Immunoconjugates and methods

Abstract

The immunoconjugates of formula (I) include a linking group for linking an antibody targeting ligand (Ab) to a drug (D). Such immunoconjugate embodiments are useful for delivering drugs to selected cells or tissues, for example, for the treatment of cancer.

Description

[Technical Field]

[0001] Incorporation by reference of any priority application All applications for which a claim of foreign or domestic priority is identified in an Application Data Sheet filed with this application are expressly incorporated herein by reference under 37 CFR 1.57.

[0002] The present application relates to conjugates that include linking groups for linking antibody targeting ligands to cell-killing moieties (such as drugs), methods of making such conjugates, and methods of using such conjugates to deliver cell-killing moieties to selected cells or tissues, for example, for the treatment or inhibition of cancer. [Background technology]

[0003] Numerous antibody-drug conjugates (ADCs) have been developed for medical use. See, for example, Non-Patent Document 1. The antibody in an 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 U.S. Food and Drug Administration (FDA) has approved several ADC formulations, including inotuzumab ozogamicin (trade name BESPONSA®), gemtuzumab ozogamicin (trade name MYLOTARG®), brentuximab vedotin (trade name ADCETRIS®), and ado-trastuzumab emtansine (trade name KADCYLA®).

[0004] Patent Document 1 discloses an ADC in which an antitumor compound is conjugated to an anti-HER2 antibody via a linker. See also Patent Documents 2 and 3. Trastuzumab deruxtecan is an example of an ADC in which an anti-HER2 antibody (trastuzumab) is linked to an antitumor compound (deruxtecan) via a cleavable maleimide tetrapeptide linker. The FDA has approved a formulation known as fam-trastuzumab deruxtecan-nxki (trade name ENHERTU®) for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2-based regimens in the metastatic setting. Figure 1 shows how a linker may connect an antibody (mAb) to a drug moiety.

[0005] Although FDA approval represents a milestone in the ongoing development of therapeutic ADCs, there remains a need for improved ADCs that will help address the long-standing need for additional options for treating cancer and / or delivering therapeutic payloads to selected cells or tissues. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 10,155,821 [Patent Document 2] US Patent Application Publication No. 2020 / 0385486 [Patent Document 3] US Patent Application Publication No. 2019 / 0077880 [Non-patent literature]

[0007] [Non-Patent Document 1] Nejadmoghaddam, M. et al., “Antibody-Drug Conjugates: Possibilities and Challenges”, Avicenna J Med Biotech 11(1), 3-23 (2019).

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[0008] Some embodiments provide an immunoconjugate of Formula (I) comprising an antibody or antigen-binding fragment (Ab), a drug moiety (D), and a linker connecting the Ab to D. In one embodiment, the immunoconjugate of Formula (I) comprises a drug moiety of Formula (II).

[0009] One embodiment provides an immunoconjugate having formula (I): Ab-[SL 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n (I) During the ceremony, Ab is an antibody or antigen-binding fragment; L 1 teeth,

[0010] [ka]

[0011] and L 2 does not exist or

[0012] [ka]

[0013] and Z 1 and Z 2 are each independently hydrogen, halogen, NO, —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 an integer 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 from 0 to 6, n 3 is an integer between 0 and 2, L 6 does not exist, or

[0014] [ka]

[0015] and

[0016] [ka]

[0017] L 7 does not exist, D is a drug moiety, and n is an integer from 1 to 10.

[0018] In one embodiment, D in Formula (I) is a drug moiety of Formula (II) having the structure:

[0019] [ka]

[0020] During the ceremony, R 1 and R 2 are each independently hydrogen, halogen, -CN, or -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; 1 and R 2 together to form a ring, R 3 is hydrogen or substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t It's CY3, R 4 is hydrogen, substituted or unsubstituted -(C1-C6 alkyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted -(C1-C6 alkenyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 , substituted or unsubstituted -(C1-C6 alkynyl)-X 2 or substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 and X 1 -O-, -S(O n6 )-, -NH-, -O-(C=O)-, -NH-(C=O)-, -NH-(C=O)-O-, -NH(C=O)-NH-, or -NH-S(On6 )- and X 2 is OR 9 or -SR 9 , or -NHR 9 and R 5 and R 6 are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t It's CY3, m is 1 or 2; n 4 and n 5 are each independently 0, 1 or 2, provided that n 4 and n 5 and are never 0, n 6 is 0, 1 or 2, each Y is independently H or halogen; each p is individually 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 H, -COR 8 , -CO2R 8 , -(CO)-NHR8 , L 4 , L 5 , L 6 or L 7 where R 7 and R 9 is L 4 , L 5 , L 6 or L 7 and each X 3 is independently —H, —OH, —SH, or —NH 2 .

[0021] One embodiment provides a compound of formula (IV) having the structure:

[0022] [ka]

[0023] During the ceremony, R 1 and R 2 are each independently hydrogen, halogen, -CN, or -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; 1 and R 2 together to form a ring, R 3 is hydrogen or substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t It's CY3, R 4is hydrogen, substituted or unsubstituted -(C1-C6 alkyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted -(C1-C6 alkenyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 , substituted or unsubstituted -(C1-C6 alkynyl)-X 2 or substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 and X 1 -O-, -S(O n6 )-, -NH-, -O-(C=O)-, -NH-(C= O)-, -NH-(C=O)-O-, -NH(C=O)-NH-, or -NH-S(O n6 )- and X 2 is -OH, -SH, or -NR 5 R 6 and R 5 and R 6 are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t It's CY3, 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 0(CY2)q]tCY3, m is 1 or 2; n 4 and n 5 are each independently 0, 1 or 2, provided that n 4 and n 5 and are not both 0, n 6 is 0, 1 or 2, each Y is independently H or halogen; each p is individually 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, 2, 3, 4, 5, or 6; and each t is independently 1, 2, 3, 4, 5, or 6; However, formula (IV) does not represent deluktecan or exatecan.

[0024] 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.

[0025] 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.

[0026] One embodiment provides the use of an effective amount of an immunoconjugate described herein, a drug compound described herein, or a pharmaceutically active salt thereof, or a pharmaceutical composition described herein in the manufacture of a medicament for treating cancer or a tumor.

[0027] Some embodiments provide a conjugate of formula (III) comprising a functional group Mi, a drug moiety (D) and a linker connecting Mi to D. In one embodiment, the conjugate of formula (III) comprises a drug moiety of formula (II).

[0028] One embodiment provides a conjugate having formula (III): Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D (III) During the ceremony, Mi

[0029] [ka]

[0030] and L 2 does not exist or

[0031] [ka]

[0032] and Z 1 and Z 2 are each independently hydrogen, halogen, NO, —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 an integer 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 from 0 to 6, n 3 is an integer between 0 and 2, L 6 does not exist, or

[0033] [ka]

[0034] and

[0035] [ka]

[0036] and L 7 does not exist, D is a drug moiety.

[0037] One embodiment provides a process for producing an immunoconjugate comprising reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with a complex described herein under reaction conditions effective to form an immunoconjugate described herein.

[0038] Preferred alternatives include:

[0039] 1. An antibody or antigen-binding fragment thereof, a) VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1; An amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2 VHCDR 2, including columns, and VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, and b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8; VLCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of AAS, and VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain comprising Including, The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1). An antibody or antigen-binding fragment thereof.

[0040] 2. The antibody or antigen-binding fragment of Alternative 1, comprising 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.

[0041] 3. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1 or 2, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 6, 11, or 13.

[0042] 4. A host cell comprising one or more nucleic acids of alternative 3.

[0043] 5. An immunoconjugate according to any one of the preceding embodiments, wherein Ab is an antibody or antigen-binding fragment according to claim option 1 or 2.

[0044] 6. An antibody or antigen-binding fragment thereof, a) VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; VHCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, and VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, and b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22; VLCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAY; and VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24 and a light chain comprising Including, The antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1). An antibody or antigen-binding fragment thereof.

[0045] 7. The antibody or antigen-binding fragment of Alternative 6, comprising 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.

[0046] 8. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of any one of embodiments 6 or 7, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 18, 20, 25, or 27.

[0047] 9. A host cell comprising one or more nucleic acids of alternative 8.

[0048] 10. The immunoconjugate of any one of the preceding embodiments, wherein Ab is the antibody or antigen-binding fragment of embodiment 6 or 7.

[0049] 11. An antibody or antigen-binding fragment thereof, a) VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29; VHCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 30, and VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31, and b) VLCDR1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36; VLCDR2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAS; and VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38 and a light chain comprising Including, The antibody or antigen-binding fragment specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1). An antibody or antigen-binding fragment thereof.

[0050] 12. The antibody or antigen-binding fragment of Alternative 11, comprising 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.

[0051] 13. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of any one of embodiments 11 or 12, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 32, 34, 39, or 41.

[0052] 14. A host cell comprising one or more nucleic acids according to option 13.

[0053] 15. The immunoconjugate of any one of the preceding embodiments, wherein Ab is the antibody or antigen-binding fragment of embodiment 11 or 12.

[0054] 16. An antibody or binding fragment thereof, wherein the heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 6 and the light chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 13, or said antibody or binding fragment thereof A composition comprising the fragment.

[0055] 17. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 16, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID NO:7 and the light chain comprises the polypeptide sequence of SEQ ID NO:14.

[0056] 18. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to any one of alternatives 16 or 17, wherein said antibody or binding fragment thereof is conjugated to a molecule.

[0057] 19. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 18, wherein the molecule is a drug, a toxin, or a cytokine.

[0058] 20. An immunoconjugate according to any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to any one of alternatives 16 or 17.

[0059] 21. A method of using an antibody or binding fragment thereof according to any one of alternatives 16-19, or a composition comprising said antibody or binding fragment thereof, such as an immunoconjugate according to alternative 20, to inhibit or treat a disease, such as cancer, said method comprising administering the antibody or binding fragment thereof according to any one of alternatives 16-19 or said composition to a subject in need thereof, optionally selecting the subject to be treated for said disease, such as cancer, and / or optionally determining the inhibition of said disease, such as cancer, following administration of said antibody or binding fragment thereof.

[0060] 22. The antibody or binding fragment thereof of any one of Alternatives 16 to 19, or a composition comprising said antibody or binding fragment thereof, such as the immunoconjugate of Alternative 20, for use as a pharmaceutical, e.g. for the purpose of inhibiting or treating a disease such as cancer.

[0061] 23. An antibody or binding fragment thereof, wherein the heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 20 and the light chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 27, or a composition comprising said antibody or binding fragment thereof.

[0062] 24. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 23, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID NO: 21 and the light chain comprises the polypeptide sequence of SEQ ID NO: 28.

[0063] 25. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to any one of alternatives 23 or 24, wherein said antibody or binding fragment thereof is conjugated to a molecule.

[0064] 26. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 25, wherein the molecule is a drug, a toxin, or a cytokine.

[0065] 27. An immunoconjugate according to any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to either alternative 23 or 24.

[0066] 28. A method of using an antibody or binding fragment thereof according to any one of alternatives 23-26, or a composition comprising said antibody or binding fragment thereof, such as an immunoconjugate according to alternative 27, to inhibit or treat a disease such as cancer, comprising administering to a subject in need thereof an antibody or binding fragment thereof or said composition according to any one of claims alternatives 23-26. optionally selecting a subject to be treated for said disease, such as cancer, and / or optionally determining inhibition of said disease, such as cancer, after administration of said antibody or binding fragment thereof.

[0067] 29. The antibody or binding fragment thereof of any one of Alternatives 23 to 26, or a composition comprising said antibody or binding fragment thereof, such as the immunoconjugate of Alternative 27, for use as a medicament, for example for the purpose of inhibiting or treating a disease such as cancer.

[0068] 30. An antibody or binding fragment thereof, wherein the heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 34 and the light chain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 41, or a composition comprising said antibody or binding fragment thereof.

[0069] 31. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 30, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID NO: 35 and the light chain comprises the polypeptide sequence of SEQ ID NO: 42.

[0070] 32. The antibody or binding fragment thereof of any one of alternatives 30 or 31 or a composition comprising said antibody or binding fragment thereof, wherein said antibody or binding fragment thereof is conjugated to a molecule.

[0071] 33. The antibody or binding fragment thereof or a composition comprising said antibody or binding fragment thereof according to alternative 32, wherein the molecule is a drug, a toxin, or a cytokine.

[0072] 34. An immunoconjugate according to any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to any one of alternatives 30 or 31.

[0073] 35. A method of using an antibody or binding fragment thereof according to any one of alternatives 30-33, or a composition comprising said antibody or binding fragment thereof, such as an immunoconjugate according to alternative 34, to inhibit or treat a disease, such as cancer, said method comprising administering the antibody or binding fragment thereof according to any one of alternatives 30-33 or said composition to a subject in need thereof, optionally selecting the subject to be treated for said disease, such as cancer, and / or optionally determining the inhibition of said disease, such as cancer, following administration of said antibody or binding fragment thereof.

[0074] 36. An antibody or binding fragment thereof according to any one of alternatives 30 to 33, or a composition comprising said antibody or binding fragment thereof, such as the immunoconjugate of alternative 34, for use as a medicament, for example for the purpose of inhibiting or treating a disease such as cancer. [Brief explanation of the drawings]

[0075] [Figure 1] 1 shows a trastuzumab delactecan antibody-drug conjugate. [Figure 2] The reaction scheme for preparing a compound of formula (IV) is shown below. Under acidic or basic hydrolysis conditions, IV-2 is formed from IV1. Friedlander reaction of IV-2 and IV-3 gives IV-4. Removal of the protecting group (Pg) in IV-4 gives IV-5. Alkylation, esterification, or amidation of IV-5 gives IV. [Figure 3] The reaction scheme for preparing the compound of formula (IV-1a) is shown below. Heck reaction between IV-6 and IV-7 gives IV-8. Hydrogenation of IV-8 gives IV-9, which is then hydrolyzed to give IV-10. Intramolecular Friedel-Crafts reaction of IV-10 gives IV-11a. Alpha-alkylation of IV-11a gives IV-12, which is then treated under either alpha-hydroxylation or alpha-amination conditions to give IV-1a (when n5=0). [Figure 4] The reaction scheme for preparing the compound of formula (IV-1b) is shown below. Dehydrogenation of IV-11b gives IV-13. IV-13 undergoes a Michael reaction followed by dehydrogenation to give IV-14. IV-14 undergoes a Michael reaction using an oxygen- or nitrogen-containing nucleophile to give IV1b (when n5=1). [Figure 5] 1 shows a reaction scheme for preparing a conjugate of formula (III), which comprises attaching a linking moiety to a compound of formula (IV). Reaction of IIIA-1 and IIIA-2 under heating conditions gives IIIA-3. Reaction of IIIA-3 with N-hydroxysuccinimide gives IIIA-4. Reaction of IIIA-4 with IIIA-5 gives IIIA-6. Reaction of IIIA-6 with IIIA-7 under amide coupling conditions gives IIIA-8. [Figure 6] The reaction scheme for making IIIA-13 is shown: Reaction of IIIA-9 with N-hydroxysuccinimide (IIIA-10) gives IIIA-11, which reacts with IIIA-12 to give IIIA-13. [Figure 7]The reaction scheme for preparing IIIA-5 is shown. IIIA-14 is reacted with Pb(OAc) to give IIIA-15. IIIA-15 and IIIA-16 are treated in the presence of NaOH to give IIIA-17. IIIA-17 is treated with DBU to give IIIA-18, which is coupled with IIIA-19 to give IIIA-20. IIIA-20 is hydrogenated to give IIIA-5. [Figure 8] The reaction scheme for preparing IIIB-4 is shown. Reaction of IIIA-4 with IIIB-1 in the presence of base gives IIIB-2, which is reacted with 4-aminobenzyl alcohol and EEDQ to produce IIIB-3. Treatment of IIIB-3 with 4-nitrophenyl chloroformate gives IIIB-4. [Figure 9] The reaction scheme for making IIIB-13 is shown. IIIB-5 is reacted with DHP to produce IIIB-6. IIIB-6 is reacted with oxalyl chloride and catalytic DMF to produce IIIB-7. Reaction of IIIB-7 with IIIA-7 gives IIIB-8, which is reacted with 4-nitrophenyl chloroformate to give IIIB-9. Reaction of IIIB-9 and IIIB-10 in the presence of base gives IIIB-11. Treatment of IIIB-11 with TFA gives IIIB-12. Combination of IIIB-12 and IIIB-4 in the presence of base gives IIIB-13. [Figure 10] The reaction scheme for preparing IIIC-7 is shown. IIIC-1 is reacted with Pb(OAc) to generate IIIC-2, which is reacted with IIIC-3 in the presence of ZnCl to give IIIC-4. Treatment of IIIC-4 with DBU gives IIIC-5. Amide coupling of IIIC-5 and IIIA-3 gives IIIC-6, which is reacted with HF-pyridine to give IIIC-7. [Figure 11] 1 shows a reaction scheme for making an immunoconjugate of formula (I), which involves coupling an Ab to a conjugate of formula (III). Michael reaction of a cysteine ​​thiol group from an antibody with the maleimide in formula III produces formula I. [Figure 12] The reaction scheme for making compounds 1-14 is shown. [Figure 13] The reaction scheme for making compound 2-30 is shown. [Figure 14] The reaction scheme for making compounds 3-34 and 3-35 is shown. [Figure 15] The reaction scheme for making compounds 4-36 and 4-37 is shown. [Figure 16] The reaction scheme for making compounds 5-47 and 5-48 is shown. [Figure 17] The reaction scheme for making compounds 3-34 and 3-35 is shown. [Figure 18] 1 shows the reaction scheme for making compounds 7-59 and 7-60. [Figure 19] The reaction scheme for making compounds 8-71 and 8-72 is shown. [Figure 20] The reaction scheme for making compounds 9-74 is shown. [Figure 21] 1 shows the reaction scheme for making compound 11-80. [Figure 22] 1 shows the reaction scheme for making compounds 13-84 and 13-85. [Figure 23] 1 shows the reaction scheme for making compounds 14-91 and 14-92. [Figure 24] 1 shows the reaction scheme for making compound 15-94. [Figure 25-1] 1 shows the reaction scheme for making compounds 18-112. [Figure 25-2] 1 shows the reaction scheme for making compounds 18-112. [Figure 26] 1 shows the reaction scheme for making compounds 21-120. [Figure 27]Figure 1 shows measurement of binding saturation data for anti-ROR-1 antibodies generated by the methods described herein. The ROR-1-positive cell line JeKo-1 was incubated in a titration series with 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, and cell binding saturation was detected by flow cytometry and reported as mean fluorescence intensity (MFI). [Figure 28] ROR-1 receptor internalization data for the anti-ROR-1 antibodies ATX-875, ATX-P-885, and ATX-P-890 are shown. 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 washed and incubated at 37°C for four different time points (30 min, 1 h, 2 h, and 4 h), after which internalization was stopped by placing the cells on ice. Receptor internalization was determined by flow cytometry and reported as the percentage of receptor internalization relative to time 0. [Figure 29A]Figure 29 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 evaluate whether ATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. (29A) shows the staining profile for antibodies binding to the same epitope. (29B) shows the staining profile for 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 (29C) or 4A5 (29D) and analyzed by flow cytometry, comparing the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals with the UC961 or 4A5 signals. [Figure 29B] Figure 29 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 evaluate whether ATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. (29A) shows the staining profile for antibodies binding to the same epitope. (29B) shows the staining profile for 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 (29C) or 4A5 (29D) and analyzed by flow cytometry, comparing the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals with the UC961 or 4A5 signals. [Figure 29C]Figure 29 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 evaluate whether ATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. (29A) shows the staining profile for antibodies binding to the same epitope. (29B) shows the staining profile for 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 (29C) or 4A5 (29D) and analyzed by flow cytometry, comparing the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals with the UC961 or 4A5 signals. [Figure 29D] Figure 29 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 evaluate whether ATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A5. (29A) shows the staining profile for antibodies binding to the same epitope. (29B) shows the staining profile for 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 (29C) or 4A5 (29D) and analyzed by flow cytometry, comparing the ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals with the UC961 or 4A5 signals. [Figure 30]AC-SINS data are shown 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 potential self-interactions. Rituximab and infliximab were used as controls to demonstrate low and high shifts, respectively. Assay results for ATX-P-875, ATX-P-885, and ATX-P-890 fell within the range determined by the control antibodies. [Figure 31] 1 shows biochemical binning data by SPR for anti-ROR1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 compared to control anti-ROR-1 antibodies UC961 (ATX-P-453) and 4a5. [Figure 32] Linker and payload combinations conjugated to three unique antibodies are shown (mAb A = ATX-P-875, mAb B = ATX-P-885, mAb C = ATX-P-890). Antibodies, mAb A (ATX-P-875), mAb B (ATX-P-885), and mAb C (ATX-P-890), were conjugated to six distinct novel linkers / payloads (18-112, 19-113, 20-114, 21-120, 22-121, and 23-122) to establish antibody-drug conjugates (ADCs). [Figure 33] A CTG assay is shown in which ROR+ (JeKo-1) cells were incubated with ADCs generated as described in Figure 32 at serial 3-fold dilutions and cell viability was assessed after 72 hours. [Figure 34-1] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-2] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-3] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-4] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-5] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-6] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-7] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-8] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. [Figure 34-9] The nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0076] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless otherwise stated. In the event that there are multiple definitions for a term herein, those in this section prevail unless stated otherwise.

[0077] 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) linked together by a chemical bond. Examples of conjugates include antibody-drug conjugates (which may optionally include a linker moiety), drug-linker conjugates, and antibody-linker conjugates. An "immunoconjugate" is a conjugate comprising an immunological substance such as an antibody.

[0078] As used herein, an "antibody" (Ab) is a protein produced by the immune system that binds to a specific site on a cell or tissue, or a synthetic variant thereof. 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 therapy, monoclonal antibodies can kill cancer cells directly, they can block the development of tumor blood vessels, or they can help the immune system kill cancer cells.

[0079] Whenever a group is described as being "optionally substituted," the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as being "unsubstituted or substituted," if substituted, the substituents may be selected from one or more of the indicated substituents. If no substituents are indicated, it means that the indicated "optionally substituted" or "substituted" group may 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, monosubstituted amine group, disubstituted amine group, monosubstituted amine(alkyl) group, and disubstituted amine(alkyl).

[0080] As used herein, "C" refers to a group of integers where "a" and "b" are integers. a ~C b " refers to the number of carbon atoms in the group. The designated group can contain from "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(CH 3)-, and (CH3)3C-. When "a" and "b" are not specified, the broadest range described by these definitions is to be assumed.

[0081] When two "R" groups are described as being "together," the R groups and the atoms to which they are attached can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic ring. For example, but not limited to, ortho R groups on a phenyl ring 1 and R 2 The substituent is -O-(CR 5 R 6 ) m -Os and R 1 and R 2 When "combined" to form a ring, -O-(CR 5 R 6 ) m -O is R 1 and R 2 It is covalently bonded to a phenyl ring at the : position to form a heterocycle.

[0082] [ka]

[0083] As used herein, the term "alkyl" refers to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight-chain. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, sec-butyl, t-butyl, and the like. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and the like. The alkyl group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as "1 to 30" refers to each integer within the given range; for example, "1 to 30 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 30 carbon atoms; this definition also extends to the appearance 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 requires otherwise. For example, one skilled in the art would recognize that a C1-C6 alkyl group can be represented by the following formula: -(C1-C6 alkyl)-X 2 We recognize that it is bivalent in

[0084] 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:

[0085] [ka]

[0086] Next comes the number of carbon atoms, followed by " * For example,

[0087] [ka]

[0088] represents ethylene. The alkylene group may have 1 to 30 carbon atoms (wherever it appears herein, a numerical range such as "1 to 30" refers to each integer within the given range, e.g., "1 to 30 carbon atoms" means that the alkyl group may have 1 carbon atom, 2 carbon atoms, or (This means that the alkylene group may consist of 1 to 3 carbon atoms, 3 carbon atoms, etc., and may consist of up to 30 carbon atoms, but this definition also extends to the occurrence of the term "alkylene" where no numerical range is specified.) An alkylene group may also be a medium-sized alkyl having 1 to 12 carbon atoms. An alkylene group may also be a lower alkyl having 1 to 4 carbon atoms. An alkylene group may be substituted or unsubstituted. For example, a lower alkylene group may be a C3-6 monocyclic cycloalkyl group (e.g.,

[0089] [ka]

[0090] ) by replacing one or more hydrogens on a lower alkylene group and / or by replacing both hydrogens on the same carbon.

[0091] As used herein, the term "alkenyl" refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon double bond, 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.

[0092] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon triple bond, including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl, etc. Alkynyl groups can be unsubstituted or substituted.

[0093] As used herein, the term "halogen atom" or "halogen" means any one of the radiostable atoms in column 7 of the periodic table of the elements, such as fluorine, chlorine, bromine, and iodine.

[0094] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen (e.g., monohaloalkyl, dihaloalkyl, trihaloalkyl, 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.

[0095] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms is replaced by a halogen (eg, mono-haloalkyl, di-haloalkyl, tri-haloalkyl, and polyhaloalkyl).

[0096] As used herein, "haloalkynyl" refers to an alkynyl group in which one or more of the hydrogen atoms has been replaced with a halogen (eg, mono-haloalkynyl, di-haloalkynyl, tri-haloalkynyl and polyhaloalkynyl).

[0097] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more of the hydrogen atoms has been replaced by a halogen (e.g., mono-haloalkoxy, di-haloalkoxy, and tri-haloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. Haloalkoxy can be substituted or unsubstituted.

[0098] Where the number of substituents is not specified (e.g., haloalkyl, haloalkenyl, haloalkynyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may include one or more of the same or different alkoxy groups containing 1, 2, or 3 atoms.

[0099] As used herein, a radical refers to a species having a single unpaired electron such that the radical-containing species can be covalently bonded to another species. Thus, in this context, a radical is not necessarily a free radical. Rather, a radical refers to a specific portion of a larger molecule. The term "radical" may be used interchangeably with the term "group."

[0100] 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 abolish 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 (such as 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. With respect to 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., NH becomes NH3). + ion), the positive charge can be a negatively charged counterion (Cl - Understand that balance can be achieved by

[0101] 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 compounds, enantiomerically enriched compounds, racemic mixtures, diastereomerically pure compounds, diastereomerically enriched compounds, or stereoisomeric mixtures. Additionally, in any compound described herein having one or more double bonds that produce geometric isomers that can 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 in any compound described, all tautomeric forms are also intended to be included.

[0102] Where the compounds disclosed herein have unfilled valences, it is understood that the valences are filled with hydrogen or an isotope thereof, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0103] It is understood that the compounds described herein can be isotopically labeled. Substitution with isotopes such as deuterium can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Each chemical element may include any isotope of that element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position in the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, reference to a compound herein encompasses all possible isotopic forms unless the context clearly dictates otherwise.

[0104] 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, etc. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and may be formed during the crystallization process with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0105] When a range of values ​​is provided, it is understood that the upper and lower limits, and every intervening value between the upper and lower limits of that range, are encompassed within an embodiment.

[0106] Terms and phrases used in this application, and variations thereof, particularly in the appended claims, should be construed as open-ended rather than limiting, unless expressly stated. As an example above, the term "including" should be construed to mean "including without limitation," "including but not limited to," etc. As used herein, the term "comprising" is synonymous with "including," "containing," or "featuring" and is inclusive or open-ended, not excluding additional, unrecited elements or method steps. The term "having" should be construed as "having at least." The term "including" should be construed as "including, but not limited to." The term "example" is used to provide illustrative examples rather than an exhaustive or exclusive list of items under discussion. The use of terms such as "preferably," "preferred," "desired," or "desirable," and words of similar import, should not be understood as implying that a particular feature is critical, essential, or even important to its structure or function, but rather is intended merely to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Additionally, 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 recited features or components, but may include additional features or components.

[0107] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate depending on 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 used to advantage. Any reference signs in the claims should not be construed as limiting the scope thereof.

[0108] compound Various embodiments disclosed herein relate to compounds of formula (IV) having the following structure, or a pharmaceutically acceptable salt thereof:

[0109] [ka]

[0110] In various embodiments, R in formula (IV) 1 and R 2 are each independently hydrogen, halogen, -CN, or -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-, and R 1 and R 2 are taken together to form a ring, and in some embodiments, R 1 and R 2 At least one of R is hydrogen. 1 and R 2At least one of R is halogen. For example, in some embodiments, 1 and R 2 At least one of R is fluoro. 1 and R 2 At least one of R is -CN. 1 and R 2 At least one of -OR 5 where 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 is methoxy.

[0111] In one embodiment, R of formula (IV) 1 and R 2 At least one of the following is -NR 5 R 6 where R 5 and R 6 are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3. In one embodiment, R 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 1 and R 2 At least one of R is methyl. 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 At least one of R is difluoromethyl. 1 and R 2At least one of R is substituted or unsubstituted —O—(C1-C6 alkyl). For example, in one embodiment, R 1 and R 2 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 R 1 and R 2 together, -O-(CR 5 R 6 ) m The end of -O is R of formula (IV) 1 and R 2 The heterocyclic ring is formed by covalently bonding the phenyl ring at the aryl position to form a heterocyclic ring.

[0112] In one embodiment, R in formula (IV) 1 and R 2 is hydrogen and R 1 and R 2 The other of R is halogen. 1 and R 2 One of R may be hydrogen; 1 and R 2 The other of R may be substituted or unsubstituted C1-C6 alkyl. 1 and R 2 One of the is hydrogen and R 1 and R 2 The other of R is unsubstituted C1-C6 haloalkyl. 1 and R 2 One of the hydrogen, R 1 and R 2 The other of R is unsubstituted —O—(C1-C6 alkyl. In one embodiment, R 1 and R 2 and R are hydrogen.1 MoR 2 is not hydrogen either.

[0113] In one embodiment, R of formula (IV) 1 and R 2 One of the groups is a halogen, and R 1 and R 2 and the other is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 One of the is hydrogen and R 1 and R 2 and the other of R is unsubstituted C1-C6 haloalkyl. 1 and R 2 One of the groups is a halogen, and R 1 and R 2 and the other is substituted or unsubstituted —O—(C1-C6 alkyl). In one embodiment, R 1 and R 2 and R are independently halogen. 1 MoR 2 It's not a halogen either.

[0114] In one embodiment, R in formula (IV) 1 and R 2 is a substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other is a substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 is a substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 alkyl. 1 MoR 2 is not a substituted or unsubstituted C1-C6 alkyl.

[0115] In one embodiment, R in formula (IV) 1 and R 2is a substituted or unsubstituted C1-C6 haloalkyl, and R 1 and R 2 and the other is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 haloalkyl. 1 MoR 2 is not a substituted or unsubstituted C1-C6 haloalkyl.

[0116] In one embodiment, R in 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 MoR 2 In one embodiment, R 1 and R 2 is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O and 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; R 1 and R 2 together form a ring.

