Antibody-drug conjugates targeting glypican-3 and methods of use thereof

Abstract

Described herein are antibody-drug conjugates (ADCs) comprising an antibody construct bound to human phosphatidylinositol proteoglycan 3 (GPC3) conjugated to a camptothecin analogue of formula (I). The ADCs are useful as therapeutic agents, in particular in the treatment of cancer.

Description

Technical Field

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

[0002] Glypican-3 (GPC3) is a glycosyl-phosphatidylinositol (GPI)-anchored oncofetal protein that is expressed on the surface of the placenta and fetal tissues such as the liver, lung, and kidney. GPC3 expression is downregulated or silenced in normal adult tissues but is expressed in hepatocellular carcinoma, melanoma, squamous cell lung cancer, and hepatoblastoma.

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

[0004] Some GPC3-targeting antibodies have been clinically tested in a monospecific format (i.e., as bivalent IgG). Cotara (also known as GC33 or RG-7686) has been clinically trial studied in adults with hepatocellular carcinoma (HCC) and, in combination with other therapeutic agents, has proven to have limited efficacy despite showing good safety. BMS-986183 is an antibody-drug conjugate (ADC) of the anti-GPC3 antibody BMS-986182 (also known as GPC3.1 (BMS) or 4A6 (Medarex)) conjugated to a tubulin drug moiety and has initiated a clinical trial in patients with advanced HCC, but no efficacy was observed and the trial was terminated.

[0005] Fu et al. (Hepatology. August 2019;70(2):563–576) have described an ADC of the anti-GPC3 antibody YP7 conjugated to the DNA-damaging agent duocarmycin SA and pyrrolobenzodiazepine (PBD) (see Fu et al. (2019) Hepatology, 70(2):563–576). YP7 conjugated to a PBD dimer showed potency in in vitro cancer cell models and caused tumor regression in a mouse tumor model, but the ADC has not 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 TM ), 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 TM ), 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. WO 2019 / 195665, WO 2019 / 236954, WO 2020 / 200880, and WO 2020 / 219287.

[0008] The purpose of providing this background information is to make known information that the applicant believes may be relevant to the present disclosure. It is not necessarily intended to admit, nor should it be construed as, any of the foregoing information constituting prior art against the claimed invention. SUMMARY OF THE INVENTION

[0009] Antibody-drug conjugates that target glypican-3 (GPC3) and methods of using them are described herein. One aspect of the present disclosure relates to an antibody-drug conjugate having formula (X):

[0010] T-[L-(D) m n

[0011] (X)

[0012] Wherein:

[0013] m is an integer between 1 and 4;

[0014] n is an integer between 1 and 10;

[0015] T is an anti-GPC3 (glypican-3) antibody construct that includes an antigen-binding domain that binds to human GPC3, the antigen-binding domain comprising:

[0016] ​a) A heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO: 6, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO: 7, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO: 8, and

[0017] b) A light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO: 18, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO: 17;

[0018] L is a linker, and

[0019] D is a compound of formula I:

[0020]

[0021] Wherein:

[0022] R 1 Is selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2, and

[0023] R 2 Is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3,

[0024] And wherein:

[0025] When R 1 Is -NH2, then R is R 3 Or R 4 , And when R 1 Is not -NH2, then R is R 4 ;

[0026] R 3 Is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0027] R 4 Is selected from:

[0028] R 5Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl and –(C1-C6 alkyl)-aryl;

[0029] R 6 and R 7 are each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl and -C(O)R 17 ;

[0030] R 8 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0031] Each R 9 is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0032] Each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0033] R 10’ is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0034] R 11 is selected from: -H and -C1-C6 alkyl;

[0035] R 12 is selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and

[0036] R 13 is selected from: -H and -C1-C6 alkyl;

[0037] R 14 and R 14’ are each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0038] R 16 is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0039] R 17 selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0040] R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-O-R 5 ;

[0041] R 24 、R 25 and R 26 each is -C1-C6 alkyl;

[0042] X a and X b each independently selected from: NH, O and S, and

[0043] X c selected from: O, S and S(O)2,

[0044] provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione.

[0045] Another aspect of the present disclosure relates to an antibody-drug conjugate having the following structure:

[0046]

[0047] wherein:

[0048] n is between 1 and 10, and

[0049] T is an anti-GPC3 (glypican-3) antibody construct comprising an antigen-binding domain that binds to human GPC3, said antigen-binding domain comprising:

[0050] a) a heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:8, and

[0051] b) i) A light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:71, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:17; or

[0052] ii) A light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:74, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:17; or

[0053] iii) A light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:77, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:17.

[0054] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0055] Another aspect of the present disclosure relates to a method of inhibiting cancer cell proliferation, which comprises contacting the cells with an effective amount of an antibody-drug conjugate as described herein.

[0056] Another aspect of the present disclosure relates to a method of killing cancer cells, which comprises contacting the cells with an effective amount of an antibody-drug conjugate as described herein.

[0057] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, which comprises administering to the subject an effective amount of an antibody-drug conjugate as described herein.

[0058] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in the treatment of cancer.

[0059] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.

[0060] Another aspect of the present disclosure relates to a kit, which comprises an antibody-drug conjugate as described herein and a label and / or package insert containing instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1AShows the Caliper electrophoresis results of v37575 (pertuzumab), v37574 (M3-H18L6), and v33624 (BMS-986182) under reducing (R) and non-reducing (NR) conditions. Figure 1B Shows the UPLC-SEC profiles of v37574 and v37575 (after SEC purification) and v33624 (after Protein A purification).

[0062] Figure 2 Shows the evaluation of the binding cross-reactivity of the humanized antibody M3-H1L1 (v36180) with GPC1, GPC2, GPC3, and GPC5 as evaluated by ELISA.

[0063] Figure 3A Shows the binding of v36180 (M3-H1L1), v37574 (M3-H18L6), and pertuzumab in HepG2 cells compared to the control palivizumab. Figure 3B Depicts the binding of these same antibodies in JHH-7 cells.

[0064] Figure 4A Depicts the cytotoxicity of anti-GPC3 ADC relative to a non-targeted control in GPC3-high HepG2 cells. Figure 4B Depicts the cytotoxicity of anti-GPC3 ADC relative to a non-targeted control in GPC3-medium JHH-7 cells.

[0065] Figure 5A Shows the cytotoxicity of M3-H18L6 ADC compared to a non-targeted control in GPC3-high HepG2 spheroids. Figure 5B Shows the cytotoxicity of M3-H18L6 ADC compared to a non-targeted control in GPC3-medium NCI-H446 spheroids compared to a non-targeted control.

[0066] Figure 6A Shows the cytotoxicity of M3-H18L6 ADC compared to BMS-986182 ADC and a non-targeted antibody ADC in JHH-7 cells. Figure 6B Shows the cytotoxicity of M3-H18L6 ADC compared to BMS-986182 ADC and a non-targeted antibody ADC in JHH-7 spheroid cells.

[0067] Figure 7 Depicts the stability of M3-H1L1 and BMS-986182 ADC in mouse plasma.

[0068] Figure 8Shows the pharmacokinetic (PK) profiles of the M3-H18L6 and M3-H1L1 antibodies and the ADCs of these antibodies in the Tg32 mouse model.

[0069] Figure 9A Shows a comparison of the efficacy of the ADCs of BMS-986182 and M3-H1L1 in a xenograft model derived from the JHH-7 cell line. Figure 9B Shows a comparison of the efficacy of the same ADCs in a xenograft model derived from the NCI-H446 cell line.

[0070] Figure 10A Shows the PK profile of the M3-H1L1 ADC in the NCI-H446 xenograft model. Figure 10B Shows the PK profile of the M3-H1L1 ADC in the NCI-H446 xenograft model and in a xenograft model derived from the JHH-7 cell line.

[0071] Figure 11A Shows the efficacy of the M3-H1L1 and M3-H18L6 ADCs in a xenograft model derived from the JHH-7 cell line. Figure 11B Shows the efficacy of the M3-H1L1 and M3-H18L6 ADCs in a xenograft model derived from the NCI-H446 cell line.

[0072] Figure 12A Depicts the efficacy of the M3-H18L6 ADC in the HepG2 xenograft model. Figure 12B Depicts the efficacy of the M3-H18L6 ADC in the Hep3B xenograft model. Figure 12C Depicts the efficacy of the M3-H18L6 ADC in the Huh-7 xenograft model. Figure 12D Depicts the efficacy of the M3-H18L6 ADC in the PLC / PRF / 5 xenograft model.

