Glypican-3-targeted antibody-drug conjugates and methods of use

Abstract

Described herein are antibody-drug conjugates (ADCs) comprising an antibody construct that binds to human glypican 3 (GPC3) conjugated to a camptothecin analog of formula (I). The ADCs are useful as therapeutic agents, particularly in the treatment of cancer.

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of immunotherapeutics, and in particular to antibody-drug conjugates that target glypican-3 (GPC3). [Background technology]

[0002] background Glypican-3 (GPC3) is a glycosylphosphatidylinositol (GPI)-anchored oncofetal protein expressed on the surface of placental and fetal tissues, including liver, lung, and kidney. GPC3 expression is downregulated or silenced in normal adult tissues, but is expressed in hepatocellular carcinoma, melanoma, squamous cell lung carcinoma, and hepatoblastoma.

[0003] Numerous antibodies that bind to human GPC3 have been reported. Many of these antibodies have been developed as T cell engagers, NK cell engagers, chimeric antigen receptor (CAR) T cells or NK cells, or bispecific antibody therapeutics for cancer treatment. International Patent Publication No. WO2021 / 226321 (Phanes Therapeutics) (Patent Document 1) describes several anti-GPC3 paratopes that specifically bind to human GPC3.

[0004] Several antibodies targeting GPC3 have been clinically tested in monospecific formats, i.e., as bivalent IgG. Codrituzumab, also known as GC33 or RG-7686, has been tested in combination with other therapeutic agents in clinical trials in adults with hepatocellular carcinoma (HCC) and demonstrated limited efficacy, although with a favorable safety profile. Clinical trials of BMS-986183, an antibody-drug conjugate (ADC) of the anti-GPC3 antibody BMS-986182 (also known as GPC3.1 (BMS) or 4A6 (Medarex)) conjugated to a tubulysin drug moiety, were initiated in patients with advanced HCC, but no efficacy was observed and the trial was terminated.

[0005] Fu et al. (Hepatology. 2019 August; 70(2):563-576) (Non-Patent Document 1) described an ADC of the anti-GPC3 antibody YP7 conjugated to the DNA damaging agent duocarmycin SA and a pyrrolobenzodiazepine (PBD) (see Fu et al. (2019) Hepatology, 70(2):563-576 (Non-Patent Document 1)). YP7 conjugated to a PBD dimer showed efficacy in in vitro cancer cell models and induced tumor remission in mouse tumor models, but this ADC has not yet been clinically evaluated.

[0006] Camptothecin analogs have been developed as payloads for ADCs. Two such ADCs have been approved for the treatment of cancer: trastuzumab deruxtecan (Enhertu™), in which the camptothecin analog deruxtecan (Dxd) is conjugated to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™), in which the camptothecin analog SN-38 is conjugated to the anti-Trop-2 antibody sacituzumab via a hydrolyzable pH-sensitive linker.

[0007] Other camptothecin analogs and derivatives, and ADCs containing them, have been described, see, for example, International (PCT) Publication Nos. WO2019 / 195665, WO2019 / 236954, WO2020 / 200880, and WO2020 / 219287.

[0008] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2021 / 226321 [Patent Document 2] WO2019 / 195665 [Patent Document 3] WO2019 / 236954 [Patent Document 4] WO2020 / 200880 [Patent Document 5] WO2020 / 219287 [Non-patent literature]

[0010] [Non-Patent Document 1] Fu et al.(Hepatology.2019 August;70(2):563-576) Summary of the Invention

[0011] overview Described herein are antibody-drug conjugates targeting glypican-3 (GPC3) and methods of use. One aspect of the disclosure is an antibody-drug conjugate of formula (X): T-[L-(D) m ] n (X) In another aspect, the invention relates to an antibody-drug conjugate having the formula: m is an integer from 1 to 4, n is an integer from 1 to 10, T is an anti-GPC3 antibody construct comprising an antigen-binding domain that binds to human GPC3 (glypican-3), wherein the antigen-binding domain is: a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 6, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 7, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 8; and b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 18, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17 Including, L is a linker, D is a compound of formula I: TIFF2025535240000002.tif53165, wherein R 1 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 1 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000003.tif27165-CO2R 8 , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025535240000004.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5, —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from -H and -C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025535240000005.tif22165, R 13 is selected from -H and -C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0012] Another aspect of the present disclosure is a compound having the following structure: TIFF2025535240000006.tif63165, wherein: n is 1 to 10, and T is an anti-GPC3 antibody construct comprising an antigen-binding domain that binds to human GPC3 (glypican-3), wherein the antigen-binding domain is: a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 6, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 7, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 8; and b) i) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 71, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17; or ii) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 74, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17; or iii) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 77, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17 Includes:

[0013] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or diluent.

[0014] Another aspect of the present disclosure relates to a method of inhibiting the proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

[0015] Another aspect of the present disclosure relates to a method of killing cancer cells comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

[0016] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody-drug conjugate described herein.

[0017] Another aspect of the present disclosure pertains to the antibody-drug conjugates described herein for use in the treatment of cancer.

[0018] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer.

[0019] Another aspect of the present disclosure relates to a kit comprising an antibody-drug conjugate described herein and a label and / or package insert containing instructions for use. [Brief explanation of the drawings]

[0020] [Figure 1A] 1 shows the results of Caliper electrophoresis under reducing (R) and non-reducing (NR) conditions for v37575 (codrituzumab), v37574 (M3-H18L6), and v33624 (BMS-986182). [Figure 1B] UPLC-SEC profiles of v37574 and v37575 (after SEC purification) and v33624 (after Protein A purification) are shown. [Figure 2] Figure 1 shows an assessment of the binding cross-reactivity of humanized antibody M3-H1L1 (v36180) to GPC1, GPC2, GPC3, and GPC5 as assessed by ELISA. [Figure 3A] 1 shows the binding of v36180 (M3-H1L1), v37574 (M3-H18L6), and codrituzumab compared to the control palivizumab in HepG2 cells. [Figure 3B] Binding of these same antibodies on JHH-7 cells is illustrated. [Figure 4A] 1 illustrates the cytotoxicity of anti-GPC3 ADCs compared to non-targeting controls in GPC3-high HepG2 cells. [Figure 4B] 1 illustrates the cytotoxicity of anti-GPC3 ADCs compared to non-targeting controls in GPC3 and JHH-7 cells. [Figure 5A] 1 shows the cytotoxicity of M3-H18L6 ADC compared to non-targeting control in GPC3-high HepG2 spheroids. [Figure 5B]1 shows the cytotoxicity of M3-H18L6 ADC compared to a non-targeting control in NCI-H446 spheroids in GPC3 compared to a non-targeting control. [Figure 6A] 1 shows the cytotoxicity of M3-H18L6 ADC compared to BMS-986182 ADC and non-targeting antibody ADC in JHH-7 cells. [Figure 6B] 1 shows the cytotoxicity of M3-H18L6 ADC compared to BMS-986182 ADC and non-targeting antibody ADC in JHH-7 spheroid cells. [Figure 7] 1 illustrates the stability of M3-H1L1 and BMS-986182 ADCs in mouse plasma. [Figure 8] 1 shows the pharmacokinetic (PK) profiles of the M3-H18L6 and M3-H1L1 antibodies and ADCs of these antibodies in the Tg32 mouse model. [Figure 9A] 1 shows a comparison of the efficacy of ADCs BMS-986182 and M3-H1L1 in a xenograft model derived from the JHH-7 cell line. [Figure 9B] 1 shows a comparison of the efficacy of the same ADC in a xenograft model derived from the NCI-H446 cell line. [Figure 10A] 1 shows the PK profile of the M3-H1L1 ADC in the NCI-H446 xenograft model. [Figure 10B] 1 shows the PK profile of the M3-H1L1 ADC in a JHH-7 cell line-derived xenograft model and in an NCI-H446 xenograft model. [Figure 11A] 1 shows the efficacy of M3-H1L1 and M3-H18L6 ADCs in a xenograft model derived from the JHH-7 cell line. [Figure 11B] 1 shows the efficacy of M3-H1L1 and M3-H18L6 ADCs in a xenograft model derived from the NCI-H446 cell line. [Figure 12A] 1 illustrates the efficacy of the M3-H18L6 ADC in a HepG2 xenograft model. [Figure 12B]1 illustrates the efficacy of the M3-H18L6 ADC in a Hep3B xenograft model. [Figure 12C] 1 illustrates the efficacy of the M3-H18L6 ADC in the Huh-7 xenograft model. [Figure 12D] 1 illustrates the efficacy of the M3-H18L6 ADC in the PLC / PRF / 5 xenograft model. [Figure 13A] 1 illustrates the efficacy of the M3-H18L6 ADC in the LI1025 patient-derived xenograft model. [Figure 13B] 1 illustrates the efficacy of the M3-H18L6 ADC in the LI1037 patient-derived xenograft model. [Figure 14A] Figure 1 shows the bystander effect of ADCs v37574 (M3-H18L6) and v37575 (codrituzumab) in co-culture with GPC3-high HepG2 cells. [Figure 14B] Figure 1 shows the bystander effect of ADCs v37574 (M3-H18L6) and v37575 (codrituzumab) in co-culture with JHH-5 cells in GPC3. [Figure 15] 1 shows the results of Membrane Proteome Array™ screening of humanized variant v38592 in HEK293T cells. [Figure 16A] 1 depicts the binding of M3-H18L6 antibody and ADC to human GPC3-transfected CHO cells. [Figure 16B] 1 depicts the binding of M3-H18L6 antibody and ADC to CHO cells transfected with cynomolgus GPC3. [Figure 17A] 1 illustrates the binding of the M3-H18L6 antibody and ADC to HepG2 cells. [Figure 17B] 1 depicts binding of M3-H18L6 antibody and ADC to JHH-7 cells. [Figure 17C] 1 depicts the binding of M3-H18L6 antibody and ADC to JHH-5 cells. [Figure 17D] 1 depicts binding of M3-H18L6 antibody and ADC to SNU-601 cells. [Figure 18A] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the JHH-7 CDX model. [Figure 18B] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the Hep3B CDX model. [Figure 18C] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the JHH-5 CDX model. [Figure 19A] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI0050 PDX model of hepatocellular carcinoma (HCC). [Figure 19B] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI1005 PDX model of HCC. [Figure 19C] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI1069 PDX model of HCC. [Figure 19D] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI1097 PDX model of HCC. [Figure 19E] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI6610 PDX model of HCC. [Figure 19F] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI6619 PDX model of HCC. [Figure 19G] 1 illustrates the in vivo efficacy of the M3-H18L6 ADC in the LI6677 PDX model of HCC. [Figure 20] 1 illustrates the pharmacokinetic (PK) profile of v38592-MC-GGFG-AM-Compound 139 in DAR4. [Figure 21] Illustrates the pharmacokinetic (PK) profile of v38592-MC-GGFG-AM-Compound 139 in DAR8. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct that binds to human glypican-3 GPC3 (anti-GPC3 antibody construct) conjugated to a camptothecin analog of formula (I) described herein. The ADCs of the present disclosure may find use, for example, as therapeutic agents, particularly in the treatment of cancer.

[0022] 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.

[0023] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0024] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0025] Where a range of values ​​is given herein, for example, where a value is defined as from an upper limit to a lower limit, it is understood that the range includes both the upper and lower limits, as well as each value therebetween.

[0026] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. When used herein in connection with a composition, use, or method, the term "consisting essentially of" means that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. When used herein in connection with a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including particular elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not those embodiments are specifically mentioned.

[0027] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) may help maintain the proper conformation of the CDRs to promote binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required for antigen binding. However, in some cases, even a single variable domain can confer antigen-binding specificity. Furthermore, as is known in the art, in some cases, antigen binding may also occur through a combination of at least one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0028] Several different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, as will be readily apparent to one skilled in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that the disclosure of a VH herein includes disclosure of the associated (unique) heavy chain CDRs (HCDRs), as defined by any of the known numbering systems. Similarly, the disclosure of a VL herein includes disclosure of the associated (unique) light chain CDRs (LCDRs), as defined by any of the known numbering systems.

[0029] [Table 1]

[0030] The term "identical" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have the same percentage of amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95%, or about 98% identity over a designated region) when compared and aligned for maximum correspondence over a comparison window or over a designated region, as measured using one of the commonly used sequence comparison algorithms known to those skilled in the art or by manual alignment and visual inspection. For sequence comparison, a test sequence is typically compared to a designated reference sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0031] A "comparison window" refers to a segment of a sequence that includes contiguous amino acid or nucleotide positions, for example, about 10 to about 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, and a test sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison purposes are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI)), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0032] The term "acyl," as used herein, refers to the group --C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0033] The term "acyloxy" refers to the group --OC(O)R, where R is alkyl.

[0034] The term "alkoxy," as used herein, refers to the group --OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0035] The term "alkyl," as used herein, refers to a straight-chain or branched saturated hydrocarbon group containing the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, t-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.

[0036] The term "alkylaminoaryl," as used herein, refers to an alkyl group, as defined herein, substituted with an aminoaryl group, as defined herein.

[0037] The term "alkylheterocycloalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one heterocycloalkyl group, as defined herein.

[0038] The term "alkylthio," as used herein, refers to the group --SR, where R is an alkyl group.

[0039] The term "amide," as used herein, refers to the group --C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0040] The term "amino," as used herein, refers to the group --NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0041] The term "aminoalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more amino groups, for example, 1, 2, or 3 amino groups.

[0042] The term "aminoaryl," as used herein, refers to an aryl group, as defined herein, substituted with an amino group.

[0043] The term "aryl," as used herein, refers to a 6- to 12-membered monocyclic or bicyclic hydrocarbon ring system in which at least one ring is aromatic. Examples of aryl include, but are not limited to, phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8-tetrahydro-naphthalenyl, indanyl, and the like.

[0044] The term "carboxy," as used herein, refers to the group --C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0045] The term "cyano," as used herein, refers to the group --CN.

[0046] The term "cycloalkyl," as used herein, refers to a monocyclic or bicyclic saturated hydrocarbon containing the specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, and the like.

[0047] The term "haloalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more halogen atoms.

[0048] The terms "halogen" and "halo" as used herein refer to fluorine (F), bromine (Br), chlorine (Cl) and iodine (I).

[0049] The term "heteroaryl," as used herein, refers to a 6- to 12-membered monocyclic or bicyclic ring system in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S, and N. Examples of heteroaryls include, but are not limited to, pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

[0050] The term "heterocycloalkyl," as used herein, refers to a monocyclic or bicyclic non-aromatic ring system containing the specified number of atoms, wherein at least one ring atom is a heteroatom, such as O, S, or N. The heterocyclyl substituent can be attached via any available ring atom, such as a ring carbon or ring nitrogen. Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.

[0051] The terms "hydroxy" and "hydroxyl," as used herein, refer to the group --OH.

[0052] The term "hydroxyalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more hydroxy groups.

[0053] The term "nitro," as used herein, refers to the group --NO.sub.2.

[0054] The term "sulfonyl," as used herein, refers to the group --S(O)2R, where R is H, alkyl, or aryl.

[0055] The term "sulfonamide," as used herein, refers to the group --NH--S(O)2R, where R is H, alkyl, or aryl.

[0056] The terms "thio" and "thiol" as used herein refer to the group --SH.

[0057] Unless specifically stated as "unsubstituted," any alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein is understood to be "optionally substituted," i.e., each such reference includes both unsubstituted and substituted forms of those groups. For example, a reference to "-C1-C6 alkyl" includes both unsubstituted -C1-C6 alkyl and -C1-C6 alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group referred to herein is optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0058] The "substituted" chemical groups described herein may contain one substituent or multiple substituents up to the maximum valence of substitution for that group. For example, a methyl group can contain one, two, or three substituents, and a phenyl group can contain one, two, three, four, or five substituents. When a group is substituted with multiple substituents, the substituents may be the same or different.

[0059] The term "subject," as used herein, refers to an animal, in some embodiments, a mammal, that is the object of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, non-human primate, etc.). In certain embodiments, the subject is a human.

[0060] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0061] Particular features, structures, and / or characteristics described in connection with an embodiment disclosed herein may be combined in any suitable manner with features, structures, and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0062] It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for the exclusion of that feature in an alternative embodiment. For example, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically mentioned.

[0063] Antibody-drug conjugates The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-GPC3 antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) wherein T is an anti-GPC3 antibody construct described herein, L is a linker, D is a camptothecin analog having formula (I): m is an integer from 1 to 4, n is an integer from 1 to 10.

[0064] The components of formula (X) are described below.

[0065] Anti-GPC3 antibody construct “T” The ADCs of the present disclosure include anti-GPC3 antibody constructs. In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides that contains one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When an antibody construct contains two or more antigen-binding domains, each of the antigen-binding domains may bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may further include a scaffold, and one or more antigen-binding domains may be fused or covalently linked to the scaffold, optionally via a linker.

[0066] According to the present disclosure, an anti-GPC3 antibody construct comprises at least one antigen-binding domain that specifically binds to human GPC3 (hGPC3). "Specifically binds" to hGPC3 means that the antibody construct binds to hGPC3 but does not exhibit significant binding to human glypican-1 (GPC1), glypican-2 (GPC2), glypican-4 (GPC4), glypican-5 (GPC5), or glypican-6 (GPC6). In one embodiment, the anti-GPC3 antibody construct binds to GPC3 but does not exhibit significant binding to GPC1, GPC2, or GPC5. In certain embodiments, the anti-GPC3 antibody construct of the present disclosure can bind to GPC3 from one or more non-human species. In certain embodiments, the anti-GPC3 antibody construct of the present disclosure can bind to cynomolgus monkey GPC3.

[0067] Human GPC3 is also known as "glypican proteoglycan 3" or "heparan sulfate proteoglycan." Protein sequences of GPC3 from various sources are known in the art and readily available from publicly accessible databases such as GenBank or UniProtKB. Exemplary hGPC3 sequences include those provided under NCBI reference numbers P51654, NP_001158091.1, NP_001158090.1, NP_001158089.1, NP_004475.1, and AAA98132.1. An exemplary hGPC3 protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI Reference Sequence: P51654). An exemplary cynomolgus monkey GPC3 protein sequence is also provided in Table 2 (SEQ ID NO: 2; UniProt ID: A0A2K5VK50).

[0068] [Table 2] TIFF2025535240000009.tif128165

[0069] Specific binding of an antigen-binding domain to a target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or conventional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as binding to a non-target protein (such as GPC1, GPC2, or GPC5) that is less than about 10% of the binding to hGPC3, as measured, for example, by ELISA or flow cytometry.

[0070] Dissociation constant (K D or K dThe term "K" as used herein is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, a ligand-protein interaction refers to, but is not limited to, a protein-protein interaction or an antibody-antigen interaction. D measures the tendency of two proteins (e.g., AB) complexed with each other to reversibly dissociate into their components (A+B), which is called the “off rate (k off The dissociation and association rates, also called "on-rates (k on )" Therefore, K D is k off / k on and is expressed as molar concentration (M). D The smaller the K, the stronger the binding affinity. D A decrease in K indicates an increase in affinity. D has a K of 1 nM D The affinity is expressed as K A or K a It is sometimes measured as K D or K d The K of the antibody construct D The K value can be determined using methods well established in the art. D One method for determining K is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. Isothermal titration calorimetry (ITC) can be used to determine K D The Octet™ system may also be used to measure the affinity of an antibody for a target antigen.

[0071] In certain embodiments, the specific binding of the antibody construct to GPC3 has a dissociation constant (K) of <1 μM, e.g., <500 nM, <250 nM, <100 nM, <50 nM, or <10 nM. DIn certain embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M or less, e.g., 10 -7 M or less, or 10 -8 The dissociation constant (K D In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M~10 -9 M, e.g., 10 -7 M~10 -9 Dissociation constant of M (K D )

[0072] In some embodiments, the antigen-binding domain of the anti-GPC3 antibody construct has a higher K than the reference antibody codrituzumab as measured by SPR. D Thus, in these embodiments, the anti-GPC3 antibody construct of the ADCs of the disclosure comprises an antigen-binding domain with a lower affinity for human GPC3 than the reference antibody codrituzumab.

[0073] Anti-GPC3 antibody constructs are internalized by GPC3-expressing cells. Antibody internalization can be measured using methods known in the art, for example, by direct internalization according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or by commercially available fluorescent dyes such as pHAb dye (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dye (ThermoFisher Scientific Corporation, Waltham, MA) and Incucyte® Fabfluor-pH antibody labeling reagent (Sartorius AG, Goettingen, Germany), and analytical techniques such as microscopy, FACS, high-content imaging or other plate-based assays.

[0074] In some embodiments, the anti-GPC3 antibody construct is internalized to a similar extent to the reference antibody codrituzumab in cells expressing high levels of GPC3, such as HepG2 cells or JHH-7 cells. In some embodiments, the amount of internalized antibody is determined after an incubation period of at least 5 hours. In some embodiments, conjugation of the anti-GPC3 antibody construct to a camptothecin analog does not affect the internalization of the anti-GPC3 antibody construct.

[0075] As noted throughout this disclosure, GPC3 expression varies depending on the cell type, and GPC3 expression levels are sometimes referred to herein as "high," "intermediate," "low," or "negative." These terms are used for reference to generally describe GPC3 expression levels, as specified in Table 12.1 of Example 12, and are not intended to be limited to specific values ​​of average GPC3 protein per cell contained therein. Alternatively, GPC3 expression levels in cells or tumors can be assessed by immunohistochemistry (IHC) according to methods known in the art. For example, IHC can be used to stain GPC3 in tumor tissue samples from xenograft models, cell lines (CDX), or patients (PDX). Tissue samples can be examined, and an H-score can be calculated as known in the art and described, for example, in Example 33 herein. The higher the H-score, the higher the expression of GPC3 in the tissue sample.

[0076] antigen-binding domain The anti-GPC3 antibody construct of the present disclosure comprises at least one antigen-binding domain capable of binding to hGPC3. The at least one antigen-binding domain capable of binding to hGPC3 is typically an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fabs (scFabs), single-chain Fvs (scFvs), and single-domain antibodies (sdAbs).