[0117] In various embodiments, R in formula (IV) 3 is hydrogen or substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3, wherein each Y is independently H or halogen. In one embodiment, R 3 is hydrogen. In some embodiments, R3 is a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3 is methyl. In some embodiments, R 3 is a substituted or unsubstituted C1-C6 haloalkyl. 3 is -[(CY2) p O(CY2) q ] t CY3, where each Y is independently H or halogen.

[0118] In various embodiments, R in formula (IV) 4 is hydrogen, substituted or unsubstituted -(C1-C6 alkyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted -(C1-C6 alkenyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 , substituted or unsubstituted -(C1-C6 alkynyl)-X 2 or substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 and X 2 is -OH, -SH or -NR 5 R 6 In one embodiment, R 4 is hydrogen. In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 alkyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 alkenyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 alkynyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2is.

[0119] In various embodiments, X in formula (IV) 1 is -O-, -S(On6)-, -NH-, -O-(C=O)-, -NH-(C=O)-, -NH-(C=O)-O-, -NH-(C=O)-NH- or -NH-S(On6)-, wherein n 6 is 0, 1 or 2. In one embodiment, X 1 is —O—. In one embodiment, X 1 -S(O n6 In one embodiment, X 1 is -NH-. In one embodiment, X 1 is —O—(C═O)—. In one embodiment, X 1 is —NH—(C═O)—. In one embodiment, X 1 is —NH—(C═O)—O—. In one embodiment, X 1 is —NH—(C═O)—NH—. In one embodiment, X 1 is -NH-S(O n6 )-.

[0120] In various embodiments, in formula (IV), X 2 is -OH, -SH, or -NR 5 R 6 where R 5 and R 6 are each independently hydrogen, halogen, or substituted or unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, -[(CY2) p O(CY2) q ] t CY3. In one embodiment, X 2 is —OH. In one embodiment, X 2 is -SH. In one embodiment, X 2 is -NR 5 R 6 is.

[0121] In various embodiments, R in formula (IV) 5 and R 6are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3, where each Y is independently H or halogen, and the variables p, q, and t are as described elsewhere herein. 5 and R 6 are each independently hydrogen or substituted or unsubstituted C1-C6 alkyl. 5 and R 6 are both hydrogen. In one embodiment, R 5 and R 6 are each independently substituted or unsubstituted C1 to C6 alkyl.

[0122] In various embodiments, in formula (IV), R 7 H, -COR 8 , -CO2R 8 or -(CO)-NHR 8 where R 8 is described elsewhere herein. In one embodiment, R 7 is H. In one embodiment, R 7 HA-COR 8 In one embodiment, R 7 Ha-CO2R 8 In one embodiment, R 7 is -(CO)-NHR 8 is.

[0123] 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 described elsewhere herein. 8 is a substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 8is a substituted or unsubstituted C1-C6 haloalkyl. 8 is -[(CY2) p O(CY2) q ] t It's CY3.

[0124] In various embodiments, m in formula (IV) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

[0125] In various embodiments, n in formula (IV) 4 and n 5 are each independently 0, 1 or 2, provided that n 4 and n 5 and are not both 0. In one embodiment, n 4 and n 5 are both 1. In one embodiment, n 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.

[0126] In various embodiments, n in formula (IV) 6 is 0, 1 or 2. In one embodiment, n 6 is 0, in this case, X 1 is -S or -NH-S-. In one embodiment, n 6 is 1, in this case, X 1 is -S(=O)- or -NH-S(=O)-. In one embodiment, n 6 is 2, in this case, X 1 is -S(=O) 2- Or -NH-S(=O)2-.

[0127] 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, -CH2F is CH3. In one embodiment, -CY3 is CF3.

[0128] In various embodiments, each p in formula (IV) is individually 1, 2, 3, 4, 5, or 6. In one embodiment, p is 1. In one embodiment, p is 2.

[0129] 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.

[0130] In various embodiments, each t in formula (IV) is individually 1, 2, 3, 4, 5, or 6. In one embodiment, t is 1. In one embodiment, p is t.

[0131] In various embodiments, formula (IV) does not represent deruxtecan or exatecan.

[0132] In various embodiments, the compound of formula (IV) is represented by formula (IVa):

[0133] [ka]

[0134] In formula (IVa), the variables are the same as defined elsewhere herein for formula (IV).

[0135] In various embodiments, the compound of formula (IV) is represented by formula (IVb):

[0136] [ka]

[0137] In formula (IVb), the variables are the same as defined elsewhere herein for formula (IV).

[0138] In various embodiments, the compound of formula (IV) is represented by formula (IVc):

[0139] [ka]

[0140] In formula (IVc), the variables are the same as defined elsewhere herein for formula (IV).

[0141] In various embodiments, the compound of formula (IV) is represented by a structure selected from the following, or a pharmaceutically acceptable salt thereof:

[0142] [ka]

[0143] [ka]

[0144] [ka]

[0145] [ka]

[0146] Conjugates Various embodiments disclosed herein relate to a conjugate of formula (III) having the following structure: Mi-L 2 -L 3 -L 4 -L 5 -L6 -L 7 -D (III)

[0147] In various embodiments, Mi in formula (III) is

[0148] [ka]

[0149] where D is a drug moiety and -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - is the linker connecting Mi to D.

[0150] In various embodiments, L in formula (III) 2 does not exist or

[0151] [ka]

[0152] wherein Z 1 and Z 2 are each independently hydrogen, halogen, NO2, —O—(C1-C6 alkyl), or C1-C6 alkyl. 2 In one embodiment, L in formula (III) is absent. 2 teeth,

[0153] [ka]

[0154] In one embodiment, in formula (III), L 2 teeth,

[0155] [ka]

[0156] is.

[0157] 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 is -O-(C1-C6 alkyl). For example, in one embodiment, Z 1 and Z 2 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.

[0158] In various embodiments, in formula (III), L 3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-, where n 1 are independently an integer 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 3is —CHC(═O)—. In embodiments, n 1 is an integer of 1 to 12, for example, 1 to 6 or 1 to 3.

[0159] In various embodiments, L in formula (III) 4 is a tetrapeptide residue. For example, in one embodiment, L 4 is a tetrapeptide selected from GGFG (gly-gly-phe-gly), EGGF (glu-gly-gly-phe), SGGF (ser-gly-gly-phe), and KGGF (lys-gly-gly-phe).

[0160] In various embodiments, L in formula (III) 5 is absent or -[NH(CH2)n 2 ]n 3 - in which 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 not present. In one embodiment, L 5 is -[NH(CH2)n 2 ]n 3 For example, in one embodiment, L 5 is -NH-. In another embodiment, L 5 is -NHCH2-.

[0161] In various embodiments, L in formula (III) 6 does not exist, or

[0162] [ka]

[0163] In one embodiment, L 6 is absent. In another embodiment, L 6 teeth,

[0164] [ka]

[0165] is.

[0166] In various embodiments, L7 in formula (III) is absent,

[0167] [ka]

[0168] In one embodiment, L 7 In one embodiment, L 7 teeth,

[0169] [ka]

[0170] In one embodiment, L 7 teeth,

[0171] [ka]

[0172] In one embodiment, L 7 teeth,

[0173] [ka]

[0174] In one embodiment, L 7 teeth,

[0175] [ka]

[0176] In various embodiments, D in the conjugate of formula (III) is a drug moiety as described herein, e.g., below under the heading "Drug Moiety." In one embodiment, D is a cytotoxic anticancer drug moiety.

[0177] In various embodiments, the conjugate of formula (III) is represented by a structure selected from the following: 1 and Z 2 are each independently selected from hydrogen, fluoro, chloro, —NO 2 , and —OCH 3 .

[0178] [ka]

[0179] [ka]

[0180] [ka]

[0181] [ka]

[0182] [ka]

[0183] Drug portion In various embodiments, the immunoconjugate of formula (I) or the conjugate of formula (III) D in the group is a drug moiety. The drug moiety is connected to the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7The compound may be any compound of formula (IV) described herein (e.g., described above under the heading "Compounds") appropriately modified so that - is attached to D. For example, in various embodiments, the drug moiety D is a compound of formula (II) having the following structure:

[0184] [ka]

[0185] Those skilled in the art will appreciate that compounds of formula (II) may be 2 -L 3 -L 4 -L 5 -L 6 -L 7 -R 4 via (X 2 and therefore R 9 ), or R as follows: 7 It will be understood that the connection is via

[0186] In various embodiments, R in formula (II) 1 and R 2 are each independently hydrogen, halogen, -CN, or -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-, and R 1 and R 2 are taken together to form a ring, and in some embodiments, R 1 and R 2 At least one of R is hydrogen. 1 and R 2 At least one of R is halogen. For example, in some embodiments,1 and R 2 At least one of R is fluoro. 1 and R 2 At least one of R is -CN. 1 and R 2 At least one of -OR 5 where 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 is methoxy.

[0187] In one embodiment, R of formula (II) 1 and R 2 At least one of the following is -NR 5 R 6 where R 5 and R 6 are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3. In one embodiment, R 1 and R 2 At least one of R is a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 1 and R 2 At least one of R is methyl. 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 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 R2 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 R 1 and R 2 together, -O-(CR 5 R 6 ) m The end of -O is R of formula (IV) 1 and R 2 The heterocyclic ring is formed by covalently bonding the phenyl ring at the aryl position to form a heterocyclic ring.

[0188] In one embodiment, R in formula (II) 1 and R 2 is hydrogen and R 1 and R 2 The other of R is halogen. 1 and R 2 One of R may be hydrogen; 1 and R 2 The other of R may be substituted or unsubstituted C1-C6 alkyl. 1 and R 2 One of the is hydrogen and R 1 and R 2 The other of R is unsubstituted C1-C6 haloalkyl. 1 and R 2 One of the is hydrogen and R 1 and R 2 The other of R is unsubstituted —O—(C1-C6 alkyl. In one embodiment, R 1 and R 2 and R are hydrogen. 1 MoR 2 is not hydrogen either.

[0189] In one embodiment, R of formula (II) 1 and R 2 One of the groups is a halogen, and R 1 and R 2 and the other is substituted or unsubstituted C1-C6 alkyl. 1 and R 2 One of the groups is a halogen, and R 1 and R 2 and the other of R is unsubstituted C1-C6 haloalkyl. 1 and R 2 One of the groups is a halogen, and R 1 and R 2 and the other is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently halogen. 1 MoR 2 It's not a halogen either.

[0190] In one embodiment, R in formula (II) 1 and R 2 is a substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other is a substituted or unsubstituted C1-C6 haloalkyl. 1 and R 2 is a substituted or unsubstituted C1-C6 alkyl, and R 1 and R 2 and the other is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 alkyl. 1 MoR 2 is not a substituted or unsubstituted C1-C6 alkyl.

[0191] In one embodiment, R in formula (II) 1 and R 2 is a substituted or unsubstituted C1-C6 haloalkyl, and R 1 and R 2and the other is substituted or unsubstituted —O—(C1-C6 alkyl). 1 and R 2 and R are independently substituted or unsubstituted C1-C6 haloalkyl. 1 MoR 2 is not a substituted or unsubstituted C1-C6 haloalkyl.

[0192] In one embodiment, R in 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 MoR 2 In one embodiment, R 1 and R 2 is a substituted or unsubstituted -O-(CR 5 R 6 ) m -O and 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; R 1 and R 2 together form a ring.

[0193] In various embodiments, R in formula (II) 3 is hydrogen or substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3, wherein each Y is independently H or halogen. In one embodiment, R 3 is hydrogen. In some embodiments, R 3 is a substituted or unsubstituted C1-C6 alkyl. For example, in one embodiment, R 3is methyl. In some embodiments, R 3 is a substituted or unsubstituted C1-C6 haloalkyl. 3 is -[(CY2) p O(CY2) q ] t CY3, where each Y is independently H or halogen.

[0194] In various embodiments, R in formula (II) 4 is hydrogen, substituted or unsubstituted -(C1-C6 alkyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 , substituted or unsubstituted - (C1-C6 alkenyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 , substituted or unsubstituted -(C1-C6 alkynyl)-X 2 , substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 where X 2 -OR 9 -SR 9 or -NHR 9 and R 9 H, non-existent, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 or L 7 In one embodiment, R 4 is hydrogen, in this case, X 2 is absent and compounds of formula (II) contain R 7 via linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -Bind to.

[0195] In other embodiments, the compound of formula (II) is R 4 is X 2 Contains R 9L 4 , L 5 , L 6 or L 7 If R 4 via linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 In some embodiments, R 4 is a substituted or unsubstituted -(C1-C6 alkyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 alkenyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkenyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 alkynyl)-X 2 In one embodiment, R 4 is a substituted or unsubstituted -(C1-C6 haloalkynyl)-X 2 R 4 is X 2 In each such embodiment, R 9 L 4 , L 5 , L 6 or L 7 Therefore, R 4 The compound of formula (II) is linked to a linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -You are given the option to connect to

[0196] In various embodiments, X in formula (II) 1is -O-, -S(On6)-, -NH-, -O-(C=O)-, -NH-(C=O)-, -NH-(C=O)-O-, -NH-(C=O)-NH- or -NH-S(On6)-, wherein n 6 is 0, 1 or 2. In one embodiment, X 1 is —O—. In one embodiment, X 1 -S(O n6 In one embodiment, X 1 is -NH-. In one embodiment, X 1 is —O—(C═O)—. In one embodiment, X 1 is —NH—(C═O)—. In one embodiment, X 1 is —NH—(C═O)—O—. In one embodiment, X 1 is —NH—(C═O)—NH—. In one embodiment, X 1 is -NH-S(O n6 )-.

[0197] In various embodiments, R in formula (II) 5 and R 6 are each independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, or -[(CY2) p O(CY2) q ] t CY3, where each Y is independently H or halogen, and the variables p, q, and t are as described elsewhere herein. 5 and R 6 are each independently hydrogen or substituted or unsubstituted C1-C6 alkyl. 5 and R 6 are both hydrogen. In one embodiment, R 5 and R 6 are each independently substituted or unsubstituted C1 to C6 alkyl.

[0198] In various embodiments, in formula (II), R 7 H, -COR 8 , -CO2R 8 , -(CO)-NHR8 , L 4 , L 5 , L 6 or L 7 where each R 8 are independently 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 HA-COR 8 In one embodiment, R 7 Ha-CO2R 8 In one embodiment, R 7 is -(CO)-NHR 8 Those skilled in the art will recognize that R 7 H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 If the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - is bonded to the compound of formula (II) by R 4 You will understand that through

[0199] In various embodiments, each R in formula (II) 8 are each a substituted or unsubstituted C1-C6 alkyl-X 3 , substituted or unsubstituted C1-C6 haloalkyl-X 3 or -[(CY2) P O(CY2) t CY2-X 3 where X 3 is -H, -OH, -SH, or -NH2 In one embodiment, each R 8 are individually substituted or unsubstituted C1-C6 alkyl-X 3 In one embodiment, each R8 are independently substituted or unsubstituted C1-C6 haloalkyl-X 3 In one embodiment, each R 8 are independently -[(CY2) p O(CY2)q] t CY2-X 3 is.

[0200] In various embodiments, X in formula (II) 2 -OR 9 , -SR 9 , or -NHR 9 where 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.

[0201] In various embodiments, in formula (II), R 9 H, -COR 8 , -CO2R 8 , -(CO)-NHR 8 , L 4 , L 5 , L 6 or L 7 where 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 H, -COR 8 , -CO2R 8 , or -(CO)-NHR 8 If the linker-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 - is bonded to the compound of formula (II) by R 7 You will understand that through

[0202] 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 L 4 , L 5 , L 6 , or L 7 when the linker -L of the compound of formula (II) is 2 -L 3 -L 4 -L 5 -L 6 -L 7 The linkage to - is R 4 In one embodiment, R 7 and R 9 Exactly one of the 4 , L 5 , L 6 , or L 7where a single 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.

[0203] In various embodiments, each X in formula (II) 3 are individually -H, -OH, -SH, or -NH. 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.

[0204] In various embodiments, m in formula (II) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

[0205] In various embodiments, n in formula (II) 4 and n 5 are each independently 0, 1 or 2, provided that n 4 and n 5 and are not both 0. In one embodiment, n 4 and n 5 are both 1. In one embodiment, n 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.

[0206] In various embodiments, n in formula (II) 6 is 0, 1 or 2. In one embodiment, n 6 is 0, in this case, X 1 is -S or -NH-S-. In one embodiment, n6 is 1, in this case, X 1 is -S(=O)- or -NH-S(=O)-. In one embodiment, n 6 is 2, in this case, X 1 is -S(=O) 2- Or -NH-S(=O)2-.

[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, In one embodiment, -CY3 is CH3. In one embodiment, -CY3 is CHF2. In one embodiment, -CH2F is CH3. In one embodiment, -CY3 is CF3.

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

[0211] In various embodiments, formula (II) does not represent deruxtecan or exatecan.

[0212] Immunoconjugates Various embodiments disclosed herein relate to immunoconjugates of formula (I) having the following structure: Ab-[SL 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7-D] n (I)

[0213] In various embodiments, L in formula (III) 1 is L 1 is

[0214] [ka]

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

[0216] [ka]

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

[0218] [ka]

[0219] In one embodiment, L in formula (III) 2 teeth,

[0220] [ka]

[0221] is.

[0222] In various embodiments, Z in formula (III) 1 and Z 2are 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 is -O-(C1-C6 alkyl). For example, in one embodiment, Z 1 and Z 2 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.

[0223] In various embodiments, in formula (III), L 3 is -(CH2)n 1 -C(=O)- or -(CH2CH2O)n 1 -(CH2)n 1 C(=O)-, where n 1 are independently an integer 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 embodiments, n 1 is an integer of 1 to 12, for example, 1 to 6 or 1 to 3.

[0224] In various embodiments, L in formula (III) 4is a tetrapeptide residue. For example, in one embodiment, L 4 is a tetrapeptide selected from GGFG (gly-gly-phe-gly), EGGF (glu-gly-gly-phe), SGGF (ser-gly-gly-phe), and KGGF (lys-gly-gly-phe).

[0225] In various embodiments, L in formula (III) 5 is absent or -[NH(CH2)n 2 ]n 3 - in which 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 not present. In one embodiment, L 5 is -[NH(CH2)n 2 ]n 3 For example, in one embodiment, L 5 is -NH-. In another embodiment, L 5 is -NHCH2-.

[0226] In various embodiments, L in formula (III) 6 does not exist, or

[0227] [ka]

[0228] In one embodiment, L 6 is absent. In another embodiment, L 6 teeth,

[0229] [ka]

[0230] is.

[0231] In various embodiments, L in formula (III) 7 does not exist or

[0232] [ka]

[0233] In one embodiment, L 7 is not present. In one embodiment, L 7 teeth,

[0234] [ka]

[0235] In one embodiment, L 7 teeth,

[0236] [ka]

[0237] In one embodiment, L 7 teeth,

[0238] [ka]

[0239] In one embodiment, L 7 teeth,

[0240] [ka]

[0241] is.

[0242] In various embodiments, D in the immunoconjugate of Formula (I) is a drug moiety as described herein (e.g., under the heading "Drug Moieties" above). In one embodiment, D is a cytotoxic anticancer drug moiety. In one embodiment, the drug moiety is exatecan.

[0243] In various embodiments, Ab in 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). In one embodiment, the Ab binds to the surface of a cancer cell. In one embodiment, the Ab is an anti-HER2 antibody.

[0244] In various embodiments, the immunoconjugate of Formula (I) is represented by a structure selected from the following: Z 1 and Z 2 are each independently selected from hydrogen, fluoro, chloro, —NO 2 , and —OCH 3 .

[0245] [ka]

[0246] [ka]

[0247] [ka]

[0248] Pharmaceutical Composition Some embodiments described herein relate to pharmaceutical compositions that can include an effective amount of one or more compounds described herein (e.g., an immunoconjugate compound of Formula (I), a drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0249] 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 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 generally tailored to the specific intended route of administration.

[0250] The term "physiologically acceptable" refers to a carrier that does not neutralize the biological activity and properties of the compound or cause substantial damage or injury to the animal to which the composition is intended to be delivered. Define a carrier, diluent, or excipient.

[0251] As used herein, "carrier" refers to a compound that facilitates the incorporation of a compound into cells or tissues. For example, and without limitation, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.

[0252] As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that has no apparent pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to bulk a potent drug whose mass is too small for manufacture and / or administration. It may 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 a buffered aqueous solution, such as, without limitation, phosphate-buffered saline, which mimics the pH and isotonicity of human blood.

[0253] 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, consistency, stability, binding ability, lubrication, disintegration ability, 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.

[0254] The pharmaceutical compositions described herein can be administered to human patients either by themselves or in pharmaceutical compositions in which they are mixed with other active ingredients, such as in combination therapy, or with carriers, diluents, excipients, or combinations thereof. The appropriate formulation depends on the selected route of administration. Techniques for formulating and administering the compounds described herein are known to those skilled in the art.

[0255] The pharmaceutical compositions disclosed herein can be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, dragee-making, elutriating, emulsifying, encapsulating, entrapping, or tabletting processes. Additionally, 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 may be provided as salts with pharmaceutically compatible counterions.

[0256] Multiple techniques of administering compounds, salts, and / or compositions exist in the art, including, but not limited to, oral, rectal, intrapulmonary, topical, aerosol, injection, infusion, and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be administered orally.

[0257] The compounds, salts, and / or compositions may also be administered in a local rather than systemic manner, for example, by injecting or implanting the compound directly into the affected area, often as a depot or sustained-release formulation. Additionally, the compounds can be administered to specific cell or tissue types in targeted drug delivery systems, for example, in liposomes coated with a targeting ligand. The liposomes are targeted and taken up selectively by the targeted cells or tissues.

[0258] The composition may be provided in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, 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 have a notice associated with the container in a format prescribed by government agencies regulating the manufacture, use, or sale of pharmaceuticals. The label may be accompanied by notice reflecting approval by an agency of the drug form for human or animal administration. Such notice may be, for example, 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 with a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0259] Uses and Treatment Methods Some embodiments described herein relate to methods of treating cancer or tumors described herein, 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 a cancer or tumor described herein. Still other embodiments described herein relate to 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 an effective amount to treat a cancer or tumor described herein.

[0260] Examples of cancers and tumors include, but are not limited to, lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0261] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and particularly 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 particularly 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.

[0262] As used herein, the terms "treat," "treating," "treatment," "therapeutic," and "therapy" do not necessarily imply a complete cure or elimination of a disease or condition. Any alleviation, to any extent, of any undesirable signs or symptoms of a disease or condition can be considered treatment and / or therapy. Moreover, treatment can include actions that may worsen a subject's overall feeling of health or appearance.

[0263] The terms "therapeutically effective amount" and "effective amount" are used to refer to an amount of an active compound or pharmaceutical agent that elicits a described 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 can occur in a tissue, system, animal, or human, and includes alleviation of the signs or symptoms of the disease or condition being treated. An effective amount Such a determination 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 will depend on the route of administration, the type of animal, such as a human, being treated, and the physical characteristics of the particular animal under consideration. Dosages can be adjusted to achieve the desired effect and will depend on factors such as body weight, diet, concurrent medications, and other factors that one skilled in the medical field would recognize.

[0264] For example, an effective amount of a compound is an amount that results in (a) reduction, alleviation, or elimination of one or more symptoms caused by cancer, (b) reduction in tumor size, (c) elimination of the tumor, and / or (d) long-term disease stabilization (growth inhibition) of the tumor. In the treatment of lung cancer (such as non-small cell lung cancer), a therapeutically effective amount is an amount that reduces or eliminates cough, shortness of breath, and / or pain.

[0265] The amount of immunoconjugate compound of Formula (I), drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof required for therapeutic use will vary depending not only on the particular compound or salt selected, but also on 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 is ultimately at the discretion of the attending physician or clinician. 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 that exceed, or even far exceed, the dosage ranges set forth herein to effectively and aggressively treat, particularly advanced diseases or conditions.

[0266] Generally, however, suitable doses will often be within the range of about 0.05 mg / kg to about 10 mg / kg. For example, suitable doses may be within the range of about 0.10 mg to about 7.5 mg per kg of body weight per day, e.g., about 0.15 mg to about 5.0 mg per kg of recipient body weight per day, about 0.2 mg to 4.0 mg per kg of recipient body weight per day, or any amount therebetween. The compound may be administered in unit dosage form, which may contain, for example, 1 to 500 mg, 10 to 100 mg, 5 to 50 mg, or any amount therebetween of active ingredient per unit dosage form.

[0267] The desired dose may conveniently be presented in a single dose or as 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.

[0268] As will be readily apparent to those skilled in the art, the useful in vivo dosage and specific administration method will vary depending on the age, weight, severity of the affliction, the mammalian species being treated, the specific compound used, and the specific application for which these compounds are used. Determination of effective dosage levels, i.e., the dosage levels necessary to achieve the desired results, can be achieved by those skilled in the art using routine methods, such as human clinical trials, in vivo studies, and in vitro studies. For example, useful dosages of the immunoconjugate compound of Formula (I), the drug compound of Formula (IV), or a pharmaceutically acceptable salt thereof, can be determined by comparing their in vitro and in vivo activity in animal models. Such comparisons can be made by comparison with established drugs such as cisplatin and / or gemcitabine.

[0269] Dosage amount and interval may be adjusted individually to provide plasma concentrations sufficient to maintain the active moiety's modulatory effect or minimal effective concentration (MEC). The MEC varies for each compound but can be estimated from in vivo and / or in vitro data. The dosage required to achieve the MEC will depend 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 most preferably 50-90%. In cases of local administration or selective uptake, the effective local concentration of drug may not be related to plasma concentration.

[0270] It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity). The magnitude of the dose administered in the management of the disease of interest will vary with the severity of the disease or condition to be treated and the route of administration. The severity of the disease or condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps dosing frequency, will also vary with the age, weight, and response of the individual patient. Programs equivalent to those discussed above can be used in veterinary medicine.

[0271] 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 certain chemical moiety can be established by evaluating in vitro toxicity on cell lines, such as mammalian cell lines, preferably human cell lines. The results of such studies often predict toxicity in animals, such as mammals, or particularly 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 for determining efficacy, those skilled in the art can use the state of the art as a guide to select the appropriate model, dose, route of administration, and / or regimen.

[0272] synthesis The drug compounds of formula (IV), or pharmaceutically acceptable salts thereof, can be made in a variety of ways by those skilled in the art using known techniques 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 2-4.

[0273] 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 5-10. While exemplified 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.

[0274] 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 11. In one embodiment, a method for making an immunoconjugate described herein comprises reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with a conjugate 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]

[0275] Further embodiments, which in no way limit the scope of the claims, are disclosed in more detail in the examples below.

[0276] Example 1 N-((1S,8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-14) (Figure 12)

[0277] [ka]

[0278] 1-Bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2): A mixture of 2-fluoro-1-methyl-4-nitrobenzene (1-1) (25.0 g, 322 mmol, 1.0 equiv) and HSO (250 mL) in heptane (250 mL) was heated at 70 °C. Then, N-bromosuccinimide (68.84 g, 386.78 mmol, 1.2 equiv) was added in small portions to the above mixture at 70 °C. The resulting red suspension was stirred at 70 °C for 15 h. TLC (petroleum ether / ethyl acetate = 10 / 1, R f =0.6) indicated the formation of a new major spot. The reaction mixture was poured into ice water (1 L) and extracted with ethyl acetate (3 × 500 mL). The combined organic phases were washed with brine (500 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with petroleum ether to give 1-bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2) (17.0 g, 20% yield). 1 H NMR (400MHz, CDCl3) δppm8.27(t,J=1.79Hz,1H),7.89(dd,J=8.76,2.21Hz,1H),2.44(d,J=2.50Hz,3H).

[0279] 3-Bromo-5-fluoro-4-methylaniline (1-3): To a solution of bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2) (15.0 g, 64.1 mmol, 1.0 equiv.) in ethanol (750 mL) and water (150 mL) was added iron powder (10.7 g, 192 mmol, 3.0 equiv.) and NH4Cl (6.86 g, 128 mmol, 2.0 equiv.). The suspension was stirred at 80 °C for 2 h. TLC (petroleum ether / ethyl acetate = 10 / 1, R f=0.3) indicated the formation of a new major spot. After cooling to 25 °C, the reaction mixture was filtered through a pad of Celite and washed with ethanol (500 mL). The combined filtrates were concentrated to dryness, and the residue was purified by column chromatography on silica gel eluting with 9% ethyl acetate in petroleum ether to give 3-bromo-5-fluoro-4-methylaniline (1-3) (8.0 g, 55% yield). 1 H NMR (400MHz, CDCl3) δppm6.67-6.73(m,1H), 6.34(dd,J=10.97,2.27Hz,1H),3.65(brs,2H),2.20(d,J=2.15Hz,3H). 19 F NMR (400MHz, CDCl3) δppm-111.86.

[0280] N-(3-bromo-5-fluoro-4-methylphenyl)acetamide 1-4): 3-Bromo-5-fluoro-4-methylaniline (1- To a mixture of 3) (8.00 g, 39.2 mmol, 1.0 equiv.), triethylamine (8.13 g, 80.4 mmol, 11.2 mL, 2.05 equiv.) and acetic anhydride (5.20 g, 51.0 mmol, 4.77 mL, 1.3 equiv.) were added. The reaction mixture was stirred at 25° C. for 3 h. TLC (petroleum ether / ethyl acetate = 3 / 1, R f =0.25) indicated the formation of a new major spot. The reaction mixture was quenched with water and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (8.8 g, 91% yield). 1 H NMR (400MHz, CD3OD) δppm7.59(d,J=1.53Hz,1H),7.41(dd,J=11.55,2.02Hz,1H),2.26(d,J=2.20Hz,3H),2.11(s,3H).

[0281] tert-Butyl (E)-3-(5-acetamido-3-fluoro-2-methylphenyl)acylate (1-5): To an orange solution of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (8.00 g, 32.5 mmol, 1.0 equiv.) and tert-butyl acrylate (4.58 g, 35.8 mmol, 5.19 mL, 1.1 equiv.) in dioxane (100 mL), N-cyclohexyl-N-methylcyclohexanamine (6.99 g, 35.8 mmol, 7.58 mL, 1.1 equiv.) and Pd(t-BuP) (831 mg, 1.63 mmol, 0.05 equiv.) were added. The reaction mixture was stirred at 100 °C under a nitrogen atmosphere for 16 h. LCMS (retention time = 0.797 min) indicated the formation of the desired product. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (200 mL), dried over NaSO, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel eluting with 16% ethyl acetate in petroleum ether to give tert-butyl (E-3-(5-acetamido-3-fluoro-2-methylphenyl)acylate (1-5) (10.0 g, 94% yield). 1 H NMR(400MHz,DMSO-D6)δppm10.06(s,1H),7.72(d,J=15.77Hz,1H),7.61(s,1H),7.51(dd,J=1 1.98,1.47Hz,1H),6.22(d,J=15.77Hz,1H),2.20(d,J=1.59Hz,3H),2.04(s,3H)1.49(s,9H). 19 F NMR (400MHz, CDCl3) δppm-115.02. LCMS(ESI+)m / z:[MH] + C 16 H 21 FNO3 + Calculated value: 294.1, measured value: 294.0.