[0073] Figure 13A Depicts the efficacy of the M3-H18L6 ADC in a patient-derived xenograft model of LI1025. Figure 13B Depicts the efficacy of the M3-H18L6 ADC in a patient-derived xenograft model of LI1037.

[0074] Figure 14A Shows the bystander effect of the ADCs of v37574 (M3-H18L6) and v37575 (cortuximab) in co-cultures with GPC3-high HepG2 cells. Figure 14BShows the bystander effect of the ADCs of v37574 (M3-H18L6) and v37575 (trastuzumab deruxtecan) in co-cultures with GPC3-medium JHH-5 cells.

[0075] Figure 15 Shows the Membrane Proteome Array screening results of the humanized variant v38592 in HEK293T cells. TM Screening results.

[0076] Figure 16A Depicts the binding of the M3-H18L6 antibody and ADC to CHO cells transfected with human GPC3. Figure 16B Depicts the binding of the M3-H18L6 antibody and ADC to CHO cells transfected with cynomolgus monkey GPC3.

[0077] Figure 17A Depicts the binding of the M3-H18L6 antibody and ADC to HepG2 cells. Figure 17B Depicts the binding of the M3-H18L6 antibody and ADC to JHH-7 cells. Figure 17C Depicts the binding of the M3-H18L6 antibody and ADC to JHH-5 cells. Figure 17D Depicts the binding of the M3-H18L6 antibody and ADC to SNU-601 cells.

[0078] Figure 18A Depicts the in vivo efficacy of the M3-H18L6 ADC in the JHH-7 CDX model. Figure 18B Depicts the in vivo efficacy of the M3-H18L6 ADC in the Hep3B CDX model. Figure 18C Depicts the in vivo efficacy of the M3-H18L6 ADC in the JHH-5 CDX model.

[0079] Figure 19A Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI0050 PDX model of hepatocellular carcinoma (HCC). Figure 19B Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI1005 PDX model of HCC. Figure 19C Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI1069 PDX model of HCC. Figure 19D Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI1097 PDX model of HCC. Figure 19E Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI6610 PDX model of HCC. Figure 19F Depicts the in vivo efficacy of the M3-H18L6 ADC in the LI6619 PDX model of HCC.Figure 19G Depicts the in vivo efficacy of M3-H18L6 ADC in the LI6677 PDX model of HCC.

[0080] Figure 20 Depicts the pharmacokinetic (PK) profile of v38592-MC-GGFG-AM-compound 139 at DAR4.

[0081] Figure 21 Depicts the pharmacokinetic (PK) profile of v38592-MC-GGFG-AM-compound 139 at DAR8. Detailed Description

[0082] The present disclosure relates to an antibody-drug conjugate (ADC) comprising an antibody construct (anti-GPC3 antibody construct) that binds to human glypican-3 GPC3 conjugated to a camptothecin analogue of formula (I) as described herein. The ADCs of the present disclosure can be used, for example, as therapeutic agents, particularly in the treatment of cancer.

[0083] Definitions

[0084] 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.

[0085] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

[0086] When used in conjunction with the term "comprising" herein, the use of the word "a" or "an" can mean "one", but it also conforms to the meaning of "one or more", "at least one", and "one or more than one".

[0087] Where ranges of values are provided herein, for example, when a value is defined as being "between" an upper limit and a lower limit, it should be understood that the range encompasses the upper and lower limits as well as each intermediate value.

[0088] As used herein, the terms "comprising," "having," "including," and "containing" and their grammatical variants are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. When used in combination with a composition, use, or method herein, the term "consisting essentially of" means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of" when used in combination with a composition, use, or method herein does not include the presence of additional elements and / or method steps. A composition, use, or method described herein as comprising certain 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 recited.

[0089] A "complementary determining region" or "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity. The "framework" region (FR) can help maintain the correct conformation of the CDRs to facilitate binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, the variable light chain (VL) and variable heavy chain (VH) of an antibody typically contain 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. The CDRs provide most of the contact residues for the binding of an antibody to an antigen or epitope. Generally, three heavy chain CDRs and three light chain CDRs are required to bind an antigen. However, in some cases, even a single variable domain can confer antigen-binding specificity. In addition, as is known in the art, in some cases, antigen binding can also occur through a combination of at least one or more CDRs (e.g., HCDR3) selected from the VH and / or VL domains.

[0090] Many 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), as well as the definitions of IMGT, AbM (University of Bath), and Contact (MacCallum et al., 1996, J Mol Biol, 262(5):732-745). For example, the CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, it is obvious to those skilled in the art that the exact numbering and placement of CDRs can vary based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes the disclosure of relevant (inherent) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, the disclosure of VL herein includes the disclosure of relevant (inherent) light chain CDRs (LCDRs) as defined by any known numbering system.

[0091] Table 1: General CDR Definitions 1

[0092]

[0093] 1 The Kabat or Chothia numbering system can be used for HCDR2, HCDR3, and light chain CDRs of all definitions except Contact, which uses the Chothia numbering.

[0094] 2 Using the Kabat numbering. The positions in the Kabat numbering scheme that delimit the ends of the Chothia and IMGT CDR-H1 loops vary according to the length of the loop, as Kabat places the insertions outside those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be clearly defined using the Chothia numbering. CDR-H1 definition using the Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0095] In the context of two or more polynucleotide or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to obtain maximum correspondence as measured over a comparison window or over a specified region using one of the commonly used sequence comparison algorithms known to those of ordinary skill in the art or by manual alignment and visual inspection, the sequences are "substantially identical" if they have a certain percentage of identical amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95% or about 98% identity within the specified region). 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 input 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 specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0096] "Comparison window" refers to a segment of a sequence that encompasses contiguous amino acid or nucleotide positions, which can be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions, at which positions, after optimal alignment of two sequences, a test sequence can be compared to a reference sequence at the same number of contiguous positions. Sequence alignment methods for comparison are known to those of ordinary skill in the art. Optimal sequence alignments for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482; the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, (Supplement 1995), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining the 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 through the website of the National Center for Biotechnology Information (NCBI).

[0097] As used herein, the term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0098] The term "acyloxy" refers to the group -OC(O)R, where R is alkyl.

[0099] As used herein, the term "alkoxy" refers to the group -OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0100] As used herein, the term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group containing a specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.

[0101] As used herein, the term "alkylaminoaryl" refers to an alkyl group as defined herein that is substituted with an aminoaryl group as defined herein.

[0102] As used herein, the term "alkylheterocycloalkyl" refers to an alkyl group as defined herein that is substituted with a heterocycloalkyl group as defined herein.

[0103] As used herein, the term "alkylthio" refers to the group -SR, where R is an alkyl group.

[0104] As used herein, the term "amido" refers to the group -C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0105] As used herein, the term "amino" refers to the group -NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0106] As used herein, the term "aminoalkyl" refers to an alkyl group as defined herein that is substituted with one or more amino groups (e.g., one, two, or three amino groups).

[0107] As used herein, the term "aminoaryl" refers to an aryl group as defined herein that is substituted with an amino group.

[0108] As used herein, the term "aryl" 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, naphthyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, indanyl, and the like.

[0109] As used herein, the term "carboxy" refers to the group -C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0110] As used herein, the term "cyano" refers to the group -CN.

[0111] As used herein, the term "cycloalkyl" refers to a monocyclic or bicyclic saturated hydrocarbon containing a 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.

[0112] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen atoms.

[0113] As used herein, the terms "halogen" and "halo" refer to fluorine (F), bromine (Br), chlorine (Cl), and iodine (I).

[0114] As used herein, the term "heteroaryl" 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 heteroaryl include, but are not limited to: pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

[0115] As used herein, the term "heterocycloalkyl" refers to a monocyclic or bicyclic non-aromatic ring system containing a specified number of atoms and in which at least one ring atom is a heteroatom (e.g., O, S, or N). Heterocyclic substituents can be attached via any available ring atom (e.g., ring carbon or ring nitrogen). Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.

[0116] As used herein, the terms "hydroxy" and "hydroxyl" refer to the group -OH.

[0117] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein that is substituted with one or more hydroxyl groups.

[0118] As used herein, the term "nitro" refers to the group -NO2.

[0119] As used herein, the term "sulfonyl" refers to the group -S(O)2R, where R is H, alkyl, or aryl.

[0120] As used herein, the term "sulfonamido" refers to the group -NH-S(O)2R, where R is H, alkyl, or aryl.

[0121] As used herein, the terms "thiol" and "mercapto" refer to the group -SH.