[0077] A "Fab fragment" contains the light and heavy chain variable domains (VL and VH, respectively), as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab fragments may also be single-chain Fab molecules, i.e., Fab molecules in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, in a single-chain Fab molecule, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain.

[0078] An "scFv" comprises an antibody heavy chain variable domain (VH) and light chain variable domain (VL) in a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For example, an scFv may comprise a VL connected from the C-terminus of the VL to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may comprise a VH connected via the C-terminus of the VH to the N-terminus of the VL by a polypeptide linker (see review in Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0079] The "sdAb" format refers to a single immunoglobulin domain. sdAbs may, for example, be of camelid origin. Camelid antibodies lack light chains and their antigen-binding site consists of a single domain, termed "VHH." sdAbs form three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and are easily expressed, for example, as fusions with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0080] In those embodiments in which the anti-GPC3 antibody construct of the ADC comprises two or more antigen-binding domains, each additional antigen-binding domain can independently be an immunoglobulin-based domain, e.g., an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, e.g., a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule, e.g., a natural or engineered ligand, capable of specifically binding to its target. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers.

[0081] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises at least one antigen-binding domain that specifically binds to hGPC3, wherein the antigen-binding domain is derived from MAb clone M3 described in WO2021 / 226321. Thus, in certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a set of HCDRs and a set of LCDRs identified according to IMGT, Kabat, Chothia AbM, or Contact numbering, as set forth in Table 3 below.

[0082] [Table 3]

[0083] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises an antigen-binding domain comprising three HCDR amino acid sequences and three LCDR amino acid sequences of v36180 (M3-H1L1) or v37574 (M3-H18L6), as defined by the IMGT, Kabat, Chothia, or AbM numbering systems.

[0084] In certain embodiments, the anti-GPC3 antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having a VH amino acid sequence comprising the three HCDR amino acid sequences of v36180 (M3-H1L1) and a VL amino acid sequence comprising the three LCDR amino acid sequences of v36180 (M3-H1L1). In certain other embodiments, the anti-GPC3 antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having a VH amino acid sequence comprising the three HCDR amino acid sequences of v37574 (M3-H18L6) and a VL amino acid sequence comprising the three LCDR amino acid sequences of v37574 (M3-H18L6).

[0085] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 18, 19, and 17, as defined by Kabat numbering.

[0086] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences set forth in SEQ ID NOs: 16 and 17, and the LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0087] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 18, 19, and 17, as defined by Chothia numbering.

[0088] In certain embodiments, the anti-GPC3 antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 20, 21, and 22, as defined by Contact numbering.

[0089] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 11, 12, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 18, 19, and 17, as defined by AbM numbering.

[0090] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without losing the antibody's ability to bind to its target. Candidate amino acid substitutions can be identified by computer modeling or techniques known in the art, such as alanine scanning, and the resulting variants are tested for binding activity by standard techniques. Thus, in certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) that have 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity with the set of CDRs of v36180 (M3-H1L1) or v37574 (M3-H18L6), where the % sequence identity is calculated across all six CDRs, and the antigen-binding domain retains the ability to bind to hGPC3.

[0091] In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a set of HCDRs and a set of LCDRs set forth in any one of Table 3A, Table 3B, or Table 3C below.

[0092] [Table 3A]

[0093] [Table 3B]

[0094] [Table 3C]

[0095] In certain embodiments, the anti-GPC3 antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having a VH amino acid sequence comprising the three HCDR amino acid sequences of LC-modified variant 40206 and a VL amino acid sequence comprising the three LCDR amino acid sequences of v40206, as defined by one of the IMGT, Kabat, Chothia, AbM, or Contact numbering systems. In certain other embodiments, the anti-GPC3 antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain having a VH amino acid sequence comprising the three HCDR amino acid sequences of LC-modified variant 40207 and a VL amino acid sequence comprising the three LCDR amino acid sequences of LC-modified variant 40207, as defined by one of the IMGT, Kabat, Chothia, AbM, or Contact numbering systems. In yet other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having a VH amino acid sequence comprising the three HCDR amino acid sequences of the LC-modified variant 40208 and a VL amino acid sequence comprising the three LCDR amino acid sequences of the LC-modified variant 40208, as defined by one of the IMGT, Kabat, Chothia, AbM, or Contact numbering systems.

[0096] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises the antigen-binding domain of LC-modified variant 40206 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 71, 19, and 17, as defined by Kabat numbering.

[0097] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40206 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences set forth in SEQ ID NOs: 70 and 17, and the LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0098] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40206 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 71, 19, and 17, as defined by Chothia numbering.

[0099] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40206 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 72, 21, and 22, as defined by Contact numbering.

[0100] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40206 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 11, 12, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 71, 19, and 17, as defined by AbM numbering.

[0101] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises the antigen-binding domain of LC-modified variant 40207 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 74, 19, and 17, as defined by Kabat numbering.

[0102] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40207 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences set forth in SEQ ID NOs: 73 and 17, and the LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0103] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises the antigen-binding domain of LC-modified variant 40207 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 74, 19, and 17, as defined by Chothia numbering.

[0104] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40207 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 75, 21, and 22, as defined by Contact numbering.

[0105] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40207 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 11, 12, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 74, 19, and 17, as defined by AbM numbering.

[0106] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises the antigen-binding domain of LC-modified variant 40208 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 77, 19, and 17, as defined by Kabat numbering.

[0107] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40208 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences set forth in SEQ ID NOs: 76 and 17, and the LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0108] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40208 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 77, 19, and 17, as defined by Chothia numbering.

[0109] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises the antigen-binding domain of LC-modified variant 40208 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 78, 21, and 22, as defined by Contact numbering.

[0110] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises the antigen-binding domain of LC-modified variant 40208 having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 11, 12, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 77, 19, and 17, as defined by AbM numbering.

[0111] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH amino acid sequence of v36180 (M3-H1L1), and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL amino acid sequence of v36180 (M3-H1L1), wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0112] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises a VH sequence having the three HCDRs of v36180(M3-H1L1) and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of v36180(M3-H1L1), and the three LCDRs of v36180(M3-H1L1) and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of v36180(M3-H1L1). and an antigen-binding domain comprising a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL amino acid sequence of hGPC3 (H1L1), wherein the three HCDRs and three LCDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system, and wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0113] In other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH amino acid sequence of v37574 (M3-H18L6), and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL amino acid sequence of v37574 (M3-H18L6), wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0114] In other embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises a VH sequence having the three HCDRs of v37574(M3-H18L6) and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of v37574(M3-H18L6), and 4(M3-H18L6) and an antigen-binding domain comprising a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL amino acid sequence of v37574(M3-H18L6), wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0115] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH amino acid sequence of LC-modified variant 40206, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL amino acid sequence of LC-modified variant 40206, wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0116] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises a VH sequence having the three HCDRs of LC-modified variant 40206 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40206, and a VH sequence having the three LCDRs of LC-modified variant 40206 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40206. and an antigen-binding domain comprising a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 40206, wherein the three HCDRs and three LCDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system, and wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0117] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH amino acid sequence of LC-modified variant 40207, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL amino acid sequence of LC-modified variant 40207, wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0118] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises a VH sequence having the three HCDRs of LC-modified variant 40207 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40207, and a VH sequence having the three LCDRs of LC-modified variant 40207 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40207. and an antigen-binding domain comprising a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL amino acid sequence of 40207, wherein the three HCDRs and three LCDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system, and wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0119] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH amino acid sequence of LC-modified variant 40208, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL amino acid sequence of LC-modified variant 40208, wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0120] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the disclosure comprises a VH sequence having the three HCDRs of LC-modified variant 40208 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40208, and a VH sequence having the three LCDRs of LC-modified variant 40208 and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH amino acid sequence of LC-modified variant 40208. and an antigen-binding domain comprising a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL amino acid sequence of 40208, wherein the three HCDRs and three LCDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system, and wherein the antigen-binding domain retains the ability to bind to hGPC3.

[0121] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 27, and an antigen-binding domain comprising a VL amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 28.

[0122] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29, and an antigen-binding domain comprising a VL amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 30.

[0123] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29, and an antigen-binding domain comprising a VL amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 68.

[0124] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29, and an antigen-binding domain comprising a VL amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 64.

[0125] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises a VH amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29, and an antigen-binding domain comprising a VL amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 60.

[0126] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising (i) the VH amino acid sequence set forth in SEQ ID NO: 27 and the VL amino acid sequence set forth in SEQ ID NO: 28, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 29 and the VL amino acid sequence set forth in SEQ ID NO: 30. In other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising the VH amino acid sequence set forth in SEQ ID NO: 29 and the VL amino acid sequence set forth in SEQ ID NO: 68. In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising the VH amino acid sequence set forth in SEQ ID NO: 29 and the VL amino acid sequence set forth in SEQ ID NO: 64. In yet other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising the VH amino acid sequence set forth in SEQ ID NO: 29 and the VL amino acid sequence set forth in SEQ ID NO: 60.

[0127] Exemplary VH and VL sequences are provided in the Examples and Sequence Listing.

[0128] In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequence set forth in SEQ ID NO: 50 and two light chains comprising the sequence set forth in SEQ ID NO: 53 (v37574 M3-H18L6). In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequence set forth in SEQ ID NO: 56 and two light chains comprising the sequence set forth in SEQ ID NO: 53 (v38592). In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequence set forth in SEQ ID NO: 44 and two light chains comprising the sequence set forth in SEQ ID NO: 47 (v36180 M3-H1L1). In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequence set forth in SEQ ID NO: 50 and two light chains comprising the sequence set forth in SEQ ID NO: 66 (v40206). In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequences set forth in SEQ ID NO: 50 and two light chains comprising the sequences set forth in SEQ ID NO: 62 (v40207). In one embodiment, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises two heavy chains comprising the sequences set forth in SEQ ID NO: 50 and two light chains comprising the sequences set forth in SEQ ID NO: 58 (v40208).

[0129] format The anti-GPC3 antibody construct of the ADC can have various formats. The minimum component of the anti-GPC3 antibody construct is an antigen-binding domain that binds to hGPC3. The anti-GPC3 antibody construct can optionally further comprise one or more additional antigen-binding domains and / or scaffolds. In those embodiments in which the anti-GPC3 antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within hGPC3, a different epitope within hGPC3, or a different antigen. Thus, the anti-GPC3 antibody construct can be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0130] In certain embodiments, the anti-GPC3 antibody construct comprises at least one antigen-binding domain that binds to hGPC3 and a scaffold, wherein the antigen-binding domain is operably linked to the scaffold. As used herein, the term "operably linked" means that the described components are in a relationship that allows them to function in their intended manner. Suitable scaffolds are described below.

[0131] In certain embodiments, the anti-GPC3 antibody construct comprises two antigen-binding domains operably linked to a scaffold. In some embodiments, the anti-GPC3 antibody construct may comprise three or four antigen-binding domains, and optionally a scaffold. In these formats, when a scaffold is included, at least the first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domain(s) may each be independently operably linked to the scaffold or the first antigen-binding domain, or, if more than two antigen-binding domains are present, may be linked to another antigen-binding domain.

[0132] Scaffold-free anti-GPC3 antibody constructs may contain a single antigen-binding domain in a suitable format, such as an sdAb, or they may contain two or more antigen-binding domains, optionally operably linked by one or more linkers. In such anti-GPC3 antibody constructs, the antigen-binding domain may be in the form of an scFv, Fab, sdAb, or a combination thereof. For example, using scFv as the antigen-binding domain allows for the construction of formats such as tandem scFv ((scFv)2 or taFv), in which scFvs are connected together by a flexible linker. scFvs can also be used to construct diabody formats containing two scFvs connected by a short linker (usually about 5 amino acids in length). The limited length of the linker allows for dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFv can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between the VL and VH by introducing an additional cysteine ​​residue in each chain (e.g., at position 44 in the VH and position 100 in the VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0133] Similarly, in some embodiments, a format comprising two sdAbs, such as VH or VHH, connected together via a suitable linker may be used. Other examples of scaffold-less anti-GPC3 antibody construct formats include those based on Fab fragments, such as Fab2 and F(ab')2 formats, in which the Fab fragments are connected via a linker or IgG hinge region.

[0134] Combinations of different forms of antigen-binding domains can also be used to create alternative scaffold-less formats, for example, an scFv or sdAb can be fused to the C-terminus of either or both the light and heavy chains of a Fab fragment, resulting in a bivalent (Fab-scFv / sdAb) construct.

[0135] In certain embodiments, the anti-GPC3 antibody construct may be in an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as the full-size antibody format (FSA) or MAb format, and includes anti-GPC3 antibody constructs comprising two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-GPC3 antibody construct may be based on an IgG class immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-GPC3 antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-GPC3 antibody construct is based on a particular immunoglobulin isotype, this means that the anti-GPC3 antibody construct comprises all or part of the constant region of that particular immunoglobulin isotype. For example, an anti-GPC3 antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and, optionally, an Ig hinge region from the same or a different isotype. It should be understood that in some embodiments, the anti-GPC3 antibody construct may also comprise a hybrid of isotypes and / or subclasses. It should also be understood that the Fc region and / or hinge region may be optionally modified to confer one or more desirable functional properties known in the art. Thus, in certain embodiments, the anti-GPC3 antibody construct comprises a VH amino acid sequence (i.e., CH1, CH2, CH3 amino acid sequence) fused to an IgG1 constant domain amino acid sequence and a VL amino acid sequence (i.e., CL amino acid sequence) fused to a kappa or lambda constant amino acid sequence domain. Exemplary amino acid sequences are provided in the Examples and Sequence Listing.

[0136] In some embodiments, the anti-GPC3 antibody construct may be derived from two or more immunoglobulins from different species, for example, the anti-GPC3 antibody construct may be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from multiple species.

[0137] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.

[0138] A "humanized antibody" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity and affinity for the target antigen. This technique for producing humanized antibodies is often referred to as "CDR grafting."

[0139] In some cases, additional modifications are made to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains of humanized antibodies contain all or substantially all of the hypervariable regions from the non-human immunoglobulin and all or substantially all of the FRs from the human immunoglobulin sequence. Humanized antibodies are described in further detail in, for example, Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0140] Several approaches for selecting the most appropriate human framework for grafting nonhuman CDRs are known in the art. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the nonhuman antibody providing the CDRs (the "fixed framework" approach). More recent approaches use variable regions with high amino acid sequence identity with the variable regions of the nonhuman antibody providing the CDRs (the "homology matching" or "best-fit" approach). An alternative approach is to select fragments of framework sequences within each light or heavy chain variable region from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding nonhuman-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0141] Alternatively or in addition to these traditional approaches, more recent techniques may be used to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on a human germline sequence or consensus sequence may be used as the acceptor human framework, rather than a human framework with somatic mutation(s). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen-binding activity are grafted onto a human germline framework. This method can help improve the "humanness" of humanized antibodies (i.e., similarity to human germline sequences), thus reducing the risk of immunogenicity of the variable regions. These techniques are described in various publications (see, e.g., Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0142] scaffold In certain embodiments, the anti-GPC3 antibody construct comprises one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerization peptides (e.g., leucine zippers, heterodimer-forming "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0143] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance.When the scaffold is a polypeptide, each antigen-binding domain of the anti-GPC3 antibody construct can be linked to either the N-terminus or C-terminus of the polypeptide scaffold.In certain embodiments, the anti-GPC3 antibody construct also includes a polypeptide scaffold in which one or more antigen-binding polypeptide constructs are linked to a region other than the N-terminus or C-terminus, for example, via the side chain of an amino acid with or without a linker.

[0144] In embodiments where the anti-GPC3 antibody construct comprises a peptide or polypeptide scaffold, the antigen binding domain(s) can be linked to the scaffold by genetic fusion or chemical conjugation.Typically, when the scaffold is a peptide or polypeptide, the antigen binding domain(s) can be linked to the scaffold by genetic fusion.In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen binding domain(s) can be linked to the scaffold by chemical conjugation.

[0145] Several protein domains containing selective pairs of two different polypeptides are known in the art and can be used to form scaffolds. One example is the leucine zipper domains, such as Fos and Jun, which selectively pair (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287:43331-43339 (2012)). Other selectively pairing molecular pairs include, for example, the barnase barnase pair (Deyev, et al., Nat Biotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(1-2):23-26 (1999)), and split fluorescent protein pairs (International Patent Application Publication No. WO2011 / 135040).

[0146] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Mueller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U.S. Patent Application Publication No. 2009 / 0285816).

[0147] For example, fusing an antigen-binding moiety, such as an scFv, diabody, or single-chain diabody, to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Mueller et al., ibid.). The antigen-binding moiety may be fused to the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0148] Derivatives of albumin have been described in the form of heteromultimers containing two transporter polypeptides obtained by segmenting the albumin protein so that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer contains four termini and can therefore be fused, optionally via linkers, to up to four different antigen-binding moieties.

[0149] In certain embodiments, the anti-GPC3 antibody construct of the ADC may comprise a protein scaffold. In some embodiments, the anti-GPC3 antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. In some embodiments, the anti-GPC3 antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0150] Fc area As used herein, the terms "Fc region," "Fc," or "Fc domain" refer to the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0151] In certain embodiments, the anti-GPC3 antibody construct of the ADC may comprise a scaffold based on an immunoglobulin Fc region. The Fc region may be dimeric and comprise two Fc polypeptides, or alternatively, the Fc region may comprise a single polypeptide.

[0152] An "Fc polypeptide" in the context of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0153] An Fc region may comprise a CH3 domain, or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0154] In some embodiments, the anti-GPC3 antibody construct of the ADC may comprise a scaffold based on the IgG Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on the human IgG Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on the IgG1 Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on the human IgG1 Fc region.

[0155] In certain embodiments, the anti-GPC3 antibody construct may comprise an IgG Fc region-based scaffold, which is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence and optionally a CH2 sequence, wherein the first and second Fc polypeptides are different. In some embodiments, the anti-GPC3 antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-GPC3 antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications.

[0156] In some embodiments, the anti-GPC3 antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, the modified CH3 domain being an asymmetrically modified CH3 domain containing one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification, such as a substitution or insertion, in which an amino acid at a particular position in a first CH3 or CH2 sequence is different from the amino acid at the same position in a second CH3 or CH2 sequence. These asymmetric amino acid modifications may result in only one of the two amino acids at the same respective amino acid position in each sequence being modified, or in both amino acids at the same respective position in each of the first and second CH3 or CH2 sequences being modified differently. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may contain one or more asymmetric amino acid modifications.

[0157] In some embodiments, the anti-GPC3 antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, the modified CH3 domain comprising one or more amino acid modifications that promote the formation of a heterodimeric Fc over the formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0158] Amino acid modifications that can be made to the CH3 domain of an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies to produce stable asymmetrically modified Fc regions, such as those described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. In certain embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

[0159] Table 4 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain. Table 4 also lists amino acid modifications in the CH3 domain that promote the formation of heterodimeric Fc, as described in International Patent Application Publication Nos. WO2012 / 058768 and WO2013 / 063702.

[0160] In certain embodiments, the anti-GPC3 antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain containing any one of variant 1, variant 2, variant 3, variant 4, or variant 5 modifications, as shown in Table 5.

[0161] [Table 4]

[0162] In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications. Modifications in the CH2 domain can affect Fc binding to Fc receptors (FcR), such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0163] In some embodiments, the anti-GPC3 antibody construct comprises an IgG Fc-based scaffold having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0164] Many amino acid modifications to the CH2 domain that selectively alter the affinity of Fc for various Fcγ receptors are known in the art. Amino acid modifications that increase binding and those that decrease binding can each be useful in specific indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased lysis of target cells. Similarly, decreasing binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations. Reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable in certain indications. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0165] Among the amino acid modifications in the CH2 domain, examples of amino acid modifications that alter Fc binding via the Fcγ receptor include S298A / E333A / K334A and S298A / E333A / K334A / K326A (improved affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (improved affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), and F243L / R292P / Y300L / L235V / P396L (improved affinity for FcγRIIIa) (Nordstrom JL, et al. al., 2011, Breast Cancer Res, 13(6):R123), F243L (improved affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8), S298A / E333A / K334A (improved affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604), S239D / I332E / A330L and S239D / I332E (improved affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (improved affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter Fc binding by FcγRIIb are described in International Publication No. WO2021 / 232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, page 283).

[0166] In certain embodiments, the anti-GPC3 antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, which contains one or more amino acid modifications that reduce or eliminate binding of the entire Fc region to Fcγ receptors (i.e., a "knockout" variant).

[0167] Various publications describe strategies used to engineer antibodies to generate "knockout" variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0168] Examples of mutations that can be introduced into the hinge or CH2 domain to generate a "knockout" variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0169] In certain embodiments, the anti-GPC3 antibody constructs described herein may comprise a scaffold based on IgG Fc with modified native glycosylation. As known in the art, glycosylation of Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a deglycosylated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0170] Conversely, removal of fucose from the oligosaccharide attached to heavy chain N297 has been shown to enhance ADCC due to improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to carbohydrates linked to N297 (International Publication No. WO 03 / 035835), or in other cells that produce defucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002 (ibid.); and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, International Publication No. WO2009 / 135181 describes the addition of fucose analogs to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody.

[0171] Other methods for producing antibodies with little or no fucose on the Fc glycosylation site (N297) are known in the art, e.g., GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0172] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which biantennary oligosaccharides attached to the Fc region of an antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include those with at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, e.g., International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0173] Preparation of anti-GPC3 antibody constructs The anti-GPC3 antibody constructs described herein may be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0174] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-GPC3 antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. The polynucleotide(s) encoding the anti-GPC3 antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be understood by those skilled in the art, the number of polynucleotides required for the expression of an anti-GPC3 antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, if the anti-GPC3 antibody construct is a monospecific mAb or FSA format, two polynucleotides (one encoding a light chain polypeptide and one encoding a heavy chain polypeptide) will be required. If multiple polynucleotides are required, they may be incorporated into one vector or into multiple vectors.