[0282] tert-Butyl 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6): To a solution of tert-butyl (E-3-(5-acetamido-3-fluoro-2-methylphenyl)acrylate (1-5) (2.80 g, 9.55 mmol, 1.0 equiv.) in dichloromethane (20 mL) and methanol (20 mL) was added Pd / C (10 wt%) (1.01 g, 0.1 equiv.) under an argon atmosphere. The suspension was degassed under vacuum and purged with H2 three times. The mixture was stirred under H2 (15 psi) at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 3 / 1, R f = 0.2) indicated the formation of a new major spot. After replacing the H atmosphere with argon, it was filtered through a pad of Celite, and the filter cake was washed with methanol (100 mL). The combined filtrate was concentrated under reduced pressure to give tert-butyl 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6) (2.6 g, 83% yield). 1 H NMR(400MHz,CD3OD)δppm7.33(dd,J=11.80,2.02Hz,1H),7.06(s,1H),2.88(t,J=7. 64Hz, 2H), 2.49 (t, J = 7.70Hz, 2H), 2.17 (d, J = 1.96Hz, 3H), 2.10 (s, 3H), 1.41 (s, 9H).

[0283] 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoic acid (1-7): To a solution of tert-butyl 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6) (2.60 g, 8.80 mmol, 1.0 equiv.) in dichloromethane (30 mL) was added trifluoroacetic acid (10 mL) at 25° C. The reaction mixture was stirred at 25° C. for 12 hours. TLC (petroleum ether / ethyl acetate=3 / 1, R f =0.01) indicated the formation of a new major spot. The reaction mixture was concentrated to give 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoic acid 1-7 (2.1 g, 89% yield). 1H NMR(400MHz,CD3OD)δppm7.35(dd,J=11.86,1.83Hz,1H),7.04(s,1H),2.91(t ,J=7.76Hz,2H),2.55(t,J=7.83Hz,2H),2.17(d,J=1.83Hz,3H),2.10(s,3H). 19 F NMR (400MHz, CDCl3) δppm -117.33, -77.77.

[0284] N-(6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8): To a solution of 3-(5-acetamido-3-fluoro-2-methyl-phenyl)propanoic acid (1-7) (2.10 g, 8.78 mmol, 1.0 equiv.) in trifluoroacetic acid (7 mL) was added trifluoroacetic anhydride (7.37 g, 35.1 mmol, 4.88 mL, 4.0 equiv.). The reaction mixture was stirred at 60 °C for 15 h. LCMS indicated the formation of the desired product. The reaction mixture was diluted with 50% acetonitrile / HO solution (100 mL), and the pH was adjusted to approximately 7 by adding 25% aqueous NaOH at 0 °C. The mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give N-(6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8) (1.5 g, 69% yield). 1 H NMR (400MHz, CD3OD) δppm8.02(d,J=12.57Hz,1H),3.01-3.08(m,2H),2.72-2.79(m,2H),2.18-2.23(m,6H). LCMS(ESI+)m / z:[MH] + C 12 H 13 FNO2 + Calculated value: 222.1, measured value: 222.0.

[0285] N-(6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-9): A solution of potassium tert-butoxide (1.19 g, 10.58 mmol, 1.3 equiv.) in tetrahydrofuran (12 mL), ethanol (2.4 mL), and butanol (2.4 mL) was stirred at 0° C. for 0.5 h. Isoamyl nitrite (1.43 g, 12.2 mmol, 1.64 mL, 1.5 equiv.) and N-(6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8) (1.80 g, 8.14 mmol, 1.0 equiv.) were added to the above mixture. The reaction mixture was stirred at 20° C. for 3 h. TLC (petroleum ether / ethyl acetate=1 / 1, R f =0.4) indicated the formation of a new major spot. The resulting red suspension was cooled to 0 °C, quenched with 1 N hydrochloric acid solution (50 mL), and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give N-(6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-9) (2.0 g, 88% yield). 1 H NMR (400MHz, DMSO-D6) δppm 12.81 (s, 1H), 10.33 (s, 1H), 8.03 (d, J = 12.57Hz, 1H), 3.71 (s, 2H), 2.20 (s, 3H), 2.16 (s, 3H).

[0286] N-(2-amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide hydrochloride (1-10): To a solution of N-(6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-9) (2.00 g, 6.98 mmol, 1 equiv.) in methanol (100 mL) was added Pd / C (10 wt%) (1.48 g, 0.2 equiv.) and hydrochloric acid (12 M, 1.74 mL, 3 equiv.) under a nitrogen atmosphere. The suspension was degassed under vacuum and purged with H2 three times. The mixture was stirred under H2 (15 psi) at 20 °C for 3 h. TLC (petroleum ether / ethyl acetate = 1 / 1, R f= 0) indicated the formation of a new major spot. After replacing the H atmosphere with nitrogen, it was filtered through a pad of Celite and washed with methanol (200 mL). The combined filtrate was concentrated to dryness to give N-(2-amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide hydrochloride (1-10) (1.5 g, 71% yield). This material was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6)δppm9.99(s,1H),8.02(d,J=12.59Hz,1H),4.33(brs,1H),3.98(brs,2 H),3.50(dd,J=16.99,8.19Hz,1H),3.00(dd,J=17.18,4.46Hz,1H),2.21(s,3H),2.18(s,3H). 19 F NMR (400MHz, CDCl3) δppm-102.81.

[0287] N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (I-11) To a mixture of N-(2-amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide hydrochloride (1-10) (1.50 g, 6.35 mmol, 1.0 equiv.) in dichloromethane (45 mL), triethylamine (1.93 g, 19.0 mmol, 2.65 mL, 3.0 equiv.) and acetic anhydride (778 mg, 7.62 mmol, 0.714 mL, 1.2 equiv.) were added. The reaction mixture was stirred at 25° C. for 2 hours. TLC (petroleum ether / ethyl acetate=1 / 1, R f =0.1) indicated the formation of a new major spot. The reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (3 x 50 mL). The combined organic layers were washed with 50 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue.

[0288] The gum was dissolved in HCl / MeOH (20 mL, 4 M) and stirred at 25° C. for 2 h. LCMS showed the formation of the desired product. The resulting red suspension was concentrated to give a red residue. The residue was diluted with methanol (2 mL) and purified by preparative HPLC (column: Phenomenex® luna C 18 80 * 40mm * Purification by elution with 3 μm; mobile phase: [water (HCl)-ACN], B%: 28%-48%, 7 min) gave N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (1-11) (0.70 g, 47% yield). 1 H NMR(400MHz,CD3OD)δppm6.52(d,J=11.56Hz,1H),4.48(dd,J=8.23,5.01Hz,1H),3.44(dd, J=17.05,8.23Hz,1H),2.83(dd,J=17.11,4.95Hz,1H),2.11(d,J=1.07Hz,3H),2.02(s,3H). 19 F NMR (400MHz, CDCl3) δppm-107.29. 13 C NMR(100MHz,CD3OD):201.9,172.1,167.9,165.4,153.8,153.7,143.1,143.0,117.0,112.6,112.4,101.6,101.3,55.5,31.8,20.8,8.0. LCMS(ESI+)m / z:[MH] + C 12 H 14 FN2O2 + Calculated value: 237.1, measured value: 237.0.

[0289] (RN-[7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide (1-12-P1) and (SN-[7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide (1-12-P1) [amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide (1-12-P2): N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (1-11) (0.70 g, 2.96 mmol) was dissolved in MeOH, neutralized with NHOH, and separated by chiral SFC to give (RN-7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (1-12-P1). Compound 1-12-P1 was shown. (S)-N-7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide (1-12-P2) (compound 1-12-P2 may be the opposite enantiomer of that shown) (200 mg, 28% yield) and (S)-N-7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide (1-12-P2) (compound 1-12-P2 may be the opposite enantiomer of that shown) (180 mg, 26% yield). Note: Stereochemistry is arbitrarily assigned. SFC separation method: Column: DAICEL CHIRALPAK AD (250 mm * 30mm, 10um); Mobile phase: [0.1%NH3H2O IPA], B%: 30%-30%, 12 min, SFC (1-12-P1, RT = 3.012 min) and SFC (1-12-P2, RT = 3.270 min).

[0290] Spectra for 1-12-P1: 1 H NMR(400MHz,CD3OD)δ ppm 2.01(s,3H)2.05(s,3H)2.74(dd,J=16.81,5.01Hz,1H)3.40(br d,J=8.70Hz,1H)4.50(dd,J=8.23,5.01Hz,1H)6.26(d,J=12.40Hz,1H). 19 F NMR (400MHz, CDCl3) δppm-108.05. LCMS(ESI+)m / z:[MH] + C 12 H 14 Calculated FN2O2: 237.10, Measured: 237.0.

[0291] Spectra for 12-P2: 1 H NMR (400 MHz, CD3OD) δ ppm 2.01(s,3H)2.05(s,3H)2.74(dd,J=17.11,4.95Hz,1H)3.40(br d,J=8.82Hz,1H)4.50(dd,J=8.29,5.07Hz,1H)6.26(d,J=12.40Hz,1H). 19 F NMR (400MHz, CDCl3) δppm-108.03. LCMS(ESI+)m / z:[MH] + C 12 H 14 Calculated FN2O2: 237.10, Measured: 237.1.

[0292] N-((1S,8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-14) A mixture of N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (1-11) (50 mg, 0.21 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (111.4 mg, 0.42 mmol), and TsOH (36.2 mg, 0.21 mmol) in xylene (2 mL) was heated at 140 °C for 12 h. All volatiles were removed under high vacuum, and the residue was first subjected to chromatography followed by chiral SFC separation to give 1-14a and 1-14b. LCMS (ESI+) m / z: [M−H] + C 25 H 23 FN3O5: 464.2, Measured: 464.3.

[0293] Example 2 N-((1S,9S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15 hexahydro-12H-cyclopenta[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (2-30) (Figure 13)

[0294] [ka]

[0295] 2-Bromo-6-methoxy-4-nitrophenol (2-16): To a solution of 2-methoxy-4-nitrophenol (2-15) (50.0 g, 296 mmol, 1.0 equiv.) in glacial acetic acid (500 mL), bromine (52.0 g, 325 mmol, 16.8 mL, 1.1 equiv.) was slowly added using a dropping funnel at 20 °C. The resulting mixture was stirred at 20 °C for 2 h. TLC (petroleum ether:ethyl acetate = 2:1) showed that the starting material was consumed and a major spot with lower polarity was formed. The reaction mixture was slowly poured into water (1.5 L) with stirring, and the resulting mixture was stirred at 20 °C for an additional 10 min. The mixture was filtered and concentrated to dryness under reduced pressure. The residue was triturated with water (2 × 500 mL), and the resulting product was collected by filtration and dried in a vacuum oven to give 2-bromo-6-methoxy-4-nitrophenol (2-16) (60.0 g, 73% yield). 1 H NMR (400MHz, CDCl3): δppm8.13(d,J=2.4Hz,1H),7.74(d,J=2.4Hz,1H),6.61(s,1H),4.03(s,3H). LCMS(ESI-)m / z:[MH] - C7H5BrNO4 - Calculated value: 245.9, Measured value: 245.9.

[0296] 3-Bromo-5-nitrobenzene-1,2-diol 2-17: To a solution of 2-bromo-6-methoxy-4-nitrophenol (2-16) (30.0 g, 121 mmol, 1 equiv.) in CHCl (1.5 L) was added boron tribromide (45.4 g, 181 mmol, 17.5 mL, 1.5 equiv.) at 0 °C. The reaction mixture was warmed to 30 °C and stirred for 15 h. TLC (petroleum ether:ethyl acetate = 1:1) showed that the starting material was consumed and a new major spot with higher polarity was formed. The reaction mixture was quenched with methanol (200 mL) and concentrated under reduced pressure to give 3-bromo-5-nitrobenzene-1,2-diol (2-17) (26.9 g, 95% yield). 1 H NMR (400MHz, DMSO-D6): δppm 10.94 (s, 2H), 7.87 (d, J = 2.8 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H).

[0297] 4-Bromo-6-nitro-1,3-benzodioxole (2-18): To a solution of 3-bromo-5-nitrobenzene-1,2-diol 2-17 (30.0 g, 128 mmol, 1.0 equiv.) in N,N-dimethylformamide (1 L) was added CsCO (125 g, 385 mmol, 3 equiv.) and diiodomethane (54.9 g, 205 mmol, 16.6 mL, 1.6 equiv.) at 20 °C. The reaction mixture was stirred at 100 °C for 12 h. TLC (petroleum ether:ethyl acetate = 3:1) showed that the starting material was consumed and a new major spot with lower polarity was formed. The reaction mixture was cooled to 20 °C, poured into ice water (1.5 L), and extracted with ethyl acetate (2 × 1.5 L). The combined organic layers were washed with brine (2 × 1 L), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a brown oil. The oil was triturated with toluene (20 mL) and the resulting product was collected by filtration and dried in a vacuum oven to give 4-bromo-6-nitro-1,3-benzodioxole (2-18) (17 g, 48% yield). 1 H NMR (400MHz, CDCl3): δppm8.06(d,J=2.0Hz,1H),7.63(d,J=2.4Hz,1H),6.23(s,2H).

[0298] 7-Bromobenzo[d][1,3]dioxol-5-amine (2-19): To a solution of 4-bromo-6-nitro-1,3-benzodioxole (2-18) (10.0 g, 40.6 mmol, 1.0 equiv.) in ethanol (500 mL) and water (100 mL) was added iron (6.81 g, 122 mmol, 3.0 equiv.) and NH4Cl (4.35 g, 81.3 mmol, 2.0 equiv.) at 20 °C. The mixture was stirred at 80 °C for 2 h. TLC (petroleum ether:ethyl acetate = 1:1) showed that the starting material was consumed and a new major spot with lower polarity was formed. After cooling to 20 °C, the reaction mixture was filtered through a pad of Celite and washed with ethanol (500 mL). The filtrate was concentrated under reduced pressure, and crushed ice was added. The resulting product was collected by filtration, washed with water, and dried in a vacuum oven to give 7-bromobenzo[d][1,3]dioxol-5-amine (2-19) (7.5 g, 76% yield). 1 H NMR (400MHz, CDCl3): δppm 6.29 (s, 1H), 6.21 (s, 1H), 5.94 (s, 2H), 3.51 (s, 2H).

[0299] N-(7-bromobenzo[d[1,3]dioxol-5-yl)acetamide (2-20): To a solution of 7-bromo-1,3-benzodioxol-5-amine (2-19) (6.00 g, 27.78 mmol, 1.0 equiv.) in ethyl acetate (50 mL) was added acetic anhydride (3.40 g, 33.33 mmol, 3.12 mL, 1.2 equiv.) and triethylamine (8.43 g, 83.3 mmol, 11.6 mL, 3.0 equiv.) at 20 °C, and the resulting mixture was stirred for 10 h. TLC (ethyl acetate:methanol = 4:1) showed that the starting material was consumed and a new major spot with higher polarity was formed. The reaction mixture was quenched at 0 °C by the addition of saturated NaHCO (50 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (eluted with petroleum ether / ethyl acetate = 1 / 3 to 1 / 4) to give N-(7-bromobenzo[d][1,3]dioxol-5-yl)acetamide (2-20) (5.5 g, yield 69%). 1 H NMR (400MHz, CDCl3): δppm7.19(s,1H),7.13(d,J=1.6Hz,1H),7.02(d,J=2.0Hz,1H),6.02(s,2H),2.15(s,3H). LCMS(ESI+)m / z:[MH] + C9H9BrNO3 + Calculated value: 258.0, measured value: 257.9.

[0300] tert-Butyl (E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21): To a solution of N-7-bromo-1,3-benzodioxol-5-yl(acetamide 2-20) (2.00 g, 7.75 mmol, 1.0 equiv.) and tert-butyl acrylate (1.09 g, 8.52 mmol, 1.24 mL, 1.1 equiv.) in dioxane (20 mL), N-cyclohexyl-N-methyl-cyclohexanamine (1.67 g, 8.52 mmol, 1.81 mL, 1.1 equiv.) and bis(tri-tert-butylphosphine)palladium(0) (198 mg, 0.387 mmol, 0.05 equiv.) were added under a nitrogen atmosphere. The mixture was stirred at 100 °C for 5 h. TLC (petroleum ether:ethyl acetate = 2:1, R f =0.4) and LCMS showed that the starting material had been consumed and the desired product had formed. After cooling to room temperature, the reaction mixture was filtered, and the filtrate was concentrated to give the product, which was then triturated with ethyl acetate (40 mL) at 20 °C for 10 min, then filtered, and the filtrate was concentrated under reduced pressure to give tert-butyl (E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21) (2.0 g, 76% yield). 1 H NMR(400MHz,CDCl3)δppm7.44(d,J=16.09Hz,1H)7.23(d,J=1.91Hz,1H)7.05(brs,1H )6.86(d,J=1.91Hz,1H)6.55(d,J=15.97Hz,1H)6.06(s,2H)2.17(s,3H)1.53(s,9H). LCMS(ESI+)m / z:[M-56] + C 16 H 20 NO5-C4H9( i Bu)+H ]+ Calculated value: 249.1, measured value: 249.9.

[0301] tert-Butyl 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22): To a suspension of Pd / C (2.90 g, 2.46 mmol, 10% purity, 0.5 equiv.) in MeOH (20 mL) was added a solution of tert-butyl (E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21) (1.50 g, 4.91 mmol, 1.0 equiv.) in MeOH (20 mL) at 20° C. The mixture was hydrogenated under a hydrogen atmosphere at 15 psi at 20° C. for 2 hours. TLC (petroleum ether / ethyl acetate=1 / 1, R f =0.48) indicated that the starting material had been consumed and a new spot had formed. After replacing the H atmosphere with nitrogen, the reaction mixture was filtered, and the filtrate was concentrated to give tert-butyl 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22) (1.4 g, 83% yield), which was used in the next step without further purification. 1 H NMR(400MHz,CDCl3)δppm7.09(d,J=1.96Hz,1H),6.98(brs,1H),6.64(s,1H),5. 94(s,2H),2.83(t,J=7.76Hz,2H),2.51-2.59(m,2H),2.14(s,3H),1.43(s,9H). LCMS(ESI+)m / z:[M+Na] + C 16 H 21 NO5Na + Calculated value: 330.1, Measured value: 330.1.

[0302] 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoic acid (2-23): To a solution of tert-butyl 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22) (1.40 g, 4.56 mmol, 1.0 equiv) in CHCl (15 mL) was added TFA (3 mL) at 20 °C, and the mixture was stirred for 2 h. TLC (petroleum ether:ethyl acetate = 0:1, R f= 0.48), indicating that the starting material had been consumed and a new spot had formed. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluting with petroleum ether / ethyl acetate = 1 / 0 to 1 / 9) to give 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoic acid (2-23) (0.85 g, 71% yield). 1 H NMR (400MHz, CD3OD) δppm7.08(d,J=1.91Hz,1H),6.76(d,J=1.79Hz,1H),5.93(s,2H),2.80-2.88(m,2H),2.57-2.65(m,2H),2.07(s,3H). LCMS(ESI+)m / z:[MH] + C 12 H 14 No. 5 + Calculated value: 252.1, measured value: 251.9.

[0303] N-(6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-24): To a solution of 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoic acid (2-23) (16.0 g, 63.7 mmol, 1.0 equiv.) in TFA (64 mL) was added TFAA (53.5 g, 255 mmol, 35.4 mL, 4 equiv.) dropwise at 20 °C. The resulting solution was heated to 60 °C for 10 h. TLC (petroleum ether:ethyl acetate = 2:1, R f =0.40) and LCMS indicated the reaction was complete. After cooling to room temperature, the reaction mixture was poured into a solution of acetonitrile (100 mL) and water (100 mL). After cooling to 0 °C, the pH of the mixture was adjusted to 7 with 25% aqueous sodium hydroxide (150 mL), and water (100 mL) was added. The resulting mixture was extracted with ethyl acetate (500 mL x 3). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was triturated with ethyl acetate (100 mL), filtered, and the filter cake was collected to give N-(6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-24) (10.4 g, 59% yield).1 H NMR (400MHz, DMSO-D6) δppm 10.47 (s, 1H), 7.85 (s, 1H), 6.15 (s, 2H), 2.84-3.04 (m, 2H), 2.60-2.74 (m, 2H), 2.17 (s, 3H). LCMS(ESI+)m / z:[MH] + C 12 H 12 No. 4 + :234.1, Actual value:234.0.

[0304] N-(7-hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-25): A solution of potassium tert-butoxide (1.0 M, 45.0 mL, 2.1 equiv.) in THF (41.6 mL), EtOH (6.7 mL), and n-BuOH (6.7 mL) was stirred at 0° C. for 30 minutes. Isopentyl nitrite (3.01 g, 25.7 mmol, 3.46 mL, 1.2 equiv.) and N-(6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-24) (5.00 g, 21.4 mmol, 1.0 equiv.) were added to the above solution at 0° C. The resulting mixture was stirred at 20° C. for 3 hours. LCMS indicated the formation of the desired product. The reaction mixture was quenched with 1.0 N hydrochloric acid (200 mL), and ethyl acetate (200 mL) was added. The resulting white precipitate was collected by filtration. The filtrate was extracted with ethyl acetate (3 × 500 mL), and the combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting product was combined with the filter cake to give N-(7-hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-25) (5.6 g, 99% yield). 1 H NMR(400MHz,DMSO-D6)δ ppm 12.71(s,1H),10.53(s,1H),7.89(s,1H),6.19(s,2H),3.63(s,2H),2.17(s,3H).LCMS(ESI+)m / z:[MH] + C 12 H 11 N2O5 +Calculated value: 263.1, measured value: 263.0.

[0305] N-(7-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide hydrochloride (2-26): To a mixture of N-(7-hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-25) (550 mg, 2.10 mmol, 1.0 equiv.) and HCl (12 M, 0.26 mL, 1.5 equiv.) in MeOH (50 mL), Pd / C (10 wt %) (300 mg) was added. The reaction mixture was purged with hydrogen gas three times and stirred under a hydrogen atmosphere of 15 psi at 20 °C for 2 h. LCMS indicated the reaction was complete. After replacing the H atmosphere with nitrogen, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to give N-(7-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide hydrochloride (2-26) (500 mg, 83% yield). This was used in the next step without further purification. 1 H NMR(400MHz,DMSO-D6)δppm10.09(brs,1H),9.05(brs,2H),8.82(brs,2H),7.8 5(s,1H),6.23-6.70(m,2H),3.98-4.39(m,2H),3.35-3.48(m,1H),2.16(s,3H). LCMS(ESI+)m / z:[MH] + C 12 H 13 N2O4 + Calculated value: 249.1, measured value: 249.0.

[0306] N-(7-acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-27): To a solution of N-(7-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide hydrochloride (2-26) (4.50 g, 15.8 mmol, 1.0 equiv.) in DCM (150 mL) was added TEA (4.80 g, 47.4 mmol, 6.60 mL, 3.0 equiv.) and acetic anhydride (1.94 g, 19.0 mmol, 1.78 mL, 1.2 equiv.) at 20 °C, and the mixture was stirred for 3 h. TLC (ethyl acetate, R f =0.40) and LCMS indicated the reaction was complete. The mixture was diluted with DCM (500 mL) and washed with water (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1 to 0 / 1, eluted with 5% THF) to give N-(7-acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-27) (2.55 g, 50% yield). 1 H NMR(400MHz,DMSO-D6)δppm10.35(s,1H),8.53(d,J=7.51Hz,1H),7.87(s,1H),6.13-6.19(m,2H) ,4.30-4.37(m,1H),3.17-3.32(m,1H),2.79(dd,J=16.75,5.07Hz,1H),2.14(s,3H),1.85(s,3H). LCMS(ESI+)m / z:[MH] + C 14 H 15 N2O5 + Calculated value: 291.1, measured value: 291.0.

[0307] N-(5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide (2-28): To a solution of N-(7-acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-27) (2.55 g, 8.78 mmol, 1.0 equiv) in MeOH (110 mL) was added HCl / MeOH (4 M, 110 mL, 50 equiv) at 20 °C, and the solution was stirred for 3 h. LCMS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in MeOH (10 mL), the pH was adjusted to 7 with saturated NaHCO3, and then extracted with ethyl acetate (20 mL × 3). The combined organic phase was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give N-(5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide (2-28) (2.0 g, 91% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.40(d,J=7.89Hz,1H),6.21(s,1H),5.97(d,J=15.79Hz,2H),4.35(td, J=8.17,5.37Hz,1H),3.20(dt,J=16.55,8.17Hz,1H),2.62(dd,J=16.66,5.26Hz,1H),1.84(s,3H). 13 C NMR(100MHz,DMSO-D6):δ199.6,169.1,154.3,145.6,133.4,127.7,111.3,101.3,93.5,54.5,28.7,22.3.LCMS(ESI+)m / z:[MH] + C 12 H 13 N2O4 + Calculated value: 249.1, measured value: 249.0.

[0308] N-[(7R)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide (2-29-P1) and N-[(7S)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide (2-29-P2): N-(5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide (2-28) (1.0 g) was dissolved in MeOH and separated by chiral SFC to give N-[(7R)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide (2-29-P1) (compound 22-9-P1 may be the opposite enantiomer of that shown) (250 mg, 24% yield) and N-[(7S)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide (2-29-P2) (compound 2-29-P2 may be the opposite enantiomer of that shown) (250 mg, 24% yield).

[0309] SFC separation method: column (column: Phenomenex®-Cellulose-2 (250 mm × 30 mm, 10 μm); mobile phase: [Neu-ETOH]; B%: 40% -40%, 5 min). Compounds (2-29-P1, RT = 2.719 min) and (2-29-P2, RT = 2.942 min) were separated by chiral SFC. Note: Stereochemistry is arbitrarily assigned for 2-29-P1 and 2-29-P2.

[0310] Spectra for 2-29-P1: 1 H NMR(400MHz,DMSO-D6)δ ppm 8.30(br d,J=7.95Hz,1H),6.59(s,2H),6.17(s,1H),5.96(d,J=15.89Hz,2H),4.36(td,J=8.16,5.32H z,1H),3.16-3.25(m,1H),2.61(dd,J=16.69,5.20Hz,1H),1.84(s,3H).LCMS(ESI+)m / z:[MH] + C 12 H 13 N2O4 + Calculated value: 249.1, measured value: 248.9.

[0311] Spectra for 2-29-P2: 1H NMR(400MHz,DMSO-D6)δ ppm 8.30(br d,J=7.95Hz,1H),6.59(s,2H),6.17(s,1H),5.96(d,J=15.89Hz,2H),4.36(td,J=8.19,5.26Hz,1H) ,3.21(dd,J=16.69,8.50Hz,1H),2.61(dd,J=16.69,5.20Hz,1H),1.85(s,3H).LCMS(ESI+)m / z:[MH] + C 12 H 13 N2C4 + Calculated value: 249.1, measured value: 248.9.

[0312] A mixture of N-[(7R-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide (2-29-P1) (30 mg, 0.12 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (63.6 mg, 0.24 mmol), and TsOH (20.7 mg, 0.12 mmol) in xylene (2 mL) was heated at 140 °C for 12 h. All volatiles were removed under high vacuum, and the residue was chromatographed to give 2-30. LCMS (ESI+) m / z: [M−H] + C 25 H 22 Calculated value of N3O7: 476.5, Measured value: 476.4.

[0313] Example 3 1 R,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35). (Figure 14)

[0314] [ka]

[0315] (S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32): To a solution of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (100 mg, 0.230 mmol, 1 equiv.) in methanol (20 mL) was added basic resin (300 mg) at 25 °C, and the mixture was stirred under ultrasound for 30 min. The mixture was filtered, and the filtrate was concentrated to give the free base.

[0316] To a solution of the free base obtained above in methanol (2 mL) was added 3,5-ditert-butyl-1,2-benzoquinone (101 mg, 0.459 mmol, 2 equiv.). The reaction mixture was stirred at 60°C for 10 minutes. The color of the reaction mixture changed from red to dark yellow. The reaction mixture was stirred at 25°C for an additional 3 hours. TLC (ethyl acetate:methanol = 8:1) showed that approximately 10% unreacted starting material remained and a new spot had formed. The reaction mixture was quenched at 25°C by the addition of oxalic acid (2M solution in tetrahydrofuran / HO = 3:1, 0.5 mL), and the mixture was stirred for 3 hours. The reaction mixture was diluted with tetrahydrofuran (5 mL), filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl condition) to give S-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32) (70 mg, 25% yield). 1H NMR(400MHz,DMSO-D6)δppm7.94(d,J=10.6Hz,1H),7.35(s,1H),5.44(s,2H),5.39(s,2H),3 .55-3.47(m,2H),3.09(t,J=7.0Hz,2H),2.46(s,3H),1.92-1.79(m,2H),0.91-0.85(m,3H). LCMS(ESI)m / z:[M+H + ]C 24 H2OFN2O5 + Calculated value: 435.1, measured value: 435.2.

[0317] Preparative HPLC method: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: HCl / H2O=0.040%v / v; B: CH3CN Column: Phenomenex® Gemini-NX 80 x 30 mm x 3 μm Flow rate: 25mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 30 8.0 60 8.1 60 8.2 100 10.2 100 10.3 30 11.5 30

[0318] (9S)-9-Ethyl-5-fluoro-1,9-dihydrodioxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano-[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33): To a solution of the resulting (S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32) (70 mg, 0.16 mmol, 1.0 equiv.) in methanol (2 mL) was added AcOH (27.6 mg, 0.460 mmol, 26.3 μL, 10 equiv.) and NaBHCN (20.1 mg, 0.322 mmol, 2.0 equiv.). The mixture was stirred at 0 °C for 10 min and then at 20 °C for 12 h. TLC (ethyl acetate:methanol = 8:1) showed the starting material was consumed and a new major spot had formed. The reaction mixture was quenched with HO (0.5 mL) at 25 °C and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (33) (20 mg, 29% yield).

[0319] Chiral SFC separation of compound 3-33 gave (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (3-34). (Compound 3-34 may be the opposite diastereomer of that shown.) (2.9 mg, 4% yield) (SFC time 1.429 min). (Peak 1 in SFC at 1.534 min) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (3-35) (compound 3-35 may be the opposite diastereomer to that shown) (4.7 mg, 7% yield) (SFC peak 2 at 1.534 min). Note: Stereochemistry is arbitrarily assigned.

[0320] Spectrum of (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (3-34): 1 H NMR(400MHz,DMSO-D6)δ ppm 7.73(dd,J=10.8,1.8Hz,7.31(s,1H),6.50(s,1H),5.97(d,J=16.0Hz,1H),5.42(s,2H),5.41(d,J=19.2Hz,1H),5.28(d,J=18.6Hz,1H),5.12-5 .17(m,1H),3.20-3.26(m,1H),2.98-3.05(m,1H),2.31-2.35(m,1H),2. 33(s,3H),1.95-2.00(m,1H),1.84-1.90(m,2H),0.88(t,J=7.2Hz,3H). 19 F NMR (400MHz, DMSO-D6): δppm-111.8(s, 1F). LCMS(ESI)m / z:[MH + ]C 24 H 22 FN2O5 + Calculated value: 437.1, Measured value: 437.1.