[0122] Unless expressly indicated as "unsubstituted", any alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group mentioned herein should be understood as "optionally substituted", i.e., each such mention includes both the unsubstituted and substituted forms of these groups. For example, mention of "-C1-C6 alkyl" includes 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, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group mentioned herein is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azido, alkylthio, thio, sulfonyl and sulfonamido.

[0123] A chemical group described herein as "substituted" may include one substituent or multiple substituents up to the full valence of substitution of the group. For example, a methyl group may include 1, 2 or 3 substituents, and a phenyl group may include 1, 2, 3, 4 or 5 substituents. When a group is substituted with more than one substituent, the substituents may be the same or they may be different.

[0124] As used herein, the term "subject" refers to an animal, which in some embodiments is a mammal, that is the object of treatment, observation or experiment. The animal may be a human, a non-human primate, a companion animal (such as a dog, a cat, etc.), a farm animal (such as a cow, a sheep, a pig, a horse, etc.) or a laboratory animal (such as a rat, a mouse, a guinea pig, a non-human primate, etc.). In certain embodiments, the subject is a human.

[0125] It is contemplated that any embodiment discussed herein can be implemented by any method, use or composition disclosed in the present invention, and vice versa.

[0126] Specific features, structures and / or characteristics described in connection with one embodiment disclosed herein can be combined with the features, structures and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more other embodiments.

[0127] It should also be understood that an affirmative statement of a feature in one embodiment is the basis for excluding that feature in another embodiment. For example, in the case where a list of options is presented for a given embodiment or claim, it should be understood that one or more options can be removed from the list, and the shortened list can form an alternative embodiment, regardless of whether such alternative embodiment is specifically mentioned.

[0128] Antibody-drug conjugate

[0129] The present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-GPC3 antibody construct conjugated to a camptothecin analogue of formula (I). In certain embodiments, the ADC has formula (X):

[0130] T-[L-(D) m n

[0131] (X)

[0132] Wherein:

[0133] T is an anti-GPC3 antibody construct as described herein;

[0134] L is a linker;

[0135] D is a camptothecin analogue of formula (I);

[0136] m is an integer between 1 and 4, and

[0137] n is an integer between 1 and 10.

[0138] The components of formula (X) are described below.

[0139] Anti-GPC3 antibody construct, "T"

[0140] The ADCs of the present disclosure comprise an anti-GPC3 antibody construct T. In this context, the term "antibody construct" refers to a polypeptide or group of polypeptides comprising one or more antigen-binding domains, wherein each antigen-binding domain in the one or more antigen-binding domains specifically binds an epitope or antigen. In cases where the antibody construct comprises two or more antigen-binding domains, each antigen-binding domain may bind the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may also comprise a scaffold, and the one or more antigen-binding domains may be fused or covalently linked to the scaffold, optionally via a linker.

[0141] ​According to the present disclosure, an anti-GPC3 antibody construct comprises at least one antigen-binding domain that specifically binds to human GPC3 (hGPC3). The term "specifically binds" to hGPC3 means that the antibody construct binds to hGPC3 but does not exhibit significant binding to any one of 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 any one of GPC1, GPC2, or GPC5. In certain embodiments, the anti-GPC3 antibody construct of the present disclosure is capable of binding to GPC3 from one or more non-human species. In certain embodiments, the anti-GPC3 antibody construct of the present disclosure is capable of binding to cynomolgus monkey GPC3.

[0142] Human GPC3 is also referred to as "glypican 3" or "heparan sulfate proteoglycan". The protein sequences of hGPC3 from various sources are known in the art and can be readily obtained from publicly accessible databases such as GenBank or UniProtKB. Examples of hGPC3 sequences include, for example, those provided with 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).

[0143] Table 2: Human and cynomolgus monkey GPC3 protein sequences

[0144]

[0145] 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 (using, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or traditional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as, for example, a degree of binding to a non-target protein (such as GPC1, GPC2, or GPC5) measured by ELISA or flow cytometry that is less than about 10% of the binding to hGPC3.

[0146] As used herein, the term "dissociation constant (K D or K d )" is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, ligand-protein interaction refers to, but is not limited to, protein-protein interaction or antibody-antigen interaction. K D measures the tendency of two proteins (such as AB) complexed together to dissociate reversibly into their constituent components (A + B), and is defined as the ratio of the dissociation rate (also referred to as the "dissociation rate (k off )") to the association rate or "association rate (k on )". Thus, K D is equal to k off / k on , and is expressed as molar concentration (M). It can be seen that the smaller the K D , the stronger the binding affinity, and thus a decrease in K D indicates an increase in affinity. Thus, compared to a K D of 1 nM, a K D of 1 mM indicates a weak binding affinity. Affinity is sometimes measured as K A or K a , which is the reciprocal of K D or K d . The K D value of an antibody construct can be determined using well-established methods in the art. One method for determining the K D of an antibody construct is by using surface plasmon resonance (SPR), typically using a biosensor system such as system. Isothermal titration calorimetry (ITC) is another method that can be used to measure K D . The Octet TM system can also be used to measure the affinity of an antibody for a target antigen.

[0147] In certain embodiments, specific binding of an antibody construct to GPC3 can be characterized by the dissociation constant (KD ) ≤ 1 μΜ, for example ≤ 500 nM, ≤ 250 nM, ≤ 100 nM, ≤ 50 nM or ≤ 10 nM. In certain embodiments, specific binding of an antibody construct to a particular antigen or epitope can be determined by the dissociation constant (K D ) of 10 -6 M or less, for example 10 -7 M or less or 10 -8 M or less. In some embodiments, specific binding of an antibody construct to a particular antigen or epitope can be determined by a dissociation constant (K D ) between 10 -6 M and 10 -9 M, for example between 10 -7 M and 10 -9 M.

[0148] In some embodiments, as measured by SPR, the antigen-binding domain of an anti-GPC3 antibody construct binds to human GPC3 with a K D higher than that of the reference antibody, trastuzumab. Thus, in these embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain with a lower affinity for human GPC3 than the affinity of the reference antibody, trastuzumab.

[0149] The anti-GPC3 antibody construct is internalized by cells expressing GPC3. Antibody internalization can be measured using methods known in the art, for example, by direct internalization assays according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57: 1879-1890, or using commercially available fluorescent dyes such as pHAb dye (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red dye (ThermoFisher Scientific Corporation, Waltham, MA), and Fabfluor-pH antibody labeling reagent (Sartorius AG, Germany), and analytical techniques such as microscopy, FACS, high-content imaging or other plate-based assays.

[0150] In some embodiments, the anti-GPC3 antibody construct is internalized to a similar extent as the reference antibody, trastuzumab, in cells that highly express GPC3 (e.g., in HepG2 cells or JHH-7 cells). In some embodiments, the amount of internalized antibody is measured after an incubation period of at least 5 hours. In some embodiments, the conjugation of the anti-GPC3 antibody construct with a camptothecin analogue does not affect the internalization of the anti-GPC3 antibody construct.

[0151] GPC3 expression varies according to the cell type indicated throughout the present disclosure, and the level of GPC3 expression is sometimes referred to herein as "high", "medium", "low", or "negative". These terms are used for reference to describe the general GPC3 expression level according to Table 12.1 in Example 12, and are not intended to be limited to the specific numerical values of the average GPC3 of each cell included therein. Alternatively, the expression level of GPC3 in a cell or tumor can be evaluated 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 line-derived (CDX), or patient-derived (PDX) sources. Tissue samples can be examined, and the H score can be calculated as known in the art and as described, for example, in Example 33 herein. The higher the H score, the higher the expression of GPC3 in the tissue sample.

[0152] Antigen-binding domain

[0153] 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 generally 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 Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAb).

[0154] A "Fab fragment" contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab′ fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are linked by a peptide linker. For example, the C-terminus of the Fab light chain can be linked to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0155] "scFv" contains the variable heavy domain (VH) and the variable light domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further contain a polypeptide linker between the VH and VL domains, enabling the scFv to form the structure required for antigen binding. For example, the scFv may include a VL that is connected from its C-terminus to the N-terminus of the VH via a polypeptide linker. Alternatively, the scFv may contain a VH that is connected to the N-terminus of the VL via its C-terminus by a polypeptide linker (see the review by Pluckthun in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269 - 315 (1994)).

[0156] The "sdAb" format refers to a single immunoglobulin domain. The sdAb can be, for example, of camel origin. Camel antibodies lack a light chain, and their antigen-binding site consists of a single domain called "VHH". The sdAb contains three CDRs / highly variable loops CDR1, CDR2, and CDR3 that form the antigen-binding site. The sdAb is quite stable and easy to express, for example, expressed as a fusion with the antibody Fc chain (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13 - 22).

