chain containing an antibody or antigen-binding fragment thereof comprising The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1); An immunoconjugate according to any one of claims 1 to 58, a pharmaceutical composition according to claim 99, a method according to claim 100 or 101, a use according to claim 102 or 103, or a process according to claim 155 or 156.

158. The immunoconjugate, pharmaceutical composition, method, use or process of claim 157, wherein the antibody or antigen-binding fragment comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 7, 12 or 14.

159. Ab is, a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17 and a heavy chain comprising b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 24 light chain containing an antibody or antigen-binding fragment thereof comprising The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1); An immunoconjugate according to any one of claims 1 to 58, a pharmaceutical composition according to claim 99, a method according to claim 100 or 101, a use according to claim 102 or 103, or a process according to claim 155 or 156.

160. 160. The immunoconjugate, pharmaceutical composition, method, use, or process of claim 159, wherein the antibody or antigen-binding fragment comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19, 21, 26, or 28.

161. Ab is, a) a VHCDR1 comprising an amino acid sequence having at least 95% sequence identity with 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 VHCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 31 and a heavy chain comprising b) a VLCDR1 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 36; a VLCDR2 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of DAS; and VLCDR3 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 38 light chain containing an antibody or antigen-binding fragment thereof comprising The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1); An immunoconjugate according to any one of claims 1 to 58, a pharmaceutical composition according to claim 99, a method according to claim 100 or 101, a use according to claim 102 or 103, or a process according to claim 155 or 156.

162. 162. The immunoconjugate, pharmaceutical composition, method, use or process of claim 161, wherein the antibody or antigen-binding fragment comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 33, 35, 40 or 42.

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