[0321] Spectrum of (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-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 (3-35): 1H NMR(400MHz,DMSO-D6)δ ppm 7.73(dd,J=10.8,1.8Hz,7.31(s,1H),6.50(s,1H),5.97(d,J=16.0Hz,1H),5.42(s,2H),5.41(d,J=19.2Hz,1H),5.28(d,J=18.6Hz,1H),5.12-5 .17(m,1H),3.20-3.26(m,1H),2.98-3.05(m,1H),2.31-2.35(m,1H),2. 33(s,3H),1.95-2.00(m,1H),1.84-1.90(m,2H),0.88(t,J=7.2Hz,3H). 19 F NMR (400MHz, DMSO-D6): δppm-111.8 (s, 1F). LCMS(ESI)m / z:[MH + ]C 24 H 22 FN2O5 + Calculated value: 437.1, measured value: 437.1.

[0322] Fractionation HPLC method: Machine: ギルソン 281 セミ HPLC システム Mobile phase: A: HCl / H2O = 0.040% v / v; B: CH3CN Karamu:Phenomenex (registered trademark) Luna 80 * 30mm * 3 um Flow rate: 25mL / min モニターWavelength: 220 and び254nm Time B% 0.0 20 8.0 50 8.1 50 8.2 100 10.2 100 10.3 20 11.5 20

[0323] SFC separation method: Machine: Waters SFC150AP SFC カラム:DAICEL CHIRALCEL OD(250mm * 30mm, 10 um) Mobile phase: A is for CO2, B is for EtOH Gradient: B% = 45% isocratic elution mode Flow rate: 70g / min Wavelength: 220nm Column temperature: 35℃ Back pressure: 120 bar

[0324] Example 4 (1R,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) and (1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-37) (Figure 15).

[0325] [ka]

[0326] N-(8S-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo2,8,9,11,12,14-hexahydrato-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-14) To a mixture of N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (100 mg, 0.423 mmol, 1.0 equiv.) (1-11) and (S-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizinc-3,6,10(4H)-trione (1-13) (223 mg, 0.847 mmol, 2.0 equiv.) in xylene (10 mL) at 140 °C was added 4-methylbenzenesulfonic acid (29.1 mg, 0.169 mmol, 0.4 equiv.). The mixture was stirred in a 40 mL sealed tube at 140° C. for 36 h. It was cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel eluting with 9% MeOH in dichloromethane to give N-((8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (I-14) (8.50 mg, 4.3% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.65(d,J=8.11Hz,1H),7.72(d,J=11.56Hz,1H),7.34(s,1H),6.51(d,J=2.27Hz,1H),5.95(brd,J=5.01Hz,1H),5.43(s, 2H),5.09-5.13(m,2H),3.82-3.93(m,1H),3.30(brs,1H),2.38(s,3H),1. 94(d,J=2.86Hz,3H),1.87(brd,J=7.39Hz,2H),0.87(brd,J=4.41Hz,3H). LCMS(ESI+)m / z:[MH] + C 25 H 23 Calculated value of FN3O5: 464.1, Measured value: 464.2.

[0327] (1R,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]-pyrano[3',4':6,7-indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) and (1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-37). A solution of N-((8S-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-h]quinolin-1-yl)acetamide (1-14) (150 mg, 0.323 mmol, 1.0 equiv) in 6 N aqueous HCl (15 mL) was stirred at 60 °C for 15 h in a sealed tube. After cooling to 25 °C, The reaction mixture was concentrated under reduced pressure, and the residue was diluted with methanol (5 mL) and purified by preparative HPLC to give 1R,8S-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]-pyrano[3',4':6,7]indolizino[1,2-h]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) (5.0 mg, 3.3% yield). Compound 4-36 was prepared in the opposite manner as shown. (This may be the enantiomer) and (1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14 dihydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-h]quinoline-9,12(2H,8H)-dione hydrochloride (4-37) (3.0 mg, 1.4% yield) were obtained (compound 4-37 may be the opposite enantiomer of that shown). Note: Stereochemistry is arbitrarily assigned.

[0328] Preparative HPLC method: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: HCl / H20 = 0.040% v / v; B: CH3CN Karamu:Phenomenex (registered trademark) luna C18 80 * 40mm * 3um Flow rate: 40mL / min モニターWavelength: 220 and び254nm Time B% 0.0 13 7.0 38 7.1 38 7.2 100 9.2 100 9.3 13 10.5 13

[0329] Spectra of 4-36: 1 H NMR (400 MHz, D2O) δ ppm 7.33-7.40(m,2H),5.65(br d,J=5.72Hz,1H),5.44-5.52(m,1H),5.33(br d,J=17.17Hz,2H),5.18-5.26(m,1H),4.00(br dd,J=18.06,7.81Hz,1H),3.45(br d,J=18.84Hz,1H),2.30(s,3H),1.90(q,J=7.27Hz,2H),0.87(t,J=7.27Hz,3H). 19 F NMR (376MHz, D2O) δppm-106.41. LCMS(ESI+)m / z:[MH] + C 23 H 21 FN3O4 + Calculated value: 422.1, measured value: 422.0.SFC (RT=1.208 points).

[0330] Spectra of 4-37:NMR(400MHz,D2O)δ ppm 7.43(s,1H),7.24(br d,J=11.26Hz,1H),5.58(br dd,J=8.00,3.25Hz,1H),5.54(d,J=16.26Hz,1H),5.37-5.45(m,2H),5.22(br d,J=19.01Hz,1H),4.05(br dd,J=18.01,8.13Hz,1H),3.52-3.61(m,1H),2.36(s,3H),1.95(q,J=7.30Hz,2H),0.93(t,J=7.38Hz,3H). 19 F NMR(376MHz,D2O)δppm-106.53. LCMS(ESI+)m / z:[MH] + C 23 H 21 FN3O4 + Calculated value: 422.1, measured value: 422.0.SFC (RT = 1.252 min).

[0331] Example 5 (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxy Synthesis of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-47) and (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48) (Figure 16)

[0332] [ka]

[0333] 3-Bromo-5-fluoro-4-methyl-aniline (1-3): To a solution of 1-bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2) (100 g, 427 mmol, 1.0 equiv.) in ethyl acetate (1.50 L) was added Pt / C (10 wt%, 10.0 g) under an argon atmosphere. The resulting suspension was degassed under vacuum and purged with H (15 psi) three times, after which it was stirred under H at 60 °C for 4 h. The H atmosphere was then replaced with argon, and the combined reaction mixture was filtered through a pad of Celite, and the filter cake was washed with ethyl acetate (10.0 L). (18 additional reactions were set up as above, and all 19 reaction mixtures were combined.) The combined filtrate was concentrated under reduced pressure to give 3-bromo-5-fluoro-4-methyl-aniline (1-3) (1.8 kg, 71% purity, 77% yield). 1 H NMR (400MHz, DMSO-d6) δppm 6.79 (s, 1H), 6.50 (dd, J = 11.74, 2.08Hz, 1H), 2.10 (d, J = 2.08Hz, 3H). 19 F NMR(376MHz,CD3OD)δ=-112.13. LCMS(ESI+)m / z:[MH] + C7H8BrFN + Calculated value: 205.0, measured value: 205.8.

[0334] N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4): To a solution of 3-bromo-5-fluoro-4-methyl-aniline (1-3) (300 g, 1.47 mol, 1.0 equiv.) in ethyl acetate (4.50 L) was added triethylamine (420 mL, 3.02 mol, 2.1 equiv.) and acetic anhydride (179 mL, 1.91 mol, 1.3 equiv.), and the mixture was stirred at 15 °C for 12 h. (Five additional vials were set up as above, and all six reaction mixtures were combined.) The combined mixture was quenched with saturated NH4Cl solution (15.0 L) and extracted with ethyl acetate (3 x 5.0 L). The combined organic layers were washed with brine (8.0 L) and diluted with Na2SO4. 4,The residue was dissolved in dichloromethane (2.00 L) and purified by silica gel column chromatography eluting with 15% ethyl acetate in petroleum ether to give N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (1.2 kg, 55% yield). 1 H NMR (400MHz, CD3OD) δ=7.59(t,J=1.5Hz,1H),7.41(dd,J=2.0,11.6Hz,1H),2.26(d,J=2.2Hz,3H),2.11(s,3H). 19 F NMR(376MHz,CD3OD)δ=-112.95. LCMS(ESI+)m / z:[MH] + C9H 10 BrFNO + Calculated value: 247.0, measured value: 247.8.

[0335] (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (5-38): To a mixture of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (200 g, 813 mmol, 1.0 equiv) in tetrahydrofuran (1.00 L) and water (200 mL), diisopropylethylamine (566 mL, 3.25 mol, 4.0 equiv), tris-o-tolylphosphane (49.5 g, 163 mmol, 0.20 equiv), but-3-enoic acid (168 g, 1.95 mol, 2.4 equiv), and Pd(OAc) (18.2 g, 81.3 mmol, 0.10 equiv) were added under N, and the reaction mixture was stirred at 75 °C for 16 h. (Five additional reactions were set up as above, and all six reaction mixtures were combined.) The combined reaction mixture was diluted with water (2.0 L) and adjusted to pH = 2 by the addition of 3N HCl. The mixture was filtered through a Celite pad and the filtrate cake was washed with ethyl acetate (4.0 L). The mixture was extracted with water (4.0 L) and the aqueous phase was extracted with ethyl acetate (3 x 1.80 L). The combined organic layers were washed with brine (4.0 L), dried over anhydrous Na2SO4, filtered, concentrated, and the residue was purified by silica gel column chromatography eluting with 70% ethyl acetate in petroleum ether to give (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (5-38) (400 g, 49% yield). 1 H NMR(400MHz,DMSO-d6)δ=12.32(brs,1H),10.02(s,1H),7.48(ddd,J=1.6,8.3,12 .0Hz,1H),7.39(s,1H),7.08-6.98(m,1H),6.94-6.83(m,1H),6.75-6.50(m,1H),6 .13(td,J=7.2,15.7Hz,1H),5.69(d,J=15.5Hz,1H),3.59(ddd,J=2.5,4.1,6.5Hz ,1H),3.56-3.46(m,1H),3.31-3.21(m,1H),2.16-2.06(m,3H),2.05-1.99(m,3H). 19 F NMR (376MHz, DMSO-d6)δ=-115.3. LCMS(ESI+)m / z:[MH] + C 13 H 15 FNO3 + Calculated value: 252.1, measured value: 252.0.

[0336] 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (5-39): To a solution of (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (5-38) (100 g, 398 mmol, 1.0 equiv.) in methanol (1.50 L) was added Pd / C (30.0 g, 39.8 mmol, 10 wt.%, 0.10 equiv.) under an argon atmosphere. The resulting suspension was degassed under vacuum, purged with H2 three times, and stirred at 35 °C for 12 h and under H2 (15 psi) for 12 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) After replacing the H2 atmosphere with argon, the combined mixture was filtered through a pad of Celite, and the filter cake was washed with methanol (8 L). The combined filtrate was concentrated under reduced pressure to give 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (5-39) (330 g, 82% yield), which was used directly in the next step without purification. 1 H NMR(400MHz,DMSO-d6)δ=12.77-11.04(m,1H),9.98(s,1H),7.42(dd,J=1.7,12.2Hz,1H),7.04(s,1H), 2.59-2.53(m,2H),2.27(t,J=7.2Hz,2H),2.09(d,J=1.8Hz,3H),2.01(s,3H),1.71(quin,J=7.5Hz,2H). 19 F NMR (376MHz, DMSO-d6)δ=-115.64. LCMS(ESI+)m / z:[MH] + C 13 H 17 FNO3 + Calculated value: 254.1, measured value: 254.0.

[0337] N-(7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-40): To a solution of 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (5-39) (110 g, 434 mmol, 1.0 equiv.) in trifluoroacetic acid (330 mL) was added trifluoroacetic anhydride (121 mL, 869 mmol, 2.0 equiv.) under N at 0 °C, and the mixture was stirred at 15 °C for 15 h. (Two additional reactions were set up as above, and all three reaction mixtures were combined.) The combined reaction mixture was poured into 50% aqueous acetonitrile (6.0 L) at 0 °C and stirred at 0 °C for 0.5 h. The resulting suspension was adjusted to pH = 7 with 25% aqueous NaOH at 0 °C. The mixture was filtered, and the residue was washed with water (1.00 L), methyl tert-butyl ester (2.00 L), and then dried under high vacuum. The residue was triturated with methyl tert-butyl ester (600 mL) and filtered to give N-(7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-40) (300 g, 88% yield). 1 H NMR(400MHz,DMSO-d6)δ=12.18(s,1H),8.28(d,J=13.2Hz,1H),2.89(t,J=6.1Hz,2H),2.68-2.60(m,2H),2.18-2.08(m,6H),1.99(quin,J=6.4Hz,2H). 19 F NMR (376MHz, DMSO-d6)δ=-103.89. LCMS(ESI+)m / z:[MH] + C 13 H 15 FNO2 + Calculated value: 236.1, measured value: 236.0.

[0338] N-(7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41): To a solution of N-(7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-40) (75.0 g, 319 mmol, 1.0 equiv.) in methanol (1.20 L) was added a solution of KOH (53.7 g, 956 mmol, 3 equiv.) in methanol (600 mL) and (diacetoxyiodo)benzene (113 g, 351 mmol, 1.1 equiv.) under argon at 0° C., and the mixture was stirred at 15° C. for 3 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined mixture was adjusted to pH 4 by the addition of 1 N HCl and concentrated at 35° C. to remove most of the methanol, and the mixture was extracted with dichloromethane (3×1.0 L). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 5 / 1 to 4 / 1) to give N-(7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41) (210 g, 66% yield). 1 H NMR(400MHz,DMSO-d6)δ=11.92(s,1H),8.26(d,J=13.1Hz,1H),5.90-4.94(m,1H),4.28(dd,J=5.0,12.7Hz,1H), 3.07-2.97(m,1H),2.96-2.84(m,1H),2.29-2.20(m,1H),2.16(s,3H),2.10(d,J=1.2Hz,3H),1.95-1.82(m,1H). 19 F NMR (377MHz, DMSO-d6)δ=-104.3. LCMS(ESI+)m / z:[MH] + C 13 H 15 FNO3 + Calculated value: 252.1, measured value: 252.0.

[0339] N-(3-allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42): To a solution of N-(7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41) (70.0 g, 279 mmol, 1.0 equiv.) in acetonitrile (1.40 L) was added a solution of AgO (129 g, 557 mmol, 2.0 equiv.) and 3-iodoprop-1-ene (140 g, 836 mmol, 76.3 mL, 3.0 equiv.) in acetonitrile (280 mL) at 15 °C, and the mixture was stirred at 40 °C for 5 h. (Two additional reactions were set up as above, and all three reaction mixtures were combined.) The combined mixture was filtered through a pad of Celite, and the filter cake was washed with dichloromethane (5.00 L). The combined filtrate was concentrated, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 9 / 1 to 4 / 1) to give the product. This material was triturated with methyl tert-butyl ester (500 mL) and filtered to give N-(3-allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42) (198 g, 81% yield). 1 H NMR(400MHz,DMSO-d6)δ=11.81(s,1H),8.27(d,J=13.1Hz,1H),5.93(tdd,J=5.3,10.5,17.2Hz,1H),5.29(qd,J=1.8,17.2Hz,1H),5.16( dd,J=1.7,10.5Hz,1H),4.32-4.06(m,3H),3.12-2.83(m,2H),2.32-2.23(m,1H),2.17(s,3H),2.11(d,J=1.5Hz,3H),2.09-1.98(m,1H). 19 F NMR (376MHz, DMSO-d6)δ=-104.2. LCMS(ESI+)m / z:[MH] + Cl6H 19 FNO3 + Calculated value: 292.1, measured value: 291.9.

[0340] N-[7-fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide (5-43): A mixture of N-(3-allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42) (42.0 g, 144 mmol, 1.0 equiv.) in dichloromethane (840 mL) and methanol (420 mL) was cooled to -70 °C, and ozone (6.92 g, 144 mmol, 1.0 equiv.) was bubbled through the mixture for 60 minutes, followed by O2 for 30 minutes. Methylsulfanylmethane (26.5 mL, 360 mmol, 2.5 equiv.) was then added to the mixture at -70 °C, which was then warmed to 15 °C and stirred at 15 °C for 1 hour. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was quenched with water (5.0 L) and extracted with dichloromethane (3 × 1.0 L). The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated to give N-[7-fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide (5-43) (168 g, 59% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,CDCl3)δ=11.94(brs,1H),9.80(s,1H),8.44(d,J=12.7Hz,1H),4.55-4.30(m,1H),3.85-3.69(m,2H),3.51- 3.48(m,1H),3.18-3.03(m,1H),2.96-2.78(m,1H),2.52-2.34(m,1H),2.26-2.21(m,3H),2.14(brdd,J=1.7,3.9Hz,3H). 19 F NMR (376MHz, CDCl3)δ=-100.7. LCMS(ESI+)m / z:[MH] + C 15 H 17 FNO4 + Calculated value: 294.1, measured value: 293.9.

[0341] N-[7-fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide (5-44): To a solution of N-[7-fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide (5-43) (42 g, 85.9 mmol, 60% purity, 1.0 equiv) in THF (850 mL) and HO (425 mL) was added NaBH (975 mg, 25.8 mmol, 0.30 equiv) in portions at 0 °C, and the mixture was stirred at 0 °C for 10 min and quenched with cold water (2.0 L). (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was extracted with dichloromethane (3 × 1.0 L), and the organic layer was washed with brine, dried over NaSO, filtered, concentrated, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 1 to 1 / 3) to give N-[7-fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide (5-44) (70 g, 61% yield). 1 H NMR(400MHz,CDCl3)δ=11.96(brs,1H),8.45(d,J=12.7Hz,1H),4.06(dd,J=4.6,12.0Hz,1H),4.01-3.62(m, 4H), 3.11(td,J=4.6,17.6Hz,1H),3.03-2.79(m,2H),2.45-2.36(m,1H),2.24(s,3H),2.15(d,J=1.5Hz,4H). 19 F NMR (376MHz, CDCl3)δ=-100.9. LCMS(ESI+)m / z:[MH] + C 15 H 19 FNO4 + Calculated value: 296.1, Measured value: 296.1.

[0342] 8-Amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-tetralin-1-one (5-45): To a solution of N-[7-fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide (5-44) (21.0 g, 71.0 mmol, 1.0 equiv) in methanol (400 mL) under argon, HCl (2 N, 630 mL, 18 equiv) was added, and the mixture was stirred at 15 °C for 18 h and adjusted to pH = 7-8 by the addition of saturated NaHCO. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was extracted with ethyl acetate (3 × 2.0 L), and the organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was triturated with methyl tert-butyl ester / dichloromethane (1:2, 300 mL) and filtered to give 8-amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-tetralin-1-one (5-45) (42 g, 81% yield). 1 H NMR(400MHz,CD3OD)δ=6.30(d,J=12.3Hz,1H),4.06(dd,J=4.4,11.2Hz,1H),3.83-3.67(m, 4H), 3.04 (td, J=4.9, 17.5Hz, 1H), 2.85-2.73 (m, 1H), 2.40-2.30 (m, 1H), 2.09-1.93 (m, 4H). 19 F NMR(376MHz,CD3OD)δ=-108.7. LCMS(ESI+)m / z:[MH] + C 13 H 17 FNO3 + Calculated value: 254.1, Measured value: 254.1.

[0343] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-46): To a mixture of 8-amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (5-45) (10.5 g, 41.4 mmol, 1.0 equiv.) and (5)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (12.0 g, 45.6 mmol, 1.1 equiv.) in toluene (525 mL) was added o-cresol (31.5 mL, 303 mmol, 7.3 equiv.) and pyridin-1-ium 4-methylbenzenesulfonate (1.56 g, 6.23 mmol, 0.15 equiv.) at 120 °C under argon, and the mixture was stirred at 120 °C for 13 h. (Three additional reactions were set up as above, and the four reaction mixtures were combined.) The combined reaction mixtures were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 13% methanol in ethyl acetate to give (9S-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline 10,13(1H,9H)-dione (5-46) (34.2 g, 43% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.58(dd,J=10.8,1.91Hz,1H),7.23(d,J=6.8Hz,1H), 6.49(s,1H),5.40(s,2H),4.95-5.24(m,2H),4.88(dt,J=8.4,4.4Hz,1H),4.69-4 .81(m,1H),3.78-3.91(m,1H),3.56-3.76(m,3H),3.10(d,J=16.4Hz,1H),2.77-2 .92(m,1H),2.33-2.45(m,1H),2.21(s,3H),1.83-2.04(m,3H),0.77-1.00(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.57. LCMS(ESI+)m / z:[ M+H] + C 26 H 26 FN2O6 + Calculated value: 481.2, measured value: 481.0.

[0344] (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione 5-47 and (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48). (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-46) (31.0 g, 64.3 mmol, 1.0 equiv.) was purified by chiral SFC (instrument: Waters SFC350 preparative SFC; column: DAICHEF CHIRAFCEF OD (250 mm) * 50 mm, 10 μm); mobile phase: A is CO2, B is MeOH; gradient: B% = 60% isocratic elution mode; flow rate: 200 g / min; wavelength: 220 nm; column temperature: 40 °C. system back pressure: 100 bar) to give (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-47) (13.5 g, 43% yield). Compound 5-47 is the opposite enantiomer of that shown. (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline 10,13(1H,9H)-dione (5-48) (10.0 g, 32% yield) (compound 5-48 may be the opposite enantiomer of that shown). Note: Stereochemistry assigned arbitrarily.

[0345] Spectra of 5-47: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.64(d,J=10.88Hz,1H),7.25(s,1H),6.50(s,1H),5.13-5.52(m,4H),4.95(br dd,J=8.25,3.75Hz,1H),4.74(br t,J=5.00Hz,1H),3.84(dt,J=9.72,4.96Hz,1H),3.59-3.76(m,3H),3.10-3.21(m,1H), 2.84-2.99(m,1H),2.23-2.46(m,4H),2.04(m,1H),1.87(m,2H),0.88(t,J=7.32Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.59. LCMS(ESI+)m / z:[M+H] + C 26 H 26 FN2O6 + Calculated value: 481.2, measured value: 481.0. キラルSFC: RT=1.48 points.

[0346] Spectra of 5-48: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.74(d,J=10.97Hz,1H),7.30(s,1H),6.51(s,1H),5.31-5.46(m,4H),5.03 (dd,J=8.11,3.70Hz,1H),4.74(t,J=5.36Hz,1H),3.81-3.89(m,1H),3.59-3 .75(m,3H),3.21(dt,J=17.02,5.38Hz,1H),2.94-3.07(m,1H),2.31-2.45( m,4H),2.08(m,1H),1.88(dt,J=13.23,6.62Hz,2H),0.89(t,J=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.53. LCMS(ESI+)m / z:[M+H] + C 26 H 26 FN2O6 +Calculated value: 481.2, Found value: 481.0. Chiral SFC: RT=1.61 min.

[0347] Example 6 Synthesis of (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4']:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35) (Figure 17).

[0348] [ka]

[0349] 2-(Allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (6-49): To a mixture of N-(7-(allyloxy)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5-42) (600 mg, 2.06 mmol, 1.0 equiv) in methanol (6.0 mL) under argon at 0° C., HCl / methanol (6.0 mL, 4 M, 11.65 equiv) was added, and the mixture was stirred at 25° C. for 2 h, cooled to 0° C., and the pH was adjusted to 8 by the addition of saturated NaHCO. This was extracted with dichloromethane (3 × 40 mL), and the combined organic layers were washed with brine, dried over NaSO, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 10% ethyl acetate in petroleum ether to give 2-(allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one (6-49) (415 mg, 81% yield). 1H NMR(400MHz,CDCl3)δppm6.24-6.68(m,2H),6.20(d,J=11.62Hz,1H),5.91-6.03(m ,1H),5.33(dq,J=17.24,1.63Hz,1H),5.20(dq,J=10.38,1.35Hz,1H),4.36(m,1H), 4.17(m,1H),3.97(dd,J=10.15,4.16Hz,1H),3.01(dt,J=17.45,5.33Hz,1H),2.75 (m,1H),2.27(dq,J=13.17,5.02Hz,1H),2.09-2.19(m,1H),2.04(d,J=1.71Hz,3H). 19 F NMR (376MHz, CDCl3) δppm-106.28. LCMS(ESI+)m / z:[MH] + C 14 H 17 FNO2 + Calculated value: 250.1, Measured value: 250.1.

[0350] (9S)-1-(allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50): A mixture of 2-(allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1(1H)-one (6-49) (200 mg, 0.802 mmol, 1.0 equiv.) and (5)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (232 mg, 0.883 mmol, 1.1 equiv.) in toluene (10 mL) was treated with o-cresol (0.609 mL, 5.86 mmol, 7.3 equiv.) and pyridinium 4-methylbenzyl ether (4-methylbenzyl ether) under argon at 120 °C. Benzene sulfonate (30.2 mg, 0.120 mmol, 0.15 equiv) was added, and the mixture was stirred at 120 °C for 32 h, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 55% ethyl acetate in petroleum ether to give (9S)-1-(allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50) (100 mg, 26% yield). 1 H NMR(400MHz,CDCl3)δppm7.58-7.67(m,2H),6.03-6.16(m,1H),5.74(dd,J=16.26,1.83H z,1H),5.39-5.52(m,2H),5.21-5.36(m,3H),4.94(m,1H),4.38-4.47(m,1H),4.26(m,1H) ),3.87(d,J=19.32Hz,1H),3.23-3.36(m,1H),2.99(brt,J=13.02Hz,1H),2.45-2.57(m, 1H),2.40(brs,3H)2.11-2.25(m,1H),1.82-1.98(m,2H),1.04(td,J=7.37,2.87Hz,3H). 19 F NMR(376MHz,CDCl3)δppm-110.35.LCMS(ESI+)m / z:[MH] + C 27 H 26 FN2O5 + Calculated value: 477.2, Measured value: 477.2.

[0351] (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33): To a mixture of (9S)-1-(allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50) (300 mg, 0.63 mmol, 1.0 equiv) in tetrahydrofuran (15 mL) was added ZnCl (110 mg, 0.82 mmol, 1.3 equiv) under argon at 25 °C. After 0.25 h, Pd(PPh) (58.0 mg, 0.157 mmol, 0.25 equiv) was added, followed by BuSnH (3.33 mL, 12.6 mmol, 20 equiv) after another 0.25 h. The mixture was stirred at 50 °C for 1.5 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 5% methanol in ethyl acetate to give a residue, which was further purified by prep-HPLC to give (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33) (90.0 mg, 32% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.72(d,J=10.97Hz,1H),7.30(d,J=2.27Hz,1H),6.50(d ,J=0.72Hz,1H),5.98(dd,J=5.90,2.92Hz,1H),5.36-5.46(m,3H),5.23-5.32(m,1H ),5.14(dt,J=9.60,4.86Hz,1H),3.23(dt,J=16.90,4.60Hz,2H),2.96-3.06(m,1H) ,2.35(s,3H),1.94-2.06(m,1H),1.80-1.93(m,2H),0.88(td,J=7.30,1.49Hz,3H). 19F NMR(376MHz,DMSO-D6)δppm-111.78.LCMS(ESI+)m / z:[MH] + C 24 H 22 FN2O5 + Calculated value: 437.2, measured value: 437.0.

[0352] Preparative HPLC method: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: H2O; B: MeOH Column: Phenomenex® Gemini-NX 80 x 30 mm x 3 μm Flow rate: 25mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 45 8.0 70 8.1 70 8.2 100 10.2 100 10.3 45 11.5 45

[0353] (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione 3-34 and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35). (9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33) (90.0 mg, 0.206 mmol, 1.0 equiv.) was purified by chiral SFC (instrument: Waters SFC 80 preparative SFC; column: DAICEL CHIRALCEL OD (250 mm *30 mm, 10 μm); mobile phase: A is CO2, B is EtOH; gradient: B% = 55% isocratic elution mode; flow rate: 60 g / min; wavelength: 220 nm; column temperature: 40 °C; system back pressure: 100 bar) to obtain (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) (26 This afforded (1.1 mg, 29% yield) (compound 3-34 may be the opposite enantiomer to that shown) and (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35) (25.1 mg, 28% yield) (compound 3-35 may be the opposite enantiomer to that shown). Note: The stereochemistry of the two products is assigned arbitrarily.

[0354] Spectra of 3-34: 1 H NMR (400 MHz, DMSO-D6) δ ppm 7.73(d,J=10.97Hz,1H),7.30(s,1H),6.50(s,1H),5.98(d,J=5.84Hz,1H),5.37-5.47(m,3H),5.26-5.35(m,1H),5.10-5.2 0(m,1H),3.20-3.30(m,2H),2.97-3.07(m,1H),2.35(s,3H),1.95-2.05(m,1H),1.78-1.94(m,2H),0.88(t,J=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.78. LCMS(ESI+)m / z:[MH] + C 24 H 22 FN2O5 + Calculated value: 437.2, Found value: 437.0. Chiral SFC: RT=1.43 min.

[0355] Spectra of 3-35: 1H NMR (400 MHz, DMSO-D6) δ ppm 7.73(d,.J=11.09Hz,1H),7.30(s,1H),6.50(s,1H),5.97(d,J=5.84Hz,1H),5.34-5.48(m,3H),5.21-5.32(m,1H),5.09-5.20 (m,1H),3.20-3.24(m,2H),2.96-3.08(m,1H),2.35(s,3H),1.99(d,J=9.54Hz,1H),1.80-1.93(m,2H),0.89(t,J=7.09Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.80. LCMS(ESI+)m / z:[MH] + C 24 H 22 FN2O5 + Calculated value: 437.2, Found value: 437.0. Chiral SFC: RT=1.54 min.

[0356] Example 7 (1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) and (1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16 tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60) (Figure 18).

[0357] [ka]

[0358] 5-(5-acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid (7-51): To a solution of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (50.0 g, 203 mmol, 1.0 equiv) in THF (250 mL) and HO (50.0 mL) was added pent-4-enoic acid (49.9 mL, 487 mmol, 2.4 equiv), diisopropylethylamine (155 mL, 894 mmol, 4.4 equiv), tris-o-tolylphosphane (12.4 g, 40.6 mmol, 0.20 equiv), and palladium(II) diacetate (4.56 g, 20.3 mmol, 0.10 equiv) under a N atmosphere, and the mixture was stirred at 75 °C for 24 h and cooled to 30 °C. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined mixture was filtered through a pad of Celite, and the filter cake was washed with water (2.0 L) and ethyl acetate (3.0 L). The mixture was adjusted to pH 4-5 with HCl solution (5N) and extracted with ethyl acetate (3 × 800 mL). The organic layer was dried over NaSO and concentrated, and the residue was purified by silica gel flash column chromatography eluting with 30% ethyl acetate in petroleum ether to give 5-(5-acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid (7-51) (0.20 kg, 83% yield). 1 H NMR (400 MHz, DMS O-d6)δ=12.19(brs,1H),9.99(s,1H),7.51-7.28(m,1H),7.00(s,1H),6.17- 5.36(m,2H),3.35-3.24(m,2H),3.19-2.93(m,2H),2.07(s,3H),2.01(s,3H) 19 F NMR (376MHz, DMSO-d6) δppm-115.96.