[0175] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector(s) along with one or more control elements, such as transcriptional elements required for efficient transcription of the polynucleotide. Examples of such control elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the selection of control elements will depend on the host cell selected for expression of the antibody construct and that such control elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal or integrating vector.

[0176] Suitable host cells for cloning or expressing anti-GPC3 antibody constructs include various prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces cells or Pichia cells). Prokaryotic host cells include, for example, E. coli cells, A. salmonicida cells, or B. subtilis cells.

[0177] In certain embodiments, anti-GPC3 antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed. Humana Press, Totowa, NJ, 2003.

[0178] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0179] Suitable host cells for the expression of glycosylated anti-GPC3 antibody constructs are usually eukaryotic cells.For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe the PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants.Mammalian cell lines adapted to grow in suspension can be particularly useful for the expression of antibody constructs. Examples include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod., 23:243-251); monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci, 383: 44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR - Exemplary mammalian host cell lines suitable for the production of antibody constructs include, but are not limited to, CHO cells; see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed. Humana Press, Totowa, NJ, 2003).

[0180] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0181] Host cells containing expression vector(s) encoding anti-GPC3 antibody constructs can be cultured using conventional methods to produce anti-GPC3 antibody constructs. Alternatively, in some embodiments, host cells containing expression vector(s) encoding anti-GPC3 antibody constructs can be used therapeutically or prophylactically to deliver anti-GPC3 antibody constructs to a subject, or polynucleotides or expression vectors can be administered ex vivo to cells from a subject, and then the cells can be returned to the subject's body.

[0182] Typically, anti-GPC3 antibody constructs are purified after expression. Proteins may be isolated or purified in various ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, sizing chromatography, gel filtration chromatography, and reversed-phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can be used to purify specific antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can also be enabled by specific fusion partners. For example, the antibody may be attached to a glutathione resin if a GST fusion is used, or to a Ni tag if a His tag is used. +2 Purification may be performed using affinity chromatography or, if a flag tag is used, using an immobilized anti-flag antibody. The degree of purification required varies depending on the use of the anti-GPC3 antibody construct. In some cases, no purification may be required.

[0183] In certain embodiments, the anti-GPC3 antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used in reference to the anti-GPC3 antibody constructs described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as in natural cells, or in the case of recombinantly produced constructs, in host cells. In certain embodiments, a substantially pure anti-GPC3 antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0184] Certain embodiments of the present disclosure relate to methods for producing anti-GPC3 antibody constructs, comprising culturing host cells into which one or more polynucleotides encoding the anti-GPC3 antibody constructs or one or more expression vectors encoding the anti-GPC3 antibody constructs have been introduced under conditions suitable for expression of the anti-GPC3 antibody constructs, and optionally recovering the anti-GPC3 antibody constructs from the host cells (or host cell culture medium).

[0185] Post-translational modifications In certain embodiments, the anti-GPC3 antibody constructs described herein may contain one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-GPC3 antibody construct from a host cell.

[0186] Post-translational modifications include various modifications known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., TECreighton, WH Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pgs.1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. NY Acad. Sci., 663:48-62). In embodiments where the anti-GPC3 antibody construct contains one or more post-translational modifications, the construct may contain the same type of modification at one or more sites, or may contain different modifications at different sites.

[0187] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage (by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0188] Other examples of post-translational modifications include, for example, the addition or removal of N- or O-linked glycans, chemical modification of N- or O-linked glycans, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to enable detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0189] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0190] Camptothecin analogues Camptothecin analogs encompassed by the ADCs of the present disclosure have formula (I): TIFF2025535240000015.tif53165, wherein R 1 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 1 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000016.tif27165-CO2R 8 , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025535240000017.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025535240000018.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19together with the N atom to which they are attached, halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0191] In some embodiments, the camptothecin analog is a compound of formula (I), except that R 1 If is NH2, R 2 is other than H.

[0192] In some embodiments, in the compound of Formula (I), R 1 is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 and NH 2 .

[0193] In some embodiments, in the compound of Formula (I), R 1 is NH2.

[0194] In some embodiments, in the compound of Formula (I), R 1 is selected from —H, —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0195] In some embodiments, in the compound of Formula (I), R 1 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0196] In some embodiments, in the compound of Formula (I), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0197] In some embodiments, in the compound of Formula (I), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0198] In some embodiments, in the compound of Formula (I), R 2 is selected from -H, -F, -Br and -Cl.

[0199] In some embodiments, in the compound of Formula (I), R 2 is selected from -F, -Br and -Cl.

[0200] In some embodiments, in the compound of Formula (I), R 3 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000019.tif27165-CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0201] In some embodiments, in the compound of Formula (I), R 4 teeth, Selected from TIFF2025535240000020.tif58165.

[0202] In some embodiments, in the compound of Formula (I), R 5is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0203] In some embodiments, in the compound of Formula (I), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from.

[0204] In some embodiments, in the compound of Formula (I), R 8 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0205] In some embodiments, in the compound of Formula (I), each R 9 is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0206] In some embodiments, in the compound of Formula (I), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0207] In some embodiments, in the compound of Formula (I), each R 9is independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0208] In some embodiments, in the compound of Formula (I), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0209] In some embodiments, in the compound of Formula (I), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0210] In some embodiments, in the compound of Formula (I), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0211] In some embodiments, in the compound of Formula (I), R 10’ is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0212] In some embodiments, in the compound of Formula (I), R 11is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0213] In some embodiments, in the compound of Formula (I), each R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 is selected from.

[0214] In some embodiments, in the compound of Formula (I), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, -S(O)2R 16 , and Selected from TIFF2025535240000021.tif22165.

[0215] In some embodiments, in the compound of Formula (I), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0216] In some embodiments, in the compound of Formula (I), R 14 and R 14’ are each independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0217] In some embodiments, in the compound of Formula (I), R 16 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0218] In some embodiments, in the compound of Formula (I), R 16 is selected from unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0219] In some embodiments, in the compound of Formula (I), R 17 is selected from unsubstituted -C1-C6 alkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0220] In some embodiments, in the compound of Formula (I), R 18 and R 19 together with the N atom to which they are attached, represent halogen, unsubstituted C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0221] In some embodiments, in the compound of formula (I), X a and X b are each independently selected from NH and O.

[0222] Combinations of any of the foregoing embodiments for compounds of formula (I) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0223] In certain embodiments, the compound of formula (I) has formula (II): TIFF2025535240000022.tif53165, wherein R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000023.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000024.tif69165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 10’is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025535240000025.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; Xc is selected from O, S, and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0224] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0225] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0226] In some embodiments, in the compound of Formula (II), R 2 is selected from F and Cl.

[0227] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000026.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000027.tif58165.

[0228] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000028.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000029.tif58165.

[0229] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535240000030.tif58165.

[0230] In some embodiments, in the compound of Formula (II), R 20 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000031.tif27165-CO2R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, Selected from TIFF2025535240000032.tif68165.

[0231] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000033.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000034.tif58165.

[0232] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000035.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000036.tif58165.

[0233] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535240000037.tif58165.

[0234] In some embodiments, in the compound of Formula (II), R 5 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0235] In some embodiments, in the compound of Formula (II), R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0236] In some embodiments, in the compound of Formula (II), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from.

[0237] In some embodiments, in the compound of Formula (II), R 6 is H and R7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0238] In some embodiments, in the compound of Formula (II), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from.

[0239] In some embodiments, in the compound of Formula (II), R 8 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0240] In some embodiments, in the compound of Formula (II), each R 9 is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0241] In some embodiments, in the compound of Formula (II), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0242] In some embodiments, in the compound of Formula (II), each R 9 is independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0243] In some embodiments, in the compound of Formula (II), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0244] In some embodiments, in the compound of Formula (II), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0245] In some embodiments, in the compound of Formula (II), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0246] In some embodiments, in the compound of Formula (II), R 10’ is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0247] In some embodiments, in the compound of Formula (II), R 11 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0248] In some embodiments, in the compound of Formula (II), R12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 is selected from.

[0249] In some embodiments, in the compound of Formula (II), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, -S(O)2R 16 , and Selected from TIFF2025535240000038.tif22165.

[0250] In some embodiments, in the compound of Formula (II), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0251] In some embodiments, in the compound of Formula (II), R 14 and R 14’ are each independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0252] In some embodiments, in the compound of Formula (II), R 16 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0253] In some embodiments, in the compound of Formula (II), R 16is selected from unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0254] In some embodiments, in the compound of Formula (II), R 17 is —C1 to C6 alkyl.

[0255] In some embodiments, in the compound of Formula (II), R 17 is selected from unsubstituted -C1-C6 alkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0256] In some embodiments, in the compound of Formula (II), R 18 and R 19 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0257] In some embodiments, in the compound of formula (II), X a and X b are each independently selected from NH and O.

[0258] Combinations of any of the foregoing embodiments for compounds of formula (II) are also contemplated, with each combination forming a separate embodiment for purposes of this disclosure.

[0259] In certain embodiments, the compound of formula (I) has formula (III): TIFF2025535240000039.tif58165, wherein R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 15 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 4 teeth, Selected from TIFF2025535240000040.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025535240000041.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2.

[0260] In some embodiments, in the compound of Formula (III), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0261] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F, and -Cl.

[0262] In some embodiments, in the compound of Formula (III), R 15 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0263] In some embodiments, in the compound of Formula (III), R 15 is selected from —CH3 and —OCH3.

[0264] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F, and -Cl; R 15 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0265] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F, and -Cl; R 15 is selected from —CH3 and —OCH3.

[0266] In some embodiments, in the compound of Formula (III), R 4 teeth, Selected from TIFF2025535240000042.tif58165.

[0267] In some embodiments, in the compound of Formula (III), R 5 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0268] In some embodiments, in the compound of Formula (III), R 8 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0269] In some embodiments, in the compound of Formula (III), each R 9is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0270] In some embodiments, in the compound of Formula (III), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0271] In some embodiments, in the compound of Formula (III), each R 9 is independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0272] In some embodiments, in the compound of Formula (III), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0273] In some embodiments, in the compound of Formula (III), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0274] In some embodiments, in the compound of Formula (III), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0275] In some embodiments, in the compound of Formula (III), R 10’ is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0276] In some embodiments, in the compound of Formula (III), R 11 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0277] In some embodiments, in the compound of Formula (III), R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 is selected from.

[0278] In some embodiments, in the compound of Formula (III), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, -S(O)2R 16 , and Selected from TIFF2025535240000043.tif22165.

[0279] In some embodiments, in the compound of Formula (III), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0280] In some embodiments, in the compound of Formula (III), R 14 and R 14’ are each independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0281] In some embodiments, in the compound of Formula (III), R 16 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0282] In some embodiments, in the compound of Formula (III), R 16 is selected from unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0283] In some embodiments, in the compound of Formula (III), R 18 and R 19 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0284] In some embodiments, in the compound of formula (III), X a and X b are each independently selected from NH and O.

[0285] Combinations of any of the foregoing embodiments for compounds of formula (III) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0286] In certain embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (I), (II), or (III) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (I), (II), or (III) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0287] In certain embodiments, the camptothecin analog included in an ADC according to the present disclosure is a compound having formula (I) and is selected from the compounds shown in Tables 5 and 6.

[0288] In certain embodiments, the camptothecin analog is a compound having formula (II): In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 ,or TIFF2025535240000044.tif27165. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 or TIFF2025535240000045.tif27165, R 5 is H and R 18and R 19 together with the N atom to which they are attached form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 Ha-(C1~C6)-OR 5 and R 5 is H. In certain embodiments, the camptothecin analog is a compound having formula (II) and is selected from the compounds shown in Table 5.

[0289] In certain embodiments, the camptothecin analog is a compound having the formula (III): In certain embodiments, the camptothecin analog is a compound having the formula (III), wherein R 2 is F and R 15 is -CH3 and R 4 teeth TIFF2025535240000046.tif27165, -R 9 is -C1-C6 hydroxyalkyl, and X a and X b are each O. In certain embodiments, the camptothecin analog is a compound having formula (III) and is selected from the compounds shown in Table 6.

[0290] In certain embodiments, the camptothecin analog included in an ADC according to the disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog included in an ADC according to the disclosure is Compound 139 or Compound 141.

[0291] [Table 5] TIFF2025535240000048.tif223165TIFF2025535240000049.tif234165TIFF2025535240000050.tif223165 TIFF2025535240000051.tif199165TIFF2025535240000052.tif229165TIFF2025535240000053.tif142165

[0292] [Table 6] TIFF2025535240000055.tif199165TIFF2025535240000056.tif236165TIFF202 5535240000057.tif205165TIFF2025535240000058.tif205165TIFF20255352400 00059.tif200165TIFF2025535240000060.tif193165TIFF2025535240000061.t if199165TIFF2025535240000062.tif204165TIFF2025535240000063.tif152165

[0293] It will be understood that references throughout this disclosure to compounds of formula (I) include, in various embodiments, compounds of formula (II) and formula (III), as well as the individual compounds set forth in Tables 5 and 6, to the same extent as if the embodiments individually reciting each of these formulas or compounds were specifically recited.

[0294] Antibody-drug conjugates As mentioned above, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-GPC3 antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) wherein T is an anti-GPC3 antibody construct described herein, L is a linker, D is a camptothecin analog having formula (I): m is an integer from 1 to 4, n is an integer from 1 to 10.

[0295] In certain embodiments, in the conjugate of formula (X), m is 1 to 2. In some embodiments, m is 1.

[0296] In some embodiments, in the conjugate of formula (X), n is 1 to 8, for example, 2 to 8. In some embodiments, n is 2 to 4.

[0297] In certain embodiments, in the conjugate of formula (X), m is 1 to 2 and n is 2 to 8, or 4 to 8. In some embodiments, in the conjugate of formula (X), m is 1 and n is 2 to 8, or 4 to 8.

[0298] As described above and reflected by the parameters m and n in formula (X), the anti-GPC3 antibody construct "T" can be conjugated with multiple compounds "D" of formula (I). While any particular anti-GPC3 antibody construct T is conjugated with an integer number of compounds D, those skilled in the art will understand that analysis of a conjugate preparation to determine the ratio of compound D to anti-GPC3 antibody construct T may yield non-integer results reflecting statistical averages. This ratio of compound D to targeting moiety T is sometimes commonly referred to as the drug-to-antibody ratio, or "DAR". Thus, conjugate preparations with non-integer DARs are intended to be encompassed by formula (X).

[0299] In certain embodiments, in the conjugate of Formula (X), D is a compound of Formula (II) or Formula (III). In certain embodiments, in the conjugate of Formula (X), D is a compound selected from the compounds shown in Tables 5 and 6. In certain embodiments, in the conjugate of Formula (X), D is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, in the conjugate of Formula (X), D is Compound 139 or Compound 141.

[0300] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (IV): TIFF2025535240000064.tif63165, wherein R 1a is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S-, or -NH-, and R 4a teeth, TIFF2025535240000065.tif68165, where * is the point of attachment to X and where p is 1, 2, 3 or 4; or X is O and R 4a -X- is Selected from TIFF2025535240000066.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9aare independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000067.tif22165, Each R 10a’ is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a , and Selected from TIFF2025535240000068.tif22165, R 13a is selected from —H and —C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16ais selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 21 is -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; TIFF2025535240000069.tif17165 shows the attachment point to the linker L.

[0301] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 , and —NH 2 .

[0302] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0303] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —OCH 3 and NH 2 .

[0304] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH3 and —OCH3.

[0305] In some embodiments, in the compound of formula (IV), R 2a is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0306] In some embodiments, in the compound of formula (IV), R 2a is selected from -H, -F, and -Cl.

[0307] In some embodiments, in the compound of formula (IV), R 2a is -F.

[0308] In some embodiments, in the compound of formula (IV), X is —O—, —S—, or —NH—, and R 4a teeth, Selected from TIFF2025535240000070.tif63165.

[0309] In some embodiments, in the compound of Formula (IV), X is —O— or —NH—.

[0310] In some embodiments, in the compound of Formula (IV), each R 9a is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0311] In some embodiments, in the compound of Formula (IV), each R 9a is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0312] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000071.tif22165.

[0313] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000072.tif22165.

[0314] In some embodiments, in the compound of formula (IV), R 12a is -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 is selected from.

[0315] In some embodiments, in the compound of formula (IV), R 13a is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0316] In some embodiments, in the compound of formula (IV), R 14a’ is selected from H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl.

[0317] In some embodiments, in the compound of formula (IV), R 16a is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0318] In some embodiments, in the compound of formula (IV), R 22 and R 23 are each independently selected from -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, and -C3-C8 cycloalkyl.

[0319] In some embodiments, in the compound of formula (IV), Xa and X b are each independently selected from NH and O.

[0320] In some embodiments, in the compound of formula (IV), X a and X b are O respectively.

[0321] In some embodiments, in the compound of Formula (IV), X is O and R 4a teeth TIFF2025535240000073.tif27165, X a and X b are O and R 9a is -C1 to C6 alkyl.

[0322] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, X is O, and R 4a teeth, TIFF2025535240000074.tif27165, X a and X b are O and R 9a is —C1 to C6 alkyl.

[0323] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, and R 2a is H or F, X is O, and R 4a teeth, TIFF2025535240000075.tif32165, X a and X b are O and R, respectively. 9a is —C1 to C6 alkyl.

[0324] Other combinations of any of the foregoing embodiments relating to compounds of formula (IV) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0325] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (V): TIFF2025535240000076.tif53165, wherein R 2a is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000077.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000078.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and -NR 14 R 14’ is selected from Each R 10’is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 , and Selected from TIFF2025535240000079.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; TIFF2025535240000080.tif17165 shows the attachment point to the linker L.

[0326] In some embodiments, in the compound of Formula (V), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0327] In some embodiments, in the compound of Formula (V), R 2a is selected from —CF3, —F, —Cl and —OCH3.

[0328] In some embodiments, in the compound of Formula (V), R 2a is F.

[0329] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000081.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000082.tif58165.

[0330] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000083.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000084.tif58165.

[0331] In some embodiments, in the compound of Formula (V), R20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000085.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000086.tif58165.

[0332] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535240000087.tif58165.

[0333] In some embodiments, in the compound of Formula (V), R 20a is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535240000088.tif27165-CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, Selected from TIFF2025535240000089.tif71165.

[0334] In some embodiments, in the compound of Formula (V), R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0335] In some embodiments, in the compound of Formula (V), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5, —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from.

[0336] In some embodiments, in the compound of Formula (V), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0337] In some embodiments, in the compound of Formula (V), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl, and —C(O)R 17 is selected from.

[0338] In some embodiments, in the compound of Formula (V), R 8 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0339] In some embodiments, in the compound of Formula (V), each R 9 is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0340] In some embodiments, in the compound of Formula (V), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0341] In some embodiments, in the compound of Formula (V), each R 9is independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0342] In some embodiments, in the compound of Formula (V), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0343] In some embodiments, in the compound of Formula (V), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0344] In some embodiments, in the compound of Formula (V), R 11 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0345] In some embodiments, in the compound of Formula (V), R 12 is -H, -C1-C6 alkyl, -aryl, -(C1-C 6 -S(O)R 16 is selected from.

[0346] In some embodiments, in the compound of Formula (V), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, -S(O)2R16 , and Selected from TIFF2025535240000090.tif22165.

[0347] In some embodiments, in the compound of Formula (V), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0348] In some embodiments, in the compound of Formula (V), R 14 and R 14’ are each independently selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0349] In some embodiments, in the compound of Formula (V), R 16 is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0350] In some embodiments, in the compound of Formula (V), R 16 is selected from unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0351] In some embodiments, in the compound of Formula (V), R 17 is selected from unsubstituted -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, unsubstituted -aryl, -hydroxyaryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0352] In some embodiments, in the compound of Formula (V), R 18 and R 19 together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0353] In some embodiments, in the compound of Formula (V), R 17 is —C1 to C6 alkyl.

[0354] In some embodiments, in the compound of formula (V), X a and X b are each independently selected from NH and O.

[0355] In some embodiments, in the compound of formula (V), X a and X b are O respectively.

[0356] In some embodiments, in the compound of Formula (V), R 20a is -(C1-C6 alkyl)-OR 5 is.

[0357] In some embodiments, in the compound of Formula (V), R 20a is -(C1-C6 alkyl)-OR 5 and R 5 is H.

[0358] In some embodiments, in the compound of Formula (V), R 2a is F and R 20a is -(C1-C6 alkyl)-OR 5 and R 5 is H.

[0359] Other combinations of any of the foregoing embodiments for compounds of formula (V) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0360] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (VI): TIFF2025535240000091.tif63165, wherein R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S-, or -NH-, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, TIFF2025535240000092.tif99165, where * is the point of attachment to X, and where p is 1, 2, 3, or 4; or X is O and R 25 -X- is Selected from TIFF2025535240000093.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6a is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 7a is -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5a , —C3-C8 heterocycloalkyl, and —C(O)R 17a is selected from R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9a are independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000094.tif22165, Each R 10a’ is independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a , and Selected from TIFF2025535240000095.tif22165, R 13a is selected from —H and —C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16ais selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 21 is -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 , and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S, and S(O)2; TIFF2025535240000096.tif17165 shows the attachment point to the linker L.

[0361] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0362] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 , and —OCF 3 .

[0363] In some embodiments, in the compound of formula (VI), R 2a is selected from F and Cl.

[0364] In some embodiments, in the compound of formula (VI), R 2a is F.

[0365] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, TIFF2025535240000097.tif63165, or X is O and R 25 -X- is Selected from TIFF2025535240000098.tif37165.

[0366] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, Selected from TIFF2025535240000099.tif63165.