[0157] In those embodiments where the anti-GPC3 antibody construct of the ADC contains two or more antigen-binding domains, each additional antigen-binding domain can independently be an immunoglobulin-based domain (such as an antigen-binding antibody fragment) or a non-immunoglobulin-based domain (such as a non-immunoglobulin antibody mimic), or other polypeptides or small molecules capable of specifically binding to its target (e.g., a natural or engineered ligand). Non-immunoglobulin antibody mimic forms include, for example, anticalin, fynomer, affimer, alphabody, DARPin, and avimer.

[0158] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure contains at least one antigen-binding domain that specifically binds to hGPC3, wherein the antigen-binding domain is derived from the MAb clone M3 described in WO2021 / 226321. Thus, in certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure contains a set of HCDRs and a set of LCDRs identified according to IMGT, Kabat, Chothia AbM, or Contact numbering, as shown in Table 3 below.

[0159] Table 3: CDR Amino Acid Sequences of MAb Clone M3

[0160]

[0161] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising the 3 HCDR amino acid sequences and 3 LCDR amino acid sequences of v36180 (M3-H1L1) or v37574 (M3-H18L6), as defined by the IMGT, Kabat, Chothia, or AbM numbering systems.

[0162] In certain 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 3 HCDR amino acid sequences of v36180 (M3-H1L1) and a VL amino acid sequence comprising the 3 LCDR amino acid sequences of v36180 (M3-H1L1). In certain 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 3 HCDR amino acid sequences of v37574 (M3-H18L6) and a VL amino acid sequence comprising the 3 LCDR amino acid sequences of v37574 (M3-H18L6).

[0163] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NO: 6, 7, and 8 and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NO: 18, 19, and 17, as defined by Kabat numbering.

[0164] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NO: 3, 4, and 5 and a light chain CDR amino acid sequence (LCDR1 and LCDR3) comprising the sequences shown in SEQ ID NO: 16 and 17 and an LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0165] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 9, 10, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 18, 19, and 17, as defined by Chothia numbering.

[0166] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 13, 14, and 15, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 20, 21, and 22, as defined by Contact numbering.

[0167] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 11, 12, and 8, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 18, 19, and 17, as defined by AbM numbering.

[0168] 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 the antibody losing its ability to bind its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art such as alanine scanning, and the binding activity of the resulting variants can be tested by standard techniques. Thus, in certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain comprising a set of CDRs having 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity to a set of CDRs of v36180 (M3-H1L1) or v37574 (M3-H18L6) (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3), wherein the % sequence identity is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0169] 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 as shown in any one of Table 3A, Table 3B, or Table 3C below:

[0170] Table 3A: CDR Amino Acid Sequences of Light Chain Modified Variants of MAb Clone M3 (v40206, G34R)

[0171]

[0172] Table 3B: CDR Amino Acid Sequences of Light Chain Modified Variants of MAb Clone M3 (v40207, G34K)

[0173]

[0174] Table 3C: CDR Amino Acid Sequences of Light Chain (LC) Modified Variants of MAb Clone M3 (v40208, G34Q)

[0175]

[0176] In certain 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 3 HCDR amino acid sequences of LC modified variant 40206 and a VL amino acid sequence comprising the 3 LCDR amino acid sequences of v40206, as defined by one of IMGT, Kabat, Chothia, AbM, or Contact numbering. In certain 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 3 HCDR amino acid sequences of LC modified variant 40207 and a VL amino acid sequence comprising the 3 LCDR amino acid sequences of LC modified variant 40207, as defined by one of IMGT, Kabat, Chothia, AbM, or Contact numbering. In 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 3 HCDR amino acid sequences of LC modified variant 40208 and a VL amino acid sequence comprising the 3 LCDR amino acid sequences of LC modified variant 40208, as defined by one of IMGT, Kabat, Chothia, AbM, or Contact numbering.

[0177] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40206, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 6, 7, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 71, 19, and 17, as defined by Kabat numbering.

[0178] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40206, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 3, 4, and 5 and light chain CDR amino acid sequences (LCDR1 and LCDR3) and LCDR2 amino acid sequence KVS comprising the sequences shown in SEQ ID NOs: 70 and 17, as defined by IMGT numbering.

[0179] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40206, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 9, 10, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 71, 19, and 17, as defined by Chothia numbering.

[0180] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40206, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 13, 14, and 15 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 72, 21, and 22, as defined by Contact numbering.

[0181] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40206, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 11, 12, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 71, 19, and 17, as defined by AbM numbering.

[0182] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40207, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 6, 7, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 74, 19, and 17, as defined by Kabat numbering.

[0183] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40207, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 3, 4, and 5 and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences shown in SEQ ID NOs: 73 and 17 and the LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0184] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC modified variant 40207, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 9, 10, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 74, 19, and 17, as defined by Chothia numbering.

[0185] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC-modified variant 40207, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 13, 14, and 15 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 75, 21, and 22, as defined by Contact numbering.

[0186] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC-modified variant 40207, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 11, 12, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 74, 19, and 17, as defined by AbM numbering.

[0187] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC-modified variant 40208, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 6, 7, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 77, 19, and 17, as defined by Kabat numbering.

[0188] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises the antigen-binding domain of LC-modified variant 40208, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 3, 4, and 5 and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences shown in SEQ ID NOs: 76 and 17 and LCDR2 amino acid sequence KVS, as defined by IMGT numbering.

[0189] 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, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 9, 10, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 77, 19, and 17, as defined by Chothia numbering.

[0190] 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, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 13, 14, and 15 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 78, 21, and 22, as defined by Contact numbering.

[0191] 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, which has heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences shown in SEQ ID NOs: 11, 12, and 8 and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences shown in SEQ ID NOs: 77, 19, and 17, as defined by AbM numbering.

[0192] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain that comprises a VH 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% identity to the VH amino acid sequence of v36180 (M3-H1L1), and 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% identity to the VL amino acid sequence of v36180 (M3-H1L1), wherein the antigen-binding domain retains the ability to bind hGPC3.

[0193] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises three HCDRs having v36180 (M3-H1L1) and a VH 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% identity to the VH amino acid sequence of v36180 (M3-H1L1), and three LCDRs having v36180 (M3-H1L1) and 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% identity to the VL amino acid sequence of v36180 (M3-H1L1), wherein the three HCDRs and the three LCDRs are defined by the IMGT, Kabat, Chothia or AbM numbering systems, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0194] In other embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises a VH 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% identity to the VH amino acid sequence of v37574 (M3-H18L6), and 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% identity to the VL amino acid sequence of v37574 (M3-H18L6), and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0195] In other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain that comprises 3 HCDRs having v37574 (M3-H18L6) and a VH 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 VH amino acid sequence of v37574 (M3-H18L6), and 3 LCDRs having v37574 (M3-H18L6) and 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 hGPC3.

[0196] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain that comprises a VH 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% identity to the VH amino acid sequence of LC modification variant 40206, and 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% identity to the VL amino acid sequence of LC modification variant 40206, wherein the antigen-binding domain retains the ability to bind hGPC3.

[0197] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain that comprises 3 HCDRs having the LC modification variant 40206 and a VH 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% identity to the VH amino acid sequence of the LC modification variant 40206, and 3 LCDRs having the LC modification variant 40206 and 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% identity to the VL amino acid sequence of the LC modification variant 40206, wherein the 3 HCDRs and the 3 LCDRs are defined by the IMGT, Kabat, Chothia or AbM numbering system, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0198] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain that comprises a VH 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% identity to the VH amino acid sequence of the LC modification variant 40207, and 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% identity to the VL amino acid sequence of the LC modification variant 40207, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0199] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises three HCDRs having LC modification variant 40207 and a VH 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% identity to the VH amino acid sequence of LC modification variant 40207, and three LCDRs having LC modification variant 40207 and 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% identity to the VL amino acid sequence of LC modification variant 40207, wherein the three HCDRs and the three LCDRs are defined by the IMGT, Kabat, Chothia or AbM numbering systems, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0200] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises a VH 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% identity to the VH amino acid sequence of LC modification variant 40208, and 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% identity to the VL amino acid sequence of LC modification variant 40208, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0201] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises three HCDRs with an LC-modified variant 40208 and a VH sequence that has 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% identity to the VH amino acid sequence of the LC-modified variant 40208, and three LCDRs with the LC-modified variant 40208 and a VL sequence that has 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% identity to the VL amino acid sequence of the LC-modified variant 40208, wherein the three HCDRs and the three LCDRs are defined by the IMGT, Kabat, Chothia or AbM numbering systems, and wherein the antigen-binding domain retains the ability to bind hGPC3.