[0359] 5-(5-acetamido-3-fluoro-2-methylphenyl)pentanoic acid (7-52): To a mixture of 5-(5-acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid (7-51) (50.0 g, 170 mmol, 90% purity, 1.0 equiv.) in methanol (500 mL) under an argon atmosphere, Pd / C (25.0 g, 170 mmol, 10 wt.%, 1.0 equiv.) was added under an argon atmosphere. The mixture was evacuated and filled with H three times, and then stirred under H at 25 °C for 12 h (15 Psi). (Three additional reactions were set up as above, and all four reaction mixtures were combined.) After replacing the H atmosphere with argon, it was filtered through a pad of Celite, the filter cake was washed with methanol (6.00 L), and the filtrate was concentrated to give 5-(5-acetamido-3-fluoro-2-methylphenyl)pentanoic acid (7-52) (190 g, 94% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-d6)δ=9.97(s,1H),7.41(dd,J=1.7,12.2Hz,1H),7.03(s,1H),2.54(brt, J=7.4Hz,2H),2.23(t,J=7.0Hz,2H),2.08(d,J=1.7Hz,3H),2.01(s,3H),1.59-1.43(m,4H). 19 F NMR(376MHz, CDCl3)δppm-115.806. LCMS(ESI+)m / z:[MH] + CI4H 19 FNO3 + Calculated value: 268.1, measured value: 268.0.

[0360] N-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-1-yl)acetamide (7-53): A mixture of 5-(5-acetamido-3-fluoro-2-methylphenyl)pentanoic acid (7-52) (38.0 g, 142 mmol, 1.0 equiv) in polyphosphoric acid (500 mL) was stirred at 110 °C for 3 h. (Four additional reactions were set up as above, and all five reaction mixtures were combined.) The combined reaction mixture was slowly poured into stirred ice-water (10.0 L), the pH was adjusted to 7 with saturated NaHCO solution, and the mixture was extracted with ethyl acetate (3 × 1.00 L). The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography eluting with 15% ethyl acetate in petroleum ether to give N-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-l-yl)acetamide (7-53) (91 g, 64% yield). 1 H NMR(400MHz,DMSO-d6)δ=9.83(s,1H),7.19(d,J=11.6Hz,1H),2.69(t,J=6.3Hz ,2H),2.58-2.52(m,2H),2.16(d,J=2.0Hz,3H),1.95(s,3H),1.74-1.58(m,4H). 19 F NMR (376MHz, DMSO-d6) δppm-111.49. LCMS(ESI+)m / z:[MH] + CI4H 17 FNO2 + Calculated value: 250.12, measured value: 250.0.

[0361] (Z)-N-(3-fluoro-8-(hydroxyimino)-4-methyl-9-oxo-6,7,8,9-tctrahvdro-5H-benzo[7]annulcan-1-yl)acetamide (7-54): To a mixture of potassium tert-butoxide (1 M in tetrahydrofuran, 191 mL, 2.1 equiv.) in tetrahydrofuran (194 mL), ethanol (31.4 mL), and n-butanol (31.4 mL) was added N-(2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydrobenzo[7]annulen-4-yl)acetamide (7-53) (22.7 g, 90.9 mmol, 1.0 equiv.) and isopentyl nitrite (14.8 mL, 109 mmol, 1.2 equiv.) at 0° C., and the mixture was stirred at 20° C. for 3 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was cooled to 0° C., quenched with 0.5 N hydrochloric acid (1.00 L), extracted with ethyl acetate (3×300 mL), and the organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was triturated with methyl tert-butyl ester (500 mL) and filtered to give (ZN-(3-fluoro-8-(hydroxyimino)-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide (7-54) (80.0 g, 88% yield). 1 H NMR(400MHz,DMSO-d6)δ=12.69-11.95(m,1H),10.10(s,1H),7.28(d,J=11.6Hz,1H),2.68(br t,J=6.6Hz,2H),2.57(t,J=6.8Hz,2H),2.17(d,J=1.8Hz,3H),1.96(s,3H),1.78-1.75(m,2H) 19 F NMR (376MHz, DMSO-d6) δppm -107.85, -109.67, -110.39. LCMS(ESI+)m / z:[MH] + C 14 H 16 FN2O3 + Calculated value: 279.1, Measured value: 279.1.

[0362] N-(8-amino-3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide hydrochloride (7-55): To a mixture of N-[(5E)-2-fluoro-5-hydroxyimino-1-methyl-6,7,8,9-tetrahydrobenzo[7]annulen-4-yl]acetamide (7-54) (20.0 g, 75.7 mmol, 1.0 equiv.) in methanol (240 mL) under a N atmosphere, hydrochloric acid (12 M, 18.9 mL, 3.0 equiv.) and Pd / C (4.00 g, 75.7 mmol, 10 wt.%, 1.0 equiv.) were added. The mixture was evacuated and filled with H three times and stirred under H (15 Psi) at 20 °C for 3 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) After replacing the H atmosphere with argon, the reaction mixtures were combined, diluted with methanol (800 mL), filtered through a pad of Celite, the filter cake was washed with methanol (10.0 L), and the combined filtrates were concentrated to dryness under reduced pressure. The residue was triturated with methyl tert-butyl ether (800 mL) and filtered to give N-(8-amino-3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide hydrochloride (7-55) (75 g, 98% yield). 1 H NM showed that this material contained approximately 25% de-Ac by-product. 1 H NMR(400MHz,DMSO-d6)δ=10.34(s,1H),8.75-8.40(m,3H),7.31-7.20(m,1H),4.44-4.21(m,1H),2.98(dt,J= 6.1,7.9Hz,1H),2.85-2.75(m,1H),2.47(brs,1H),2.27-2.11(m,3H),2.10-1.94(m,3H),1.94-1.50(m,3H). 19 F NMR (376MHz, DMSO-d6) δppm -107.88, -110.63. LCMS(ESI+)m / z:[MH] + C 14 H 16 FN2O3 + Calculated value: 265.1, measured value: 265.0.

[0363] N.N'-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,8-yl)diacetamide (7-56): To a suspension of N-(5-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-4-yl)acetamide hydrochloride (7-55) (25.0 g, 87.2 mmol, 1.0 equiv) in dichloromethane (225 mL) was added triethylamine (36.4 mL, 262 mmol, 3.0 equiv) and acetic anhydride (9.80 mL, 105 mmol, 1.2 equiv), and the mixture was stirred at 20° C. for 3 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was extracted with saturated NH4Cl solution (2.00 L), the aqueous phase was extracted with ethyl acetate (3 x 500 mL), the combined organic layers were dried over Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 30% ethyl acetate / petroleum ether to give N,N'-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5 / 7-benzo[7]annulene-1,8-diyl)diacetamide (7-56) (54.0 g, 53% yield) and N-(4-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulene-5-yl)acetamide (7-57) (9.00 g, 10% yield).

[0364] Spectra of 7-56: 1 H NMR(400MHz,DMSO-d6)δ=9.61(br s,1H),8.69(br d,J=4.5Hz,1H),7.69(d,J=12.2Hz,1H),4.56-4.39(m,1H),2.93(br dd,J=7.0,12.3Hz,1H),2.43(br dd,J=10.3,14.7Hz,1H),2.14(d,J=1.2Hz,3H),2.01(s,4H),1.93(s,4H),1.81-1.61(m,2H). 19 F NMR (376MHz, DMSO-d6) δppm-111.86. LCMS(ESI+)m / z:[M+H] + C 16 H 20 FN2O3 + Calculated value: 307.1, Measured value: 307.1.

[0365] Spectra of 7-57: 1 H NMR(400MHz,DMSO-d6)δ=8.25(br d,J=7.1Hz,1H),6.45-6.33(m,3H),4.57(td,J=6.7,11.5Hz,1H),2.97-2.86(m,1H),2.81-2.70( m,1H),2.07-1.97(m,4H),1.95-1.88(m,1H),1.84(s,3H),1.70-1.59(m,1H),1.57-1.42(m,1H). 19 F NMR (376MHz, DMSO) δppm-110.23. LCMS(ESI+)m / z:[M+H] + CI4H 18 FN2O3 + Calculated value: 265.1, Measured value: 265.1.

[0366] N-(4-amino-2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)acetamide (7-57): To a mixture of N,N'-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,8-diyl)diacetamide (7-56) (18.0 g, 61.6 mmol, 1.0 equiv.) in methanol (180 mL) was added hydrochloric acid / methanol (4 M, 180 mL, 11.7 equiv.), and the mixture was stirred at 25 °C for 2 h. (Three additional reactions were set up as above, and all four reaction mixtures were combined.) The combined reaction mixture was concentrated under reduced pressure, and the residue was extracted with saturated NaHCO solution (2.00 L) and ethyl acetate / methanol (10 / 1, 3 × 800 mL). The combined organic phases were washed with brine, dried over anhydrous NaSO, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 30% ethyl acetate in petroleum ether to give N-(4-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-yl)acetamide (7-57) (25 g, 54% yield). 1H NMR(400MHz,DMSO-d6)δ=8.25(brd,J=7.1Hz,1H),6.45-6.33(m,3H),4.57(td,J=6.7,11.5Hz,1H),2.97-2.86(m, 1H),2.81-2.70(m,1H),2.07-1.97(m,4H),1.95-1.88(m,1H),1.84(s,3H),1.70-1.59(m,1H),1.57-1.42(m,1H). 19 F NMR (376 MHz, CDCl 3 )δppm-111.22. LCMS(ESI+)m / z:[M+H] + C 14 H 18 FN2O3 + Calculated value: 265.1, Measured value: 265.1.

[0367] N-((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-1 1,14-Dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]-quinolin-1-yl)acetamide (7-58): To a mixture of N-(4-amino-2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)acetamide (7-57) (230 mg, 0.870 mmol, 1.0 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (458 mg, 1.74 mmol, 2.0 equiv.) in toluene (23 mL) was added pyridinium 4-methylbenzenesulfonate (87.4 mg, 0.348 mmol, 0.4 equiv.), and the mixture was stirred at 120 °C in a sealed tube for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 50% ethyl acetate in petroleum ether to give N-((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indole-dino[1,2-b]quinolin-1-yl)acetamide (7-58) (240 mg, 54% yield). 1 H NMR(400MHz,DMSO-D6)δ=8.71(brd,J=6.2Hz,1H),7.73(d,J=10.5Hz,1H),7.29 (s,1H),6.52(d,J=7.4Hz,1H),5.62-5.50(m,1H),5.46-5.31(m,3H),5.22-5.1 1(m,1H),3.27-3.20(m,2H),3.17(d,J=4.8Hz,1H),2.42(s,3H),2.31-2.20(m, 1H),2.14-2.02(m,2H),1.97(s,3H),1.84-1.60(m,2H),0.87(q,J=7.4Hz,3H). LCMS(ESI+)m / z:[M−H] + C 27 H 27 FN3O5 + Calculated value: 492.2, measured value: 492.1.

[0368] (1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) and (1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60). N-((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (7-58) (160 mg, 0.101 mmol, 1.0 equiv) in 2-methoxyethanol (0.8 mL) was added to methylcyclohexane (0.8 mL), HO (0.75 mL), and methanesulfonic acid (0.25 mL) under argon, and the mixture was stirred at 100 °C for 8 h, concentrated, and the residue was purified by preparative HPLC to give (1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13, 16-Tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) (5.5 mg, 2.4% yield) (compound 7-59 may be the opposite enantiomer to that shown) and (1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60) (6.4 mg, 3.6% yield) (compound 7-60 may be the opposite enantiomer to that shown). Note: Stereochemistry is assigned arbitrarily.

[0369] Fractionation HPLC method: Machine: ギルソン 281 セミ HPLC システム Mobile phase: A: H2O; B: MeOH Color:Phenomenex(registered trademark) Gemini-NX 80×30mm×3um Flow rate: 25mL / min モニターWavelength: 220 and び254nm Time B% 0.0 40 6.0 75 6.1 75 8.2 100 10.2 100 10.3 40 11.5 40

[0370] Spectra of 7-59: 1 H NMR (400 MHz, D2O) δ ppm 7.38-7.49(m,2H),5.52-5.62(m,1H),5.35-5.46(m,3H),5.16(t,J=4.4Hz,1H),3.40-3.51(m,1H),2.99-3.13(m,1H),2.77(s,6 H),2.48-2.57(m,2H),2.38(d,J=1.6Hz,3H),2.22-2.33(m,1H),2.06-2.20(m,1H),1.94(q,J=7.5Hz,2H),0.90(t,J=7.2Hz,3H). 19 F NMR (376MHz, D2O) δppm-111.53. LCMS(ESI+)m / z:[MH] + C 25 H 25 FN3O4 + Calculated value: 450.2, measured value: 450.0.

[0371] Spectra of 7-60: 1 H NMR (400 MHz, D2O) δ ppm 7.40-7.51(m,2H),5.44-5.60(m,2H),5.33-5.43(m,2H),5.09-5.14(m,1H),3.37-3.48(m,1H),3.01-3.14(m,1H),2.77(s ,3H),2.43-2.54(m,1H),2.39(s,3H),2.27-2.35(m,1H),2.06-2.23(m,2H),1.95(q,J=7.6Hz,2H),0.92(t,J=7.2Hz,3H). 19 F NMR(376MHz,D2O)δppm-109.21. LCMS(ESI+)m / z:[MH] + C 25 H 25 FN3O4 + Calculated value: 450.2, measured value: 450.0.

[0372] Example 8 (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)dione hydrochloride (8-72) (Figure 19).

[0373] [ka]

[0374] 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (8-61): To a mixture of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (10.0 g, 40.6 mmol, 1.0 equiv) and 2-methylbut-3-enoic acid (13.0 g, 130 mmol, 3.2 equiv) in tetrahydrofuran (40 mL) and water (10 mL), N-ethyl-N,N-diisopropylamine (38.2 mL, 219 mmol, 5.4 equiv), tris-o-tolylphosphane (2.47 g, 8.13 mmol, 0.2 equiv), and diacetoxypalladium (912 mg, 4.06 mmol, 0.1 equiv) were added under nitrogen. The mixture was stirred at 75 °C for 5 h, quenched with water (50 mL), and the pH was adjusted to approximately 3 by adding 3 N hydrochloric acid at 0 °C. It was filtered through a pad of Celite, the filtrate was extracted with ethyl acetate (3 × 150 mL), the organic layer was washed with brine, dried over NaSO, filtered, concentrated, and the residue was purified by silica gel flash column chromatography eluting with 50% ethyl acetate in petroleum ether to give 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (8-61) (6.00 g, 56% yield). 1 H NMR(400MHz,DMSO-D6)δppm12.38(s,1H),10.02(d,J=4.0Hz,1H),7.46-7.52(m,1H),6.99-7.38(m,1H),6.65-6.69(m,1H), 6.17(dd,J=15.6,8.0Hz,1H),3.49(d,J=7.2Hz,1H),2.11(dd,J=13.2,1.6Hz,3H),2.02(d,J=4.8Hz,3H),1.24-1.88(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-115.68. LCMS(ESI+)m / z:[MH] + C 14 H 17 FNO3 + Calculated value: 266.1, Measured value: 266.1.

[0375] 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (8-62): To a mixture of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (1) (5.00 g, 18.8 mmol, 1.0 equiv.) in methanol (20 mL) under nitrogen, Pd / C (10 wt %) (2.40 g, 0.12 equiv.) was added. The suspension was degassed under vacuum, purged with H three times, and stirred under H (15 psi) at 25 °C for 10 h. After replacing the H atmosphere with argon, it was filtered through a Celite pad and the filter cake was washed with methanol (300 mL). The combined filtrate was concentrated under reduced pressure to give 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (8-62) (4.4 g, 87% yield). 1 H NMR(400MHz,DMSO-D6)δppm10.0(s,1H),7.42(dd,J=12.4,1.6Hz,1H),7.04(s,1H),2.52- 2.58(m,2H),2.24(t,J=6.8Hz,2H),2.08(d,J=1.6Hz,3H),2.01(s,3H),1.40-1.65(m,4H). 19 F NMR (376MHz, DMSO-D6) δppm-125.77. LCMS(ESI+)m / z:[MH] + C 14 H 19 FNO3 + Calculated value: 268.1, Measured value: 268.1.

[0376] N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (8-63): To a mixture of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (8-62) (5.00 g, 18.7 mmol, 1.0 equiv) in trifluoroacetic acid (10 mL) was added trifluoroacetic anhydride (5.20 mL, 37.4 mmol, 2.0 equiv) at 0 °C. The mixture was stirred at 0 °C for 2 h, quenched with ice water (100 mL), and the pH was adjusted to about 7 by adding 25% aqueous NaOH at 0 °C. It was extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography eluting with 8% ethyl acetate in petroleum ether to give N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (8-63) (3.50 g, 75% yield). 1 H NMR(400MHz,DMSO-D6)δppm12.13(s,1H),8.28(d,J=13.2Hz,1H),2.93-3.04(m,1H),2.81-2.92( m,1H),2.63-2.75(m,1H),2.07-2.18(m,7H),1.74(qd,J=12.0,4.8Hz,1H),1.15(d,J=6.4Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-104.64. LCMS(ESI+)m / z:[MH] + C 14 H 17 FNO2 + Calculated value: 250.1, Measured value: 250.1.

[0377] Di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1234-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (8-64): To a mixture of N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (8-63) (35.1 g, 140 mmol, 1.0 equiv) in toluene (700 mL) under nitrogen at 0° C., sodium bis(trimethylsilyl)amide (309 mL, 1 M, 2.2 equiv) was added dropwise. The mixture was cooled to −40° C., and a solution of di-tert-butyldiazene-1,2-dicarboxylate (42.1 g, 183 mmol, 1.3 equiv) in toluene (350 mL) was added dropwise. The reaction mixture was warmed to 25° C., stirred at 25° C. for 4 h, cooled to 0° C., diluted with water (1 L), and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 20% ethyl acetate in petroleum ether to give di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (8-64) (41.0 g, 60% yield). 1 H NMR(400MHz,DMSO-D6)δppm11.61-11.84(m,1H),8.26(d,J=12.8Hz,1H),7.96-8.15(m,1 H),2.96-3.16(m,2H),2.68-2.84(m,1H),2.12(s,4H),2.08(s,3H),1.35-1.46(m,21H). 19 F NMR (376MHz, DMSO-D6) δppm-105.29. LCMS(ESI+)m / z:[MH] + C 24 H 35 FN3O6 + Calculated value: 480.2, Found value: 502 (MS+Na).

[0378] N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-66): Di-tert-butyl 1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2- To a solution of dicarboxylate 8-64 (41.0 g, 85.5 mmol, 1.0 equiv) in dichloromethane (820 mL) at 25° C., trifluoroacetic acid (410 mL) was added, and the mixture was stirred at 25° C. for 1 h. Acetone (480 mL) was added, and the mixture was stirred for an additional 0.5 h at 25° C. The mixture was concentrated to give N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide 8-66 (18.1 g, 66% yield). 1 H NMR(400MHz,DMSO-D6)δppm11.90(s,1H),8.31(d,J=12.8Hz,1H),3.05-3.14(m,1H),2.9 0-3.02(m,1H),2.11-2.19(m,7H),2.05-2.08(m,2H),1.98(d,J=2.0Hz,6H),1.34(s,3H). 19 F NMR (376MHz, DMSO-D6) δppm-75.04. LCMS(ESI+)m / z:[MH] + C 17 H 23 FN3O2 + Calculated value: 320.1, Measured value: 320.1.

[0379] N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-67): To a mixture of N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-66) (15.1 g, 47.3 mmol, 1.0 equiv) in acetic acid (302 mL) was added zinc powder (40.8 g, 624 mmol, 13.2 equiv) in portions, the mixture was stirred at 20 °C for 2 h, filtered, and the filtrate was concentrated under reduced pressure to give N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-67) (23.8 g, crude), which was used directly in the next step without further purification. 1 H NMR (400MHz, DMSO-D6) δppm 11.56 (s, 1H), 8.26-8.30 (m, 1H), 3.01-3.09 (m, 2H), 2.12-2.19 (m, 10H), 1.43 (s, 3H). 19 F NMR (376MHz, DMSO-D6) δppm-73.57. LCMS(ESI+)m / z:[MH] + C 14 H 18 FN2O2 + Calculated value: 265.1, Measured value: 265.1.

[0380] N,N'-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (8-68): To a mixture of N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-67) (23.8 g, 73.3 mmol, 1.0 equiv) in dichloromethane (414 mL) was added acetic anhydride (8.28 mL, 88.0 mmol, 1.2 equiv) and triethylamine (30.6 mL, 220 mmol, 3.0 equiv), and the mixture was stirred at 25 °C for 12 h, quenched with water (500 mL), and extracted with dichloromethane (3 x 200 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 70% ethyl acetate in petroleum ether to give N,N'-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (8-68) (7.5 g, 51% yield). 1 H NMR(400MHz,DMSO-D6)δppm11.81(s,1H),8.20-8.37(m,2H),2.94-3.03(m,1H),2.77 -2.88(m,1H),2.65(m,1H),2.06-2.18(m,6H),1.85(m,1H),1.79(s,3H),1.32(s,3H). 19 F NMR (376MHz, DMSO-D6) δppm-105.03.

[0381] N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (8-69): To a mixture of N,N'-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (8-68) (7.50 g, 24.4 mmol, 1.0 equiv) in methanol (105 mL) was added HCl / MeOH (105 mL, 4 M), the mixture was stirred at 25 °C for 2 h, concentrated under reduced pressure, and the residue was diluted with dichloromethane (300 mL) and extracted with saturated aqueous NaHCO (2 × 200 mL). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (8-69) (5.50 g, 89% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.91(s,1H),7.39(s,2H),6.36(d,J=12.4Hz,1H),2.78-2.89(m, 1H),2.67-2.75(m,2H),1.97(d,J=1.20Hz,3H),1.83-1.88(m,1H),1.80(s,3H),1.27(s,3H). 19 F NMR (376MHz, DMSO-D6) δppm-108.38. LCMS(ESI+)m / z:[MH] + C 14 H 18 FN2O2 + Calculated value: 265.1, Measured value: 265.1.

[0382] N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (8-70): To a mixture of N-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (500 mg, 1.89 mmol, 1.0 equiv) (8-69) and (5)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (547 mg, 2.08 mmol, 1.1 equiv) in toluene (25 mL) was added pyridine 4-methylbenzenesulfonate (71.3 mg, 0.283 mmol, 0.15 equiv) and o-cresol (1.44 mL, 13.8 mmol, 7.3 equiv) under argon at 120 °C, and the mixture was stirred at 130 °C for 13 h using a Dean-Stark trap to remove the formed water. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 7% methanol in dichloromethane to give N-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (8-70) (362 mg, 39% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.78(d,J=11.2Hz,1H),7.30(s,1H),6.53(d,J=10.0Hz,1H),5.25-5.54(m,4H),4.81-4.90(m,1H),3.24- 3.29(m,1H),2.86-3.11(m,3H),2.39(s,3H),1.95(d,J=3.6Hz,3H),1.84-1.89(m,2H),1.50(d,J=4.8Hz,3H),0.87(d,J=5.2Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.94. LCMS(ESI+)m / z:[MH] + C 27 H 27 FN3O5 + Calculated value: 492.1, Measured value: 492.2

[0383] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrohcnzo[de]pymno[3',4':6,7]indolizino[1,2b]quinoline-10,13(1H,9H)-dione hydrochloride (8-72). A mixture of N-((9)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) adjet-amide (8-70) (600 mg, 1.22 mmol, 1.0 equiv) in dioxane (6 mL) and concentrated hydrochloric acid (6 mL, 12 M) was stirred at 100 °C for 24 h in a sealed tube under argon. This was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (61.0 mg, 10% yield) and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-72) (85.0 mg, 14% yield). Note: Stereochemistry arbitrarily assigned.

[0384] Preparative HPLC conditions: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: HCl / H2O=0.040%v / v; B: ACN Column: Phenomenex® Luna 80 * 30mm* 3um Flow rate: 25mL / min モニターWavelength: 220 and び254nm Time B% 0.0 15 8.0 30 8.1 30 8.2 100 10.2 100 10.3 5 11.5 5

[0385] Spectra of 8-71: 1 H NMR (400 MHz, DMSO-D6) δ ppm 8.97(s,3H),7.89(d,J=10.8Hz,1H),7.36(s,1H),5.73-5.81(m,1H),5.56-5.65(m,1H),5.41-5 .49(m,2H),3.22(d,J=4.4Hz,2H),2.34-2.44(m,5H),1.82-1.94(m,5H),0.88(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.24. LCMS(ESI+)m / z:[MH] + C 25 H 25 FN3O4 + Calculated value: 450.1, measured value: 450.1

[0386] Spectra of 8-72: 1 H NMR (400 MHz, DMSO-D6) δ ppm 8.98(s,3H),7.89(d,J=10.8Hz,1H),7.36(s,1H),5.71-5.79(m,1H),5.57-5.64(m,1H),5. 46(s,2H),3.18-3.26(m,2H),2.36-2.44(m,5H),1.81-1.94(m,5H),0.87(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.22. LCMS(ESI+)m / z:[MH] + C 25 H 25 FN3O4+ Calculated value: 450.1, Measured value: 450.1

[0387] Example 9 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl 10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (9-74) (Figure 20)

[0388] [ka]

[0389] 2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (9-73): A mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (40.0 mg, 0.089 mmol, 1.0 equiv.)), N-ethyl-N-isopropylpropan-2-amine (46.5 μL, 0.266 mmol, 3.0 equiv.) in N,N-dimethylformamide (2 mL) was stirred at 25 °C for 0.5 h, cooled to 0 °C, and 2-chloro-2-oxoethyl acetate (14.6 mg, 11.5 μL, 0.106 mmol, 1.2 equiv.) was added dropwise over 5 min. The mixture was warmed to 25°C, stirred at 25°C for 3 hours, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with chloroform / methanol / water = 7 / 3 / 1 to give 2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (9-73) (30.0 mg, 61% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.84(s,1H),7.78(d,J=10.85Hz,1H),7.30(s,1H),6.51(s,1H),5.35-5.49(m,3H),5.13(d,J=19.07Hz,1H),4.66(d ,J=14.66Hz,1H),4.50(d,J=14.66Hz,1H),2.93-3.09(m,3H),2.39(s,3 H),1.99(s,3H),1.83-1.95(m,3H),1.55(s,3H),0.88(t,J=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.87. LCMS(ESI+)m / z:[MH] + C 29 H 29 FN3O7 + Calculated value: 550.2, Measured value: 550.2.

[0390] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (9-74): 2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl A mixture of 10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (9-73) (30.0 mg, 0.055 mmol, 1.0 equiv) in tetrahydrofuran (1 mL) was added to 1 N aqueous NaOH (0.235 mL, 4.3 equiv) under argon at 25 °C. The mixture was stirred at 25° C. for 2 hours, quenched with 1N aqueous hydrochloric acid (0.273 mL, 5.0 equiv.), concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with chloroform / methanol / water (7 / 3 / 1) to give N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (9-74) (13.5 mg, 50% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.31(s,1H),7.77(d,J=10.85Hz,1H),7.29(s,1H),6.50(s,1H),5.34-5.54(m,4H),4.91(d,J=19.07Hz, 1H),3.77-4.01(m,2H),3.22-3.31(m,1H),2.87-3.13(m,2H),2.39(s,3H),1.77-2.01(m,3H),1.58(s,3H),0.87(t,7=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.92. LCMS(ESI+)m / z:[MH] + C 27 H27 FN3O6 + Calculated value: 508.2, measured value: 508.1.

[0391] Example 10 N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (10-75)

[0392] [ka]

[0393] Example 10 (10-75) was prepared in a similar manner to Example 9, using 8-72 instead of 8-71.

[0394] 1 H NMR(400MHz,DMSO-D6)δppm8.31(s,1H),7.77(d,J=10.85Hz,1H),7.29(s,1H),6.50(s,1H),5.34-5.54(m,4H),4.91(d,J=19.07Hz, 1H),3.77-4.01(m,2H),3.22-3.31(m,1H),2.87-3.13(m,2H),2.39(s,3H),1.77-2.01(m,3H),1.58(s,3H),0.87(t,J=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.88. LCMS(ESI+)m / z:[MH] + C 27 H 27 FN3O6 + Calculated value: 508.2, measured value: 508.1.

[0395] Example 11 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamide hydrochloride (11-80) (Figure 21).

[0396] [ka]

[0397] 4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77): To a mixture of sodium 4-hydroxybutanoate (11-76) (2.10 g, 16.6 mmol, 1.0 equiv) and imidazole (1.70 g, 25.0 mmol, 1.5 equiv) in N,N-dimethylformamide (31.5 mL) under argon, tert-butylchlorodiphenylsilane (5.49 g, 19.9 mmol, 1.2 equiv) was added, and the mixture was stirred at 25 °C for 1 h, poured into ice-water (60 mL), and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography eluting with 30% ethyl acetate in petroleum ether to give 4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77) (2.60 g, 41% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.61-7.63(m,6H),7.46(dd,J=7.2,4.4Hz,4H),3. 72(t,J=6.4Hz,2H),2.65(t,J=7.2Hz,2H),1.87(t,J=6.8Hz,2H),1.00(s,9H). LCMS(ESI-)m / z:[MH] - C 20 H 25 O3Si - Calculated value: 341.2, Measured value: 341.2.

[0398] 4-((tert-butyldiphenylsilyl)oxy)butanoyl chloride (11-78): To a mixture of 4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77) (1.50 g, 4.10 mmol, 1.0 equiv) in dichloromethane (75 mL) under nitrogen at 0 °C, oxalyl dichloride (1.05 g, 8.24 mmol, 2.0 equiv) and N,N-dimethylformamide (9.02 mg, 0.03 equiv) were added, and the mixture was warmed to 25 °C, stirred at 25 °C for 2 h, and concentrated under reduced pressure to give 4-((tert-butyldiphenylsilyl)oxy)butanoyl chloride (11-78) (1.50 g, crude), which was used directly in the next step without purification. 1 H NMR(400MHz,CDCl3)δppm7.63-7.71(m,6H),7.42-7.48(m,4H),3.72(dt,J=11.6,6. 0Hz,2H),2.96-3.13(m,1H),2.63(t,7=7.6Hz,1H),1.91-2.01(m,2H),1.07(s,9H).