[0367] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , Selected from TIFF2025535240000100.tif63165.

[0368] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 teeth, Selected from TIFF2025535240000101.tif63165.

[0369] In some embodiments, in the compound of Formula (VI), X is —O— or —NH—.

[0370] In some embodiments, in the compound of formula (VI), R 6a is H.

[0371] In some embodiments, in the compound of formula (VI), R 6a is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0372] In some embodiments, in the compound of formula (VI), R 7a is -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17a is selected from.

[0373] In some embodiments, in the compound of Formula (VI), each R 9a is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0374] In some embodiments, in the compound of Formula (VI), each R 9a is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0375] In some embodiments, in the compound of Formula (VI), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000102.tif22165.

[0376] In some embodiments, in the compound of Formula (VI), each R 10aare independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535240000103.tif22165.

[0377] In some embodiments, in the compound of formula (VI), R 12a -C1-C6 alkyl, -aryl, -(C1-C 6 -S(O)R 16a is selected from.

[0378] In some embodiments, in the compound of formula (VI), R 13a is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0379] In some embodiments, in the compound of formula (VI), R 14a’ is selected from H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl.

[0380] In some embodiments, in the compound of formula (VI), R 16a is selected from -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0381] In some embodiments, in the compound of formula (VI), R 17a is —C1 to C6 alkyl.

[0382] In some embodiments, in the compound of formula (VI), R 22 and R 23 are each independently selected from -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, and -C3-C8 cycloalkyl.

[0383] In some embodiments, in the compound of formula (VI), X a and X b are each independently selected from NH and O.

[0384] In some embodiments, in the compound of formula (VI), X a and X b are O respectively.

[0385] In some embodiments, in the compound of Formula (VI), X is O and R 25 is —C1 to C6 alkyl.

[0386] In some embodiments, in the compound of formula (VI), R 2a is F, X is O, and R 25 is —C1 to C6 alkyl.

[0387] Other combinations of any of the foregoing embodiments relating to compounds of formula (VI) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0388] In certain embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (IV), (V), or (VI) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (IV), (V), or (VI) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0389] In certain embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a is -CH3 and R 2a is F. In some embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a is -CH3 and R 2a is F, X is -O-, and R 4a teeth TIFF2025535240000104.tif27165, R 9a is -C1-C6 alkyl, and X a and X b are O respectively.

[0390] In certain embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R 2a is F and R 20 a is H, -(C1-C6)-OR 5 ,or TIFF2025535240000105.tif27165. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a is H, -(C1~C6)-OR 5 ,or TIFF2025535240000106.tif27165, R 5 is H and R 18 and R 19 are taken together with the N atom to which they are attached to form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a -(C1~C6)-OR 5 and R 5 is H.

[0391] In certain embodiments, in the ADC having formula (X), D is a compound of formula (VI), wherein R 2a is F, X is -O-, and R 25 is -C1 to C6 alkyl.

[0392] Linker L The conjugate of formula (X) comprises a linker L, which is a bifunctional or polyfunctional moiety capable of linking one or more camptothecin analogs D to the anti-GPC3 antibody construct T. A bifunctional (or monovalent) linker L links a single compound D to a single site on the anti-GPC3 antibody construct T, whereas a polyfunctional (or polyvalent) linker L links multiple compounds D to a single site on the anti-GPC3 antibody construct T. A linker that links one compound D to multiple sites on the anti-GPC3 antibody construct T can also be considered polyfunctional.

[0393] The linker L comprises a functional group capable of reacting with a targeting group(s) on the anti-GPC3 antibody construct T and at least one functional group capable of reacting with a targeting group on the camptothecin analog D. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Groups on the anti-GPC3 antibody construct T and the camptothecin analog D that can function as targeting groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0394] Non-limiting examples of functional groups that can react with thiols include maleimides, haloacetamides, haloacetyls, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In this context, the "self-stabilizing" maleimides described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062, are also useful.

[0395] Non-limiting examples of functional groups that can react with amines include activated esters (e.g., N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters (e.g., Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (e.g., diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the conversion of a carboxyl group to an activated ester using succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), which can then be reacted with an amine.

[0396] Non-limiting examples of functional groups that can react with electrophilic groups such as aldehyde or ketone carbonyl groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0397] In certain embodiments, the linker L may comprise a functional group that allows for cross-linking of two interchain cysteines on the anti-GPC3 antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0398] Alternatively, the anti-GPC3 antibody construct T may be modified to include a non-natural reactive group, such as an azide, that allows conjugation with a linker via a complementary reactive group on the linker. For example, conjugation of a linker to the anti-GPC3 antibody construct may utilize click chemistry, such as the azide-alkyne cycloaddition (AAC) reaction, which has been successfully used in the development of antibody-drug conjugates (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling an azide with a cyclooctyne.

[0399] The linker L may be a cleavable or non-cleavable linker. A cleavable linker is a linker that is susceptible to cleavage under certain conditions, such as intracellular conditions (e.g., in endosomes or lysosomes) or in the vicinity of target cells (e.g., tumor microenvironment). Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on the degradation of the antibody in cells, which typically results in the release of the amino acid-linker-drug moiety.

[0400] Examples of cleavable linkers include linkers containing an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix surrounding target cells, such as cancer cells, may be utilized. Examples of extracellular tumor-associated proteases include plasmin, matrix metalloproteinases (MMPs), elastase, and kallikrein-related peptidases.

[0401] For conjugates intended to be internalized by cells, the linker L may comprise an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease, examples of which include cathepsins B, C, D, H, L, and S, and legumain.

[0402] The cleavage recognition sequence may be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that may be included in a cleavable linker include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys. Examples of tripeptide and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0403] Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Disulfide-containing linkers may optionally contain additional groups adjacent to the disulfide bond to provide steric hindrance (e.g., incorporation of geminal dimethyl groups) to improve the extracellular stability of the linker. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers containing a combination of these functional groups may also be useful; for example, linkers containing both hydrazones and disulfides are known in the art.

[0404] Further examples of cleavable linkers are linkers containing β-glucuronides that can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007 / 011968). The β-glucuronide can also function to increase the hydrophilicity of the linker L.

[0405] Another example of a linker that is cleaved intracellularly to increase hydrophilicity is a linker that contains a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0406] In certain embodiments, the linker L included in the conjugate of formula (X) is a cleavable linker. In some embodiments, the linker L comprises a cleavage recognition sequence. In some embodiments, the linker L may comprise an amino acid sequence that is recognized and cleaved by a lysosomal protease.

[0407] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative group, a self-leaving group, a stretcher, or a hydrophilic moiety.

[0408] Self-immolative and self-leaving groups that find use as linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED), and hemiaminal groups. Other examples of self-immolative groups include aromatic compounds electronically similar to PAB or PABC groups, such as heterocyclic derivatives, for example, but not limited to, 2-aminoimidazole-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). The self-immolative / self-leaving group is typically attached to an amino or hydroxyl group of compound D. Self-immolative / self-leaving groups, alone or in combination, are often included in peptide-based linkers, but may also be included in other types of linkers.

[0409] Stretchers used in linkers for drug conjugates include, for example, alkylene groups and fatty acid, dibasic acid, amine, or diamine-based stretchers, such as diglycolate, malonate, caproate, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers.

[0410] PEG and mPEG stretchers can also function as hydrophilic moieties within the linker. For example, PEG or mPEG can be included in the linker either "linearly" or as a pendant group to increase the hydrophilicity of the linker (see, e.g., U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd. (Plain City, OH). Other hydrophilic groups that can optionally be incorporated into the linker L include, for example, β-glucuronide, sulfonate group, carboxylate group, and pyrophosphate diester.

[0411] In certain embodiments, an ADC of Formula (X) may comprise a cleavable linker. In certain embodiments, an ADC of Formula (X) may comprise a peptide-containing linker. In certain embodiments, an ADC of Formula (X) may comprise a protease-cleavable linker.

[0412] In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XI): TIFF2025535240000107.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-GPC3 antibody construct T, Str is a stretcher AA1 and AA2 are each independently an amino acid, where AA1-[AA2] r forms a protease cleavage site, X is a self-immolative group, q is 0 or 1, r is 1, 2, or 3; s is 0, 1, or 2; # is the attachment point to the anti-GPC3 antibody construct T, % is the point of attachment to camptothecin analogue D.

[0413] In some embodiments, q is 1 in the linker of formula (XI).

[0414] In some embodiments, in the linker of formula (XI), s is 1. In some embodiments, in the ADC of formula (XI), s is 0.

[0415] In some embodiments, in the linker of Formula (XI), r is 1. In some embodiments, in the ADC of Formula (XI), r is 3.

[0416] In some embodiments, a compound of formula (XI): In the linker, Z is TIFF2025535240000108.tif32165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0417] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025535240000109.tif53165, During the ceremony, R is H or C1-C6 alkyl; t is an integer from 2 to 10, u is an integer from 1 to 10.

[0418] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025535240000110.tif22165, t is an integer from 2 to 10, u is an integer from 1 to 10.

[0419] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XI), AA1-[AA2] rare Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0420] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XI), AA1-[AA2] r has a sequence selected from Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0421] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XI), AA1-[AA2] r has a sequence selected from Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0422] In certain embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XII): TIFF2025535240000111.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-GPC3 antibody construct T, Str is a stretcher AA1 and AA2 are each independently an amino acid, where AA1-[AA2] r forms a protease cleavage site, Y is -NH-CH-; q is 0 or 1, r is 1, 2, or 3; v is 0 or 1, # is the attachment point to the anti-GPC3 antibody construct T, % is the point of attachment to camptothecin analogue D.

[0423] In some embodiments, q is 1 in the linker of formula (XII).

[0424] In some embodiments, in the linker of Formula (XII), v is 0. In some embodiments, in the ADC of Formula (XII), s is 1.

[0425] In some embodiments, in the linker of Formula (XII), r is 1. In some embodiments, in the ADC of Formula (XII), r is 3.

[0426] In some embodiments, a compound of formula (XII): In the linker, Z is TIFF2025535240000112.tif32165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0427] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025535240000113.tif53165, During the ceremony, R is H or C1-C6 alkyl; t is an integer from 2 to 10, u is an integer from 1 to 10.

[0428] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025535240000114.tif22165, t is an integer from 2 to 10, u is an integer from 1 to 10.

[0429] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XII), AA1-[AA2] r are Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0430] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XII), AA1-[AA2] r has a sequence selected from Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0431] In some embodiments, in the linker of formula (XII), AA1-[AA2] ris a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XII), AA1-[AA2] r has a sequence selected from Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0432] In some embodiments, in the linker of formula (XII), Y is -NH-CH. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH.

[0433] In some embodiments, the ADC of formula (X) may comprise a disulfide-containing linker. In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XIII): TIFF2025535240000115.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-GPC3 antibody construct T, Q is -(CH2) p -or-(CH2CH2O) q -, and p and q are each independently an integer of 1 to 10; each R is independently H or C1-C6 alkyl; n is 1, 2, or 3; # is the attachment point to the anti-GPC3 antibody construct T, % is the point of attachment to camptothecin analogue D.

[0434] In some embodiments, an ADC of Formula (X) may comprise a b-glucuronide-containing linker.

[0435] Various non-cleavable linkers for linking drugs to targeting moieties are known in the art and may be useful in certain embodiments for the ADCs of the present disclosure. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with anti-GPC3 antibody constructs and a maleimide or haloacetyl-based moiety for reaction with camptothecin analogs, or vice versa. An example of such a non-cleavable linker is a sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC)-based linker. Sulfo-SMCC conjugation typically occurs via a maleimide group that reacts with sulfhydryls (thiols, -SH) on camptothecin analogs, while sulfo-NHS esters are reactive with primary amines (such as those found in lysines and the N-termini of proteins or peptides) on anti-GPC3 antibody constructs. Other non-limiting examples of such linkers include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) ("long chain" SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid These include linkers based on N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI).Other examples include those containing haloacetyl-based functional groups such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0436] Non-limiting examples of drug-linkers comprising camptothecin analogs of Formula (I) are shown in Tables 7, 8, and 9. Non-limiting examples of conjugates comprising these drug-linkers are shown in Tables 10, 11, and 12. In certain embodiments, an ADC of Formula (X) comprises a drug-linker selected from those shown in Tables 7, 8, and 9. In certain embodiments, an ADC of Formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, where T is an anti-GPC3 antibody construct and n is 1 to 10. In some embodiments, an ADC of Formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, where T is an anti-GPC3 antibody construct and n is 2 to 8. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, where T is an anti-FRα antibody construct and n is 4 to 8.

[0437] In certain embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m), and n is 4 or 8. In some embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m ), and n is 8.

[0438] Preparation of ADCs ADCs of formula (X) can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Additionally, various antibody-drug conjugation services are commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramel Pharma Solutions (Grangemouth, UK).

[0439] In general, preparation of an ADC involves first preparing a drug-linker DL comprising one or more camptothecin analogs of formula (I) and a linker L, and then conjugating the drug-linker DL to a suitable group on an anti-GPC3 antibody construct T. However, ligation of the linker L to the anti-GPC3 antibody construct T followed by ligation of the anti-GPC3 antibody construct-linker TL to one or more camptothecin analogs D of formula (I) remains an alternative approach available in some embodiments.

[0440] Suitable groups on compound D of formula (I) for attachment of linker L in any of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of the present disclosure, linker L is attached to the compound through a hydroxyl or amine group on compound D of formula (I).

[0441] Suitable groups on the anti-GPC3 antibody construct T for attachment of the linker L in any of the above approaches include sulfhydryl groups (e.g., in the side chain of a cysteine ​​residue), amino groups (e.g., in the side chain of a lysine residue), carboxylic acid groups (e.g., in the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

[0442] For example, the anti-GPC3 antibody construct T may contain one or more naturally occurring sulfhydryl groups that allow the anti-GPC3 antibody construct T to be bound to the linker L via the sulfhydryl group's sulfur atom. Alternatively, the anti-GPC3 antibody construct T may contain one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"), and 2-iminothiolane hydrochloride (Traut's reagent). Alternatively, the anti-GPC3 antibody construct T may contain one or more carbohydrate groups that can be chemically modified to contain one or more sulfhydryl groups.

[0443] Carbohydrate groups on the anti-GPC3 antibody construct T may also be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which may then be reacted with a linker L, for example, via a hydrazine or hydroxylamine group on the linker L.

[0444] The anti-GPC3 antibody construct T can also be modified to include additional cysteine ​​residues (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622; and 9,000,130) or unnatural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow site-specific conjugation. Alternatively, the anti-GPC3 antibody construct T can be modified to include a non-natural reactive group, such as an azide, that allows for conjugation with a linker via a complementary reactive group on the linker, e.g., click chemistry (e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to enable the attachment of linkers via metal-free click chemistry (see, e.g., European Patent No. EP2911699).

[0445] Other protocols for modifying proteins for attachment or association of a linker L are known in the art, including those described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002).

[0446] Alternatively, ADCs may be prepared using the enzyme transglutaminase, particularly bacterial transglutaminase (BTG) from Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of glutamine (typically an amine acceptor on an antibody) and an alkyleneamino group (typically an amine donor on a drug-linker), which may be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a glutamine-containing peptide, or "tag," that allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US2013 / 0230543 and International (PCT) Publication No. WO2016 / 144608).

[0447] A similar conjugation approach utilizes the enzyme sortase A. In this approach, an antibody is typically modified to contain a sortase A recognition motif (LPXTG, where X is any naturally occurring amino acid), and the drug-linker is designed to contain an oligoglycine motif (typically GGG) to enable sortase A-mediated transpeptidation (see, e.g., Beerli, et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0448] Once conjugation is complete, the average number of compounds of formula (I) conjugated to the anti-GPC3 antibody construct T (i.e., "drug-to-antibody ratio" or DAR) can be determined by standard techniques, such as UV / VIS spectroscopy, ELISA-based techniques, chromatographic techniques, such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOFMS. In addition, the distribution of drug-linked forms (e.g., the proportion of anti-GPC3 antibody construct T containing 0, 1, 2, 3, etc. compounds of formula (I) D) can also be optionally analyzed. Various techniques for assessing DAR distribution are known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or isoelectric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al., 2011, mAbs, 3:161-172).

[0449] Pharmaceutical Composition For therapeutic use, the ADCs of the present disclosure are typically formulated as pharmaceutical compositions. Accordingly, certain embodiments of the present disclosure relate to pharmaceutical compositions comprising an ADC described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

[0450] Pharmaceutical compositions may be formulated for administration to a subject, for example, by oral (e.g., buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. The term "parenteral" as used herein includes subcutaneous injection, and intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to a subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard capsule, soft capsule, pill, suppository, oily suspension, aqueous suspension, dispersible powder, dispersible granule, emulsion, injection, or solution. Pharmaceutical compositions may be provided as unit-dose formulations.

[0451] In certain embodiments, a pharmaceutical composition comprising an ADC is formulated for parenteral administration, e.g., as a lyophilized preparation or an aqueous solution. Such a pharmaceutical composition may be provided, for example, in a unit dosage injectable form.

[0452] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or These include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0453] In certain embodiments, compositions containing the ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension may contain the ADC in a non-toxic parenterally acceptable diluent or carrier. Acceptable diluents and carriers that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Additionally, sterile fixed oils may be used as carriers. For this purpose, various bland fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions. Auxiliaries such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0454] In certain embodiments, a composition comprising an ADC may be formulated for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0455] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"; Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000)).

[0456] How to use Certain embodiments of the present disclosure relate to therapeutic uses of the ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents.

[0457] Certain embodiments of the present disclosure relate to methods of inhibiting abnormal cancer or tumor cell proliferation, methods of inhibiting cancer or tumor cell proliferation, or methods of treating cancer in a subject, comprising administering an ADC described herein. In certain embodiments, the ADC described herein can be used to treat cancer. Accordingly, some embodiments of the present disclosure relate to the use of an ADC as an anticancer agent.

[0458] Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X). Some embodiments relate to methods of killing cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X).

[0459] Some embodiments relate to methods of treating a subject with cancer by administering to the subject an ADC described herein, e.g., an ADC of Formula (X). In this context, treating the subject can result in one or more of: a reduction in tumor size; a delay or prevention of an increase in tumor size; an increase in disease-free survival between the disappearance or removal and reappearance of a tumor; prevention of subsequent development of a tumor (e.g., metastasis); an increase in time to progression; a reduction in one or more adverse symptoms associated with the tumor; and / or an increase in overall survival of the subject with cancer.

[0460] Certain embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vitro. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vivo in a subject with cancer.

[0461] Examples of cancers that can be treated in certain embodiments include carcinomas, melanomas, and sarcomas, including adenocarcinomas and squamous cell carcinomas. Carcinomas and sarcomas are also often referred to as "solid tumors." Examples of commonly occurring solid tumors that can be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors, and therefore, in certain circumstances, they may also be considered solid tumors. Typically, the cancer to be treated is a GPC3-expressing cancer.

[0462] Certain embodiments relate to methods of inhibiting the growth of GPC3-positive tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X). The cells may be in vitro or in vivo. In certain embodiments, the ADC may be used in methods of treating a GPC3-positive cancer or tumor in a subject.

[0463] In some embodiments, the ADCs described herein can be used to treat subjects with cancers that overexpress GPC3. GPC3-overexpressing cancers are typically solid tumors. Examples include, but are not limited to, hepatocellular carcinoma (HCC), melanoma, lung cancer, and hepatoblastoma.

[0464] Medicine Kit Certain embodiments relate to pharmaceutical kits that include an ADC described herein, e.g., an ADC of Formula (X).

[0465] The kit typically includes a container holding the ADC and a label and / or package insert on or associated with the container. The label or package insert includes instructions customarily included in commercial packaging of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic product. The label or package insert may further include a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, such notice reflecting approval by that agency for manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle.

[0466] In addition to the container holding the ADC, the kit may optionally include one or more additional containers containing other components of the kit, for example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0467] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dried form, such as a powder or granules, and the kit may additionally include a suitable solvent for the reconstitution of the lyophilized or dried component(s).

[0468] Kits may further include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0469] Tables 7~12

[0470] [Table 7] TIFF2025535240000117.tif191165TIFF2025535240000118.tif232165

[0471] [Table 8] TIFF2025535240000120.tif176165TIFF2025535240000121.tif197165TIFF2025535240000122.tif187165 TIFF2025535240000123.tif176165TIFF2025535240000124.tif194165TIFF2025535240000125.tif140165

[0472] [Table 9] TIFF2025535240000127.tif174165TIFF2025535240000128.tif135165

[0473] [Table 10] TIFF2025535240000130.tif210165TIFF2025535240000131.tif146165TIFF2025535240000132.tif146165TIFF2025535240000133.tif197165

[0474] [Table 11] TIFF2025535240000135.tif176165TIFF2025535240000136.tif199165TIFF2025535240000137.tif82165

[0475] [Table 12] TIFF2025535240000139.tif202165TIFF2025535240000140.tif214165

[0476] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0477] Examples 1-3 below illustrate various methods for preparing camptothecin analogs of Formula (I). It is understood that those skilled in the art can produce these compounds by similar methods or by combining other methods known in the art. It is also understood that those skilled in the art can produce other compounds of Formula (I) not specifically shown below using the methods described below or similar methods by using appropriate starting components and modifying the synthetic parameters as needed. In general, the starting components can be obtained from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (Thermo Fisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or can be synthesized according to materials known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013), or can be prepared as described herein.