[0202] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises a VH amino acid sequence that has 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 shown in SEQ ID NO:27, and a VL amino acid sequence that has 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 shown in SEQ ID NO:28.

[0203] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that comprises a VH amino acid sequence that has 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 shown in SEQ ID NO:29, and a VL amino acid sequence that has 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 shown in SEQ ID NO:30.

[0204] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that 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 shown in SEQ ID NO:29, and 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 shown in SEQ ID NO:68.

[0205] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that 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 shown in SEQ ID NO:29, and 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 shown in SEQ ID NO:64.

[0206] In certain embodiments, the anti-GPC3 antibody construct of the ADCs of the present disclosure comprises an antigen-binding domain that 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 shown in SEQ ID NO:29, and 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 shown in SEQ ID NO:60.

[0207] In certain embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising (i) a VH amino acid sequence as shown in SEQ ID NO: 27 and a VL amino acid sequence as shown in SEQ ID NO: 28, or (ii) a VH amino acid sequence as shown in SEQ ID NO: 29 and a VL amino acid sequence as shown 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 a VH amino acid sequence as shown in SEQ ID NO: 29 and a VL amino acid sequence as shown 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 a VH amino acid sequence as shown in SEQ ID NO: 29 and a VL amino acid sequence as shown in SEQ ID NO: 64. In other embodiments, the anti-GPC3 antibody construct of the ADC of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence as shown in SEQ ID NO: 29 and a VL amino acid sequence as shown in SEQ ID NO: 60.

[0208] Exemplary VH and VL sequences are provided in the Examples and the Sequence Listing.

[0209] In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:50 and two light chains containing the sequence shown in SEQ ID NO:53 (v37574 M3-H18L6). In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:56 and two light chains containing the sequence shown in SEQ ID NO:53 (v38592). In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:44 and two light chains containing the sequence shown in SEQ ID NO:47 (v36180 M3-H1L1). In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:50 and two light chains containing the sequence shown in SEQ ID NO:66 (v40206). In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:50 and two light chains containing the sequence shown in SEQ ID NO:62 (v40207). In one embodiment, the anti-GPC3 construct of the ADC of the present disclosure comprises two heavy chains containing the sequence shown in SEQ ID NO:50 and two light chains containing the sequence shown in SEQ ID NO:58 (v40208).

[0210] Format

[0211] The anti-GPC3 antibody construct of the ADC can have various formats. The minimal component of the anti-GPC3 antibody construct is the antigen-binding domain that binds hGPC3. The anti-GPC3 antibody construct may also optionally 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 the same epitope within hGPC3, may bind different epitopes within hGPC3, or may bind a different antigen. Thus, the anti-GPC3 antibody construct can be, for example, monospecific, bispecific, bispecific, or multispecific.

[0212] In certain embodiments, the anti-GPC3 antibody construct comprises at least one antigen-binding domain that binds 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 permits them to function in their intended manner. Suitable scaffolds are described below.

[0213] In certain embodiments, the anti-GPC3 antibody construct comprises two antigen-binding domains optionally 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 domains may each independently be operably linked to the scaffold or the first antigen-binding domain, or when there are more than two antigen-binding domains, to another antigen-binding domain.

[0214] An anti-GPC3 antibody construct lacking a scaffold may comprise a single antigen-binding domain in a suitable format, such as an sdAb, or they may comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such anti-GPC3 antibody constructs, the antigen-binding domains may be in the format of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as the antigen-binding domain, formats such as tandem scFv ((scFv)2 or taFv) can be constructed, where the scFvs are linked together by a flexible linker. ScFv can also be used to construct diabody formats, which comprise two scFvs linked by a short linker (usually about 5 amino acids in length). The restricted linker length results in dimerization of the scFvs in a head-to-tail manner. In any of the foregoing formats, the scFv can be further stabilized by including interdomain disulfide bonds. For example, a disulfide bond can be introduced between VL and VH by introducing additional cysteine residues in each chain (e.g., at position 44 in VH and position 100 in VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond can 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).

[0215] Similarly, in some embodiments, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable linker can be employed. Other examples of the anti-GPC3 antibody construct lacking a scaffold form include those based on Fab fragments, such as Fab2 and F(ab’)2 formats, where the Fab fragments are linked by a linker or an IgG hinge region.

[0216] Combinations of antigen-binding domains of different sizes can also be employed to generate alternative scaffold-free formats. For example, scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of a Fab fragment, thereby generating a bivalent (Fab-scFv / sdAb) construct.

[0217] In certain embodiments, the anti-GPC3 antibody construct can be an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as a full-size antibody format (FSA) or Mab format and includes an anti-GPC3 antibody construct containing two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-GPC3 antibody construct can be based on IgG class immunoglobulins, such as IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the anti-GPC3 antibody construct can be based on IgG1 immunoglobulin. In the context of the present disclosure, when the anti-GPC3 antibody construct is based on a specified immunoglobulin isotype, it means that the anti-GPC3 antibody construct contains all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-GPC3 antibody construct based on a given Ig isotype can contain at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold contains 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 can also contain hybrids of isotypes and / or subclasses. It should also be understood that the Fc region and / or hinge region can optionally be modified to confer one or more desired functional properties known in the art. Thus, in certain embodiments, the anti-GPC3 antibody construct contains a VH amino acid sequence fused to an IgG1 constant domain amino acid sequence (i.e., CH1, CH2, CH3 amino acid sequences) and a VL amino acid sequence fused to a κ or λ constant amino acid sequence domain (i.e., CL amino acid sequence). Exemplary amino acid sequences are provided in the Examples and Sequence Listing.

[0218] In some embodiments, the anti-GPC3 antibody construct can be derived from two or more immunoglobulins from different species. For example, the anti-GPC3 antibody construct can 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 more than one species.

[0219] A "chimeric antibody" generally contains 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 have 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. Patent No. 4,816,567.

[0220] A "humanized antibody" is a class of chimeric antibodies that contain the minimal sequence derived from non-human antibodies. Typically, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the hypervariable regions from the recipient are replaced with the residues of the hypervariable regions from a non-human species (donor antibody) that have the desired specificity and affinity for the target antigen, such non-human species as mice, rats, rabbits or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR grafting".

[0221] In some cases, additional modifications are made to further improve antibody performance. For example, residues in the framework regions (FRs) of the human immunoglobulin are replaced with the corresponding non-human residues, or the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. In general, the variable domains in a humanized antibody will contain all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more 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.

[0222] Many methods are known in the art for selecting the most appropriate human framework in which to graft non-human CDRs. Early methods used a limited subset of well-characterized human antibodies, regardless of sequence identity to the non-human antibody providing the CDRs ("fixed framework" methods). More recent methods have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs ("homology matching" or "best fit" methods). An alternative approach is to select fragments of framework sequences from within each light or heavy chain variable region from several different human antibodies. In some cases, CDR grafting may result in partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some human-derived residues to the corresponding non-human residues. Methods for preparing humanized antibodies by these methods are well known in the art (see, e.g., 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).

[0223] Alternatively, or in addition to these traditional methods, newer techniques can be employed to further reduce the immunogenicity of humanized antibodies for CDR grafting. For example, frameworks based on germline or consensus sequences can be used as the acceptor human framework instead of a human framework with somatic mutations. Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only the specific determining residues (SDRs). In this method, only the minimum number of CDR residues required for antigen-binding activity ("SDRs") are grafted into the germline framework. This method improves the "humanity" of the humanized antibody (i.e., similarity to the germline sequence) and thus may help reduce the immunogenicity risk of the variable region. These techniques have been described in various publications (see, e.g., Almagro and 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).

[0224] scaffold

[0225] In certain embodiments, the anti-GPC3 antibody construct of the ADC comprises one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain can be one or a combination of the forms described above (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, heterodimerizing peptides (such as leucine zippers, "zipper" peptides derived from Jun and Fos that form heterodimers, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK TM (DNL TM ) technology (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0226] The scaffold can be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. When the scaffold is a polypeptide, each antigen-binding domain of the anti-GPC3 antibody construct can be linked to the N-terminus or C-terminus of the polypeptide scaffold. Also contemplated in certain embodiments are anti-GPC3 antibody constructs comprising a polypeptide scaffold, wherein one or more antigen-binding polypeptide constructs are linked to a region other than the N-terminus or C-terminus, with or without a linker, for example via a side chain of an amino acid.

[0227] In embodiments where the anti-GPC3 antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain can be linked to the scaffold by genetic fusion or chemical conjugation. Generally, when the scaffold is a peptide or polypeptide, the antigen-binding domain is linked to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen-binding domain can be linked to the scaffold by chemical conjugation.