[0399] 4-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1.2-b]quinolin-1-yl)butanamide (11-79): To a mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (100 mg, 0.206 mmol, 1.0 equiv.) and trimethylamine (166 mg, 229 μL 1.65 mmol, 8.0 equiv.) in dichloromethane (5 mL) was added 4-((tert-butyldiphenylsilyl)oxy)butanoyl chloride (11-78) (303 mg, 0.839 mmol, 2.4 equiv.), the mixture was stirred at 25 °C for 1 h, filtered, the filtrate was concentrated, and the residue was purified by silica gel flash column chromatography eluting with 60% dichloromethane in ethyl acetate. The resulting product was 4-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (11-79) (30.0 mg, 17% yield). LCMS (ESI+) m / z: [MH] + C 45 H 49 FN3O6Si + Calculated value: 774.3, measured value: 774.2.

[0400] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1.2-b]quinolin-1-yl)-4-hydroxybutanamide hydrochloride (11-80): To a stirred mixture of 4-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7-indolizino[1,2-b]quinolin-1-yl)butanamide (11-79) (30.0 mg II-79) (mg, 0.039 mmol, 1.0 equiv) in dioxane (0.15 mL) was added HCl / dioxane (0.5 mL, 8 M) dropwise under nitrogen at 25° C., and the mixture was stirred at 25° C. for 1 h, filtered, and the filter cake was collected. The material was triturated with methyl tert-butyl ether (2 mL) at 25 °C for 30 min, collected by filtration, and dried under vacuum to give N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamide hydrochloride (11-80) (10.2 mg, 46% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.61(s,1H),7.78(d,J=10.8Hz,1H),7.31(s,1H),5.35-5.48(m,3H),4.87(d,J=18.8Hz,1H),3.37(t,J=6.4Hz,3H), 3.22-3.32(m,1H),2.86-3.11(m,3H),2.39(s,3H),2.25-2.33(m,2H),1 .79-1.95(m,3H),1.56-1.66(m,2H),1.51(s,3H),0.88(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.98. LCMS(ESI+)m / z:[MH] + C 29 H 31 FN3O6 + Calculated value: 536.2, Measured value: 536.2.

[0401] Example 12 N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamide hydrochloride (12-81)

[0402] [ka]

[0403] Example 12 (12-81) was made in a similar manner to Example 11, substituting 8-72 for 8-71. 1 H NMR(400MHz,DMSO-D6)δppm8.60(s,1H),7.78(d,J=10.8Hz,1H),7.30(s,1H),5.31-5.50(m,3H),4.86(d,J=18.8Hz,1H),3.36(t,J=6.4Hz,2H),3.2 3-3.31(m,1H),2.78-3.15(m,3H),2.39(s,3H),2.26-2.34(m,2H),1.75-1 .99(m,3H),1.60(quin,J=6.8Hz,2H),1.50(s,3H),0.86(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.95. LCMS(ESI+)m / z:[MH]+C 29 H 31 FN3O6 + Calculated value: 536.2, measured value: 536.1.

[0404] Example 13 (1S,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3,4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-84) and (1R,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3,4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-85) (Figure 22).

[0405] [ka]

[0406] N-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (13-82): To a mixture of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (8-63) (1.00 g, 4.01 mmol, 1.0 equiv) and cesium carbonate (261 mg, 0.802 mmol, 0.2 equiv) in dimethyl sulfoxide (16 mL) was added triethyl phosphite (1.33 g, 8.02 mmol, 1.38 mL, 2.0 equiv) at 25 °C. The reaction mixture was evacuated and filled with O 3 three times and stirred under O 3 (15 psi) at 25 °C for 24 h. After replacing the oxygen atmosphere with nitrogen, the mixture was diluted with water (80 mL) and extracted with ethyl acetate (2 × 40 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 15% ethyl acetate in petroleum ether to give N-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-82) (620 mg, 58% yield). 1H NMR(400MHz,DMSO-D6)δppm11.97(s,1H),8.29(d,J=13.08Hz,1H),5.47(s,1H),2.94-3.04(m ,1H),2.78-2.88(m,1H),2.17(s,3H),2.11(d,J=1.59Hz,3H),1.96-2.09(m,2H),1.28(s,3H). 19 F NMR (376MHz, DMSO-D6) δppm-104.38. LCMS(ESI+)m / z:[MH] + C 14 H 17 FNO3 + Calculated value: 266.1, measured value: 266.0.

[0407] 8-Amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1(2H-one (12-83) To a mixture of N-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12-82) (300 mg, 1.13 mmol, 1.0 equiv) in methanol (6 mL) was added HCl / MeOH (6 mL, 4 M) dropwise at 25 °C, the mixture was stirred at 25 °C for 1.5 h, cooled to 0 °C, and the pH was adjusted to 7 by adding saturated NaHCO at 0 °C. It was extracted with dichloromethane (3 × 10 mL), and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give 8-amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (13-83) (250 mg, 99% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-D6)δppm7.41(brs,2H),6.37(d,J=12.59Hz,1H),5.10(s ,1H),2.81-2.92(m,1H),2.63-2.75(m,1H),1.89-2.01(m,5H),1.22(s,3H). 19 F NMR (376MHz, DMSO-D6) δppm-108.05.

[0408] (1S,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3,4':6,7-indolizino[1,2-b]quinoline-10,13-dione 13-84 and (1R,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-85). To a mixture of 8-amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1(2H-one 13-83) (54.0 mg, 241 μmol, 1.0 equiv) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (95.5 mg, 0.362 mmol, 1.5 equiv) in xylene (5.4 mL) was added 4-methylbenzenesulfonic acid (24.9 mg, 0.145 mmol, 0.6 equiv) under argon at 120° C., and the mixture was stirred in a sealed tube at 120° C. for 24 h. It was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 60% tetrahydrofuran in petroleum ether to give a residue, which was further separated by preparative HPLC and lyophilized to give (1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7-indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-84)( The resulting product was (70.1 mg, 13% yield) (compound 13-84 may be the opposite enantiomer of that shown), and (1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-85) (73.3 mg, 13% yield) (compound 13-85 may be the opposite enantiomer of that shown). Note: Stereochemistry is arbitrarily assigned.

[0409] Fractionation HPLC method: Machine: ギルソン 281 セミ HPLC システム Mobile phase: A: H2O; B: CH3OH Color:Phenomenex(registered trademark) Gemini-NX 80×30mm×3um Flow rate: 25mL / min モニターWavelength: 220 and び254nm Time B% 0.0 40 8.0 70 8.1 70 8.2 100 10.2 100 10.3 40 11.5 40

[0410] Spectra of 13-84: 1 H NMR(400MHz, DMSO-D6)δ ppm 7.76(d,J=10.88Hz,1H),7.31(s,1H),6.51(s,1H),5.79(br s,1H),5.43(s,4H),3.25(br d,J=2.57Hz,1H),2.96-3.09(m,1H),2.38(s,3H),2.24(br dd,J=13.08,3.06Hz,1H),2.11(td,J=13.24,5.69Hz,1H),1.87(m,2H),1.45(s,3H),0.88(t,J=7.34Hz,3H). 19 F NMR (376MHz, DMSO-D6) 5ppm-112.10. LCMS(ESI+)m / z:[MH] + C 25 H 24 FN2O5 + Calculated value: 451.1, measured value: 451.1. SFC: RT=1.338 points.

[0411] Spectra of 13-85: 1H NMR(400MHz,DMSO-D6)δ ppm 7.77(d,J=11.00Hz,1H),7.31(s,1H),6.50(s,1H),5.80(br s,1H),5.44(s,4H),3.25(br s,1H),2.96-3.10(m,1H),2.38(s,3H),2.24(br dd,J=12.65,3.24Hz,1H),2.12(td,J=13.14,5.38Hz,1H),1.78-1.93(m,2H),1.44(s,3H),0.87(t,J=7.27Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-112.07. LCMS(ESI+)m / z:[MH] + C 25 H 24 FN2O5 + Calculated value: 451.1, Measured value: 451.1. SFC: RT = 1.453 min.

[0412] Example 14 1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92) (Figure 23).

[0413] [ka]

[0414] N-(7-(allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-86): To a mixture of N-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (13-82) (500 mg, 1.88 mmol, 1.0 equiv) and AgO (4.37 g, 18.8 mmol, 10 equiv) in acetonitrile (10 mL) was added 3-iodoprop-1-ene (6.33 g, 3.44 mL, 37.7 mmol, 20 equiv) under argon at 20 °C, and the mixture was stirred at 40 °C for 12 h and cooled to 25 °C. (Three additional reactions were set up as above, and the four reaction mixtures were combined.) The combined reaction mixture was filtered through a Celite pad, the filter cake was washed with dichloromethane (200 mL), the combined filtrate was concentrated, and the residue was purified by silica gel flash column chromatography eluting with 5% ethyl acetate in petroleum ether to give N-(7-(allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-86) (1.30 g, 54% yield). 1 H NMR(400MHz,DMSO-D6)δppm11.82(s,1H),8.28(d,J=13.13Hz,1H),5.70-5.83(m,1H),5.14(m,1H),5.01(m,1H),3.99(m,1H),3.79(m,1H) ),3.00(m,1H),2.77-2.88(m,1H),2.36(dt,J=14.07,5.22Hz,1H),2.17(s,3H),2.11(d,J=1.63Hz,3H),2.03(m,1H),1.30-1.38(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-104.04. LCMS(ESI+)m / z:[MH] + C 17 H 21 FNO3 + Calculated value: 306.1, Measured value: 306.1.

[0415] N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-87): To a stirred mixture of N-(7-(allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (14-86) (1.30 g, 4.17 mmol, 1.0 equiv) in dichloromethane (26 mL) and methanol (13 mL) was bubbled ozone for 5 min at −78° C., followed by the addition of dimethylsulfane (648 mg, 0.766 mL, 10.4 mmol, 2.5 equiv). The reaction mixture was warmed to 25° C., stirred at 25° C. for 1 h, quenched with water (40 mL), and extracted with dichloromethane (3×40 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-87) (1.20 g, crude), which was used directly in the next reaction without further purification.

[0416] N-(3-fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-88): To a mixture of N-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (14-87) (1.20 g, 2.73 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) and HO (15 mL) at 0 °C, sodium tetrahydroborate (51.7 mg, 1.37 mmol, 0.5 equiv) was added portionwise and the mixture was stirred at 0 °C for 10 min, warmed to 25 °C, stirred at 25 °C for 20 min, quenched with water (30 mL) and extracted with dichloromethane (3 × 40 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 40% ethyl acetate in petroleum ether to give N-(3-fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-88) (800 mg, 61% yield).1 H NMR(400MHz,CDCl3)δppm12.07(brs,1H),8.46(d,J=12.76Hz,1H),3.63-3.76(m,2H),3.53-3.61(m,1H),3.44-3.52(m,1H),3. 03-3.23(m,1H),2.74-2.92(m,1H),2.35-2.46(m,1H),2.22-2.26(m,3H),2.12(brs,3H),2.03-2.11(m,1H),1.37-1.48(m,3H). 19 F NMR (376MHz, CDCl3) δppm-101.12. LCMS(ESI+)m / z:[MH] + C 16 H 21 FNO4 + Calculated value: 310.1, Measured value: 310.1.

[0417] 8-Amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H-one (14-89): To a solution of N-(3-fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-88) (200 mg, 581 μmol, 1.0 equiv) in methanol (8 mL) under argon at 20° C., 2N hydrochloric acid (8 mL) was added, and the mixture was stirred at 60° C. for 1 h and cooled to 0° C. (Three additional reactions were set up as above, and the four reaction mixtures were combined.) The combined reaction mixture was cooled to 0° C., the pH was adjusted to 8 by the addition of saturated NaHCO3, warmed to 25° C., and extracted with dichloromethane (2×100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with 40% ethyl acetate in petroleum ether to give 8-amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-89) (310 mg, 45% yield). 1H NMR(400MHz,CD3OD)δppm6.30(d,J=12.26Hz,1H),3.49-3.61(m,3H),3.39(m,1H),3. 01-3.10(m,1H),2.69-2.79(m,1H),2.32(m,1H),2.00(d,J=2.25Hz,4H),1.37(s,3H). 19 F NMR (376MHz, CD3OD) δppm-108.91. LCMS(ESI+)m / z:[MH] + C 14 H 19 FNO3 + Calculated value: 268.1, Measured value: 268.1.

[0418] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1.2-b]quinoline-10,13(1H,9H)-dione (14-90): 8-amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3 To a mixture of (S)-4-dihydro-naphthalen-1(2H)-one (14-89) (150 mg, 0.561 mmol, 1.0 equiv) and (S)-4-ethyl-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (162 mg, 0.617 mmol, 1.1 equiv) in toluene (7 mL) at 120 °C under argon, o-cresol (443 mg, 0.426 mL, 4.10 mmol, 7.3 equiv) and pyridine 4-methylbenzenesulfonate (21.1 mg, 0.084 mmol, 0.15 equiv) were added, and the mixture was stirred in a sealed tube at 120 °C for 32 h and cooled to 25 °C. (One additional reaction was set up as above, and the two reaction mixtures were combined.) The combined reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography eluting with ethyl acetate to give (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15 tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-90) (210 mg, 22% yield). 1 H NMR(400MHz,DMSO-D6)δppm7.76(dd,J=10.85,3.46Hz,1H),7.30(s,1H),6.51(d,J=2.15Hz,1H),5.32-5.62(m,4H),4.74(q,J=5.2 1Hz,1H),3.58-3.71(m,3H),2.92-3.06(m,1H),2.14-2.42(m,6H),1.81-1.91(m,2H),1.53(brd,J=6.79Hz,3H),0.81-0.94(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.89. LCMS(ESI+)m / z:[MH] + C 27 H 28 FN2O6 + Calculated value: 495.2, Measured value: 495.2.

[0419] 1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzolide]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92). (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-90) (210 mg, 0.424 mmol) was dissolved in methanol and analyzed by chiral SFC (instrument: Waters SFC 150 preparative SFC. Column: DAICEFT CHIRAFPAK AD (250 mm)). *30 mm, 10 μm); mobile phase: A is CO2, B is ethanol; gradient: B% = 50% isocratic elution mode; flow rate: 70 g / min; wavelength: 220 nm; column temperature: 40 °C; system back pressure: 120 bar) to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indo-lidino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) (65.1 mg, 31% yield). (Compound 14-91 is the opposite enantiomer to that shown.) (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92) (62.5 mg, 30% yield) (compound 14-92 may be the opposite enantiomer to that shown). Note: Stereochemistry assigned arbitrarily.

[0420] Spectra of 14-91: 1 H NMR(400MHz,DMSO-D6)δ ppm 7.77(br d,J=10.88Hz,1H),7.30(s,1H),5.30-5.63(m,4H),3.64(br s,3H),3.28-3.31,(m,2H),2.99(br s,1H),2.38(br s,3H),2.33(br s,1H),2.20(br s,1H),1.76-1.93(m,2H),1.53(br s,3H),0.81-0.92(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.87. LCMS(ESI+)m / z:[MH] + C 27 H 28 Calculated for FN2O6: 495.2, Found: 495.1. Chiral SFC: RT = 1.715 min.

[0421] Spectra of 14-92: 1H NMR(400MHz,DMSO-D6)δ ppm 7.77(br d,J=10.88Hz,1H),7.31(s,1H),5.33-5.57(m,4H),3.65(br s,3H),3.28-3.30,(m,2H),2.95(br s,1H),2.38(br s,3H),2.33(br s,1H),2.17(br s,1H),1.76-1.93(m,2H),1.53(br s,3H),0.84-0.92(m,3H). 19 F NMR (376MHz, DMSO-D6) δppm-111.90. LCMS(ESI+)m / z:[MH] + C 27 H 28 FN2O6 + Calculated: 495.2, Found: 495.3. Chiral SFC: RT=1.948 min.

[0422] Example 15 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94) (Figure 24)

[0423] [ka]

[0424] (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93): A mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15 tetrahydro-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (Exatecan) (100 mg, 0.188 mmol, 1.0 equiv) and triethylamine (19.0 mg, 0.188 mmol, 1.0 equiv) in methanol (2 mL) was stirred at 25 °C for 0.5 h, followed by the addition of 2-((tert-butyldimethylsilyl)oxy)acetaldehyde (32.8 mg, 0.188 mmol, 1.0 equiv). The reaction mixture was stirred at 25° C. for 1 h, after which sodium cyanoborohydride (17.7 mg, 0.282 mmol, 1.5 equiv) was added and stirred for another 2 h at 25° C. It was quenched by the addition of water (0.2 mL), diluted with dichloromethane (5 mL), dried over NaSO, filtered, concentrated under reduced pressure, and the residue was purified by preparative HPLC to give (1H,9H)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93) (25.0 mg, 22% yield). 1 H NMR(400MHz,DMSO-D6)δ ppm 7.74(d,J=11.13Hz,1H),7.30(s,1H),6.51(s,1H),5.34-5.47(m,4H),4.30(br s,1H),3.69(t,J=5.93Hz,2H),3.15-3.24(m,1H),2.96-3.07(m,1H),2.87(br d,J=5.14Hz,1H),2.73-2.82(m,1H),2.37(s,3H),2.22(dd,J=13.57,4.89Hz,1H),1.99-2.14(m, 2H),1.87(dt,J=17.36,7.09Hz,2H),0.87(t,J=7.34Hz,3H),0.82(s,9H),0.03(d,J=3.18Hz,6H). 19F NMR(400MHz,DMSO-D6)δ ppm-111.83.LCMS(ESI+)m / z:[MH] + C 32 H 41 FN3O5Si + Calculated value: 594.2, Measured value: 594.2.

[0425] HPLC method: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: 10mM NH4HCO3 in H2O; B: CH3CN Column: Waters Xbridge BEH C 18 100 * 30mm * 10um Flow rate: 25mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 65 10.0 95 10.1 95 10.2 100 12.2 100 12.3 65 13.5 65

[0426] 1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94): A solution of (1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93) (25 mg, 0.042 mmol, 1.0 equiv) in methanol (0.5 mL) was added to HCl / MeOH (2.5 mL) , 4M) was added dropwise under nitrogen at 25 °C, and the mixture was stirred at 25 °C for 1 hour, filtered, collected, and dried under vacuum to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15 tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94) (18.0 mg, 87% yield). 1 H NMR(400MHz,DMSO-D6)δppm8.81-9.14(m,2H),7.89(d,J=10.85Hz,1H),7.35(s ,1H),6.56(s,1H),5.54-5.67(m,1H),5.37-5.50(m,3H),5.28(brd,J=0.95Hz, 1H),5.10(brs,1H),3.71(brs,2H),3.09-3.26(m,3H),2.79(brd,J=13.95Hz,1 H),2.41(s,3H),2.09-2.25(m,1H),1.77-1.96(m,2H),0.88(t,J=7.33Hz,3H). 19 F NMR (376MHz, DMSO-D6) δppm-110.92. LCMS(ESI+)m / z:[MH] + C 26 H 27 FN3O5 + Calculated value: 480.2, Measured value: 480.2.

[0427] Example 16 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (16-95)

[0428] [ka]

[0429] Example 16 (16-95) was made in a similar manner to Example 15, substituting 8-71 for exatecan. 1 H NMR(400MHz,CD3OD)δppm7.82(d,J=10.49Hz,1H),7.68(s,1H),5.71-5.78 (m,1H),5.57-5.65(m,2H),5.41(d,J=16.33Hz,1H),3.74-3.84(m,2H),3.3 5(brs,2H),3.20-3.28(m,2H),2.74(dt,J=14.16,5.80Hz,1H),2.39-2.53( m,4H),2.09(s,3H),1.98(qd,J=7.17,3.99Hz,2H),1.02(t,J=7.33Hz,3H). 19 F NMR (376MHz, CD3OD) δppm-111.87. LCMS(ESI+)m / z:[MH] + C 27 H 29 FN3O5 + Calculated value: 494.2, measured value: 494.1.

[0430] Example 17 (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (17-96)

[0431] [ka]

[0432] Example 17 (17-96) was made in a similar manner to Example 15, substituting 8-72 for exatecan. 1 H NMR(400MHz,CD3OD)δppm7.82(d,J=10.39Hz,1H),7.68(s,1H),5.73-5.81(m,1H),5.63(d,J=3.30Hz,1H),5.58(s,1H),5.42(d,J=16.26Hz,1H),3 .74-3.87(m,2H),3.34(brs,2H),3.22-3.28(m,2H),2.70-2.82(m,1H),2 .36-2.56(m,4H),2.10(s,3H),1.89-2.04(m,2H),1.01(t,J=7.34Hz,3H). 19 F NMR (376MHz, CD3OD) δppm-111.90. LCMS(ESI+)m / z:[MH] + C 27 H 29 FN3O5 + Calculated value: 494.2, measured value: 494.1.

[0433] Example 18 S-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamido)-N-(2-(((2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-112 (Figure 25).

[0434] [ka]

[0435] 2,5-Dioxopyrrolidin-1-yl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98): To a mixture of 2-[[2-(benzyloxycarbonylamino)acetyl]amino]acetic acid (18-97) (55.0 g, 206 mmol, 1.0 equiv.) in acetonitrile (550 mL) was added 1-hydroxypyrrolidine-2,5-dione (26.1 g, 227 mmol, 1.1 equiv.) and dN 1 -((ethylimino)methylene)-N 3 ,N 3 -Dimethylpropane Benzene-1,3-diamine hydrochloride (47.5 g, 247 mmol, 1.2 equiv) was added, and the mixture was stirred at 25 °C for 4 h and cooled to -10 °C, resulting in the formation of a white material. The suspension was diluted with water (50 mL), stirred, filtered, and the filter cake was dried under vacuum to give 2,5-dioxopyrrolidin-1-yl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98) (85.0 g, 175 mmol, 85% yield), which was used directly in the next step without purification. 1 H NMR(400MHz,DMSO-d6)δppm2.81(s,4H),2.93-3.12(m,2H),3.69(d,J=6.11Hz,2H),4.27(d,J =5.87Hz,2H),5.04(s,2H),7.24-7.45(m,5H),7.57(t,J=6.11Hz,1H),8.58(t,J=5.87Hz,1H). LCMS(ESI+)m / z:[MH] + C 16 H 18 N3O7 + Calculated value: 364.1, Measured value: 364.1.

[0436] (S)-11-benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (18-99): To a mixture of 2,5-dioxopyrrolidin-1-yl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98) (85.0 g, 175 mmol, 1.0 equiv) in acetonitrile (425 mL) and HO (425 mL) was added (2S)-2-amino-3-phenyl-propanoic acid (34.7 g, 210 mmol, 1.2 equiv) and trimethylamine (26.8 mL, 193 mmol, 1.1 equiv) and the mixture was stirred at 25 °C for 2 h, cooled to 0 °C, and hydrochloric acid (12 M, 16.2 mL, 1.1 equiv) was added and stirred at 0 °C for 6 h. The resulting suspension was warmed to 25 °C, filtered, and the filter cake was dried under vacuum to give (S)-11-benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (18-99) (50.0 g, 108 mmol, 62% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,DMSO-d6)δppm2.59(s,1H),2.88(dd,J=13.77,9.00Hz,1H),3.04-3.11(m,1H),3.60-3.79(m,4H),4.43(td,J=8.43,5.19Hz,1 H),5.03(s,2H),7.15-7.40(m,10H),7.50(t,J=6.02Hz,1H),8.04(brt,J=5.60Hz,1H),8.16(d,J=8.11Hz,1H),12.72(brd,J=5.01Hz,1H). LCMS(ESI+)m / z:[MH] + C 21 H 24 N3O6 + Calculated value: 414.2, Measured value: 414.2.

[0437] (2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (18-101): To a mixture of 2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]acetic acid (18-100) (100 g, 282 mmol, 1.0 equiv.) in tetrahydrofuran (1.5 L) and acetic acid (300 mL), lead tetraacetate (150 g, 338 mmol, 1.2 equiv.) was added, and the mixture was stirred at 50° C. for 36 h, cooled to 25° C., and filtered. The filtrate was washed twice with aqueous trisodium citrate dihydrate (2.5 L, 20% wt.), and the organic phase was concentrated to approximately 2.0 L. Water (2.5 L) was added, and the mixture was stirred at 5° C. for 2 h. The formed precipitate was filtered, washed with a cold (5 °C) mixture of tetrahydrofuran and water (3:10, 1.2 L), and dried under vacuum to give methyl (2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (18-101) (100 g, 86% yield). 1 H NMR(400MHz,CDCl3)δppm2.09(s,3H),3.90(d,J=5.01Hz,2H),4.19-4.28(m,1H),4.41-4.52(m,2H),5.25(d,J=6.97Hz,2H), 5.41-5.60(m,1H),7.17-7.27(m,1H),7.29-7.36(m,2H),7.41(t,J=7.40Hz,2H),7.55-7.64(m,2H),7.77(d,J=7.58Hz,2H). LCMS(ESI+)m / z: [MNa] + C 20 H 20 N2O5Na + Calculated value: 391.1, Measured value: 391.1.

[0438] Benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (18-102): To a mixture of methyl (2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (18-101) (80.0 g, 173 mmol, 1.0 equiv.) in 1,2-dimethoxyethane (1.5 L) was added benzyl 2-hydroxyacetate (57.7 g, 49.3 mL, 347 mmol, 2.0 equiv.) and cooled to 0 °C with acetic acid (5.22 g, 86.8 mmol, 4.97 mL, 0.5 equiv.). The mixture was stirred at 0 °C for 1 h and then cooled to 0 °C with sodium hydroxide (10 N, 1 A solution of benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (18-102) (7.3 mL, 1.0 equiv.) was added dropwise and stirred at 0° C. for 1 h, and water (1.2 L) was added and stirred at 0° C. for 2 h. The formed white precipitate was filtered, washed with cold (5° C.) 1,2-dimethoxyethane:water (1:2, 500 mL) and methyl tert-butyl ester (500 mL), and dried under vacuum at 40° C. to give benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (18-102) (60.0 g, 69% yield). 1 H NMR(400MHz,CDCl3)δppm3.82(d,J=5.26Hz,2H),4.16-4.29(m,3H),4.45(d,J=6.80Hz,2H),4.83(d,J=7.02Hz,2H), 5.16(s,2H),5.45(t,J=5.59Hz,1H),7.08(s,1H),7.28-7.48(m,9H),7.59(d,J=7.23Hz,2H),7.77(d,J=7.45Hz,2H). LCMS(ESI+)m / z:[MNa] + C 27 H 26 N2O6Na + Calculated value: 497.2, measured value: 497.1.

[0439] Benzyl 2-((2-aminoacetamido)methoxy)acetate HOBT salt (18-103): To a mixture of benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (18-102) (60.0 g, 126 mmol, 1.0 equiv) in acetonitrile (1.08 L) was added DBU (9.53 mL, 63.2 mmol, 0.5 equiv) at 0° C. The mixture was stirred at 25° C. for 4 h, cooled to 0° C., and HOBt (34.1 g, 252 mmol, 2.0 equiv) was added and stirred at 0° C. for 1.5 h. It was warmed to 25 °C, filtered, and the filter cake was washed with cold (5 °C) acetonitrile (200 mL) and dried under vacuum to give benzyl 2-((2-aminoacetamido)methoxy)acetate·HOBt (18-103) (46.0 g, 84% yield). 1 H NMR(400MHz,METHANOL-d4)δ ppm 3.68(s,2H),4.21(s,2H),4.79(s,2H),5.18(s,2H),7.22-7.42(m,7H),7.62-7.74(m,2H).LCMS(ESI-)m / z:[MH]-C 18 H 33 N5O6 + Calculated value: 269.1, measured value: 269.0.

[0440] (S)-Benzyl 11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate (18-105): To a mixture of benzyl 2-((2-aminoacetamido)methoxy)acetate HOBt (18-103) (46.0 g, 118 mmol, 1.0 equiv.) in acetonitrile (500 mL) was added (S)-11-benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid (18-104) (45.0 g, 108 mmol, 0.9 equiv.) and N 1 -((ethylimino)methylene)-N 3 ,N 3To the resulting mixture was added 1,3-dimethylpropane-1,3-diamine hydrochloride (25.5 g, 133 mmol, 1.1 equiv.) at 0° C. The mixture was stirred at 0° C. for 3.5 h, water (200 mL) was added, and the mixture was stirred at 0° C. for 2 h. The precipitate formed was filtered and washed with cold (5° C.) acetonitrile. The solid was purified by washing with water (1:2, 201 mL) and dried under vacuum to give (S)-benzyl 11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate (18-105) (30.0 g, 34% yield). 1 H NMR(400MHz,DMSO-d6)δppm2.79(dd,J=13.67,9.92Hz,1H),3.06(dd,J=13.67,4.41Hz,1H ),3.56-3.66(m,3H),3.69-3.81(m,3H),4.15(s,2H),4.51(td,J=8.76,4.52Hz,1H),4.63 (d,J=6.62Hz,2H),5.03(s,2H)5.13-5.17(m,2H),7.03-7.46(m,15H),7.49-7.55(m,1H), 8.00-8.10(m,1H),8.18(d,J=7.94Hz,1H),8.36(t,J=5.62Hz,1H),8.62(t,J=6.73Hz,1H).

[0441] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oic acid (18-106): To a mixture of (S)-benzyl 11benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate (18-105) (10.0 g, 15.4 mmol, 1.0 equiv.) in tetrahydrofuran (210 mL) and HO (140 mL), Pd / C (10 wt%) (4.40 g, 1.2 equiv.) was added under argon at 25 °C, and the suspension was evacuated and filled with H three times and stirred under H (15 psi) for 2.5 h at 25 °C. After the H atmosphere was replaced with argon, it was filtered through a Celite pad, and the filter cake was washed with water (80 mL) and ethanol (150 mL). The filtrate was concentrated to approximately 30 mL, ethanol (120 mL) was added, and the mixture was stirred for 2 h at 0° C. The formed precipitate was collected by filtration, washed with ethanol (30 mL), and dried under vacuum to give (5)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oic acid (18-106) (4.00 g, 58% yield). 1 H NMR (400MHz, D2O) δppm 2.88-3.26 (m, 2H), 3.64-4.16 (m, 8H), 4.55-4.71 (m, 3H), 7.11-7.50 (m, 5H). LCMS(ESI-)m / z:[MH] - C 18 H 24 N5O7 - Calculated value: 422.2, measured value: 422.1.

[0442] (E)-4-((4-(2-carboxyethyl)phenyl)amino)-4-oxobut-2-enoic acid (18-108): To a mixture of furan-2,5-dione (4.16 g, 42.3 mmol, 1.0 equiv) in ether (42 mL) was added a solution of 3-(4-aminophenyl)propanoic acid (18-107) (7.00 g, 42.3 mmol, 1.0 equiv) and 2,6-dimethylpyridine (4.58 g, 4.98 mL, 42.3 mmol, 1.0 equiv) in tetrahydrofuran (70 mL) at 25 °C. The reaction mixture was stirred at 65 °C for 0.5 h, cooled to 25 °C, filtered, and the filter cake was washed with methyl tert-butyl ether (150 mL) and dried under vacuum to give (£)-4-((4-(2-carboxyethyl)phenyl)amino)-4-oxobut-2-enoic acid (18-108) (11.0 g, 98% yield). 1 H NMR(400MHz,DMSO-d6)δppm12.12-13.09(m,2H),10.37(s,1H),7.52(d,J=8.4Hz,2H),7.18(d,J =8.4Hz,2H),6.46(d,J=12.0Hz,1H),6.30(d,J=12.0Hz,1H),2.78(t,J=7.6Hz,2H),2.52(s,2H). LCMS(ESI-)m / z:[MH] - C 13 H 12 No. 5 - Calculated value: 262.0, measured value: 261.9.