[0478] Abbreviation The following abbreviations are used throughout the Examples section: BCA: bicinchoninic acid, Boc: di-tert-butyl dicarbonate, CE-SDS: capillary electrophoresis sodium dodecyl sulfate, DCM: dichloromethane, DTPA: diethylenetriaminepentaacetic acid, DIPEA: N,N-diisopropylethylamine, DMF: dimethylformamide, DMM™: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride, EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Fmoc: fluorenylmethyloxycarbonyl, HATU: azabenzotriazole tetramethylwt. tetrafluorophenylalanine, HCl: hydrophobic interaction chromatography, HOAt: 1-hydroxy-7-azabenzotriazole, HPLC: high performance liquid chromatography, LC / MS: liquid chromatography mass spectrometry, MC: maleimidocaproyl, MT: maleimidotriethylene glycolate, NMM: N-methylmorpholine, PNP: p-nitrophenol, RP-UPLC-MS: reversed-phase ultra-performance liquid chromatography mass spectrometry, SEC: size exclusion chromatography, TCEP: tris(2-carboxyethyl)phosphine, Tfp: tetrafluorophenyl, TLC: thin layer chromatography, TFA: trifluoroacetic acid.

[0479] General Chemistry Procedures General Procedure 1: Conversion of Chlorides to Amine To a stirred solution of the chloride compound in dimethylformamide (0.05-0.1 M) was added the appropriate secondary amine (3 equiv.). Upon completion (as determined by LC / MS, typically 1-3 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0480] General Procedure 2: Conversion of amines to amides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added triethylamine (1.2 equiv.), the appropriate carboxylic acid (1.1 equiv.), followed by a solution of DMMTM (2 equiv.) in water (1 M). Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0481] General Procedure 3: Conversion of amines to sulfonamides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added DIPEA (3 equiv.), followed by the appropriate sulfonyl chloride. Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0482] General Procedure 4: Two-Step Conversion of Amines to Ureas (Synthetic Scheme IV; Figure 1D) Step 1: To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 equivalent), followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the reaction mixture was concentrated to dryness and then purified by reverse-phase HPLC to yield the desired PNP-carbamate intermediate after lyophilization. This intermediate may be used to generate a single analog or may be split into multiple batches to generate multiple analogs in a second step. Step 2: To the PNP-carbamate intermediate in dimethylformamide (0.1-0.2 M) was added the appropriate primary amine (3 equivalents). Upon completion (as determined by LC / MS, typically 1 hour), the reaction mixture was purified by reverse-phase HPLC to yield the desired product after lyophilization.

[0483] General Procedure 5: Conversion of amines to carbamates To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 equivalent), followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the appropriate alcohol was added to the resulting PNP-carbamate intermediate. Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was purified by reverse-phase HPLC to yield the desired product after lyophilization.

[0484] General Procedure 6: Removal of the Boc protecting group To a stirred solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20% by volume). Upon completion (as determined by LC / MS, typically 1 h), the reaction mixture was concentrated in vacuo to give a crude solid, or purified as described in General Procedure 9.

[0485] General Procedure 7: Copper-Mediated Amide Coupling To a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 equiv.) and HOAt (3 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added EDC (HCl salt, 3 equiv.). After 5 min, a solution (0.02 M) of the amine-containing payload (1 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added, followed immediately by CuCl (4 equiv.). Upon completion (as determined by LC / MS, typically 1-16 h), the reaction mixture was concentrated in vacuo to give a crude solid or purified by preparative HPLC to give the desired product after lyophilization.

[0486] General Procedure 8: MT Installation To a stirred solution (approximately 0.02 M) of the amine compound (1 equivalent) in dimethylformamide was added a solution (approximately 0.02 M) of MT-OTfp (1.2-1.5 equivalents) in acetonitrile, followed by DIPEA (10 μL, 4 equivalents). Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was concentrated in vacuo to give a crude solid, which was purified by preparative HPLC to give the desired product after lyophilization.

[0487] General Procedure 9: Compound Purification Flash chromatography: Crude reaction products were purified using Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC) eluting with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane on a Biotage® Isolera™ automated flash system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was performed using Biotage® Snap Ultra C18 columns (12, 30, 60, or 120 g) eluting with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. Purified compounds were isolated either by removal of organic solvents by rotary evaporation or by lyophilization of the acetonitrile / water mixture.

[0488] Preparative HPLC: Reverse-phase HPLC of crude compounds was performed on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) using a Luna® 5-μm C18 100 Å (150 × 30 mm) column (Phenomenex, Torrance, CA) eluted with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. Purified compounds were isolated by lyophilization of the acetonitrile / water mixture.

[0489] General Procedure 10: Compound Analysis LC / MS: Reaction completion was monitored, and purified compounds were analyzed on an Agilent 1290 HPLC / 6120 Single Quad LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) using a Kinetex® 2.6-μm C18 100 Å (30 × 3 mm) column (Phenomenex, Torrance, CA) eluted with a 10–100% linear gradient of 0.1% formic acid in acetonitrile / 0.1% formic acid in water.

[0490] NMR: 1 H NMR spectra were collected using a Bruker AVANCE III 300 Spectrometer (300 MHz) (Bruker Corporation, Billerica, Mass.) Chemical shifts are reported in parts per million (ppm).

[0491] Example 1: Preparation of camptothecin analogs bearing methyl at C10 position 1.1: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 1.1) TIFF2025535240000141.tif42165 The title compound was prepared according to the procedure set forth in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0492] 1.2: (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 1.2) TIFF2025535240000142.tif47165 The title compound was prepared according to the procedure set forth in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0493] 1.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 100) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0494] LC / MS:C 26 H 26 Calculated m / z for FN3O5 = 479.2, observed [M+H] + =480.4.

[0495] 1 H NMR (300 MHz, CDCl3) δ 8.20 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 10.4 Hz, 1H), 7.67 (s, 1H), 5.77 (d, J = 16.4 Hz, 1H), 5.42 (s, 2H), 5.33 (d, J = 16.4 Hz, 1H), 4.26 (s, 2H), 3.81 (t, J = 4.7 Hz, 4H), 2.82 - 2.76 (m, 4H), 2.57 (d, J = 1.7 Hz, 3H), 1.99 - 1.82 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H).

[0496] 1.4: ((S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 102) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0497] LC / MS:C 32 H 31 Calculated m / z for FN4O6 = 618.2, observed [M+H] + =619.4.

[0498] 1 H NMR (300 MHz, CDCl3) δ 8.07 (d, J = 7.9 Hz, 1H), 7.88 - 7.44 (m, 7H), 5.73 (d, J = 16.4 Hz, 1H), 5.33 (s, 2H), 5.33 - 5.26 (m, 1H), 4.19 (s, 2H), 3.12 (s, 4H), 2.80 (s, 4H), 2.54 (s, 3H), 1.90 (dt, J = 11.6, 7.0 Hz, 2H), 1.04 (t, J = 7.3 Hz, 3H).

[0499] 1.5: (S)-11-((4-((4-aminophenyl)sulfonyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 104) TIFF2025535240000145.tif63165 The title compound was prepared according to general procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was performed as described in general procedure 9 eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0500] LC / MS:C 32 H 32 Calculated m / z for FN5O6 = 633.2, found [M+H] + =634.4.

[0501] 1 H NMR (300 MHz, MeOD) δ 8.32 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 10.5 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 5.61 (d, J = 16.5 Hz, 1H), 5.44 (s, 2H), 5.41 (d, J = 16.5 Hz, 1H), 4.51 (s, 2H), 3.22 - 3.07 (m, 8H), 2.58 (s, 3H), 2.03 - 1.93 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H).

[0502] 1.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 106) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

[0503] LC / MS:C 27 H 29 Calculated m / z for FN4O4 = 492.2, found [M+H] + =493.4.

[0504] 1.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 108) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0505] LC / MS:C 32 H 32 Calculated m / z for FN5O4 = 569.2, observed [M+H] + =570.4.

[0506] 1H NMR (300 MHz, MeOD) δ 8.39 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 10.6 Hz, 1H), 7.21 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 5.62 (d, J = 16.4 Hz, 1H), 5.49 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.45 (s, 2H), 3.44 - 3.38 (m, 4H), 3.06 - 3.00 (m, 4H), 2.58 (d, J = 1.8 Hz, 3H), 2.00 - 1.89 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0507] 1.8: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 110) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0508] LC / MS:C 27 H 28 Calculated m / z for FN3O4 = 477.2, observed [M+H] + =478.2.

[0509] 1H NMR (300 MHz, MeOD) δ 8.34 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 10.3 Hz, 1H), 7.70 (s, 1H), 5.63 (d, J = 16.4 Hz, 1H), 5.52 (s, 2H), 5.44 (d, J = 16.5 Hz, 1H), 4.99 (s, 2H), 3.73 - 3.46 (m, 4H), 2.64 (s, 3H), 2.03 - 1.90 (m, 2H), 1.90 - 1.84 (m, 6H), 1.03 (t, J = 7.4 Hz, 3H).

[0510] 1.9: tert-Butyl (S)-4-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazine-1-carboxylate (compound 111) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

[0511] LC / MS:C 31 H 35 Calculated m / z for FN4O6 = 578.2, observed [M+H] + =579.4.

[0512] 1.10: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 112) TIFF2025535240000150.tif53165 The title compound was prepared according to general procedure 6 starting from compound 111 (5.0 mg) to afford the title compound as an off-white solid (TFA salt, 4.4 mg).

[0513] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [M+H] + =479.2.

[0514] 1.11: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(((R)-2-(hydroxymethyl)morpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 113) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).

[0515] LC / MS:C 27 H 28 Calculated m / z for FN3O6 = 509.2, found [M+H] + =510.4.

[0516] 1.12: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)thiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 114) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0517] LC / MS:C 27 H 28 Calculated m / z for FN3O5S = 525.6, found [M+H] + =526.5.

[0518] 1 H NMR (300 MHz, 10%D2O / CD3CN) 8.36 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J = 16.4 Hz, 1H), 5.52 - 5.29 (m, 3H), 5.02 (d, J = 14.6 Hz, 1H), 4.71 - 4.54 (m, 1H), 4.27 (dd, J = 12.4, 5.0 Hz, 1H), 3.98 (dd, J = 12.3, 3.4 Hz, 1H), 3.55 (s, 1H), 3.30-3.03 (m, 4H) 2.97 - 2.72 (m, 3H), 2.62 (s, 1H), 2.55 (s, 3H), 0.95 (t, J = 7.4 Hz, 3H).

[0519] 1.13: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 115) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0520] LC / MS:C 28 H 28 Calculated m / z for FN3O6 = 521.5, observed [M+H] + =522.5.

[0521] 1 H NMR (300 MHz, 10%D2O / CD3CN) δ 8.36 (d, J = 7.9 Hz, 1H), 7.86 (dd, J = 10.6, 5.0 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H), 5.63 - 5.49 (m, 2H), 5.37 (dd, J = 17.8, 14.1 Hz, 2H), 5.05 (s, 2H), 4.63 (d, J = 2.5 Hz, 1H), 4.55 (d, J = 10.7 Hz, 1H), 4.33 (s, 2H), 3.92 (d, J = 10.7 Hz, 1H), 3.36 (s, 2H), 2.57 (s, 3H), 2.41 - 2.13 (m, 2H), 1.97-1.85 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).

[0522] 1.14: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxidethiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 116) The title compound was prepared according to general procedure 1 starting from compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ-thiomorpholine-1,1-dione. Purification was carried out as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0523] LC / MS:C 27 H 28 Calculated m / z for FN3O7S = 557.6, found [M+H] + =558.4.

[0524] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.44 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.50 (s, 1H), 5.58 (d, J = 16.5 Hz, 1H), 5.45 - 5.26 (m, 3H), 4.60 (d, J = 14.9 Hz, 1H), 4.33 (d, J = 14.7 Hz, 1H), 3.88 (d, J = 4.8 Hz, 2H), 3.41-2.85 (m, 4H), 2.53 (s, 2H), 2.19 (p, J = 2.5 Hz, 2H), 1.74 (p, J = 2.5 Hz, 2H), 1.27 (s, 2H), 0.95 (t, J = 7.4 Hz, 3H).

[0525] 1.15: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]heptan-3-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 117) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0526] LC / MS:C 28 H 28 Calculated m / z for FN3O5 = 505.5, observed [M+H] + =506.6.

[0527] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.25 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 10.6 Hz, 1H), 7.50 (s, 1H), 5.65 - 5.27 (m, 4H), 4.98 (s, 2H), 4.24 (s, 1H), 3.83 - 3.57 (m, 4H), 2.54 (s, 5H), 2.01-1.86 (m, 2H), 1.70 (s, 2H), 0.95 (t, J = 7.3 Hz, 3H).

[0528] 1.16: (S)-4-Ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 118) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0529] LC / MS:C 26 H 25 Calculated m / z for F2N3O5 = 497.5, observed [M+H] + =498.4.

[0530] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.24 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J = 16.5 Hz, 1H), 5.48 - 5.28 (m, 3H), 4.98 (s, 2H), 4.44 - 4.14 (m, 4H), 3.78 (d, J = 14.9 Hz, 2H), 2.01-1.86 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).

[0531] 1.17: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)azetidin-1-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 119) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and azetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0532] LC / MS:C 26 H 26 Calculated m / z for FN3O5 = 479.5, observed [M+H] + =480.4.

[0533] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.23 ​​(d, J = 7.8 Hz, 1H), 7.90 (d, J = 10.6 Hz, 1H), 7.53 (s, 1H), 5.58 (d, J = 16.5 Hz, 1H), 5.50 - 5.28 (m, 3H), 5.01 (s, 2H), 4.31 - 4.17 (m, 2H), 4.15 - 4.00 (m, 2H), 3.62 (d, J = 3.9 Hz, 2H), 2.58 (s, 3H), 2.01-1.86 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).

[0534] 1.18: (4S)-11-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 120) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

[0535] LC / MS:C 28 H 28 Calculated m / z for F3N3O5 = 543.5, observed [M+H] + =544.4.

[0536] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.25 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 10.7, 1.4 Hz, 1H), 7.47 (s, 1H), 5.55 (d, J = 16.5 Hz, 1H), 5.42 - 5.25 (m, 3H), 4.66 (d, J = 3.2 Hz, 2H), 3.90 - 3.77 (m, 1H), 3.71 - 3.45 (m, 4H), 2.24 (q, J = 11.8, 9.2 Hz, 2H), 2.01-1.86 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H).

[0537] 1.19: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]heptan-7-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 121) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0538] LC / MS:C 29 H 30 Calculated m / z for FN3O5 = 519.6, observed [M+H] + =520.4.

[0539] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.22 (s, 1H), 7.92 (d, J = 10.7 Hz, 1H), 7.54 (s, 1H), 5.59 (dd, J = 17.6, 7.6 Hz, 2H), 5.33 (t, J = 17.4 Hz, 2H), 4.98 - 4.81 (m, 1H), 4.67 - 4.44 (m, 2H), 4.28 - 3.93 (m, 4H), 2.73 (s, 2H), 2.34 - 2.03 (m, 4H), 1.91 (d, J = 14.0 Hz, 5H), 0.96 (t, J = 7.4 Hz, 3H).

[0540] 1.20: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 122) The title compound was prepared according to general procedure 3, starting from compound 1.2 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (0.8 mg, 7% yield).

[0541] LC / MS:C 23 H 22 Calculated m / z for FN3O6S = 487.1, observed [M+H] + =488.2.

[0542] 1 H NMR (300 MHz, MeOD) δ 8.33 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.8 Hz, 1H), 7.68 (s, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.52 (s, 2H), 5.42 (d, J = 16.4 Hz, 1H), 4.87 (s, 2H), 3.06 (s, 3H), 2.59 (s, 3H), 2.06-1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0543] 1.21: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (compound 124) The title compound was prepared according to general procedure 3, starting from compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.0 mg, 17% yield).

[0544] LC / MS:C 29 H 25 Calculated m / z for FN4O8S = 608.1, observed [M+H] + =609.2.

[0545] 1 H NMR (300 MHz, CDCl3) δ 8.02 - 7.92 (m, 3H), 7.74 (d, J = 10.5 Hz, 1H), 7.65 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 5.66 (d, J = 16.8 Hz, 1H), 5.28 (d, J = 16.5 Hz, 1H), 5.14 (d, J = 5.4 Hz, 2H), 4.67 (s, 2H), 4.28 (d, J = 6.3 Hz, 2H), 3.39 (s, 3H), 2.03 - 1.83 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).

[0546] 1.22: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 125) The title compound was prepared according to general procedure 3, starting from compound 1.2 (10 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (9.8 mg, 73% yield).

[0547] LC / MS:C 28 H 24 Calculated m / z for FN3O6S = 549.6, observed [M+H] + =550.6.

[0548] 1 H NMR (300 MHz, DMSO-d6) δ 8.60 (t, J = 6.2 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.8 Hz, 1H), 7.71 (dd, J = 7.1, 1.7 Hz, 2H), 7.66 - 7.48 (m, 2H), 7.46 (dd, J = 8.3, 6.8 Hz, 2H), 7.40 - 7.27 (m, 2H), 7.18 (s, 1H), 7.01 (s, 1H), 5.45 (s, 2H), 5.33 (s, 2H), 4.63 (d, J = 6.2 Hz, 2H), 2.48 (s, 3H), 1.98 - 1.76 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0549] 1.23: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 1.23) The title compound was prepared according to general procedure 3, starting from compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. Purification of the title compound was carried out as described in general procedure 9 using a 12 g C18 column eluted with a 5 to 75% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (37.8 mg, 47% yield).

[0550] LC / MS:C 28 H 23 Calculated m / z for FN4O8S = 594.6, observed [M+H] + =595.2.

[0551] 1.24: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 127) TIFF2025535240000164.tif63165 To a solution of compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL) was added platinum 1% vanadium 2% carbon (75 mg). The flask was purged with H and then stirred under an H atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (30 mg, 84% yield).

[0552] LC / MS:C 28 H 24 Calculated m / z for FN4O6S = 564.6, observed [M+H] + =565.2.

[0553] 1 H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J = 8.2 Hz, 1H), 8.02 (t, J = 6.2 Hz, 1H), 7.88 (d, J = 10.8 Hz, 1H), 7.48 - 7.35 (m, 2H), 7.31 (d, J = 8.4 Hz, 1H), 6.63 - 6.45 (m, 2H), 5.45 (s, 2H), 5.36 (s, 2H), 4.50 (d, J = 6.3 Hz, 2H), 1.98 - 1.75 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0554] 1.25: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 129) The title compound was prepared according to general procedure 3, starting from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 25 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (1.3 mg, 13% yield).

[0555] LC / MS:C 24 H 24 Calculated m / z for FN3O7S = 517.1, observed [M+H] + =518.2.

[0556] 1 H NMR (300 MHz, DMSO-d6) δ 8.30 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 10.9 Hz, 1H), 7.84 (t, J = 6.3 Hz, 1H), 7.33 (s, 1H), 5.50-5.33 (m, 4H), 5.07 (t, J = 5.4 Hz, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.07 (s, 3H), 3.80 (dt, J = 6.3 Hz, J = 5.8 Hz, 2H), 1.86 (m, 2H), 0.87 (d, J = 7.3 Hz, 3H).

[0557] 1.26: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfamide (Compound 131) TIFF2025535240000166.tif53165 To a solution of chlorosulfonyl isocyanate (3 μL) in dichloromethane (1 mL) was added tert-butanol (3 μL). This solution was stirred for 1 hour, and then compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 μL). The reaction was stirred for 1 hour and then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was performed as described in General Procedure 9, eluting with a 10 to 50% CH3CN / HO + 0.1% TFA gradient. To the purified solid in dichloromethane (1 mL) was added trifluoroacetic acid (200 μL). The reaction was stirred for 16 hours and then concentrated to dryness to give the title compound as an off-white solid (7.5 mg, 48% yield).

[0558] LC / MS:C 22 H 21 Calculated m / z for FN4O6S = 488.1, observed [M+H] + =489.0.

[0559] 1 H NMR (300 MHz, MeOD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 10.7 Hz, 1H), 7.62 (s, 1H), 5.59 (d, J = 16.4 Hz, 1H), 5.45 (s, 2H), 5.39 (d, J = 16.4 Hz, 1H), 4.81 (s, 2H), 2.55 (d, J = 1.7 Hz, 3H), 2.07 - 1.89 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0560] 1.27: 4-Nitrophenyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 1.27) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting from compound 1.2 (24 mg). Purification was carried out as described in General Procedure 9 using a 12 g C18 column eluted with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (14 mg, 53% yield).

[0561] LC / MS:C 29 H 23 Calculated m / z for FN4O8S = 574.2, observed [M+H] + =575.2.

[0562] 1.28: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 132) The title compound was prepared according to general procedure 4, starting with compound 1.2 (25 mg) and aqueous methylamine (500 μL, 40 wt % in water) as the primary amine. In this case, the intermediate PNP carbamate was used crude. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (8.9 mg, 31% yield).

[0563] LC / MS:C 24 H 23 Calculated m / z for FN4O5 = 466.2, observed [M+H] + =467.2.

[0564] 1H NMR (300 MHz, MeOD) δ 8.26 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 10.7 Hz, 1H), 7.66 (s, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.97 (s, 2H), 2.73 (s, 3H), 2.57 (s, 3H), 2.08 - 1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0565] 1.29: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 134) The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (0.6 mg, 12% yield).

[0566] LC / MS:C 30 H 28 Calculated m / z for FN5O5 = 557.2, found [M+H] + =558.4.

[0567] 1H NMR (300 MHz, MeOD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.67 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 5.63 (d, J = 16.4 Hz, 1H), 5.48 (s, 2H), 5.43 (d, J = 16.4 Hz, 1H), 5.01 (s, 2H), 4.37 (s, 2H), 2.56 (d, J = 1.7 Hz, 3H), 2.05 - 1.94 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0568] 1.30: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 136) The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (2.4 mg, 66% yield).

[0569] LC / MS:C 25 H 25 Calculated m / z for FN4O6 = 496.2, observed [M+H] + =497.2.

[0570] 1H NMR (300 MHz, MeOD) δ 8.08 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 10.5 Hz, 1H), 7.68 (s, 1H), 5.64 (d, J = 16.4 Hz, 1H), 5.41 (s, 2H), 5.31 (d, J = 16.4 Hz, 1H), 4.96 (s, 2H), 3.63 (t, J = 5.2 Hz, 2H), 3.29 (t, J = 5.3 Hz, 2H), 2.54 (s, 3H), 1.98 - 1.87 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).