[0228] Many protein domains are known in the art that involve the selective pairing of two different polypeptides and can be used to form scaffolds. Examples are leucine zipper domains that pair selectively, such as Fos and Jun (Kostelny et al., J Immunol, 148:1547-53 (1992); Wranik et al., J. Biol. Chem., 287:43331-43339 (2012)). Other selectively paired molecular pairs include, for example, the barnase-barstar 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. WO 2011 / 135040).

[0229] Other examples of protein scaffolds include the immunoglobulin Fc region, 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, Müller 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 US Patent Application Publication No. 2009 / 0285816).

[0230] For example, it has been demonstrated that fusing an antigen-binding moiety such as an scFv, diabody or single-chain diabody to albumin can improve the serum half-life of the antigen-binding moiety (Müller et al., supra). The antigen-binding moiety can optionally be fused at the N-terminus and / or C-terminus of albumin via a linker.

[0231] Heteromeric forms of albumin derivatives have been described that contain two transport polypeptide chains obtained by albumin fragmentation such that the transport polypeptide chains self-assemble to form a native-like albumin (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). Due to albumin fragmentation, the heteromer includes four termini and can thus optionally be fused to up to four different antigen-binding moieties via linkers.

[0232] In certain embodiments, the anti-GPC3 antibody construct of the ADC can comprise a protein scaffold. In some embodiments, the anti-GPC3 antibody construct can comprise a protein scaffold based on an immunoglobulin Fc region, albumin or an albumin analogue or derivative. In some embodiments, the anti-GPC3 antibody construct can comprise a protein scaffold based on an immunoglobulin Fc region (e.g., an IgG Fc region).

[0233] Fc region

[0234] As used herein, the terms “Fc region,” “Fc,” or “Fc domain” refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in the 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).

[0235] In certain embodiments, the anti-GPC3 antibody construct of the ADC can comprise a scaffold based on an immunoglobulin Fc region. The Fc region can be dimeric and composed of two Fc polypeptides, or alternatively, the Fc region can be composed of a single polypeptide.

[0236] In the case of a dimeric Fc, "Fc polypeptide" 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 a first Fc polypeptide and a second Fc polypeptide.

[0237] The Fc region may comprise a CH3 domain or it may comprise both CH3 and CH2 domains. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise IgG CH2 domain sequences and IgG CH3 domain sequences. In such embodiments, the CH3 domain comprises two CH3 sequences, i.e., one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, i.e., one from each of the two Fc polypeptides of the dimeric Fc region.

[0238] In some embodiments, the anti-GPC3 antibody construct of the ADC may comprise a scaffold based on an IgG Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on a human IgG Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on a human IgG1 Fc region.

[0239] In certain embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on an IgG Fc region that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each of the first Fc polypeptide and the second Fc polypeptide comprising a CH3 sequence and optionally a CH2 sequence, and wherein the first Fc polypeptide and the second Fc polypeptide are different. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on an Fc region that comprises two CH3 sequences, wherein at least one of the CH3 sequences comprises one or more amino acid modifications. In some embodiments, the anti-GPC3 antibody construct may comprise a scaffold based on an Fc region that comprises two CH3 sequences and two CH2 sequences, wherein at least one of the CH2 sequences comprises one or more amino acid modifications.

[0240] In some embodiments, an anti-GPC3 antibody construct can comprise a heterodimeric Fc region containing a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification such as a substitution or insertion, wherein the amino acid at a particular position on a first CH3 or CH2 sequence is different from the amino acid at the same position on a second CH3 or CH2 sequence. These asymmetric amino acid modifications can be the result of modification of only one of two amino acids at the same corresponding amino acid position on each sequence, or the result of different modifications of each of the two amino acids at the same corresponding position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc can comprise one or more than one asymmetric amino acid modification.

[0241] In some embodiments, an anti-GPC3 antibody construct can comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote heterodimeric Fc formation relative to homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0242] Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("staple - socket"), 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 methods that combine positive and negative design strategies to produce stable asymmetrically modified Fc regions, as described in International Publications WO 2012 / 058768 and WO2013 / 063702. In certain embodiments, an anti - GPC3 antibody construct can comprise a scaffold based on a modified Fc region, as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0243] 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 contains amino acids 341 - 447 of the full-length human IgG1 heavy chain. Also shown in Table 4 are amino acid modifications in the CH3 domain that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012 / 058768 and WO2013 / 063702.

[0244] In certain embodiments, the anti-GPC3 antibody construct can comprise a heterodimeric Fc scaffold having a modified CH3 domain, the modified CH3 domain comprising the modification of any one of Variant 1, Variant 2, Variant 3, Variant 4, or Variant 5, as shown in Table 4.

[0245] Table 4: Amino acid modifications in the human IgG1 Fc sequence 1 and CH3 domain that promote heterodimer formation

[0246]

[0247]

[0248] 1 Sequence (EU number) at positions 231 - 447

[0249] In some embodiments, the anti-GPC3 antibody construct can comprise an Fc region-based scaffold that includes two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0250] In some embodiments, the anti-GPC3 antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in an altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0251] A variety of amino acid modifications that selectively alter the affinity of Fc for different Fcγ receptors in the CH2 domain are known in the art. Amino acid modifications that result in increased binding and those that result in decreased binding can each be used for certain indications. For example, increasing the binding affinity of Fc for FcγRIIIa, an activating receptor, can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn causes an increase in the lysis of target cells. Decreasing the binding to FcγRIIb, an inhibitory receptor, may also be beneficial in some cases. In certain indications, it may be desirable to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, modified CH2 domains ("knockout" variants) containing amino acid modifications that result in increased binding to FcγRIIb or that reduce or eliminate the binding of the Fc region to all Fcγ receptors may be useful.

[0252] Examples of amino acid modifications to the CH2 domain that alter the binding of Fcγ receptors to Fc include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein EngDes Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter the binding of FcγRIIb to Fc are described in International Publication No. WO 2021 / 232162. Other modifications that affect the binding of Fc 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).

[0253] In certain embodiments, the anti-GPC3 antibody construct comprises a scaffold based on an IgG Fc with a modified CH2 domain, wherein the modified CH2 domain comprises one or more amino acid modifications that result in reduced or eliminated binding of the Fc region to all Fcγ receptors (i.e., a “knockout” variant).

[0254] A variety of publications have described strategies that have been used to engineer antibodies to produce “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 reducing effector function by glycosylation modification, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 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).

[0255] Examples of mutations that can be introduced into the hinge or CH2 domain to produce a “knockout” variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0256] In certain embodiments, the anti-GPC3 antibody constructs described herein may comprise a scaffold based on an IgG Fc, wherein the native glycosylation has been modified. As is known in the art, the glycosylation of the 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 non-glycosylated Fc that lacks all effector functions (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0257] In contrast, removal of fucose from the N297-linked oligosaccharides of the heavy chain has been shown to enhance ADCC based on 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 afucosylated 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 variant CHO cell line Lec 13 with reduced ability to link fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or in other cells that produce afucosylated 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. WO 2009 / 135181 describes the addition of fucose analogs to the culture medium during antibody production to inhibit fucose incorporation into the carbohydrates on the antibody.

[0258] Other methods for generating antibodies with little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, techniques (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0259] Other glycosylation variants include those with bisecting oligosaccharides, e.g., variants in which the biantennary oligosaccharide linked to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants can have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO 2003 / 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 linked to the Fc region, which can have improved CDC function (see, e.g., International Publication No. WO 1997 / 030087, WO 1998 / 58964, and WO 1999 / 22764).

[0260] Preparation of anti-GPC3 antibody constructs

[0261] The anti-GPC3 antibody constructs described herein can be generated using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and "Antibodies: A Laboratory Manual", 2nd Edition, edited by Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0262] Generally, for the recombinant production of antibody constructs, a polynucleotide or a 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 encoding the anti-GPC3 antibody construct can be generated by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and updates, and "Antibodies: A Laboratory Manual", 2nd Edition, edited by Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be understood by those skilled in the art, the number of polynucleotides required to express the anti-GPC3 antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, when the anti-GPC3 antibody construct is in a monospecific mAb or FSA format, two polynucleotides will be required, one polynucleotide encoding the light chain polypeptide and one polynucleotide encoding the heavy chain polypeptide. When multiple polynucleotides are required, they can be incorporated into one vector or more than one vector.

[0263] Typically, for expression, a polynucleotide or a set of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the choice of regulatory elements depends on the host cell selected for expressing the antibody construct, and such regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate the 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 can be an extrachromosomal vector or an integrating vector.