[0443] 3-(4-(2.5-dioxo-2.5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid (18-109): To a mixture of (E)-4-((4-(2-carboxyethyl)phenyl)amino)-4-oxobut-2-enoic acid (18-108) (11.0 g, 41.7 mmol, 1.0 equiv) in acetic anhydride (100 mL) was added potassium acetate (2.26 g, 22.9 mmol, 0.55 equiv) under nitrogen. The suspension was evacuated and backfilled with nitrogen three times, stirred at 145 °C for 0.5 h, cooled to 20 °C, and concentrated under reduced pressure. The residue was diluted with ethyl acetate (300 mL), washed with brine, dried over NaSO, filtered, concentrated, and the residue was purified by silica gel flash column chromatography eluting with 33%-100% ethyl acetate in petroleum ether containing 20% ​​tetrahydrofuran to give 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid (18-109) (3.60 g, 29% yield). 1 H NMR(400MHz,DMSO-d6)δppm12.16(s,1H),7.33(d,J=8.4Hz,2H),7.20-7.24(m,2H),7.16(s,2H),2.86(t,J=7.6Hz,2H),2.57(t,J=7.6Hz,2H). LCMS(ESI-)m / z:[MH]C 13 H 10 No. 4 - Calculated value: 244.0, measured value: 243.9.

[0444] 2,5-Dioxopyrrolidin-1-yl 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110): To a mixture of 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid (18-109) (3.60 g, 14.6 mmol, 1.0 equiv.) in acetonitrile (72 mL) was added 1-hydroxypyrrolidine-2,5-dione (1.77 g, 15.4 mmol, 1.05 equiv.) and N,N'-methanediylidene dicyclohexanamine (3.18 g, 15.4 mmol, 1.05 equiv.). The reaction mixture was stirred at 25 °C for 3 h, filtered, and the filtrate containing 2,5-dioxopyrrolidin-1-yl 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110) was used directly in the next step without workup or purification. LCMS(ESI+)m / z:[M−H] + C 17 H 15 N2O6 + Calculated value: 343.1, Measured value: 343.1.

[0445] (S)-10-benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oic acid (18-111): To a mixture of 2,5-dioxopyrrolidin-1-yl 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110) (1.62 g, 4.72 mmol, 1.0 equiv) in acetonitrile (80 mL) was added a solution of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oic acid (18-106) (2.00 g, 4.72 mmol, 1.0 equiv) and N-ethyl-N-isopropylpropan-2-amine (0.658 mL, 3.78 mmol, 0.8 equiv) in HO (20 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 12 h, filtered, concentrated, and the residue was purified by preparative HPLC to give (S)-10-benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oic acid (18-111) (0.78 g, 24% yield). 1 H NMR(400MHz,DMSO-d6)δppm8.53-8.64(m,1H),8.31(t,J=5.6Hz,1H),8.23(t,J= 5.6Hz,1H),8.16(d,J=8.0Hz,1H),8.08(t,J=5.2Hz,1H),7.14-7.38(m,11H),4.6 1(d,J=6.8Hz,2H),4.44-4.55(m,1H),3.97(s,2H),3.64-3.80(m,5H),3.63(d,J =5.6Hz,1H),3.06(dd,J=13.6,4.4Hz,1H),2.78-2.89(m,3H),2.53-2.60(m,1H). LCMS(ESI-)m / z:[MH]C 31 H 33 NO 10 - Calculated value: 649.2, measured value: 649.3.

[0446] Preparative HPLC conditions: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: H2O; B: ACN Column: Agela DuraShell C18 250 * 70mm * 10um Flow rate: 130mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 10 20.0 40 20.1 40 20.2 100 26.2 100 26.3 10 27.5 10

[0447] S-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamide)-N-(2-(((2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1.2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-112): (S)-10-benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oic acid (18-111) (246 mg, 0.378 mmol, 1.0 equiv.), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6 ,7]Indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (170 mg, 0.378 mmol, 1.0 equiv.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate (124 mg, 0.378 mmol, 1.0 equiv.), and 4-methylmorpholine (0.166 mL, 1.51 mmol, 4.0 equiv.) in N,N-dimethylformamide (5 mL) were stirred at 25 °C for 1 hour. The mixture was filtered, and the filtrate was purified by preparative HPLC to give (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamido)-N-(2-(((2-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (18-112) (130 mg, 30% yield). 1H NMR(400MHz,DMSO-d6)δppm8.39(s,1H),8.32-8.37(m,1H),8.12-8.22(m,2H),8.04(t,J=5.6Hz,1H),7.78(d,J=10.8Hz,1H) ,7.26-7.34(m,3H),7.18-7.26(m,6H),7.16(s,2H),6.51(s,1H),5.28-5.50(m,3H),4.92(d,J=18.8Hz,1H),4.67(d,J=6.8H z,2H),4.51(d,J=4.0Hz,1H),3.90-4.05(m,2H),3.64-3.83(m,5H),3.62(d,J=5.6Hz,1H),3.21-3.27(m,1H),3.00-3.13(m, 2H),2.89-2.99(m,1H),2.76-2.87(m,3H),2.46(s,3H),2.38(s,3H),1.75-2.01(m,3H),1.58(s,3H),0.87(t,7=7.2Hz,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.87. LCMS(ESI+)m / z:[MH] + C 56 H 57 FN9O 13 + Calculated value: 1082.4, measured value: 1082.3.

[0448] HPLC conditions for fractionation: Machine: ギルソン 281 セミ HPLC システム Mobile phase: A: H2O; B: ACN KARARA:Phenomenex (registered trademark) C 18 75 * 30mm * 3um Flow rate: 25mL / min モニターWavelength: 220 and び254nm Time B% 0.0 20 8.0 50 8.1 50 8.2 100 10.2 100 10.3 20 11.5 20

[0449] Example 19 N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide (19-113)

[0450] [ka]

[0451] Example 19 (19-113) was made in a similar manner to Example 18 (18-112). 1 H NMR(400MHz,DMSO-d6)δppm8.87(t,J=5.6Hz,1H),8.69(t,J=6.4Hz,1H),8.32-8.41(m,2H),8.10-8.19(m,2H),7.90( d,J=7.6Hz,1H),7.83-7.86(m,1H),7.78(d,J=10.8Hz,1H),7.55-7.61(m,1H),7.48-7.53(m,1H),7.31(s,1H),7.18-7 .26(m,6H),7.13-7.18(m,1H),6.51(s,1H),5.30-5.47(m,3H),4.92(d,J=18.8Hz ,1H),4.67(d,J=7.2Hz,2H),4.51(dd,J=8.0,4.4Hz,1H),3.84-4.04(m,4H),3.71 -3.83(m,3H),3.54-3.68(m,1H),3.25(s,1H),2.88-3.15(m,3H),2.82(dd,J=13. 6,9.6Hz,1H),2.38(s,3H),1.73-2.04(m,3H),1.58(s,3H),0.87(t,7=7.2Hz,3H). 19F NMR (376MHz, DMSO-d6) δppm-111.87. LCMS(ESI+)m / z:[MH] + C 54 H 53 FN9O 13 + Calculated value: 1054.3, Measured value: 1054.3.

[0452] Example 20 N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (20-114)

[0453] [ka]

[0454] Example 20 (20-114) was made in a similar manner to Example 18 (18-112). 1H NMR(400MHz,DMSO-d6)δppm8.69(t,J=6.4Hz,1H),8.39(s,1H),8.34(,J=5.6Hz,1H),8.14(d,J=8. 0Hz,1H),8.04(t,J=5.6Hz,1H),7.99(t,J=5.6Hz,1H),7.78(d,J=10.8Hz,1H),7.31(s,1H),7.20- 7.28(m,4H),7.14-7.20(m,1H),6.98(s,2H),6.51(s,1H),5.35-5.48(m,3H),4.92(d,J=18.8Hz,1 H),4.67(d,J=6.8Hz,2H),4.46-4.54(m,1H),3.90-4.04(m,2H),3.70-3.85(m,3H),3.55-3.67(m, 3H),3.33-3.38(m,2H),3.26(d,J=2.0Hz,1H),2.87-3.11(m,3H),2.81(dd,J=13.6,9.6Hz,1H),4. 46-4.54(m,1H),3.90-4.04(m,2H),3.70-3.85(m,3H),3.55-3.67(m,3H),3.33-3.38(m,2H),3.26 (d,J=2.0Hz,1H),2.87-3.11(m,3H),2.81(dd,J=13.6,9.6Hz,1H),2.39(s,3H),2.08(t,J=7.6Hz, 2H),1.76-2.01(m,3H),1.58(s,3H),1.40-1.51(m,4H),1.12-1.23(m,2H),0.87(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.88. LCMS(ESI+)m / z:[MH] + C 53 H 59 FN9O 13 + Calculated value: 1048.4, measured value: 1048.5.

[0455] Example 21 S-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamido)-N-(2-(((2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (21-120) (Figure 26).

[0456] [ka]

[0457] (2S)-2-[[2-[[2-[3-[4-(2,5-dioxopyrrol-1yl)phenylpropanoyl]aminoacetyl]amino]acetyl]amino]-3-phenyl l-propionic acid (21-116): A mixture of (S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropanoic acid (21-115) (5.03 g, 14.6 mmol, 1.0 equiv) and N-ethyl-N,N-diisopropylamine (1.52 g, 11.7 mmol, 2.05 mL, 0.8 equiv) in HO (25 mL) was added dropwise to a mixture of 2,5-dioxopyrrolidin-1-yl 3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110) (4.10 g, 14.6 mmol, 1.0 equiv) in acetonitrile (75 mL). The reaction mixture was stirred at 25° C. for 5 h, filtered, and the filtrate was purified by preparative HPLC to give (2S)-2-[[2-[[2-[3-[4-(2,5-dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoic acid (21-116) (3.32 g, 40% yield). 1H NMR(400MHz,DMSO-d6)δppm8.21(t,J=5.6Hz,1H),8.07(t,J=5.6Hz,1H),7.87(d,J=7.6Hz,1H),7.32(d,J=8.4Hz,2H),7.06-7 .27(m,9H),4.29(d,J=5.2Hz,1H),3.61-3.77(m,4H),3.19(s,1H),3.03(d,J=4.8Hz,1H),2.86(t,J=8.0Hz,3H),2.67(s,1H). LCMS(ESI-)m / z:[MH] + C 26 H 25 N4O7 - Calculated value: 505.1, measured value: 505.2.

[0458] Preparative HPLC conditions: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: H2O; B: ACN Column: Phenomenex® C 18 250 * 100mm 10u Flow rate: 260mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 1 20.0 40 20.1 40 20.2 100 25.2 100 25.3 1 26.5 1

[0459] (9H-Fluoren-9-yl)methyl (2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (21-118): To a solution of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48) (1.25 g, 2.60 mmol, 1.0 equiv) in dichloromethane (37 mL) at 25 °C, scandium(III) trifluoromethanesulfonate (50.5 mg, 0.260 mmol, 0.1 equiv) and [[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate (21-117) (1.44 g, 3.90 mmol, 1.5 equiv) were added in three portions over 1.5 hours. The reaction mixture was stirred at 30° C. for 36 h, quenched with water (100 mL) and extracted with dichloromethane (3×100 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was purified by silica gel flash column chromatography eluting with 10%-100% dichloromethane in ethyl acetate to give (9H-fluoren-9-yl)methyl (2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (21-118) (1.00 g, 45% yield). 1H NMR(400MHz,DMSO-D6)δppm8.90(brt,J=6.60Hz,1H),7.80-7.91(m,2H),7.73(d,J=11.13Hz,1H),7.56-7 .63(m,2H),7.23-7.44(m,6H),6.53(s,1H),5.32-5.53(m,3H),4.96(brdd,J=9.60,4.10Hz,1H),4.62-4. 83(m,2H),4.44-4.59(m,1H),4.20-4.32(m,1H),4.05-4.19(m,3H),3.91-4.00(m,1H),3.57-3.78(m,5H) ,3.14-3.28(m,1H),2.86-3.08(m,1H),2.34(s,3H),1.85(brdd,J=7.03,4.46Hz,3H),0.78-0.94(m,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.56. LCMS(ESI+)m / z:[MH] + C 44 H 42 FN4O9 + Calculated value: 789.3, Measured value: 789.3.

[0460] 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide (21-119): To a mixture of (9H-fluoren-9-yl)methyl (2-((2-(((1S',9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (21-118) (1.00 g, 1.27 mmol, 1.0 equiv) in N,N-dimethylformamide (20 mL) was added piperidine (0.125 mL, 1.27 mmol, 1.0 equiv). The reaction mixture was stirred at 0° C. for 1 h, filtered, and the filtrate was purified by preparative HPLC to give 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide (21-119) (360 mg, 45% yield). 1 H NMR(400MHz,DMSO-d6)δppm8.78(t,J=6.8Hz,1H),7.66-7.81(m,1H),7.32(s,1H),6. 52(s,1H),5.35-5.46(m,3H),5.01(dd,J=9.2,3.6Hz,1H),4.59-4.83(m,2H),3.92-4. 03(m,1H),3.77(m,1H),3.59-3.71(m,2H),3.20-3.29(m,2H),3.16(s,2H),2.92-3.0 5(m,1H),2.59-2.84(m,1H),2.29-2.41(m,3H),1.72-2.09(m,3H),0.78-0.97(m,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.54. LCMS(ESI+)m / z:[MH] + C 29 H 32 FN4O7 + Calculated value: 567.2, measured value: 567.3.

[0461] Preparative HPLC conditions Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: 10mM NH4HCO3 in H2O; B: ACN Column: Phenomenex® C 18 75 * 30mm * 3 um Flow rate: 25mL / min Monitor wavelength: 220 and 254 nm Time B% 0.0 10 8.0 40 8.1 40 8.2 100 10.2 100 10.3 10 11.5 10

[0462] S-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamide)-N-(2-(((2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (21-120): (2S)-2-[[2-[[2-[3-[4-(2,5-dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoic acid (21-116) (160 mg, 0.317 mmol, 1.0 equiv.), 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9, 10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide (21-119) (180 mg, 317 μmol, 1.0 equiv.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate (104 mg, 0.317 mmol, 1 A mixture of (S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamide)acetamide)acetamide)-N-(2-(((( 2-(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]p-lano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenyl-propanamide (21-120) (80.0 mg, yield 23%) was obtained. 1H NMR(400MHz,DMSO-d6)δppm8.69(t,J=6.8Hz,1H),8.28(t,J=5.6Hz,1H),8.19(t,J=5.6Hz,1H),8.10(d,J=8.0Hz,1H),8.03 (t,J=5.6Hz,1H),7.76(d,J=11.2Hz,1H),7.26-7.35(m,3H),7.13-7.25(m,8H),6.52(s,1H),5.34-5.53(m,4H),5.03(dd,J =8.8,4.00Hz,1H),4.63-4.81(m,2H),4.41-4.57(m,1H),3.90-4.03(m,1H),3.74-3.84(m,3H),3.54-3.74(m,6H),3.16-3. 30(m,2H),2.93-3.07(m,2H),2.73-2.88(m,3H),2.42-2.48(m,3H),2.37(s,3H),1.80-2.03(m,3H),0.87(t,J=7.2Hz,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.53. LCMS(ESI+)m / z:[MH] + C 55 H 56 FN8O 13 + Calculated value: 1055.4, measured value: 1055.3.

[0463] HPLC conditions for fractionation Machine: ギルソン 281 セミ HPLC システム Mobile phase: A: H2O; B: ACN KARARA:Phenomenex (registered trademark) C 18 75 * 30mm * 3um Flow rate: 60mL / min モニターWavelength: 220 and び254nm Time B% 0.0 25 8.0 55 8.1 55 8.2 100 10.2 100 10.3 25 11.5 25

[0464] Example 22 N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide (22-121)

[0465] [ka]

[0466] Example 22 (22-121) was prepared in a similar manner to Example 21 (21-120). 1 H NMR(400MHz,DMSO-d6)δppm8.86(t,J=5.60Hz,1H),8.67(brt,J=6.56Hz,1H),8.29(brt,J=5.66Hz,1H),8.05-8.17(m,2H),7.89(d,J=7.99Hz,1H) ),7.83(s,1H),7.75(d,J=10.97Hz,1H),7.54-7.61(m,1H),7.47-7.53( m,1H),7.33(s,1H),7.11-7.25(m,7H),6.52(s,1H),5.34-5.48(m,4H),5 .03(brdd,J=8.82,3.58Hz,1H),4.63-4.79(m,2H),4.44-4.54(m,1H),3.96(brdd,J=7.03,4.17Hz,1H),3.88(brd,J=5.72Hz,2H),3.57-3.81(m, 7H),3.19-3.27(m,1H),2.93-3.08(m,2H),2.78(brdd,J=13.65,9.83Hz ,1H),2.45(brs,1H),2.36(s,3H),1.81-2.02(m,3H),0.83-0.91(m,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.54. LCMS(ESI+)m / z:[MH]+ C 53 H 52 FN8O 13 + Calculated value: 1027.3, Measured value: 1027.3.

[0467] Example 23 N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)oxy)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (23-122)

[0468] [ka]

[0469] Example 23 (23-122) was made in a similar manner to Example 21 (21-120). 1H NMR(400MHz,DMSO-D6)δppm8.63-8.74(m,1H),8.28(t,J=5.63Hz,1H),8.09(d ,J=8.13Hz,1H),8.04(t,J=5.63Hz,1H),7.98(t,J=5.63Hz,1H),7.77(d,J=11. 01Hz,1H),7.34(s,1H),7.11-7.26(m,5H),6.98(s,2H),6.52(s,1H),5.31-5.5 8(m,4H),5.05(brdd,J=9.13,4.00Hz,1H),4.61-4.82(m,2H),4.40-4.55(m,1H ),3.90-4.04(m,1H),3.73-3.84(m,3H),3.60-3.73(m,5H),3.53-3.60(m,1H) ,3.33-3.37(m,2H),3.21-3.27(m,1H),2.94-3.08(m,2H),2.77(dd,J=13.70,9 .82Hz,1H),2.46-2.48(m,1H),2.38(s,3H),2.05-2.12(m,2H),1.82-2.01(m,3 H),1.45(dq,J=14.13,7.00Hz,4H),1.10-1.22(m,2H),0.87(t,J=7.32Hz,3H). 19 F NMR (376MHz, DMSO-d6) δppm-111.54. LCMS(ESI+)m / z:[MH] + C 52 H 58 FN8O 13 + Calculated value: 1021.4, measured value: 1021.4.

[0470] Example 24 CTGアッセイ(Jeko-1 and びMDA-MB-468) The CTG assay is a method for determining the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells. The cell assay requires the addition of a single reagent, Cell Titer Glo, which lyses the cells and generates a luminescent signal. The luminescent signal is proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. For our assay, we ensured that cells were in logarithmic phase for either 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 μM to 0.0000508 μM 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 with the luminescence of the vehicle control and plotted in a percentage of viability versus 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. Representative assay results are summarized in Table 1.

[0471] Example 25 Human Hepatocyte Clearance (HHEP CL) Human hepatocytes (from 10 mixed-sex human donors, final concentration 0.5 x 10) in Williams' E medium 6Suspensions of 1000 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) for 90 min in a 5% CO2 and 95% humidity incubator at 37 °C with constant shaking at approximately 600 rpm. The total incubation volume was 200 μl. Samples (25 mL) were taken at TO, 15, 30, 60, and 90 min and added midway to ice-cold stop solution (acetonitrile containing 200 ng / mL tobutamide and labetalol as internal standards) (125 μl), vortexed at 500 rpm for 10 min, and centrifuged at 3220 × g for 20 min at 4 °C. The assay plate was sealed and stored at 4 °C until LCMS analysis. Hepatocyte viability upon preincubation was 84.5%. Representative assay results are summarized in Table 1.

[0472] Example 26 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). Appropriate concentrations of microsome working solutions were prepared in 100 mM potassium phosphate buffer. The reaction plate containing the compound and microsome mixture was preincubated at 37°C for 10 minutes, after which the reaction was initiated by adding 98 mL of 2 mM NADPH and 2 mM MgCl2 solution. 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 performed at 37°C for 60 minutes. Samples were removed at T0, T5, T15, T30, T45, and T60, added midway to ice-cold stop solution (acetonitrile containing 200 ng / mL tobutamide and labetalol as an internal standard) (125 μl), shaken for 10 min, and centrifuged at 4000 rpm for 20 min at 4°C. The analytical plate was analyzed by LCMS. Representative assay results are summarized in Table 1.

[0473] The human liver microsomal clearance assay evaluates metabolism by cytochrome P450 (phase I enzymes). These enzymes oxidize substrates by incorporating oxygen atoms into hydrocarbons, thus introducing a hydroxyl group or causing NO and S-dealkylation of the substrate, forming more polar products that are easier to remove. The human hepatocyte clearance assay more broadly measures the overall cellular metabolism (phase I and phase II enzymatic pathways) of the test compound. Phase II enzymes catalyze the conjugation reaction of xenobiotic metabolites with charged species, such as glutathione, sulfate, glycine, or glucuronic acid, to form more polar compounds for easier clearance.

[0474] Payloads with higher intrinsic clearance may offer better therapeutic indices due to their potentially lower systemic plasma exposure (Non-Patent Document 2). In Table 1, payloads with higher intrinsic clearance are likely to have improved safety profiles because potentially toxic payloads to healthy cells are rapidly removed from the plasma, reducing the chance of interacting with healthy cells.

[0475] Example 27 PAMPA (Parallel Artificial Membrane Permeability Assay) PAMPA is a method for determining the permeability of substances from a donor compartment through a lipid-infused artificial membrane to an acceptor compartment. See Non-Patent Document 3. A multiwell microtiter plate is used as the donor, and the membrane / acceptor compartment is placed on top. The entire assembly is commonly referred to as a "sandwich." At the start of the test, the drug is added to the donor compartment, while the acceptor compartment contains no drug. 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.

[0476] PAMPA was performed by Pion Inc. using GIT-0 lipid and 5 μM donor solution in PRISMA buffer (containing 0.05% DMSO) at pH 5.0 and pH 7.4. Higher PAMPA data are associated with better bystander killing (Non-Patent Document 4).

[0477] Representative assay results are summarized in Table 1. Higher permeability is important because it means there is a greater chance of "bystander killing." That is, once the payload neutralizes a tumor cell, a more permeable payload is more likely to escape the neutralized tumor cell and then embed itself in an adjacent tumor cell. Once there, it can neutralize the tumor cell, escape, embed itself in another adjacent tumor cell, and repeat the process.

[0478] [Table 1]

[0479] Dxd: deruxtecan; HHEP Cl: human hepatocyte intrinsic clearance, mL / min / Kg; HHEP t 1 / 2 , min. HLM: Human liver microsomal clearance, mL / min / Kg. ND: Not determined.

[0480] Example 28 We developed novel and diverse anti-ROR-1-specific monoclonal antibodies that bind to multiple regions of the ROR-1 extracellular domain (ECD) through an antibody development campaign employing three strategies: (1) mice in cohort 1 were immunized with the full-length ROR-1 ECD, (2) mice in cohorts 2 and 3 were immunized with the ROR-1 IgG-like domain, and (3) mice in cohort 4 were immunized with a short region of the human IgG-like sequence of ROR-1. Following mouse immunization, 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 transfected into HEK293 cultures. After 5 days of shaking at 37°C in 293 cell culture medium, antibodies were captured onto an agarose-based protein A resin. After several stringent washes, the antibody was eluted in a glycine solution (pH 3), neutralized with Hepes (pH 9), and buffer exchanged into PBS.

[0481] Using these approaches, several monoclonal antibodies were developed, and the resulting antibodies were subjected to further screening to evaluate the specific characteristics of the antibodies. 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, rodent ROR-1 protein, and in cell-based assays. Further screening parameters included antibody internalization, epitope binning against known anti-ROR-1 antibodies (UC961 and 4a5), binding to the human ROR-1 Ig-like domain, and thermal deactivation. Analysis of the shift and assessment of self-interaction by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) were included.

[0482] Example 29 A cell-binding saturation assay was developed to evaluate how well the anti-ROR-1 antibodies developed in Example 28 bound to extracellular ROR-1 protein endogenously expressed on cell lines. More specifically, the anti-ROR-1 monoclonal antibodies developed in Example E, 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 titrated concentrations of each antibody construct. The cells were then washed and subjected to secondary antibody staining and flow cytometric detection. The mean fluorescence (MFI) was determined by analysis with 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 UC 961 (see Figure 27). As shown in Figure 27, cell binding saturation for antibodies ATX-P-875, ATX-P-885, and ATX-P-890 was comparable to that for UC961, but a higher concentration of ATX-P-875 was required to achieve saturation compared to UC-961. ATX-P-890 and ATX-P-885 each had better or improved binding saturation concentrations compared to UC-961. The comparable saturation to UC961 demonstrates that the anti-ROR1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 have similar affinity for the human ROR-1 target as the clinically approved antibody UC-961.

[0483] Example 30 After determining a saturating concentration (74 nM) in a binding assay, the anti-ROR-1 antibodies (ATX-P-875, P-885, and P-890) developed herein were evaluated for their ability to internalize ROR-1 receptors on human ROR-1-positive cells (JeKo-1 and MDA-MB-468). Briefly, ROR-positive cell lines were incubated with the antibodies under supersaturating conditions to bind 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 antibody on the surface was detected using a labeled secondary antibody and flow cytometry. Percent internalization was calculated based on time 0, which was assumed to be when 100% of available receptors were present on the cell surface. The results in Figure 28 demonstrate 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, the internalization of two anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) was improved over the clinically used UC 961 anti-ROR-1 antibody, providing evidence that the ATX-P-875 and ATX-P-890 antibodies have improved ability to internalize the ROR-1 receptor from the surface of solid tumors.

[0484] Example 31 Cell binning was also used to determine whether the monoclonal antibodies ATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitope as the previously known anti-ROR-1 monoclonal antibodies UC 961 and 4A5 (control). In step 1 of the cell binning experiment, various amounts of the ATX-P-875, ATX-P-885, and ATX-P-890 monoclonal antibodies were separately incubated with ROR-1-expressing cells (MDA-MB-468). In step 2, a fluorescently labeled secondary antibody that recognizes the novel antibodies was incubated with the samples. 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 UC 961 (Dy 650-UC 961) 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 bound to the same epitope as the known ROR-1-binding antibodies UC961 and 4A5. Figure 29A below shows the expected staining profiles if the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bound to the same epitope as the UC 961 and 4A5 antibodies. Figure 29B shows the expected profiles of the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies if they bound to a different epitope on ROR-1 than the UC 961 or 4A5 antibodies. Briefly, when binding to the same epitope, increasing concentrations of the novel antibodies block binding of the pre-labeled competing antibody, thereby reducing the competitor's signal at higher concentrations. When the antibodies bind to separate epitopes, staining increases with increasing dose because each antibody (novel antibody and competing antibody) does not compete for binding to the receptor. Cell binning data obtained with MDA-MB-468 cells showed that ATX-P-885 significantly bound to the same epitope as UC 961, and both ATX-P-875 and ATX-P-890 significantly bound to the same epitope as 4A5 (see Figures 29C, 29D, and 30).The ability of the antibodies developed herein to bind to different ROR-1 epitopes provides the opportunity to modulate the target in a variety of ways.

[0485] Example 32 Biochemical binning by SPR was also evaluated for the anti-ROR1 antibodies (ATX-P-875, P-885, and P-890) compared with the control anti-ROR1 antibodies UC961 and 4a5. In these experiments, 10 μg / ml of purified cloned Hu / Cy / Rh ROR1-His protein was covalently coupled to an HC30M chip. Individual dilutions of each antibody at 10 pg / mL were injected over the chip, and binding was assessed by Carterra SPR. Unexpectedly, the data demonstrated the presence of three distinct binding epitopes among ATX-P-875, ATX-P-885, and ATX-P-890, with ATX-885 being the only antibody that confers partial blocking to the UC961 antibody (see Figure 31). Cell binning only assessed the ability of the antibodies to block either UC961 or 4a5, two clinically used ROR-1 antibodies. Biochemical SPR evaluation also tested the ability of the 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.

[0486] Example 33 Antibody characterization of ATX-P-875, ATX-P-885, and ATX-P-890 compared to UC961 is summarized in Figure 30 and Tables 2-7. An initial evaluation of antibody developability was performed by AC-SINS to assess the potential for self-interactions (Figure 30). The control antibodies adalimumab and rituximab exhibited the expected low shift, while infliximab exhibited the expected high shift. The anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 developed herein did not exhibit significant self-interactions, consistent with the control antibodies potentially not posing 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 2–5 provide characterization data for this antibody compared with the known ROR-1-binding antibody UC 961, including the tabulated results measured by biochemical binding to purified protein and SPR (Tables 2–5), cellular binding (EC 50 ) to the ROR-1-positive cell lines JeKo-1 and MDA-MB-468 (Table 6), and cellular internalization (% internalization) (Table 7). Of particular note, the reduced affinity of ATX-P-875 (KD: 1.09E-08) compared with UC961 and other anti-ROR-1 antibodies (ATX-P-885 and ATX-P-890) may provide unexpected therapeutic benefits. By binding less strongly to the ROR-1 epitope, the ATX-P-885 antibody may be able to penetrate further into tumors and reach more distant cells expressing the ROR-1 target.

[0487] [Table 2]

[0488] [Table 3]

[0489] [Table 4]

[0490] [Table 5]

[0491] [Table 6]

[0492] [Table 7]

[0493] Example 34 Synthesis of immunoconjugates was accomplished as described in this example. Antibodies were produced as described in Example 28 and suspended in PBS pH 7.2 at the following protein concentrations: mAb A 12.44 mg / ml, mAb B 13.29 mg / ml, mAb C at 1 4.90 mg / ml. A molecular weight of 150,000 Da was used for all antibodies for reduction and conjugation calculations.

[0494] 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, 10 mM stock of TCEP in water), and the mixture was kept at 20°C for 2 hours.

[0495] After adding DMA and gently mixing with the reduced protein solution above to achieve a final 10% v / v in the conjugation, the toxin-linker stock solution (12 equiv., 50 mM in DMA) was added and gently mixed. Bioconjugation was allowed to proceed overnight at 20 °C for approximately 16–20 h. It was completed within 2 h with extended time to allow for maleimide ring opening.

[0496] The crude conjugate was purified using a gravity-fed NAP 25 (small scale) or Flow HiPrep G The column was buffer exchanged into PBS pH 7.4 using 25 (large scale), and the column was prepared and operated according to the manufacturer's instructions (Cytivia). To remove residual toxins, a 100 mg / ml slurry of activated charcoal (Sigma / C9157) in PBS pH 7.4 was prepared and added to achieve 1 mg carbon per 1 mg starting antibody mass. This was mixed gently for 2 hours to ensure the carbon remained 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 ADC was finally filtered through an appropriately 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 color development).