[0571] 1.31: Methyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 138) The title compound was prepared according to general procedure 5, starting from compound 1.2 (50 mg) and reacting methanol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (3.5 mg, 6% yield).

[0572] LC / MS:C 24 H 22 Calculated m / z for FN3O6 = 467.2, observed [M+H] + =468.2.

[0573] 1H NMR (300 MHz, MeOD) δ 8.17 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 10.5 Hz, 1H), 7.69 (s, 1H), 5.65 (d, J = 16.5 Hz, 1H), 5.48 (s, 2H), 5.33 (d, J = 16.4 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.65 (s, 3H), 2.56 (s, 3H), 2.02 - 1.89 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0574] 1.32: 2-Hydroxyethyl (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 139) The title compound was prepared according to general procedure 5, starting from compound 1.2 (18 mg) by reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.2 mg, 19% yield).

[0575] LC / MS:C 25 H 24 Calculated m / z for FN3O7 = 497.2, observed [M+H] + =498.2.

[0576] 1H NMR (300 MHz, DMSO) δ 8.23 ​​(d, J = 8.2 Hz, 1H), 7.78 (d, J = 10.7 Hz, 1H), 7.40 (s, 1H), 5.47 (d, J = 16.5 Hz, 1H), 5.42 (s, 2H), 5.34 (d, J = 16.4 Hz, 1H), 4.77 (s, 2H), 3.99 (t, J = 4.9 Hz, 2H), 3.64 - 3.38 (m, 2H), 2.48 (s, 3H), 2.02 - 1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0577] Example 2: Preparation of camptothecin analogs with methoxy at C10 position 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethan-1-one (compound 2.1) A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0 °C. To this solution was added first 1 M BCl in DCM (71 mL, 71 mmol), followed by 1 M chloro(diethyl)almane in DCM (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated at reflux for 3 h, cooled to room temperature, and quenched by the addition of 2 M aqueous HCl. The resulting heterogeneous mixture was heated at reflux for 1 h, cooled to room temperature, and then the pH was adjusted to approximately 12 with NaCO. The layers were separated, and the aqueous layer was extracted with DCM (3 × 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and flash purified as described in general procedure 9, eluting with 0 to 20% EtOAc / hexanes, to give the title compound (6 g, 28 mmol, 39% yield).

[0578] LC / MS: m / z calculated for C9H9ClFNO2 = 217.1, found [M+H] + =218.1.

[0579] 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 9.2 Hz, 1H), 6.44 (d, J = 12.8 Hz, 1H), 4.59 (s, 2H), 3.86 (s, 3H).

[0580] 2.2: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 2.2) To a solution of compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene (200 mL) was added toluene-4-sulfonic acid (157 mg, 0.9 mmol). The solution was heated at 140° C. for 3 hours and then cooled to room temperature. The product was collected by filtration as a yellow precipitate to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0581] LC / MS:C 22 H 18 Calculated m / z for ClFN2O5 = 445.2, found [M+H] + =445.1.

[0582] 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J =12.0 Hz, 1H) 7.80 (d, J = 9.2 Hz, 1H) 7.27 (s, 1H), 6.50 (s, 1H), 5.45 (s, 2H), 5.41 (s, 2H), 5.33 (s, 2H) 4.08 (s, 3H), 1.87 - 1.83 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0583] 2.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 101) The title compound was prepared according to General Procedure 1, starting from compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.6 mg, 41% yield).

[0584] LC / MS:C 26 H 26 Calculated m / z for FN3O6 = 495.2, found [M+H] + =496.4.

[0585] 1 H NMR (300 MHz, MeOD) δ 7.84 - 7.70 (m, 2H), 7.59 (s, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.45 - 5.36 (m, 3H), 4.29 (s, 2H), 4.12 (s, 3H), 3.58 - 3.48 (m, 2H), 3.28 - 3.09 (m, 2H), 2.75 - 2.61 (m, 2H), 2.05 - 1.91 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0586] 2.4: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 103) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.5 mg, 14% yield).

[0587] LC / MS:C 32 H 31 Calculated m / z for FN4O7S = 634.2, observed [M+H] + =635.4.

[0588] 2.5: (S)-11-((4-((4-aminophenyl)sulfonyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 105) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (4.0 mg, 23% yield).

[0589] LC / MS:C 32 H 32 Calculated m / z for FN5O7S = 649.2, observed [M+H] + =650.4.

[0590] 1H NMR (300 MHz, DMSO) δ 8.08 (s, 2H), 7.90 - 7.67 (m, 2H), 7.35 (s, 1H), 7.32 - 7.26 (m, 2H), 6.67 - 6.57 (m, 2H), 5.46 (d, J = 16.5 Hz, 1H), 5.33 -5.22 (m, 3H), 3.92 (s, 3H), 3.02 - 2.72 (m, 4H), 2.75 - 2.58 (m, 4H), 1.97 - 1.70 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H).

[0591] 2.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 107) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.1 mg, 19% yield).

[0592] LC / MS:C 27 H 29 Calculated m / z for FN4O5 = 508.2, observed [M+H] + =509.4.

[0593] 2.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 109) TIFF2025535240000179.tif58165 The title compound was prepared according to general procedure 1 starting from compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in general procedure 9 eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (3.2 mg, 20% yield).

[0594] LC / MS:C 32 H 32 Calculated m / z for FN5O5 = 585.2, observed [M+H] + =586.4.

[0595] 1 H NMR (300 MHz, MeOD) δ 7.83 - 7.74 (m, 2H), 7.62 (s, 1H), 7.06 (d, J = 8.9 Hz, 2H), 6.98 (d, J = 8.9 Hz, 2H), 5.65 (d, J = 16.4 Hz, 1H), 5.36 (s, 2H), 5.27 (d, J = 16.4 Hz, 1H), 4.13 (s, 2H), 4.06 (s, 3H), 3.26 (br s, 4H), 2.79 (br s, 4H), 1.97 - 1.83 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H).

[0596] 2.8: (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 2.8) To a solution of compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL) was added hexamethylenetetramine (236 mg, 1.7 mmol), followed by iPrNEt (100 μL, 0.56 mmol). The solution was heated to reflux for 5 h, cooled to room temperature, and quenched with 12 M aqueous HCl (60 μL). The solution was concentrated to approximately half its volume, and 1 M aqueous HCl (1.5 mL) was added. The mixture was stirred for 5 min and then concentrated to give a brown residue. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the title compound as a pale yellow solid (179 mg, 75% yield).

[0597] LC / MS:C 22 H 20 Calculated m / z for FN3O5 = 425.4, observed [M+H] + =426.2.

[0598] 2.9: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 123) The title compound was prepared according to general procedure 3, starting from compound 2.8 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 65% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (8.5 mg, 91% yield).

[0599] LC / MS:C 23 H 22 Calculated m / z for FN3O7S = 503.1, observed [M+H] + =504.2.

[0600] 1 H NMR (300 MHz, DMSO-d6) δ 7.98 (d, J = 12.1 Hz, 1H), 7.89 (t, J = 6.4 Hz, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.28 (s, 1H), 5.42 (s, 2H), 5.39 (s, 2H), 4.77 (d, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.06 (s, 3H), 1.95-1.73 (m, 2H), 0.88 (d, J = 7.3 Hz, 3H).

[0601] 2.10: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 126) The title compound was prepared according to general procedure 3, starting from compound 2.8 (7.5 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 70% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.6 mg, 46% yield).

[0602] LC / MS:C 28 H 24 Calculated m / z for FN3O7S = 565.6, observed [M+H] + =566.2.

[0603] 1H NMR (300 MHz, DMSO-d6) δ 8.59 (t, J = 6.3 Hz, 1H), 7.94 (d, J = 12.2 Hz, 1H), 7.82 - 7.68 (m, 2H), 7.62 - 7.46 (m, 1H), 7.51 - 7.40 (m, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.52 (s, 1H), 5.44 (s, 1H), 5.36 (s, 1H), 4.64 (d, J = 6.3 Hz, 1H), 4.09 (s, 2H), 1.95 - 1.81 (m, 1H), 0.89 (t, J = 7.3 Hz, 2H).

[0604] 2.11: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 2.11) The title compound was prepared according to general procedure 3, starting from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride. Purification was carried out as described in general procedure 9 using a 12 g C flash column eluted with a 5 to 75% CHCN / HO + 0.1% TFA gradient to afford the title compound as a pale yellow solid (9.7 mg, 71% yield).

[0605] LC / MS:C 28 H 23 Calculated m / z for FN4O9S = 610.6, observed [M+H] + =611.5.

[0606] 2.12: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 128) TIFF2025535240000184.tif63165 To a solution of compound 2.11 (9.7 mg, 0.016 mmol) in methanol (1.6 mL) was added platinum 1% vanadium 2% carbon (15 mg). The flask was purged with H and then stirred under an H atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (1.5 mg, 16% yield).

[0607] LC / MS:C 28 H 25 Calculated m / z for FN4O7S = 580.6, observed [M+H] + =581.4.

[0608] 1 H NMR (300 MHz, MeOD) δ 7.77 (d, J = 11.0 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.39 (d, J = 16.4 Hz, 1H), 5.30 (s, 1H), 4.56 (s, 1H), 4.10 (d, J = 3.7 Hz, 3H), 2.04 - 1.91 (m, 2H), 1.31 (s, 1H), 1.02 (t, J = 7.3Hz, 3H), 0.90 (s, 1H).

[0609] 2.13: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 130) The title compound was prepared according to general procedure 3, starting from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 15 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (2.2 mg, 22% yield).

[0610] LC / MS:C 24 H 24 Calculated m / z for FN3O8S = 533.1, observed [M+H] + =534.2.

[0611] 1 H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J = 12.2 Hz, 1H), 7.89-7.79 (m, 2H), 7.29 (s, 1H), 5.43 (s, 2H), 5.40 (s, 2H), 4.76 (d, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.81 (t, J = 6.3 Hz, 2H), 3.34 (t, J = 6.3 Hz, 2H), 1.94-1.75 (m, 2H), 0.87 (d, J = 7.4 Hz, 3H).

[0612] 2.14: 4-Nitrophenyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 2.14) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting with compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as the solvent. Flash purification was performed as described in General Procedure 9 using a 12 g C12 column eluted with a 10 to 50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (61 mg, 86% yield). This intermediate was resolved and used to generate the next compound.

[0613] LC / MS:C 29 H 23 Calculated m / z for FN4O9 = 590.1, observed [M+H] + =591.2.

[0614] 2.15: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 133) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and aqueous methylamine (500 μL, 40 wt % in water) as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.8 mg, 47% yield).

[0615] LC / MS:C 24 H 23 Calculated m / z for FN4O6 = 482.2, observed [M+H] + =483.2.

[0616] 1H NMR (300 MHz, DMSO-d6) δ 8.00 - 7.87 (m, 2H), 7.31 (s, 1H), 5.48 - 5.39 (m, 3H), 4.81 (s, 3H), 2.56 (s, 3H), 1.93 - 1.81 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0617] 2.16: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 135) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

[0618] LC / MS:C 30 H 28 Calculated m / z for FN5O6 = 573.2, found [M+H] + =574.2.

[0619] 1H NMR (300 MHz, MeOD) δ 7.79 (d, J = 11.9 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.59 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 5.61 (d, J = 16.3 Hz, 1H), 5.52 - 5.35 (m, 3H), 4.98 (s, 2H), 4.39 (s, 2H), 4.01 (s, 3H), 2.03 - 1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0620] 2.17: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 137) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (1.5 mg, 12% yield).

[0621] LC / MS:C 25 H 25 Calculated m / z for FN4O7 = 512.2, observed [M+H] + =513.2.

[0622] 1H NMR (300 MHz, MeOD) δ 7.93 (d, J = 12.1 Hz, 1H), 7.88 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 5.62 (d, J = 16.2 Hz, 1H), 5.52 (s, 2H), 5.45 (d, J = 16.3 Hz, 1H), 4.98 (s, 2H), 4.17 (s, 3H), 3.59 (t, J = 5.6 Hz, 2H), 3.28 (t, J = 5.6 Hz, 2H), 2.10 - 1.91 (m, 2H), 1.05 (t, J = 7.3 Hz, 3H).

[0623] Example 3: Preparation of camptothecin analogs with an amino at the C10 position 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (compound 3.1) TIFF2025535240000190.tif2716To a stirred solution of HNO3 (121.2 mL, 67% purity, 2.0 equiv.) in H2SO4 (500 mL) at 50 °C was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 equiv.). After the addition was complete, the ice bath was removed and the reaction was stirred at 25 °C for 5 h. The mixture was poured onto ice (5 L), filtered, and then dried under reduced pressure. The title compound was obtained as a yellow solid (219 g).

[0624] 1 H NMR (400 MHz, CDCl3) δ 10.39 (s, 1H), 8.23 ​​(d, J = 6.8 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H).

[0625] 3.2: tert-Butyl (2-fluoro-5-formyl-4-nitrophenyl)carbamate (compound 3.2) A mixture of compound 3.1 (219 g, 1.0 equiv.), tert-butyl carbamate (124 g, 1.2 equiv.), CsCO (575 g, 2 equiv.), Pd(dba) (40 g, 0.05 equiv.), and XPhos (84 g, 0.2 equiv.) in toluene (2000 mL) was degassed and purged with N for three cycles. The mixture was then stirred at 90 °C under a N atmosphere for 15 h. The reaction mixture was diluted with H2O (800 mL) and extracted with EtOAc (300 mL × 2). The combined organic layers were washed with brine (200 mL × 2), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 20:1) to give the title compound as a yellow solid (140 g, 56% yield).

[0626] 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.94 (s, 1H), 8.42 (d, J=7.6 Hz, 1H), 8.16 (d, J=10.8 Hz, 1H), 1.50 (s, 9H).

[0627] 3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (compound 3.3) TIFF2025535240000192.tif27165To a solution of compound 3.2 (100 g, 1.0 equiv.) in HO (300 mL) and EtOH (1200 mL) was added NH4Cl (30.5 g, 1.62 equiv.). Iron (78.6 g, 4.0 equiv.) was added portionwise at 80 °C. The mixture was stirred at 80 °C for 6 h. The mixture was filtered, water was added to the filtrate, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1) and TLC (petroleum ether) to give the title compound as a yellow solid (19.0 g, 21% yield).

[0628] LC / MS:C 12 H 15 Calculated m / z for FN2O3 = 254.1, observed [M+H] + =255.0.

[0629] 1 H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1 H), 8.57 (s, 1 H), 7.58 (d, J = 4.8 Hz, 1 H), 7.21 (s, 2 H), 6.53 (d, J = 12.8 Hz, 1 H), 1.43 (s, 9 H).

[0630] 3.4: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.4) A mixture of compound 3.3 (4.20 g, 1.2 equiv.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 equiv.), and TsOH (monohydrate, 253 mg, 0.1 equiv.) in toluene (350 mL) was stirred at 110 °C for 2 h. The reaction solution was cooled to 25 °C, and the solid was washed with methyl t-butyl ether (30 mL) and then dried under reduced pressure. The title compound was obtained as a yellow solid (4.5 g, 62% yield).

[0631] LC / MS:C 25 H 24 Calculated m / z for FN3O6 = 481.2, observed [M+H] + =482.1.

[0632] 1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.65 (s, 1H), 8.43 (d, J =8.4 Hz, 1H), 7.95 (d, J = 12.0 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.25 (s, 2H), 1.80 - 1.92 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 7.2 Hz, 3H).

[0633] 3.5: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.5) TIFF2025535240000194.tif47165 To a mixture of compound 3.4 (4.00 g) in MeOH (360 mL) was added a solution of FeSO (heptahydrate, 1.2 g) and HSO (280 μL) in H2O (4 mL). The reaction mixture was heated to 65 °C while H2O (24 mL, 30% purity) was added dropwise over 30 min and then stirred for 0.5 h. The reaction solution was cooled to 25 °C and filtered to give the title compound as a yellow solid (1.53 g, 33.2% yield). The filtrate was added H2O (400 mL) and then quenched with saturated aqueous Na2SO3. The pH was adjusted to 7-8 with saturated aqueous Na2CO3, and the solution was concentrated and filtered. The solid was triturated with MeOH (30 mL) at 55° C. for 1 h and then filtered to give a second batch of the title compound as a brown solid (1.09 g, 26% yield).

[0634] LC / MS:C 26 H 26 Calculated m / z for FN3O7 = 511.2, observed [M+H] + =512.2.

[0635] 1H NMR (300 MHz, d6-DMSO) δ 9.47 (s, 1H), 8.47 (d, J =7.6 Hz, 1H), 7.94 (d, J =12.0 Hz, 1H), 7.29 (d, J =1.6 Hz, 1H), 6.49 (s, 1H), 5.86 - 5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J =4.4 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 6.4 Hz, 3H).

[0636] 3.6: tert-Butyl (S)-(4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.6) To a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), DCM (2.9 mL) was added, followed by Dess-Martin periodinane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). The solution was stirred at room temperature for 18 h, then diluted with DCM and washed with saturated aqueous NaHCO3 and brine. The layers were separated, and the combined organic layer was evaporated onto Celite. Flash purification was performed as described in General Procedure 9 using a 10 g silica column eluting with 0 to 10% DCM / MeOH to afford the title product as an orange powder (42.5 mg, 28%).

[0637] LC / MS:C 26 H 24 Calculated m / z for FN3O7 = 509.2, found [M+H] + =510.4.

[0638] 1H NMR (300 MHz, acetone-d6) δ 11.10 (s, 1H), 9.68 (d, J =8.6 Hz, 1H), 8.81 (s, 1H), 8.04 (d, J =11.9 Hz, 1H), 7.63 (s, 1H), 5.73 (s, 2H), 5.69 (d, J =16.2 Hz, 1H), 5.42 (d, J =16.2 Hz, 1H), 2.02-1.95 (m, 2H), 8.47 (d, J =7.6 Hz, 1H), 7.94 (d, J =12.0 Hz, 1H), 7.29 (d, J =1.6 Hz, 1H), 6.49 (s, 1H), 5.86 - 5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J =4.4 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 6.4 Hz, 3H).

[0639] 3.7: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 140) TIFF2025535240000196.tif37165 The title compound was prepared according to general procedure 6 starting from compound 3.4 (40 mg) to afford the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[0640] LC / MS:C 20 H 16 Calculated m / z for FN3O4 = 381.1, observed [M+H] + =382.2.

[0641] 1H NMR (300 MHz, DMSO) δ 8.28 (s, 1H), 7.72 (d, J = 12.5 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 5.43 (d, J = 16.2 Hz, 1H), 5.34 (d, J = 16.2 Hz, 1H), 5.17 (s, 2H), 1.92 - 1.74 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0642] 3.8: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 141) TIFF2025535240000197.tif47165 The title compound was prepared according to general procedure 6 starting from compound 3.5 (5 mg) to afford the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).

[0643] LC / MS:C 21 H 18 Calculated m / z for FN3O5 = 411.2, observed [M+H] + =412.2.

[0644] 1 H NMR (300 MHz, MeOD) δ 7.71 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.29 (d, J = 9.5 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.47 (s, 2H), 5.40 (d, J = 16.3 Hz, 1H), 5.25 (s, 2H), 2.03 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0645] 3.9: tert-Butyl (S)-(11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.9) To a stirred solution of compound 3.5 (100 mg) in dichloromethane (5 mL) was added a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL). After 1 h, additional thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added. After an additional 1 h, the reaction was diluted with dichloromethane (10 mL) and toluene (1 mL) and then concentrated in vacuo to give the title compound as a red solid, which was used in the next reaction without further purification.

[0646] LC / MS:C 26 H 25 Calculated m / z for ClFN3O6 = 529.1, found [M+H] + =530.2.

[0647] 3.10: tert-Butyl (S)-(11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.10) TIFF2025535240000199.tif47165 To compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg) followed by DIPEA (99 μL). The solution was heated at 60° C. for 16 hours and then concentrated to dryness in vacuo. Flash purification was performed as described in General Procedure 9 using a 12 g C18 column eluted with a 10 to 50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

[0648] LC / MS:C 26 H 27Calculated m / z for FN4O6 = 510.2, observed [M+H] + =511.4.

[0649] 1 H NMR (300 MHz, MeOD) δ 8.88 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 11.9 Hz, 1H), 7.62 (s, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.80 (s, 2H), 2.07 - 1.89 (m, 2H), 1.64 (s, 9H), 1.02 (t, J = 7.3 Hz, 3H).

[0650] 3.11: (S)-9-Amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 145) TIFF2025535240000200.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.10 (2.1 mg) to afford the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[0651] LC / MS:C 21 H 19 Calculated m / z for FN4O4 = 410.1, found [M+H] + =411.2.

[0652] 1H NMR (300 MHz, MeOD) δ 7.82 (d, J = 12.1 Hz, 1H), 7.60 (s, 1H), 7.37 (d, J = 9.1 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.42 (s, 2H), 5.41 (d, J = 16.3 Hz, 1H), 4.69 (s, 2H), 2.08 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0653] Example 3.12: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 3.12) The title compound was prepared according to General Procedure 1, starting from compound 3.9 (150 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a red solid (TFA salt, 103 mg, 52% yield).

[0654] LC / MS:C 30 H 33 Calculated m / z for FN4O7 = 580.2, observed [M+H] + =581.4.

[0655] 1H NMR (300 MHz, MeOD) δ 9.06 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 12.0 Hz, 1H), 7.66 (s, 1H), 5.63 (d, J = 16.3 Hz, 1H), 5.51 (s, 2H), 5.43 (d, J = 16.4 Hz, 1H), 4.92 (s, 2H), 3.84 (s, 4H), 3.10 (s, 4H), 1.99 (d, J = 5.5 Hz, 2H), 1.63 (s, 9H), 1.03 (t, J = 7.4 Hz, 3H).