[0264] Suitable host cells for cloning or expressing anti-GPC3 antibody constructs include a variety of 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 or Pichia cells). Prokaryotic host cells include, for example, Escherichia coli (E. coli), Aeromonas salmonicida, or Bacillus subtilis cells.

[0265] In certain embodiments, anti-GPC3 antibody constructs can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, edited by B.K.C. Lo, Humana Press, Totowa, N.J., 2003.

[0266] In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast can be suitable expression host cells, especially fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibody constructs with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0267] Suitable host cells for expressing glycosylated anti-GPC3 antibody constructs are typically 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 PLANTIBODIES for generating antigen-binding constructs in transgenic plants. TM Mammalian cell lines suitable for growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (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, dog kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals N.Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR - 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 generating antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (edited by B.K.C. Lo, Humana Press, Totowa, N.J., 2003).

[0268] In certain embodiments, the host cell can be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell can be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any of these cell lines. In some embodiments, the host cell can be a stable cell line that permits maturation glycosylation of the antibody construct.

[0269] Host cells comprising an expression vector encoding an anti-GPC3 antibody construct can be cultured using conventional methods to produce the anti-GPC3 antibody construct. Alternatively, in some embodiments, host cells comprising an expression vector encoding an anti-GPC3 antibody construct can be used therapeutically or prophylactically to deliver the anti-GPC3 antibody construct to a subject, or a polynucleotide or expression vector can be administered ex vivo to cells from a subject and then the cells returned to the subject.

[0270] Typically, the anti-GPC3 antibody construct is purified after expression. Proteins can be isolated or purified in a variety of ways known to those of skill in the art (see, e.g., 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, size exclusion chromatography or gel filtration, and reverse phase chromatography, using systems such as FPLC and HPLC at atmospheric or high pressure. Additional purification methods include electrophoresis, immunoprecipitation, dialysis, and chromatofocusing techniques. The combination of ultrafiltration and diafiltration techniques with protein concentration is also useful. As is well known in the art, a variety of native proteins bind Fc and antibodies, and these proteins can be used to purify certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be carried out by specific fusion partners. For example, if a GST fusion is employed, the antibody can be purified using glutathione resin, if a His tag is used, Ni +2 affinity chromatography can be used to purify the antibody, or if a flag tag is used, immobilized anti-flag antibody can be used to purify the antibody. The degree of purification required will vary depending on the use of the anti-GPC3 antibody construct. In some cases, purification may not be necessary.

[0271] In certain embodiments, the anti-GPC3 antibody construct is substantially pure. As used herein with respect to the anti-GPC3 antibody constructs described herein, the term "substantially pure" (or "substantially purified") means that the antibody construct is substantially or essentially free of components that normally accompany or interact with a protein as found in its natural environment (such as a natural cell, or in the case of a recombinantly produced construct, the host cell). 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.

[0272] Certain embodiments of the present disclosure relate to a method for preparing an anti-GPC3 antibody construct, the method comprising culturing a host cell into which one or more polynucleotides encoding the anti-GPC3 antibody construct or one or more expression vectors encoding the anti-GPC3 antibody construct have been introduced under conditions suitable for expressing the anti-GPC3 antibody construct, and optionally recovering the anti-GPC3 antibody construct from the host cell (or from the host cell culture medium).

[0273] Post-translational modification

[0274] In certain embodiments, the anti-GPC3 antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after the anti-GPC3 antibody construct has been isolated from the host cell.

[0275] Post-translational modifications include various modifications known in the art (see, e.g., Proteins - Structure and Molecular Properties, 2nd ed., T.E. Creighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, edited by B.C. Johnson, Academic Press, New York, pp. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N.Y. Acad. Sci., 663:48-62). In those embodiments in which the anti-GPC3 antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0276] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH4.

[0277] Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked carbohydrate chains, the chemical modification of N-linked or O-linked carbohydrate chains, the processing of the N-terminus or C-terminus, the attachment of chemical moieties to the amino acid backbone, and the addition or deletion of an N-terminal methionine residue produced by expression in a prokaryotic host cell. Post-translational modifications can also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to allow detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable cofactor 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.

[0278] Additional examples of post-translational modifications include acetylation, 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 a covalent crosslinking body, formation of cysteine, formation of pyroglutamate, γ-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, tetradecylation, polyethylene glycolylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to a protein such as arginylation and ubiquitination.

[0279] Camptothecin analog

[0280] The camptothecin analog contained in the ADC of the present disclosure is a compound having formula (I):

[0281]

[0282] Wherein:

[0283] R 1 is selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3, and -NH2, and

[0284] R 2 is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3,

[0285] And wherein:

[0286] When R 1 is -NH2, then R is R 3 or R 4 , and when R 1 is not -NH2, then R is R 4 ;

[0287] R 3 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0288] R 4 is selected from:

[0289] R 5 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl and –(C1-C6 alkyl)-aryl;

[0290] R 6 and R 7 are each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl and -C(O)R 17 ;

[0291] R 8 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0292] Each R 9 is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0293] Each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0294] R 10’ is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0295] R11 Selected from: -H and -C1-C6 alkyl;

[0296] R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and

[0297] R 13 Selected from: -H and -C1-C6 alkyl;

[0298] R 14 and R 14’ each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0299] R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0300] R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0301] R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-O-R 5 ;

[0302] R 24 、R 25 and R 26 each is -C1-C6 alkyl;

[0303] X a and X b each independently selected from: NH, O and S, and

[0304] X c selected from: O, S and S(O)2,

[0305] provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione.

[0306] In some embodiments, the camptothecin analog is a compound of formula (I), provided that when R 1 is NH2, R 2 is not H.

[0307] In some embodiments, in the compound of formula (I), R 1 is selected from: -CH3, -CF3, -OCH3, -OCF3, and NH2.

[0308] In some embodiments, in the compound of formula (I), R 1 is NH2.

[0309] In some embodiments, in the compound of formula (I), R 1 is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0310] In some embodiments, in the compound of formula (I), R 1 is selected from: -CH3, -CF3, -OCH3, and -OCF3.

[0311] In some embodiments, in the compound of formula (I), R 2 is selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0312] In some embodiments, in the compound of formula (I), R 2 is selected from: -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0313] In some embodiments, in the compound of formula (I), R 2 is selected from: -H, -F, -Br, and -Cl.

[0314] In some embodiments, in the compound of formula (I), R 2 is selected from: -F, -Br, and -Cl.

[0315] In some embodiments, in the compound of formula (I), R 3 is selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0316] In some embodiments, in the compound of formula (I), R4 Selected from:

[0317] In some embodiments, in the compound of formula (I), 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.

[0318] In some embodiments, in the compound of formula (I), R 6 and R 7 are each independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R 17 .

[0319] 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.

[0320] 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.

[0321] In some embodiments, in the compound of formula (I), each R 9 is independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0322] In some embodiments, in the compound of formula (I), 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.

[0323] In some embodiments, in the compound of formula (I), each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’, -aryl, and -(C1-C6 alkyl)-aryl.

[0324] In some embodiments, in the compounds of formula (I), each R 10 is independently selected from: -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0325] In some embodiments, in the compounds of formula (I), each R 10 is independently selected from: 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.

[0326] In some embodiments, in the compounds 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.

[0327] In some embodiments, in the compounds of formula (I), R 11 is selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0328] In some embodiments, in the compounds of formula (I), R 12 is selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 .

[0329] In some embodiments, in the compounds of formula (I), R 12 is selected from: -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

[0330] In some embodiments, in the compounds of formula (I), R 13Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0331] 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.

[0332] In some embodiments, in the compound of formula (I), R 16 is selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0333] 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.

[0334] 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.

[0335] In some embodiments, in the compound of formula (I), R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 .

[0336] In some embodiments, in the compound of formula (I), X a and X b are each independently selected from: NH and O.

[0337] Combinations of any of the foregoing embodiments of the compound of formula (I) are also contemplated, and each combination forms a separate embodiment for the purposes of the present disclosure.

[0338] In certain embodiments, the compound of formula (I) has the formula (II):

[0339]

[0340] Wherein:

[0341] R 2 is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3;

[0342] R 20 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl,

[0343] R 5 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0344] R 6 and R 7 are each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl and -C(O)R 17 ;

[0345] R 8 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0346] Each R 9 is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0347] Each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0348] R 10’ is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0349] R 11Selected from: -H and -C1-C6 alkyl;

[0350] R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and

[0351] R 13 Selected from: -H and -C1-C6 alkyl;

[0352] R 14 and R 14’ each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0353] R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0354] R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0355] R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-O-R 5 ;

[0356] R 24 、R 25 and R 26 each is -C1-C6 alkyl;

[0357] X a and X b each independently selected from: NH, O and S, and

[0358] X c selected from: O, S and S(O)2,

[0359] provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione.