[0497] The analytical process was carried out on an HPLC instrument using the protocols described below and in Tables 8 and 9. The analysis was performed on an Agilent 1100 or 1260.

[0498] SEC-HPLC-monomer content and ADC concentration (mg / mL) Column: TOSOH TSKgel G3000SWXL 7.8mm x 30cm The 5 pm particles (MERCK808541) were combined with a guard column (MERCK822858) equipped with a GFC 3000 4 x 3 mm cartridge (Phenomenex®). Buffer solution: 0.2M phosphate, 0.25M KCl, 10% IPA, pH 6.95±0.1 Gradient: Isocratic at 0.5 ml / min at 25°C The sample load was approximately 10 μg, and the monomer and concentration were determined from the 214 nm signal. Monomer was reported based on peak integration, and [ADC] mg / mL was reported based on the antibody calibration curve.

[0499] PLRP-HPLC-DAR determination Column: PLRP-S 2.1 mm x 5 cm, 5 μm (Agilent PL912-1502) Mobile phase A: 0.1%v / v TFA in water Mobile phase B: 0.1%v / v TFA in ACN Gradient: 1 ml / min at 50°C The sample load was 2 μg and analyzed at 214 nm.

[0500] [Table 8]

[0501] residual toxins Column Kinetex® 2.6 μm C8 100 Å, LC column 50 × 4.6 mm, (Phenomex 00B-4497-E0) Mobile phase A 0.05% TFA in water Mobile phase B 0.05% TFA in ACN Gradient: 2 ml / min at 60°C

[0502] [Table 9]

[0503] Analytical sample preparation: Samples (50 μl, ADC or PBS / PS20 matrix) were diluted with 2 μl of 5 M NaCl and 150 μl of cold MeOH (from a -20°C freezer), incubated at -20°C for 30 minutes, and centrifuged at 21,000 g and 4°C for 30 minutes. The supernatant (125 μl) was then extracted and mixed with 125 μl of WFI, of which 100 μl was injected onto a Kinetex® column. Data were analyzed at 214 nm, and residual toxin in the samples was estimated from an external calibration curve of the relevant toxin linker. Results are expressed as the percentage of free and bound, using the ADC concentration and calculated DAR to determine the amount of bound toxin.

[0504] Example 35 Novel ROR-1 antibody-drug conjugates (ADCs) were evaluated by CTG assay in a manner similar to that described in Example 24 for payload screening. A total of three unique antibodies (mAb A=875, mAb B=885, mAb C=890) were conjugated to six distinct novel linker / payloads (18-112, 19-113, 20-114, 21-120, 22-121, and 23-122) (Figure 32). Briefly, ROR+ (JeKo-1 / MDA-MB-468) or ROR- (Ramos) cells were plated into 96-well plates and treated with 3-fold serial dilutions of ADCs starting from 1 mM to 0.0000508 mM (10-point dilutions) for 72 hours. Cell viability was analyzed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions (Figure 33). The percentage of viable cells at each ADC concentration was determined by normalizing to the luminescence of the vehicle control and plotted as a percentage of viability versus dose-response curve using a nonlinear fit in GraphPad Prism software. The IC50 for each ADC was calculated as the concentration of compound that killed 50% of the cells. Representative assay results are summarized in Table 10. In ROR+ cell lines, an approximately 4-fold increase in potency was achieved with ADCs containing 22-121, 21-120, and 23-122 compared to VLS-101. In vitro data also demonstrate that mAb A (ATX-P-875) is slightly more potent than the other two ROR-1-targeting antibodies B and C (ATX-P-885 and ATX-P-895, respectively).

[0505] [Table 10]

[0506] Moreover, although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those skilled in the art that numerous and various modifications may be made thereto without departing from the spirit of the present disclosure. It should therefore be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather encompass all modifications and alternative forms consistent with the true scope and spirit of the present invention.

[0507] Sequence Listing SEQ ID NO:1 ATX-P-875 VH CDR1(Kabat) GFTFSNAW

[0508] SEQ ID NO:2 ATX-P-875 VH CDR2(Kabat) IKSKTDGGTT

[0509] SEQ ID NO:3 ATX-P-875 VH CDR3(Kabat) TTGPDDLDY

[0510] SEQ ID NO:4 ATX-P-875VH nt GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAA CAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCASEQ

[0511] SEQ ID NO:5 ATX-P-875VH AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSS

[0512] SEQ ID NO:6 ATX-P-875HC IgGl-Fc nt

[0513] SEQ ID NO:7 ATX-P-875HC IgGl-Fc AA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG

[0514] SEQ ID NO:8 ATX-P-875 VL CDR1(Kabat) QSISSY

[0515] ATX-P-875 VL CDR2(Kabat) AAS

[0516] SEQ ID NO:10 ATX-P-875 VL CDR3(Kabat) QQYDNLPIT

[0517] SEQ ID NO:11 ATX-P-875VL nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0518] Accession No.: 12 ATX-P-875VL AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIK

[0519] Accession No.: 13 ATX-P-875 Kappa LC nt GACATCCAGATGACCCAGTTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGAAAGCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCCGATCACCTTGGCCAAGGGACACGACTGGAGATTAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0520] sequence no: 14 ATX-P-875 Kappa LC AA DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0521] sequence no: 15 ATX-P-885 VH CDR1(Kabat) GGSFSGYY

[0522] SEQ ID NO:16 ATX-P-885 VH CDR2(Kabat) INHSGST

[0523] SEQ ID NO:17 ATX-P-885 VH CDR3(Kabat) AREGVYEDY

[0524] SEQ ID NO:18 ATX-P-885VH nt CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCA TAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0525] SEQ ID NO:19 ATX-P-885VH AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSS

[0526] SEQ ID NO:20 ATX-P-885HC IgGl-Fc nt

[0527] SEQ ID NO:21 ATX-P-885HC IgGl-Fc AA QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0528] SEQ ID NO:22 ATX-P-885 VL CDR1(Kabat) QSVSNY

[0529] ATX-P-885 VL CDR2(Kabat) DAY

[0530] SEQ ID NO:24 ATX-P-885 VL CDR3(Kabat) QQRSNWPLT

[0531] SEQ ID NO:25 ATX-P-885VL nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0532] Accession number: 26 ATX-P-885VL AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIK

[0533] Accession number: 27 ATX-P-885 Rappa LC nt GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGAAAGAGCCACCCTCTCCTGCAGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTGGCCAAGGGACACGACTGGAGATTAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0534] sequence no: 28 ATX-P-885 Kappa LC AA EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0535] sequence no.:29 ATX-P-890 VH CDR1(Kabat) GYTFTGYY

[0536] SEQ ID NO:30 ATX-P-890 VH CDR2(Kabat) INPNSGGT

[0537] SEQ ID NO:31 ATX-P-890 VH CDR3(Kabat) VRDQVQLERFDS

[0538] SEQ ID NO:32 ATX-P-890VH nt CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGCACAAACT ATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0539] SEQ ID NO:33 ATX-P-890VH AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSS

[0540] SEQ ID NO:34 ATX-P-890HC IgGl-Fc nt

[0541] SEQ ID NO:35 ATX-P-890HC IgGl-Fc AA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0542] SEQ ID NO:36 ATX-P-890 VL CDR1(Kabat) QDISNY

[0543] ATX-P-890 VL CDR2(Kabat) DAS

[0544] SEQ ID NO:38 ATX-P-890 VL CDR3(Kabat) QQYDNLPPT

[0545] SEQ ID NO:39 ATX-P-890VL nt GACATCCAGATGACCCAGTTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAAACAGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCCTCCACTTTCGGCCCTGGGACCAAGGTGGAAATC AAA

[0546] sequence no: 40 ATX-P-890VL AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTGFGPGTKVEIK

[0547] sequence no.:41 ATX-P-890 Kappa LC nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAAACAGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAA CGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

[0548] sequence no: 42 ATX-P-890 Kappa LC AA DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFFTISSLQPEDIATYYCQQYDNLPPTGFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An immunoconjugate having formula (I), Ab - [S - L １ -L ２ -L ３ -L ４ -L ５ -L ６ -L ７ -D] n (I) wherein Ab is an antibody or an antigen-binding fragment thereof, L １ is 【Chemical 1】 and L ２ Does not exist, or 【Chemical 2】 and Z １ and Z ２ are each independently hydrogen, halogen, NO ２ , -O-(C １ ~C ６ alkyl), or C １ ~C ６ alkyl, L ３ is -(CH ２ )n １ -C(=O)- or -(CH ２ CH ２ )n １ -(CH ２ )n １ C(=O)-, and n １ is independently an integer from 0 to 12, L ４ is a tetrapeptide residue, L ５ is non-existent or -[NH(CH ２ )n ２ ]n ３ - and n ２ is an integer from 0 to 6, and n ３ is an integer from 0 to 2, L ６ does not exist, or 【Chemical Formula 3】 and L ７ Does not exist, or [Chemical Formula 4] and D is a drug moiety, and n is an integer from 1 to 10, the immunoconjugate.

2. (i) L ２ is 【Chemical Formula 5】 and (ii) at least one of Z1 and Z2 is hydrogen, (iii) L3 is -(CH2)n1-C(=O)-, (iv) L4 is gly-gly-phe-gly (GGFG), (v) L5 does not exist, (vi) L6 does not exist, and (vii) L7 does not exist, the immunoconjugate according to Claim 1.

3. (i) L2 does not exist, (ii) at least one of Z1 and Z2 is hydrogen, (iii) L3 is -(CH2)n1-C(=O)-, (iv) L4 is gly-gly-phe-gly (GGFG), (v) L5 does not exist, (vi) L6 does not exist, and (vii) L7 does not exist, the immunoconjugate according to Claim 1.

4. L2 either does not exist or 【Chemical Formula 6】 and Z1 and Z2 are each independently hydrogen, L3 is -(CH2)n1-C(=O)-, where n1 is an integer from 1 to 6, L4 is gly-gly-phe-gly (GGFG), L5 is -[NH(CH2)n2]n3-, where n2 is an integer from 0 to 6 and n3 is an integer from 0 to 2, L6 does not exist, and L7 does not exist, the immunoconjugate according to Claim 1.

5. D is a drug moiety of formula (II) having the following structure, 【Chemical Formula 7】 wherein 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 , and substituted or unsubstituted -O-(CR ５ R ６ ) ｍ -O- and are selected from the group consisting of, R １ and R ２ together form a ring, R ３ is hydrogen, 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 and is R ４ is hydrogen, substituted or unsubstituted -(C １ ~C ６ alkyl)-X ２ substituted or unsubstituted -(C １ ~C ６ haloalkyl)-X ２ substituted or unsubstituted -(C １ ~C ６ alkenyl)-X ２ substituted or unsubstituted -(C １ ~C ６ haloalkenyl)-X ２ substituted or unsubstituted -(C １ ~C ６ alkynyl)-X ２ or substituted or unsubstituted -(C １ ~C ６ haloalkynyl)-X ２ and X １ is -O-, -S(O ｎ６ ), -NH-, -O-(C=O)-, -NH-(C=O)-, -NH-(C=O)-O-, -NH(C=O)-NH-, or -NH-S(O ｎ６ ), and X ２ is -OR ９ , -SR ９ , or -NHR ９ and R ５ and R ６ each independently represents hydrogen, halogen, 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,​ m is 1 or 2, n ４ and n ５ are each independently 0, 1, or 2, provided that neither ４ n ５ nor n is 0 n ６ is 0, 1, or 2, and each Y is independently H or halogen, each p is individually 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 is R ８ is a substituted or unsubstituted C １ -C ６ alkyl-X ３ a substituted or unsubstituted C １ -C ６ haloalkyl-X ３ or -[(CY ２ ) ｐ O(CY ２ ) ｑ CY ｔ -X ２ wherein ３ ３ R ９ is H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ or L ７ wherein only one of R ７ and R ９ is L ４ , L ５ , L ６ or L ７ and Each X ３ is independently, -H, -OH, -SH, or -NH ２ The immunoconjugate according to any one of claims 1 to 4, which is as described above. **Claim 6**: R1 and R2 are each independently selected from the group consisting of hydrogen, halogen, -OR5, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 haloalkyl, unsubstituted -O-(C1-C6 alkyl), and unsubstituted -O-(CR5R6)m-O-, R1 and R2 together form a ring, R3 is hydrogen or unsubstituted C1-C6 alkyl, R4 is unsubstituted -(C1-C6 alkyl)-X2, X1 is -O-, X2 is -OR9, R5 and R6 are each independently hydrogen, m is 1, n4 is 1 or 2, n5 is 0, R7 is H, and R9 is L4, L5, L6 or L7, the immunoconjugate according to claim 5. **Claim 7**: R1 and R2 are each independently selected from the group consisting of hydrogen, fluoro, methoxy, methyl, difluoromethyl, and -O-(CH2)-O-, R1 and R2 together form a ring, R3 is hydrogen, R4 is unsubstituted -(C1-C6 alkyl)-X2, X1 is -O-, X2 is -OR9, n4 is 2, n5 is 0, R7 is H, and R9 is L5, the immunoconjugate according to claim 5. **Claim 8** The immunoconjugate according to any one of claims 1-4, wherein Ab specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1) and / or Ab binds to the surface of cancer cells. **Claim 9**: Ab is VHCDR1 containing the amino acid sequence of SEQ ID NO: 29, VHCDR2 containing the amino acid sequence of SEQ ID NO: 30, and VHCDR3 containing the amino acid sequence of SEQ ID NO: 31 a heavy chain comprising, and VLCDR1 containing the amino acid sequence of SEQ ID NO: 36, VLCDR2 containing the amino acid sequence DAS, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 38 a light chain comprising, The immunoconjugate according to any one of claims 1-4, wherein Ab specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1). **Claim 10**: Ab is VHCDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR 2 comprising the amino acid sequence of SEQ ID NO: 2, and VH CDR 3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain comprising, and VL CDR 1 comprising the amino acid sequence of SEQ ID NO: 8, VL CDR 2 comprising the amino acid sequence AAS, and VL CDR 3 comprising the amino acid sequence of SEQ ID NO: 10 a light chain comprising, wherein the Ab specifically binds to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1), the immunoconjugate according to any one of claims 1 to 4.

11. The Ab is VH CDR 1 comprising the amino acid sequence of SEQ ID NO: 15, VH CDR 2 comprising the amino acid sequence of SEQ ID NO: 16, and VH CDR 3 comprising the amino acid sequence of SEQ ID NO: 17 a heavy chain comprising, and VL CDR 1 comprising the amino acid sequence of SEQ ID NO: 22, VL CDR 2 comprising the amino acid sequence DAY, and VL CDR 3 comprising the amino acid sequence of SEQ ID NO: 24 a light chain comprising, the immunoconjugate according to any one of claims 1 to 4.

12. a compound of formula (IV) having the following structure or a pharmaceutically acceptable salt thereof, wherein 【Chemical 8】 in the formula, R １ and R ２ each independently represents 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 -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- selected from the group consisting of, R １ and R ２ together form a ring, R ３ is hydrogen or a substituted or unsubstituted C １ ~C ６ alkyl, a substituted or unsubstituted C １ ~C ６ haloalkyl, or -[(CY ２ ) ｐ O(CY ２ ) ｑ ｔ CY ３ and is R ４ is hydrogen, substituted or unsubstituted -(C １ ~C ６ alkyl)-X ２ substituted or unsubstituted -(C １ ~C ６ haloalkyl)-X ２ substituted or unsubstituted -(C １ ~C ６ alkenyl)-X ２ substituted or unsubstituted -(C １ ~C ６ haloalkenyl)-X ２ substituted or unsubstituted -(C １ ~C ６ alkynyl)-X ２ or substituted or unsubstituted -(C １ ~C ６ haloalkynyl)-X ２ and X １ is —O—, —S(O ｎ６ )—, —NH—, —O—(C═O)—, —NH—(C═O)—, —NH—(C═O)—O—, —NH(C═O)—NH—, or —NH—S(O ｎ６ )—, and X ２ is —OH, —SH, or —NR ５ R ６ wherein R ５ and R ６ each independently represents hydrogen, halogen, substituted or unsubstituted C １ to C ６ alkyl, substituted or unsubstituted C １ to C ６ haloalkyl, or -[(CY ２ ) ｐ O(CY ２ ) ｑ ｔ CY ３ wherein R ７ is H, -COR ８ , -CO ２ R ８ , or -(CO)-NHR ８ and R ８ is a substituted or unsubstituted C １ -C ６ alkyl, a substituted or unsubstituted C １ -C ６ haloalkyl, or -[(CY ２ )pO(CY ２ ) ｑ ] ｔ CY ３ and is m is 1 or 2, n ４ and n ５ are each independently 0, 1, or 2, provided that both n ４ and n ５ are not 0 n ６ is 0, 1 or 2, and each Y is independently H or halogen, each p is individually 1, 2, 3, 4, 5, or 6, each q is independently 0, 1, 2, 3, 4, 5, or 6, and each t is independently 1, 2, 3, 4, 5, or 6, provided that formula (IV) does not represent delutecan or exatecan, a compound or a pharmaceutically acceptable salt thereof.

13. (i) R １ and R ２ wherein at least one of them is halogen (ii) at least one of R1 and R2 is substituted or unsubstituted C1-C6 alkyl, (iii) R3 is hydrogen, (iv) R4 is substituted or unsubstituted -(C1-C6 alkyl)-X2, (v) R4 is unsubstituted -(C1-C6 alkyl)-X2, (vi) X1 is -O-, and (vii) X2 is -OH, the compound or a pharmaceutically acceptable salt thereof according to claim 12.

14. R1 and R2 are each independently selected from the group consisting of hydrogen, halogen, -OR5, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 haloalkyl, unsubstituted -O-(C1-C6 alkyl), and unsubstituted -O-(CR5R6)m-O, and R1 and R2 together form a ring, R3 is hydrogen or unsubstituted C1-C6 alkyl, R4 is hydrogen or unsubstituted -(C1-C6 alkyl)-X2, X1 is -O-, X2 is -OH, R5 and R6 are each independently hydrogen, R7 is H, m is 1, n4 is 1 or 2, and n5 is 0, the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

15. The compound of formula (IV) is 【Chemical Formula 9】 【Chemical 10】 【Chemical 11】 【Chemical Formula 12】 【Chemical 13】 selected from the group consisting of, the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

16. The compound of formula (IV) is 【Chemical Formula 14】 the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

17. The compound of formula (IV) is 【Chemical 15】 selected from the group consisting of, the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

18. An immunoconjugate according to any one of claims 1 to 4, a compound according to any one of claims 12 to 17, or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof, a pharmaceutical composition.

19. A pharmaceutical composition according to claim 18 for treating cancer or a tumor.

20. 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 stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, or sarcoma, the pharmaceutical composition according to claim 19.

21. A conjugate having formula (III), Mi-L ２ -L ３ -L ４ -L ５ -L ６ -L ７ -D (III) wherein, Mi is 【Chemical 16】 is L ２ Does not exist, or 【Chemical 17】 is Z １ and Z ２ are each independently hydrogen, halogen, NO ２ , -O-(C １ ~C ６ alkyl), or C １ ~C ６ alkyl, L ３ is -(CH ２ )n １ -C(=O)- or -(CH ２ CH ２ )n １ -(CH ２ )n １ C(=O)-, and n １ is, independently, an integer from 0 to 12, L ４ is a tetrapeptide residue, L ５ is either non-existent or -[NH(CH ２ )n ２ n ３ -, and n ２ is an integer from 0 to 6, and n ３ is an integer from 0 to 2, and L ６ does not exist, or 【Chemical 18】 is L ７ Does not exist, or 【Chemical 19】 and D is a drug moiety, conjugate.

22. (i) L2 is 【Chemical 20】 is (ii) at least one of Z1 and Z2 is hydrogen, (iii) L3 is -(CH2)n1-C(=O)-, (iv) L4 is gly-gly-phe-gly (GGFG), (v) L5 is absent, (vi) L6 is absent, and (vii) L7 is absent, the conjugate according to claim 21.

23. (i) L2 is absent, (ii) At least one of Z1 and Z2 is hydrogen, (iii) L3 is -(CH2)n1-C(=O)-, (iv) L4 is gly-gly-phe-gly (GGFG), (v) L5 is absent, (vi) L6 is absent, and (vii) L7 is absent, the conjugate according to claim 21.

24. D is a compound of formula (II) having the following structure, 【Chemical 21】 wherein, 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 , and substituted or unsubstituted -O-(CR ５ R ６ ) ｍ -O- and are selected from the group consisting of, where R １ and R ２ together form a ring​ R ３ is hydrogen or a substituted or unsubstituted C １ to C ６ alkyl, a substituted or unsubstituted C １ to C ６ haloalkyl, or -[(CY ２ ｐ O(CY ２ ｑ ｔ CY ３ and​​​ R ４ is hydrogen, substituted or unsubstituted -(C １ ~C ６ alkyl)-X ２ , substituted or unsubstituted -(C １ ~C ６ haloalkyl)-X ２ , substituted or unsubstituted -(C １ ~C ６ alkenyl)-X ２ , substituted or unsubstituted -(C １ ~C ６ haloalkenyl)-X ２ , substituted or unsubstituted -(C １ ~C ６ alkynyl)-X ２ , or substituted or unsubstituted -(C １ ~C ６ haloalkynyl)-X ２ and X １ is -O-, -S(O ｎ６ ), -NH-, -O-(C=O)-, -NH-(C=O)-, -NH-(C=O)-O-, -NH(C=O)-NH-, or -NH-S(O ｎ６ ), and X ２ is -OR ９ or -SR ９ or -NHR ９ and R ５ and R ６ each independently represents hydrogen, halogen, substituted or unsubstituted C １ to C ６ alkyl, substituted or unsubstituted C １ to C ６ haloalkyl, or -[(CY ２ ) ｐ O(CY ２ ) ｑ ｔ CY ３ wherein m is 1 or 2, n ４ and n ５ are each independently 0, 1, or 2, provided that both n ４ and n ５ are not 0, n ６ is 0, 1, or 2, and each Y is independently H or halogen, each p is individually 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 Each R ８ is, independently, a substituted or unsubstituted C １ to C ６ alkyl-X ３ a substituted or unsubstituted C １ to C ６ haloalkyl-X ３ or -[(CY ２ ) ｐ O(CY ２ ) ｑ ｔ CY ２ -X ３ wherein R ９ is H, -COR ８ , -CO ２ R ８ , -(CO)-NHR ８ , L ４ , L ５ , L ６ or L ７ wherein, however, only one of R ７ and R ９ is L ４ , L ５ , L ６ or L ７ and Each X ３ is, independently, -H, -OH, -SH, or -NH2, and is the conjugate according to any one of claims 21 to 23.

25. R1 and R2 are each independently hydrogen, halogen, -OR5, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 haloalkyl, unsubstituted -O-(C1-C6 alkyl), and unsubstituted -O-(CR5R6)m-O-, selected from the group consisting of, and R1 and R2 together form a ring, R3 is hydrogen or unsubstituted C1-C6 alkyl, R4 is unsubstituted -(C1-C6 alkyl)-X2, X1 is -O-, X2 is -OR9, R5 and R6 are each independently hydrogen, m is 1, n4 is 1 or 2, n5 is 0, R7 is H, and R9 is L4, L5, L6 or L7, the conjugate according to claim 24.

26. R1 is methyl, R2 is fluoro, R3 is hydrogen, R4 is unsubstituted -(C2 alkyl)-X2, and n4 is 2, the conjugate according to claim 24.

27. Formula (III) is 【Chemical 22】 【Chemical 23】 【Chemical 24】 【Chemical 25】 selected from wherein Z １ and Z ２ are each independently hydrogen, fluoro, chloro, -NO ２ , and -OCH ３ and are selected from the group consisting of, the conjugate according to any one of claims 21 to 23.

28. A process for generating an immunoconjugate comprising reacting an effective amount of a thiol-functionalized antibody or an antigen-binding fragment thereof with the conjugate according to any one of claims 21 to 23 under reaction conditions effective to form the immunoconjugate according to any one of claims 1 to 4.

29. The process of claim 28, further comprising reducing an antibody or an antigen-binding fragment thereof under reducing conditions effective to form the thiol-functionalized antibody or antigen-binding fragment thereof.

30. The conjugate is 【Chemical 26】 selected from the group consisting of, the process of claim 28.

31. An immunoconjugate produced by the process of claim 28.

32. 【Fig. 27】 represented by

33. 【Fig. 28】 represented by

34. 【Fig. 29】 an immunoconjugate represented by wherein Ab is an antibody or an antigen-binding fragment thereof, and n is an integer from 1 to 10, immunoconjugate.

35. 【Fig. 30】 an immunoconjugate represented by wherein Ab is an antibody or an antigen-binding fragment thereof, and n is an integer from 1 to 10, immunoconjugate.

36. 【Fig. 31】 an immunoconjugate represented by wherein Ab is an antibody or an antigen-binding fragment thereof, and n is an integer from 1 to 10, immunoconjugate.

37. The immunoconjugate of claim 31, wherein Ab specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

38. Ab is a) VHCDR 1 comprising the amino acid sequence of SEQ ID NO: 1, VHCDR 2 comprising the amino acid sequence of SEQ ID NO: 2, and VHCDR 3 comprising the amino acid sequence of SEQ ID NO: 3 comprising a heavy chain, and b) VLCDR 1 comprising the amino acid sequence of SEQ ID NO: 8, VLCDR 2 comprising the amino acid sequence AAS, and VLCDR 3 comprising the amino acid sequence of SEQ ID NO: 10 comprising a light chain comprising, the immunoconjugate of claim 31.

39. a) A heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5 and retaining the amino acid sequence of VHCDR 1 described in SEQ ID NO: 1, the amino acid sequence of VHCDR 2 described in SEQ ID NO: 2, and the amino acid sequence of VHCDR 3 described in SEQ ID NO: 3, and b) A light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 and retaining the amino acid sequence of VLCDR 1 described in SEQ ID NO: 8, the amino acid sequence of VLCDR 2 AAS, and the amino acid sequence of VLCDR 3 described in SEQ ID NO: 10 comprising, the immunoconjugate of claim 31.

40. Ab is a) VHCDR 1 comprising the amino acid sequence of SEQ ID NO: 15, A VH CDR 2 comprising the amino acid sequence of SEQ ID NO: 16, and a VH CDR 3 comprising the amino acid sequence of SEQ ID NO: 17 a heavy chain comprising, and b) a VL CDR 1 comprising the amino acid sequence of SEQ ID NO: 22, a VL CDR 2 comprising the amino acid sequence DAY, and a VL CDR 3 comprising the amino acid sequence of SEQ ID NO: 24 a light chain comprising The immunoconjugate according to claim 31, comprising. **Claim 41** a) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 19, comprising the amino acid sequence of VH CDR 1 described in SEQ ID NO: 15, the amino acid sequence of VH CDR 2 described in SEQ ID NO: 16, and the amino acid sequence of VH CDR 3 described in SEQ ID NO: 17, a heavy chain, and b) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 26, comprising the amino acid sequence of VL CDR 1 described in SEQ ID NO: 22, the amino acid sequence DAY of VL CDR 2, and the amino acid sequence of VL CDR 3 described in SEQ ID NO: 24, a light chain The immunoconjugate according to claim 31, comprising. **Claim 42** The Ab is a) a VH CDR 1 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR 2 comprising the amino acid sequence of SEQ ID NO: 30, and a VH CDR 3 comprising the amino acid sequence of SEQ ID NO: 31 a heavy chain comprising, and b) a VL CDR 1 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR 2 comprising the amino acid sequence DAS, and a VL CDR 3 comprising the amino acid sequence of SEQ ID NO: 38 a light chain comprising The immunoconjugate according to claim 31, comprising. **Claim 43** a) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 33, comprising the amino acid sequence of VH CDR 1 described in SEQ ID NO: 29, the amino acid sequence of VH CDR 2 described in SEQ ID NO: 30, and the amino acid sequence of VH CDR 3 described in SEQ ID NO: 31, a heavy chain, and b) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 40, comprising the amino acid sequence of VL CDR 1 described in SEQ ID NO: 36, the amino acid sequence DAS of VL CDR 2, and the amino acid sequence of VL CDR 3 described in SEQ ID NO: 38, a light chain The immunoconjugate according to claim 31, comprising. **Claim 44** a) A heavy chain comprising VHCDR1 comprising the amino acid sequence of SEQ ID NO: 1, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and b) A light chain comprising VLCDR1 comprising the amino acid sequence of SEQ ID NO: 8, VLCDR2 comprising the amino acid sequence AAS, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 10, An antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1). **Claim 45** a) A heavy chain comprising VHCDR1 comprising the amino acid sequence of SEQ ID NO: 15, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and b) A light chain comprising VLCDR1 comprising the amino acid sequence of SEQ ID NO: 22, VLCDR2 comprising the amino acid sequence DAY, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 24, An antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1). **Claim 46** a) A heavy chain comprising VHCDR1 comprising the amino acid sequence of SEQ ID NO: 29, VHCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and VHCDR3 comprising the amino acid sequence of SEQ ID NO: 31, and b) A light chain comprising VLCDR1 comprising the amino acid sequence of SEQ ID NO: 36, VLCDR2 comprising the amino acid sequence DAS, and VLCDR3 comprising the amino acid sequence of SEQ ID NO: 38, An antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1). **Claim 47** a) A heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5 and retaining the amino acid sequences of VHCDR1, VHCDR2, and VHCDR3 as described in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and b) A light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 and retaining the amino acid sequences of VLCDR1, VLCDR2 amino acid sequence AAS, and VLCDR3 as described in SEQ ID NO: 8 and SEQ ID NO: 10, respectively An antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1). **Claim 48** a) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 19, comprising the amino acid sequence of VH CDR 1 set forth in SEQ ID NO: 15, the amino acid sequence of VH CDR 2 set forth in SEQ ID NO: 16, and the amino acid sequence of VH CDR 3 set forth in SEQ ID NO: 17, a heavy chain, and b) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 26, the amino acid sequence sequence containing the amino acid sequence of VL CDR 1 set forth in SEQ ID NO: 22, the amino acid sequence DAY of VL CDR 2, and the amino acid sequence of VL CDR 3 set forth in SEQ ID NO: 24, a light chain An antibody or antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1), comprising **Claim 49** a) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 33, comprising the amino acid sequence of VH CDR 1 set forth in SEQ ID NO: 29, the amino acid sequence of VH CDR 2 set forth in SEQ ID NO: 30, and the amino acid sequence of VH CDR 3 set forth in SEQ ID NO: 31, a heavy chain, and b) An amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 40, the amino acid sequence containing the amino acid sequence of VL CDR 1 set forth in SEQ ID NO: 36, the amino acid sequence DAS of VL CDR 2, and the amino acid sequence of VL CDR 3 set forth in SEQ ID NO: 38, a light chain An antibody or antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1), comprising

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