[0656] 3.13: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 142) TIFF2025535240000202.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.12 (45 mg) to afford the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[0657] LC / MS:C 25 H 25 Calculated m / z for FN4O5 = 480.2, observed [M+H] + =481.4.

[0658] 1 H NMR (300 MHz, MeOD) δ 7.73 (d, J = 12.0 Hz, 1H), 7.54 (s, 1H), 7.48 (d, J = 9.2 Hz, 1H), 5.60 (d, J = 16.3 Hz, 1H), 5.47 - 5.34 (m, 3H), 4.65 (s, 2H), 3.91 - 3.85 (m, 4H), 3.30 - 3.24 (m, 4H), 2.08 - 1.91 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H).

[0659] 3.14: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 148) To a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol), dichloromethane (1.45 mL) was added, followed by acetic acid (18.69 μL, 0.327 mmol), piperidine (21.52 μL, 0.218 mmol), and sodium triacetoxyborohydride (23.0 mg, 0.109 mmol). The solution was then stirred at room temperature for 2 h, quenched by the addition of water + 0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to general procedure 6 to give the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).

[0660] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [M+H] + =479.4.

[0661] 1 H NMR (300 MHz, MeOD) δ 7.78 (d, J = 12.1 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 9.1 Hz, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.47 - 5.35 (m, 3H), 4.86 (s, 2H), 3.80 - 3.68 (m, 2H), 3.28 - 3.19 (m, 2H), 2.02 - 1.68 (m, 8H), 1.01 (t, J = 7.4 Hz, 3H).

[0662] 3.15: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 149) To a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol) was added dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and N-methylpiperazine (4.90 μL, 0.044 mmol). The solution was stirred at room temperature for 4 h, followed by the addition of sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) and stirring for an additional 45 min. The excess hydride was then quenched by the addition of 0.1% aqueous TFA (0.5 mL). Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluting with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was deprotected according to general procedure 6 to give the title compound as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

[0663] LC / MS:C 26 H 28 Calculated m / z for FN5O4 = 493.2, observed [M+H] + =494.4.

[0664] 1 H NMR (300 MHz, MeOD) δ 7.68 (d, J = 12.2 Hz, 1H), 7.56 (s, 1H), 7.53 (d, J = 9.5 Hz, 1H), 5.60 (d, J = 16.3 Hz, 1H), 5.45-5.30 (m, 3H), 4.15 (s, 2H), 3.55 - 3.44 (m, 2H), 3.18 - 3.07 (m, 2H), 2.93 (s, 3H), 2.70 - 2.51 (m, 2H), 2.03 - 1.89 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0665] 3.16: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 153) The Boc-protected precursor of the title compound was prepared according to General Procedure 1, starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC was performed as described in General Procedure 9, eluting with a 35 to 44% CHCN / HO + 0.1% TFA gradient, to afford the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to afford the title compound (TFA salt, 2.4 mg, 17% yield over two steps).

[0666] LC / MS: m / z calculated for C31H30FN5O6S = 619.2, found [M+H]+ = 520.4.

[0667] 1 H NMR (300 MHz, MeOD) δ 7.81-7.60 (m, 7H), 7.34 (s, 1H), 5.51 (d, J = 16.4 Hz, 1H), 5.35 (d, J = 16.4 Hz, 1H), 5.22 (s, 2H), 4.10 (s, 2H), 3.15-3.02 (m, 4H), 2.79-2.71 (m, 4H), 2.00-1.93 (m, 2H), 1.00(t,J= 7.4 Hz, 3H).

[0668] 3.17: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 147) The title compound was prepared according to General Procedure 2 followed by General Procedure 6, starting from compound 3.10 (8 mg) and acetic acid. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.0 mg, 56% yield).

[0669] LC / MS:C 23 H 21 Calculated m / z for FN4O5 = 452.2, observed [M+H] + =453.2.

[0670] 1 H NMR (300 MHz, MeOD) δ 7.69 (d, J = 12.1 Hz, 1H), 7.56 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.44 - 5.33 (m, 3H), 4.85 (s, 3H), 2.03 (s, 3H), 2.00 - 1.84 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0671] 3.18: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 146) The title compound was prepared according to general procedure 3 followed by general procedure 6, starting from compound 3.10 (8 mg) and methanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.4 mg, 57% yield).

[0672] LC / MS:C 22 H 21 Calculated m / z for FN4O6S = 488.1, observed [M+H] + =489.2.

[0673] 1 H NMR (300 MHz, MeOD) δ 7.74 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.49 (d, J = 9.3 Hz, 1H), 5.61 (d, J = 16.2 Hz, 1H), 5.45 (s, 2H), 5.40 (d, J = 16.2 Hz, 1H), 4.78 (s, 2H), 3.05 (s, 3H), 2.08 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0674] 3.19: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 150) The title compound was prepared according to general procedure 3 followed by general procedure 6, starting from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (1 mg, 16% yield).

[0675] LC / MS:C 23 H 23 Calculated m / z for FN4O7S = 518.5, observed [M+H] + =519.5.

[0676] 1H NMR (300 MHz, 10% D2O / CD3CN) δ 7.77 - 7.61 (m, 1H), 7.48 - 7.30 (m, 2H), 5.53 (d, J = 16.3 Hz, 1H), 5.31 (d, J = 15.4 Hz, 3H), 4.69 (s, 2H), 3.97 (dd, J = 6.6, 4.9 Hz, 2H), 3.39 (t, J = 5.8 Hz, 2H), 2.93 (s, 1H), 1.99-1.83 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0677] 3.20: 4-Nitrophenyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 3.20) To a solution of compound 3.10 (10 mg, 0.02 mmol) in DMF (400 μL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 μL, 0.04 mmol). The solution was stirred at room temperature for approximately 30 minutes and then used directly in the next reaction.

[0678] 3.21: Methyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 143) The title compound was prepared by adding MeOH (100 μL) to a solution of 200 μL of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.1 mg, 47% yield) following General Procedure 6.

[0679] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.4, observed [M+H] + =468.3.

[0680] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 7.72 (d, J = 12.2 Hz, 1H), 7.41 (d, J = 18.1 Hz, 1H), 6.96 (s, 1H), 5.52 (d, J = 3.6 Hz, 1H), 5.39 - 5.23 (m, 3H), 4.82 (s, 1H), 4.73 (s, 1H), 3.63 (d, J = 1.2 Hz, 3H), 1.56 (s, 3H), 1.27 (s, 2H), 0.94 (t, J = 7.4 Hz, 3H).

[0681] 3.22: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 144) The title compound was prepared by adding methylamine hydrochloride (10 mg) to a 200 μL solution of compound 3.20, followed by iPrNEt (5 μL). The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.9 mg, 64.5% yield) following General Procedure 6.

[0682] LC / MS:C 23 H 21 Calculated m / z for FN5O5 = 467.5, observed [M+H] + =468.5.

[0683] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.13 (d, J = 9.2 Hz, 1H), 7.92 (s, 1H), 7.73 (d, J = 12.3 Hz, 1H), 7.52 - 7.35 (m, 2H), 6.94 (d, J = 9.2 Hz, 2H), 5.55 (d, J = 16.5 Hz, 2H), 5.44 - 5.27 (m, 4H), 4.85 (s, 2H), 4.78 (s, 1H), 1.56 (d, J = 2.5 Hz, 3H), 1.27 (s, 2H), 0.93 (q, J = 11.7, 9.5 Hz, 3H).

[0684] 3.23: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 151) The title compound was prepared by adding ethanolamine (100 μL) to a solution of 200 μL of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (0.5 mg, 8.5% yield) following General Procedure 6.

[0685] LC / MS:C 24 H 24 Calculated m / z for FN5O6 = 497.5, observed [M+H] + =498.5.

[0686] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 7.77 - 7.61 (m, 1H), 7.48 - 7.30 (m, 2H), 5.53 (d, J = 16.3 Hz, 1H), 5.31 (d, J = 15.4 Hz, 1H), 5.19 (s, 2H), 4.69 (s, 2H), 3.97 (dd, J = 6.6, 4.9 Hz, 2H), 3.39 (t, J = 5.8 Hz, 2H), 2.93 (s, 1H), 2.01-1.83 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0687] 3.24: (S)-9-Amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 152) To a stirred solution of compound 3.5 (100 mg) in 2 mL of dichloromethane was added thionyl chloride (35 μL, 2.5 equiv.). The solution was stirred at room temperature for 20 minutes, after which additional thionyl chloride (35 μL, 2.5 equiv.) was added. After 20 minutes, toluene (1 mL) was added, and the reaction mixture was concentrated in vacuo. The crude solid was suspended in DMSO (1 mL), and sodium azide (19 mg, 1.5 equiv.) was added. The solution was stirred at room temperature for 16 hours. Purification was carried out as described in General Procedure 9, eluting with a 5-50% CH3CN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (20 mg, 23% yield).

[0688] LC / MS:C 21 H 17 Calculated m / z for FN6O4 = 436.1, observed [M+H] + =437.2.

[0689] 1 H NMR (300 MHz, MeOD) δ 7.75 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.46 - 5.35 (m, 3H), 5.07 (s, 2H), 2.03 - 1.97 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0690] 3.25: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 164) The title compound was prepared according to general procedure 2, starting from compound 145 (10 mg) and glycolic acid. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).

[0691] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.1, observed [M+H] + =469.2.

[0692] 1 H NMR (300 MHz, MeOD) 7.70 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.42 (d, J = 9.4 Hz, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.43 (s, 2H), 5.36 (d, J = 16.2 Hz, 1H), 4.95 (d, J = 5.9 Hz, 2H), 4.08 (s, 2H), 2.04 - 1.90 (m, 1H), 1.03 (t, J = 7.4 Hz, 3H).

[0693] 3.26: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylthiourea (compound 161) To a solution of compound 145 (9 mg, 1.0 equiv.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 equiv.), followed by DIPEA (8 μL, 2.0 equiv.). The resulting solution was stirred at 25° C. for 2 h, after which complete conversion to the isothiocyanate intermediate was confirmed. Methylammonium chloride (3 mg, 2.0 equiv.) was then added, and the reaction mixture was heated at 60° C. for 30 min. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 45% CH CN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (2.3 mg, 22% yield).

[0694] LC / MS:C 23 H 22 Calculated m / z for FN5O4S = 483.1, observed [M+H] + =484.2.

[0695] 1 H NMR (300 MHz, MeOD) δ 7.70 (d, J = 12.0 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.62 (d, J = 16.2 Hz, 1H), 5.36 (s, 2H), 5.31 (d, J = 16.2 Hz, 1H), 5.30 (s, 2H), 3.04 (s, 3H), 1.99 - 1.90 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0696] 3.27: S-(2-Hydroxyethyl)-(S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamothioate (compound 160) The title compound was prepared according to general procedure 5, starting from compound 145 (10 mg) and 2-mercaptoethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).

[0697] LC / MS:C 24 H 23 Calculated m / z for FN4O6S = 514.1, observed [M+H] + =515.2.

[0698] 1 H NMR (300 MHz, MeOD) δ 7.71 (d, J = 12.1 Hz, 1H), 7.60 (s, 1H), 7.36 (d, J = 9.4 Hz, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.42 (s, 2H), 5.35 (d, J = 16.2 Hz, 1H), 4.88 (d, J = 4.6 Hz, 2H), 3.68 (t, J = 6.4 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.04 - 1.92 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0699] 3.28: (S)-9-Amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 154) TIFF2025535240000217.tif47165 To a 5 mL flask containing compound 140 (50 mg), water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg), and propionaldehyde (74 μL) were added. The resulting suspension was cooled to −15 °C using an ice-salt water bath, and then sulfuric acid (0.40 mL) was added dropwise. Hydrogen peroxide (95 μL) was then added dropwise. The mixture was stirred at −15 °C for 10 minutes, then warmed to room temperature, and stirred for 2 hours. The reaction mixture was diluted with water (30 mL), and the resulting suspension was extracted with DCM (3 × 30 mL). The organic phase was then evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25 to 70% CH3CN / H2O+0.1% TFA gradient to afford the title compound as a deep orange solid (2.4 mg, 4.4% yield).

[0700] LC / MS:C 22 H 20 Calculated m / z for FN3O4 = 410.1, found [M+H] + =410.2.

[0701] 1 H NMR (300 MHz, MeOD) δ 7.63 (d, J = 12.3 Hz, 1H), 7.55 (s, 1H), 7.36 (d, J = 9.4 Hz, 1H), 5.57 (d, J = 16.4 Hz, 1H), 5.37 (d, J = 16.4 Hz, 1H), 5.21 (s, 2H), 3.13 (q, J = 7.7 Hz, 2H), 2.02 - 1.90 (m, 2H), 1.38 (t, J = 7.7 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H).

[0702] 3.29: tert-Butyl-(S)-(11-((carbamoyloxy)methyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.29) Compound 3.5 (15 mg) was added to a 5 mL Erlenmeyer flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL) at −20° C. The resulting suspension was stirred at −20° C. for 5 minutes. Water (59 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 2 hours before being heated at 70° C. for 1 hour. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 40-55% CH3CN / HO + 0.1% TFA gradient to afford the title compound as a dark orange solid (5.1 mg, 31% yield).

[0703] LC / MS:C 27 H 27 Calculated m / z for FN4O8 = 555.2, observed [M+H] + =555.2.

[0704] 1 H NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.56 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 12.0 Hz, 1H), 7.31 (s, 1H), 7.11-6.62 (m, 2H), 6.52 (s, 1H), 5.58 (s, 2H), 5.49-5.27 (m, 4H), 1.94-1.77 (m, 2H), 1.52 (s, 9H), 1.38 (t, J = 7.7 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[0705] 3.30: (S)-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methylcarbamate (compound 169) TIFF2025535240000219.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.29 (5.1 mg) to afford the title compound as a yellow powder (TFA salt, 3.8 mg, 73% yield).

[0706] LC / MS:C 22 H 19 Calculated m / z for FN4O6 = 455.1, observed [M+H] + =455.2.

[0707] 1 H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J = 12.4 Hz, 1H), 7.29 (d, J = 9.7 Hz, 1H), 7.21 (s, 1H), 7.0-6.50 (m, 2H), 5.45 (s, 2H), 5.40 (s, 2H), 5.33 (s, 2H), 1.95-1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0708] 3.31: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 155) TIFF2025535240000220.tif47165 To a 50 mL flask containing compound 3.5 (30 mg) was added MeOH / dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL). The reaction mixture was then stirred at reflux for 24 h. The reaction mixture was concentrated, poured into water (30 mL), and extracted with DCM (3 × 50 mL). The organic phases were combined and dried over MgSO. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25-40% CHCN / H2O + 0.1% TFA gradient, to afford the title compound as a dark orange solid (5.1 mg, 16% yield).

[0709] LC / MS:C 22 H 20 FN3 Calculated m / z for O5 = 426.1, observed [M+H]+ = 426.2.

[0710] 1 H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J = 12.3 Hz, 1H), 7.24 (d, J = 9.9 Hz, 1H), 7.20 (s, 1H), 6.47 (s, 1H), 6.30-5.92 (brs, 2H), 5.40 (s, 2H), 5.24 (s, 2H), 4.93 (s, 2H), 3.43 (s, 3H), 1.95-1.75 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0711] 3.32: (4S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(((1R,5S)-6-hydroxy-3-azabicyclo[3.1.1]heptan-3-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 158) To a 5 mL Erlenmeyer flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL), followed by 3-azabicyclo[3.1.1]heptan-6-ol (10 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature, and sodium triacetoxyborohydride (9.4 mg) was added. After 1 h at room temperature, the reaction was quenched by the addition of water + 0.1% TFA and diluted with DMF. The reaction mixture was then partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient, to afford the Boc-protected title compound as a yellow powder. Deprotection was carried out according to general procedure 6, and the resulting residue was purified by preparative HPLC purification as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as a yellow powder (TFA salt, 7.1 mg, 39% yield).

[0712] LC / MS:C 27 H 27 Calculated m / z for FN4O5 = 507.2, [M+H] + =507.4.

[0713] 1 H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J = 12.1 Hz, 1H), 7.46 (d, J = 9.4 Hz, 1H), 7.23 (s, 1H), 6.64-5.85 (m, 3H), 5.60-5.25 (m, 4H), 4.85 (s, 1H), 4.10-3.95 (m, 1H), 3.68 (s, 2H), 2.45-2.33 (m, 2H), 1.96-1.72 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0714] 3.33: (S)-9-Amino-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 159) To a 5 mL Erlenmeyer flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL), followed by (3-fluoroazetidin-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature, and sodium triacetoxyborohydride (9.4 mg) was added. After 1 h at room temperature, the reaction was quenched by the addition of water + 0.1% TFA, diluted with DMF, and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20-50% CH3CN / HO + 0.1% TFA gradient, to afford the Boc-protected title compound as a yellow powder. Deprotection was then carried out according to General Procedure 6. The resulting residue was purified by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 50% CHCN / H0+0.1% TFA gradient to afford the title compound as a yellow powder (TFA salt, 1.8 mg, 10% yield).

[0715] LC / MS:C 25 H 24 Calculated m / z for F2N4O5 = 499.2, found [M+H] + =499.4.

[0716] 1 H NMR (300 MHz, DMSO-d6) δ 7.82 (d, J = 12.4 Hz, 1H), 7.45 (d, J = 9.5 Hz, 1H), 7.21 (s, 1H), 5.45-5.33 (m, 4H), 3.75-3.61 (m, 2H), 1.93-1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0717] 3.34: tert-Butyl-(S)-(4-ethyl-8-fluoro-4-hydroxy-11-((methylamino)methyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.34) To a stirred solution of compound 3.9 (210 mg) in DMF (5 mL) was added sodium iodide (5.9 mg), followed by methylammonium chloride (107 mg). The reaction mixture was then stirred at room temperature overnight. Reverse-phase purification was performed as described in General Procedure 9 using a 30 g C18 column eluted with a 10 to 65% CH3CN / HO + 0.1% TFA gradient to afford the title compound as a yellow solid (15.0 mg, 7.2% yield).

[0718] LC / MS:C 27 H 29 Calculated m / z for FN4O6 = 524.2, observed [M+H] + =525.4.

[0719] 3.35: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-N-methylacetamide (compound 165) The Boc-protected form of the title compound was prepared according to General Procedure 2, starting from compound 3.34 (6.4 mg) and glycolic acid. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient. Deprotection was then carried out according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.0 mg, 28% yield).

[0720] LC / MS:C 24 H 23 Calculated m / z for FN4O6 = 482.2, observed [M+H] + =483.2.

[0721] 1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J = 12.3 Hz, 1H), 7.27 (d, J = 9.5 Hz, 1H), 7.22 (s, 1H), 6.48 (s, 1H), 6.28-6.02 (m, 2H), 5.40 (s, 2H), 5.21 (s, 2H), 5.06-4.93 (m, 2H), 4.18 (s, 2H), 2.80 (s, 3H), 1.92-1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0722] 3.36: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-N-methylmethanesulfonamide (compound 166) The Boc-protected form of the title compound was prepared according to General Procedure 3, starting from compound 3.34 (8.0 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient. Deprotection was then carried out according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.6 mg, 34% yield).

[0723] LC / MS:C 23 H 23 Calculated m / z for FN4O6S = 502.1, observed [M+H] + =503.2.

[0724] 1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J = 12.3 Hz, 1H), 7.41 (d, J = 9.4 Hz, 1H), 7.23 (s, 1H), 6.63-5.84 (m, 2H), 5.42 (s, 2H), 5.29 (s, 2H), 4.81-4.64 (m, 2H), 3.14 (s, 3H), 2.67 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0725] 3.37: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 170) TIFF2025535240000226.tif53165 To a 10 mL round-bottom flask containing compound 3.4 (62.0 mg), water (0.89 mL), FeSO4 (heptahydrate, 18.0 mg), and 3-methoxypropanal (113.0 mg) were added. To the resulting suspension, sulfuric acid (0.495 mL) was added dropwise while stirring in an ice-salt bath at −15°C. Hydrogen peroxide (0.118 mL) was then added dropwise. The mixture was stirred at −15°C for 10 minutes, then warmed to room temperature, and stirred for 1 hour. The reaction mixture was then diluted with water (30 mL), and the resulting suspension was extracted with DCM (3 × 30 mL). The organic phase was evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25 to 45% CHCN / H0+0.1% TFA gradient to afford the title compound as a deep orange solid (TFA salt, 3.1 mg, 4.4% yield).

[0726] LC / MS:C 23 H 22 Calculated m / z for FN3O5 = 440.2, observed [M+H] + =440.2.

[0727] 1H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J = 12.4 Hz, 1H), 7.33 (d, J = 9.4 Hz, 1H), 7.20 (s, 1H), 6.60-6.42 (m, 2H), 5.40 (s, 2H), 5.25 (s, 2H), 3.69 (t, J = 6.5 Hz, 2H), 3.24 (s, 3H), 3.23 (t, J = 6.5 Hz, 2H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0728] 3.38: (S)-N-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)acetamide (Compound 171) To a 25 mL round-bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL) was added N-methylmorpholine (0.343 mL), HOAt (0.142 g), and HATU (0.435 g). After stirring at room temperature for 5 minutes, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). Immediately after stirring at room temperature for 24 hours, the solution was purified by preparative HPLC as described in General Procedure 9, eluting with a 25-45% CHCN / HO + 0.1% TFA gradient to afford the title compound as a bright yellow powder (43.0 mg, 38% yield).

[0729] LC / MS:C 22 H 18 Calculated m / z for FN3O5 = 424.1, observed [M+H] + =424.2.

[0730] 1H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.61 (s, 1H), 7.96 (d, J = 912.1 Hz, 1H), 7.29 (s, 1H), 6.60-6.42 (m, 2H), 5.41 (s, 2H), 5.21 (s, 2H), 2.20 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0731] 3.39: tert-Butyl (5-formyl-2-methoxy-4-nitrophenyl)carbamate (compound 3.39) To a solution of compound 3.2 (1.3 g, 1.0 equiv.) in MeOH (12 mL) at 50 °C was added sodium methoxide (0.74 g, 3.0 equiv.). After the addition was complete, the ice bath was removed and the resulting solution was stirred at room temperature for 72 h. The reaction was then quenched with ice water (50 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford the title compound as an orange solid (1.2 g, 89% yield).

[0732] LC / MS:C 13 H 16 Calculated m / z for N2O6 = 296.10, observed [M+H] + =297.1.

[0733] 1 H NMR (300 MHz, MeOD) δ 10.29 (s, 1H), 8.61 (s, 1H), 7.73 (s, 1H), 4.08 (s, 3H), 1.57 (s, 9H).

[0734] 3.40: tert-Butyl (4-amino-5-formyl-2-methoxyphenyl)carbamate (compound 3.40) To a solution of compound 3.39 (500 mg, 1 equiv.) in MeOH (10 mL) and HO (1 mL) was added B(OH) (454 mg, 3 equiv.). The resulting mixture was cooled to 0 °C, and 5 M aqueous NaOH (2.75 mL) was added with stirring over 10 min. The reaction mixture was stirred for an additional 5 min before being quenched by pouring the solution onto ice (40 mL). The resulting mixture was extracted with DCM (3 × 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was performed as described in General Procedure 9 using a 25 g silica column eluting with 10 to 50% hexanes / EtOAc to afford the title compound as an orange solid (386 mg, 86%).

[0735] LC / MS:C 13 H 18 Calculated m / z for N2O4 = 266.1, observed [M+H] + =297.2.

[0736] 3.41: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 168) A mixture of compound 3.40 (385 mg, 1.0 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (362 mg, 0.95 equiv.), TsOH (monohydrate, 25 mg, 0.1 equiv.), and toluene (30 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 hours. The reaction mixture was then cooled to 25° C. and concentrated under reduced pressure. Purification was carried out as described in General Procedure 9 using a 25 g silica column eluted with a 0 to 50% DCM / MeOH gradient to give the Boc-protected intermediate as a red solid. This material was then deprotected according to General Procedure 6, followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CHCN / H0+0.1% TFA gradient, to afford the title compound as a red solid (TFA salt, 300 mg, 53% yield).

[0737] LC / MS:C 21 H 19 Calculated m / z for N3O5 = 393.2, observed [M+H] + =393.2.

[0738] 1 H NMR (300 MHz, MeOD) δ 8.27 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 7.11 (s, 1H), 5.61 (d, J = 16.2 Hz, 1H), 5.38 (d, J = 16.2 Hz, 1H), 5.24 (s, 2H), 4.11 (s, 3H), 2.06 - 1.91 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).

[0739] 3.42: 5-Bromo-2-nitro-4-(trifluoromethyl)benzaldehyde (compound 3.42) TIFF2025535240000231.tif3216To a stirred solution of HNO3 (2.0 g, 1.4 mL, 67% purity, 2 equiv.) in H2SO4 (8 mL) at 50 °C was added 3-bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 equiv.). After the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 5 h. The mixture was poured onto ice (100 mL) and the precipitate was extracted with DCM (3 × 100 mL). The combined organic fractions were then washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo to afford the title compound as a yellow solid (4.4 g, 93% yield).

[0740] LC / MS: m / z calculated for C8H3BrF3NO3 = 296.90, found [M+H] + =298.0.

[0741] 1 H NMR (300 MHz, MeOD) δ 10.35 (s, 1H), 8.29 (s, 1H), 8.23 ​​(s, 1H).

[0742] 3.43: tert-Butyl (5-formyl-4-nitro-2-(trifluoromethyl)phenyl)carbamate (compound 3.43) A mixture of compound 3.42 (800 mg, 1 equiv.), tert-butyl carbamate (378 mg, 1.2 equiv.), CsCO (1.7 g, 2 equiv.), Pd(dba) (122 mg, 0.05 equiv.), and dicyclohexyl[2',4',6'-tris(propan-2-yl)[1,1'-biphenyl]-2-yl]phosphane (XPhos) (256 mg, 0.2 equiv.) in toluene (5 mL) was degassed and purged with N for three cycles. The mixture was then stirred at 90 °C under a N atmosphere for 15 h. The reaction mixture was diluted with HO (25 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (2 x 25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was performed using a 25 g silica column according to General Procedure 9, eluting with 0 to 25% DCM / MeOH to give the title compound as an orange solid (750 mg, 84% yield).

[0743] LC / MS:C 13 H 13 Calculated m / z for FN2O5 = 334.1, measured [MH] - =333.1.

[0744] 3.44: tert-Butyl (4-amino-5-formyl-2-(trifluoromethyl)phenyl)carbamate (compound 3.44) To a solution of compound 3.43 (750 mg, 1 equiv.) in MeOH (16 mL) and HO (1.6 mL) was added B(OH) (603 mg, 3 equiv.). The resulting mixture was cooled to 0 °C, and 5 M aqueous NaOH (2.75 mL) was added with stirring over 10 min. The reaction mixture was stirred for an additional 5 min before being quenched by pouring the solution onto ice (50 mL). The resulting mixture was extracted with DCM (3 × 75 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was performed as described in General Procedure 9 using a 25 g silica column eluting with 10 to 50% hexanes / EtOAc to afford the title compound as an orange solid (460 mg, 67%).

[0745] LC / MS:C 13 H 15 Calculated m / z for F3N2O3 = 304.1, observed [M+H] + =305.2.

[0746] 3.45: (S)-9-Amino-4-ethyl-4-hydroxy-8-(trifluoromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 167) A mixture of compound 3.44 (460 mg, 1 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (378 mg, 0.95 equiv.), TsOH (monohydrate, 26 mg, 0.1 equiv.), and toluene (35 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 hours. The reaction mixture was then cooled to 25° C. and concentrated under reduced pressure. Purification was carried out as described in General Procedure 9 using a 25 g silica column eluted with a 0 to 50% DCM / MeOH gradient to give the Boc-protected intermediate as a red solid. This material was then deprotected according to General Procedure 6, followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CHCN / H0+0.1% TFA gradient, to afford the title compound as a yellow solid (6.2 mg, 48%).

[0747] LC / MS:C 21 H 16 Calculated m / z for F3N3O4 = 431.1, found [M+H] + =432.2.

[0748] 1 H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.27 (s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.39 (d, J = 16.3 Hz, 1H), 5.28 (s, 2H), 2.00 - 1.89 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0749] ...

Claims

1. Formula (X): T-[L-(D) ｍ ] ｎ (X) 1. An antibody-drug conjugate having the formula: During the ceremony, m is an integer from 1 to 4, n is an integer from 1 to 10, T is an anti-GPC3 antibody construct comprising an antigen-binding domain that binds to human GPC3 (glypican-3), said antigen-binding domain comprising: c) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:8; and d) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 18, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:

17. Including, L is a linker, and D is a compound of formula I: is a compound of the formula R １ is -H, -CH ３ , -CHF ２ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , -OCF ３ , and —NH ２ is selected from R ２ is -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ and wherein: R １ Ga-NH ２ If R is R ３ or R ４ and R １ Ga-NH ２ If other than R, then R ４ and R ３ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, -(C １ ~C ６ alkyl)-O-R ５ , -CO ２ R ８ , -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ４ teeth, is selected from R ５ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ６ and R ７ are each independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, -(C １ ~C ６ alkyl)-O-R ５ , -C ３ ~C ８ Heterocycloalkyl, and —C(O)R １７ is selected from R ８ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; Each R ９ are independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; Each R １０ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, —NR １４ R １４’ , -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １０’ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １１ is -H and -C １ ~C ６ alkyl, R １２ is -H, -C １ ~C ６ Alkyl, —CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ , and is selected from R １３ is -H and -C １ ~C ６ alkyl, R １４ and R １４’ are each independently —H, C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １６ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １７ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, —C ３ ~C ８ heterocycloalkyl, -(C １ ~C ６ alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １８ and R １９ together with the N atom to which they are attached, halogens, -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ alkyl)-O-R ５ forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R ２４ , R ２５ , and R ２６ are -C respectively. １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; and X ｃ is O, S, and S(O) ２ is selected from provided that the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione; Antibody-drug conjugates.

2. The antigen-binding domain comprises: a) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29 and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; or b) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27 and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28 The antibody-drug conjugate of claim 1, comprising:

3. D is a compound of formula (IV): is a compound of the formula R １ａ is -H, -CH ３ , -CHF ２ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , -OCF ３ , and —NH ２ is selected from R ２ａ is -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ is selected from X is —O—, —S—, or —NH—, and R ４ａ teeth, wherein * is the point of attachment to X and wherein p is 1, 2, 3 or 4; or X is O and R ４ａ -X- is is selected from R ５ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ８ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; Each R ９ａ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; or R ９ａ does not exist and X ｂ =X, Each R １０ａ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, and is selected from Each R １０ａ’ are independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; Each R １０ｂ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １１ａ is not present or -C １ ~C ６ is alkyl, R １２ａ is -C １ ~C ６ Alkyl, —CO ２ R ８ａ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ａ , and is selected from R １３ａ is -H and -C １ ~C ６ alkyl, R １４ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １４ａ’ is H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １６ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ２１ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, and -(C １ ~C ６ alkyl)-O-R ５ａ is selected from R ２２ and R ２３ are each independently —H, -halogen, or —C １ ~C ６ Alkyl, and -C ３ ~C ８ cycloalkyl; R ２４ , R ２５ , and R ２６ are -C respectively. １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; X ｃ is O, S, and S(O) ２ is selected from indicates the point of attachment to the linker L, The antibody-drug conjugate according to claim 1 or 2.

4. R １ａ But -CH ３ , -CF ３ , -OCH ３ , -OCF ３ , and —NH ２ The antibody-drug conjugate of claim 3, selected from

5. R １ａ But -CH ３ , -OCH ３ , and N.H. ２ The antibody-drug conjugate of claim 3, selected from

6. R ２ａ The antibody-drug conjugate of any one of claims 3 to 5, wherein is selected from -H, -F, -Br, and -Cl.

7. X is —O—, —S—, or —NH—, and R ４ａ but, The antibody-drug conjugate according to any one of claims 3 to 6, which is selected from the group consisting of:

8. D is a compound of formula (V): is a compound of the formula R ２ａ is -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ is selected from R ２０ａ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, -(C １ ~C ６ alkyl)-O-R ５ , -CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, is selected from R ５ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ６ and R ７ are each independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, -(C １ ~C ６ alkyl)-O-R ５ , -C ３ ~C ８ Heterocycloalkyl, and —C(O)R １７ is selected from R ８ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; Each R ９ are independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; Each R １０ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, and —NR １４ R １４’ is selected from Each R １０’ are independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １１ is -H and -C １ ~C ６ alkyl, R １２ is -H, -C １ ~C ６ Alkyl, —CO ２ R ８ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ , and is selected from R １３ is -H and -C １ ~C ６ alkyl, R １４ and R １４’ are each independently —H, C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １６ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １７ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, —C ３ ~C ８ heterocycloalkyl, -(C １ ~C ６ alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １８ and R １９ together with the N atom to which they are attached, halogens, -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl and -(C １ ~C ６ alkyl)-O-R ５ forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R ２４ , R ２５ , and R ２６ are -C respectively. １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; X ｃ is O, S, and S(O) ２ is selected from indicates the point of attachment to the linker L, The antibody-drug conjugate according to claim 1 or 2.

9. R ２ａ The antibody-drug conjugate of claim 8, wherein is F.

10. R ２０ａ But -H, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ , -(C １ ~C ６ alkyl)-aryl, The antibody-drug conjugate of claim 8 or 9, selected from the group consisting of:

11. D is a compound of formula (VI): is a compound of the formula R ２ａ is -H, -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ and -OCF ３ is selected from X is —O—, —S—, or —NH—, and R ２５ is -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -CO ２ R ８ａ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, wherein * is the point of attachment to X and wherein p is 1, 2, 3, or 4; or X is O and R ２５ -X- is is selected from R ５ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ６ａ is -H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R ７ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -C ３ ~C ８ Heterocycloalkyl, and —C(O)R １７ａ is selected from R ８ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; Each R ９ａ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ alkyl)-aryl; or R ９ａ does not exist and X ｂ =X, Each R １０ａ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, and is selected from Each R １０ａ’ are independently —H, —C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; Each R １０ｂ are independently -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １１ａ is not present or -C １ ~C ６ is alkyl, R １２ａ is -C １ ~C ６ Alkyl, —CO ２ R ８ａ , -aryl, -heteroaryl, -(C １ ~C ６ alkyl)-aryl, -S(O) ２ R １６ａ , and is selected from R １３ａ is -H and -C １ ~C ６ alkyl, R １４ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １４ａ’ is H, -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, and —C ３ ~C ８ heterocycloalkyl; R １６ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R １７ａ is -C １ ~C ６ Alkyl, —C ３ ~C ８ cycloalkyl, —C ３ ~C ８ heterocycloalkyl, -(C １ ~C ６ alkyl)-C ３ ~C ８ Heterocycloalkyl, -aryl, -heteroaryl, and -(C １ ~C ６ selected from: alkyl-aryl; R ２１ is -C １ ~C ６ Alkyl, —C ３ ~C ８ Cycloalkyl, and -(C １ ~C ６ alkyl)-O-R ５ａ is selected from R ２２ and R ２３ are each independently —H, -halogen, or —C １ ~C ６ Alkyl, and -C ３ ~C ８ cycloalkyl; R ２４ , R ２５ , and R ２６ are -C respectively. １ ~C ６ is alkyl, X ａ and X ｂ are each independently selected from NH, O, and S; X ｃ is O, S, and S(O) ２ is selected from indicates the point of attachment to the linker L, The antibody-drug conjugate according to claim 1 or 2.

12. R ２ａ But -CH ３ , -CF ３ , -F, -Br, -Cl, -OH, -OCH ３ , and -OCF ３ The antibody-drug conjugate of claim 11, wherein the antibody-drug conjugate is selected from the group consisting of:

13. R ２ａ The antibody-drug conjugate of claim 11, wherein is F.

14. X is —O—, —S—, or —NH—, and R ２５ But, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -(C １ ~C ６ alkyl)-aryl, Selected from or X is O and R ２５ -X- is Selected from: The conjugate according to any one of claims 11 to 13.

15. X is —O—, —S—, or —NH—, and R ２５ But, -C １ ~C ６ Alkyl, -(C １ ~C ６ alkyl)-O-R ５ａ , -(C １ ~C ６ alkyl)-aryl, The conjugate according to any one of claims 11 to 13, selected from:

16. The antibody-drug conjugate of any one of claims 1 to 15, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

17. The antibody-drug conjugate of any one of claims 1 to 15, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

18. 3. The antibody-drug conjugate of claim 1 or 2, wherein D has the structure of any one of the compounds listed in Table 5 or Table 6.

19. The antibody-drug conjugate of claim 1 or 2, wherein D is compound 139 or compound 141.

20. The antibody-drug conjugate of any one of claims 1 to 19, wherein L is a cleavable linker.

21. 21. The antibody-drug conjugate of claim 20, wherein L is a protease-cleavable linker.

22. 22. The antibody-drug conjugate of claim 20 or 21, wherein L comprises a dipeptide, tripeptide, or tetrapeptide.

23. The antibody-drug conjugate of any one of claims 20 to 22, wherein L has the following: (a) Formula (XI) During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-GPC3 antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, where AA １ - [AA ２ ] ｒ forms a protease cleavage site, X is a self-immolative group; q is 0 or 1; r is 1, 2, or 3; s is 0, 1, or 2; # is the point of attachment to the anti-GPC3 antibody construct T; and % is the point of attachment to camptothecin analog D; or (b) Formula (XII) During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-GPC3 antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, where AA １ - [AA ２ ] ｒ forms a protease cleavage site, Y is -NH-CH ２ - and q is 0 or 1; r is 1, 2, or 3; v is 0 or 1; # is the point of attachment to the anti-GPC3 antibody construct T; and % is the point of attachment to camptothecin analogue D.

24. The antibody-drug conjugate of claim 1 or 2, wherein L-(D) in formula (X) has the structure of any one of the drug-linkers (DL) listed in Tables 7 to 9.

25. The antibody-drug conjugate of claim 1 or 2, wherein L-(D) in formula (X) has the structure of any one of the drug-linkers (DL) listed in Table 7 or Table 8.

26. L-(D) in formula (X) is as follows: The antibody-drug conjugate of claim 1 or 2,

27. The antibody-drug conjugate of any one of claims 1 to 26, wherein m is 1 to 2.

28. The antibody-drug conjugate of any one of claims 1 to 26, wherein m is 1.

29. The antibody-drug conjugate of any one of claims 1 to 28, wherein n is 2 to 8.

30. The antibody-drug conjugate of any one of claims 1 to 28, wherein n is 4 to 8.

31. The antibody-drug conjugate of any one of claims 1 to 30, wherein the anti-GPC3 antibody construct further comprises a scaffold, and the antigen-binding domain is operably linked to the scaffold.

32. The antibody-drug conjugate of claim 31 , wherein the scaffold comprises an IgG Fc region.

33. The anti-GPC3 antigen-binding construct comprises: a) two heavy chains each comprising the sequence set forth in SEQ ID NO: 35 and two light chains each comprising the sequence set forth in SEQ ID NO: 36, or b) two heavy chains each comprising the sequence set forth in SEQ ID NO: 37 and two light chains each comprising the sequence set forth in SEQ ID NO: 38 The antibody-drug conjugate of any one of claims 1 to 32, comprising:

34. L-(D) in formula (X) is as follows: The antibody-drug conjugate of claim 33, wherein

35. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 34 and a pharmaceutically acceptable carrier or diluent.

36. 35. A method for inhibiting the proliferation of cancer cells, comprising contacting said cells with an effective amount of the antibody-drug conjugate of any one of claims 1 to 34.

37. 35. A method of killing cancer cells, comprising contacting said cells with an effective amount of the antibody-drug conjugate of any one of claims 1 to 34.

38. 35. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 34.

39. Use of an effective amount of the antibody-drug conjugate of any one of claims 1 to 34 for the treatment of cancer in a subject in need thereof.

40. 35. The antibody-drug conjugate of any one of claims 1 to 34 for use in the treatment of cancer.

41. Use of the antibody-drug conjugate of any one of claims 1 to 34 in the manufacture of a medicament for the treatment of cancer.

42. A kit comprising the antibody-drug conjugate of any one of claims 1 to 34 and a label and / or package insert containing instructions for use.

43. The following structure:

1. An antibody-drug conjugate having the formula: During the ceremony, n is 4 to 8, and T is an anti-GPC3 antibody construct comprising an antigen-binding domain that binds to human GPC3 (glypican-3), said antigen-binding domain comprising: a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:8; and b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 18, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17 Including, Antibody-drug conjugates.

44. 44. The antibody-drug conjugate of claim 43, wherein the antigen-binding domain comprises a VH domain having the sequence set forth in SEQ ID NO:29 and a VL domain having the sequence set forth in SEQ ID NO:

30.

45. The following structure:

1. An antibody-drug conjugate having the formula: During the ceremony, n is 1 to 10, and T is an anti-GPC3 antibody construct comprising an antigen-binding domain that binds to human GPC3 (glypican-3), said antigen-binding domain comprising: c) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:8; and d) i) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 71, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17; or ii) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 74, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17; or iii) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 77, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 17 Including, Antibody-drug conjugates.

46. The antigen-binding domain comprises: a) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29 and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 68; or b) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29 and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 64; or c) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29 and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 60 46. ​​The antibody-drug conjugate of claim 45, comprising:

47. The antigen-binding domain comprises: a) a VH domain comprising the sequence set forth in SEQ ID NO: 29 and a VL domain comprising the sequence set forth in SEQ ID NO: 68, or b) a VH domain comprising the sequence set forth in SEQ ID NO: 29 and a VL domain comprising the sequence set forth in SEQ ID NO: 64, or c) a VH domain comprising the sequence set forth in SEQ ID NO: 29 and a VL domain comprising the sequence set forth in SEQ ID NO: 60 47. The antibody-drug conjugate of claim 46, comprising:

48. The antibody-drug conjugate of any one of claims 45 to 46, wherein n is 4 to 8.

49. The antibody-drug conjugate of any one of claims 45 to 48, wherein the anti-GPC3 antibody construct further comprises a scaffold, and the antigen-binding domain is operably linked to the scaffold.

50. 50. The antibody-drug conjugate of claim 49, wherein the scaffold comprises an IgG Fc region.

51. The anti-GPC3 antigen-binding construct comprises: a) two heavy chains each comprising the sequence set forth in SEQ ID NO: 50 and two light chains each comprising the sequence set forth in SEQ ID NO: 66, or b) two heavy chains each comprising the sequence set forth in SEQ ID NO: 50 and two light chains each comprising the sequence set forth in SEQ ID NO: 62; or c) two heavy chains each comprising the sequence set forth in SEQ ID NO: 50 and two light chains each comprising the sequence set forth in SEQ ID NO: 58 The antibody-drug conjugate of any one of claims 45 to 49, comprising:

52. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 43 to 51 and a pharmaceutically acceptable carrier or diluent.

53. 52. A method for inhibiting the proliferation of cancer cells, comprising contacting said cells with an effective amount of the antibody-drug conjugate of any one of claims 43 to 51.

54. 52. A method of killing cancer cells, comprising contacting said cells with an effective amount of the antibody-drug conjugate of any one of claims 43 to 51.

55. 52. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 43 to 51.

56. 52. Use of an effective amount of the antibody-drug conjugate of any one of claims 43 to 51 for the treatment of cancer in a subject in need thereof.

57. 52. The antibody-drug conjugate of any one of claims 43 to 51 for use in the treatment of cancer.

58. Use of the antibody-drug conjugate of any one of claims 43 to 51 in the manufacture of a medicament for the treatment of cancer.

59. A kit comprising the antibody-drug conjugate of any one of claims 43 to 51 and a label and / or package insert containing instructions for use.

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