[0360] In some embodiments, in the compound of formula (II), R 2 is selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0361] In some embodiments, in the compound of formula (II), R 2 is selected from: -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0362] In some embodiments, in the compound of formula (II), R 2 is selected from F and Cl.

[0363] In some embodiments, in the compound of formula (II), R 20 is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , –(C1-C6 alkyl)-aryl,

[0364] In some embodiments, in the compound of formula (II), R 20 is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , –(C1-C6 alkyl)-aryl,

[0365] In some embodiments, in the compound of formula (II), R 20 is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 ,

[0366] In some embodiments, in the compound of formula (II), R 20 is selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, –(C1-C6 alkyl)-aminoaryl,

[0367] In some embodiments, in the compound of formula (II), R 2Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , –(C1-C6 alkyl)-aryl,

[0368] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , –(C1-C6 alkyl)-aryl,

[0369] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 ,

[0370] In some embodiments, in the compound of formula (II), R 5 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.

[0371] In some embodiments, in the compound of formula (II), R 6 and R 7 independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 .

[0372] In some embodiments, in the compound of formula (II), R 6 is H, and R 7 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R 17 .

[0373] In some embodiments, in the compound of formula (II), R 6 is H, and R 7 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 .

[0374] In some embodiments, in the compound of formula (II), R 6 and R 7 are each independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R 17 .

[0375] 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.

[0376] 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.

[0377] In some embodiments, in the compound of formula (II), each R 9 is independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0378] 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.

[0379] In some embodiments, in the compound of formula (II), each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0380] In some embodiments, in the compound of formula (II), each R10 independently selected from: -C1-C6 alkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0381] In some embodiments, in the compound of formula (II), each R 10 is independently selected from: 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.

[0382] 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.

[0383] 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.

[0384] In some embodiments, in the compound of formula (II), R 12 is selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0385] In some embodiments, in the compound of formula (II), R 12 is selected from: -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

[0386] 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.

[0387] 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.

[0388] In some embodiments, in the compound of formula (II), R 16 is selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0389] In some embodiments, in the compound of formula (II), 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.

[0390] In some embodiments, in the compound of formula (II), R 17 is -C1-C6 alkyl.

[0391] 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.

[0392] In some embodiments, in the compound of formula (II), R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 .

[0393] In some embodiments, in the compound of formula (II), X a and X b are each independently selected from: NH and O.

[0394] Any combination of any of the foregoing embodiments of the compound of formula (II) is also contemplated, and each combination forms a separate embodiment for the purposes of the present disclosure.

[0395] In certain embodiments, the compound of formula (I) has formula (III):

[0396]

[0397] Wherein:

[0398] R 2 is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3;

[0399] R 15 is selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3;

[0400] R 4 is selected from:

[0401] R 5 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0402] R 8 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0403] Each R 9 is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0404] Each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ 、-aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0405] R 10’ is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0406] R 11 is selected from: -H and -C1-C6 alkyl;

[0407] R 12 is selected from: -H, -C1-C6 alkyl, -CO2R 8 、-aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and

[0408] R 13 Selected from: -H and -C1-C6 alkyl;

[0409] R 14 and R 14’ are each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0410] R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0411] R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 ;

[0412] R 24 、R 25 and R 26 are each -C1-C6 alkyl;

[0413] X a and X b are each independently selected from: NH, O, and S, and

[0414] X c is selected from: O, S, and S(O)2.

[0415] In some embodiments, in the compound of formula (III), R 2 is selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0416] In some embodiments, in the compound of formula (III), R 2 is selected from: -H, -F, and -Cl.

[0417] In some embodiments, in the compound of formula (III), R 15 is selected from: -CH3, -CF3, -OCH3, and -OCF3.

[0418] In some embodiments, in the compound of formula (III), R 15 is selected from: -CH3 and -OCH3.

[0419] In some embodiments, in the compound of formula (III), R 2 is selected from: -H, -F, and -Cl, and R 15Selected from: -CH3, -CF3, -OCH3, and -OCF3.

[0420] In some embodiments, in the compound of formula (III), R 2 is selected from: -H, -F, and -Cl, and R 15 is selected from: -CH3 and -OCH3.

[0421] In some embodiments, in the compound of formula (III), R 4 is selected from:

[0422] 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.

[0423] 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.

[0424] In some embodiments, in the compound of formula (III), each R 9 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0425] In some embodiments, in the compound of formula (III), each R 9 is independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0426] 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.

[0427] In some embodiments, in the compound of formula (III), each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’, -aryl, and -(C1-C6 alkyl)-aryl.

[0428] In some embodiments, in the compound of formula (III), each R 10 is independently selected from: -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0429] In some embodiments, in the compound of formula (III), each R 10 is independently selected from: 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.

[0430] 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.

[0431] 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.

[0432] In some embodiments, in the compound of formula (III), R 12 is selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 .

[0433] In some embodiments, in the compound of formula (III), R 12 is selected from: -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

[0434] In some embodiments, in the compound of formula (III), R 13Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0435] 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.

[0436] In some embodiments, in the compound of formula (III), R 16 is selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0437] 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.

[0438] In some embodiments, in the compound of formula (III), R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 .

[0439] In some embodiments, in the compound of formula (III), X a and X b are each independently selected from: NH and O.

[0440] Any combination of any of the foregoing embodiments of the compound of formula (III) is also contemplated, and each combination forms a separate embodiment for the purposes of the present disclosure.

[0441] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl as defined in any one of formulas (I), (II), or (III) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl as defined in any one of formulas (I), (II), or (III) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azide, alkylthio, thio, sulfonyl, and sulfonamido.

[0442] In certain embodiments, the camptothecin analog contained in the ADC according to the present disclosure is a compound having formula (I) and is selected from the compounds shown in Tables 5 and 6.

[0443] 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)-O-R 5 or In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F; R 20 is H, -(C1-C6)-O-R 5 or R 5 is H, and R 18 and 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; R 20 is -(C1-C6)-O-R 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.

[0444] In certain embodiments, the camptothecin analog is a compound having formula (III). In certain embodiments, the camptothecin analog is a compound having formula (III), wherein R 2 is F; R 15 is -CH3; R 4 is R9 is a -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.

[0445] In certain embodiments, the camptothecin analog included in the ADC according to the present disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog included in the ADC according to the present disclosure is Compound 139 or Compound 141.

[0446] Table 5: Exemplary camptothecin analogs of formula (II)

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453] Table 6: Exemplary camptothecin analogs of formula (III)

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462] It should be understood that throughout the present disclosure, references to the compounds of formula (I) include, in various embodiments, the compounds of formula (II) and formula (III) and the individual compounds shown in Table 5 and Table 6 to the extent as if each of these formulas or compounds were specifically enumerated in separate embodiments.

[0463] Antibody-drug conjugate

[0464] As described above, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-GPC3 antibody construct conjugated to a camptothecin analogue having formula (I). In certain embodiments, the ADC has formula (X):

[0465] T-[L-(D) m n

[0466] (X)

[0467] Wherein:

[0468] T is an anti-GPC3 antibody construct as described herein;

[0469] L is a linker;

[0470] D is a camptothecin analogue having formula (I);

[0471] m is an integer between 1 and 4, and

[0472] n is an integer between 1 and 10.

[0473] In certain embodiments, in the conjugate of formula (X), m is between 1 and 2. In some embodiments, m is 1.

[0474] In some embodiments, in the conjugate of formula (X), n is between 1 and 8, such as between 2 and 8. In some embodiments, n is between 4 and 8.

[0475] In certain embodiments, in the conjugate of formula (X), m is between 1 and 2, and n is between 2 and 8, or between 4 and 8. In some embodiments, in the conjugate of formula (X), m is 1, and n is between 2 and 8, or between 4 and 8.

[0476] As shown above and reflected by the parameters m and n in formula (X), the anti-GPC3 antibody construct "T" can be conjugated to more than one compound "D" of formula (I). Those skilled in the art will understand that although any particular anti-GPC3 antibody construct T is conjugated to an integer number of compounds D, analysis of a formulation of the conjugate to determine the ratio of compound D to the anti-GPC3 antibody construct T can yield a non-integer result, thereby reflecting a statistical average. This ratio of compound D to the targeting moiety T is commonly referred to as the drug-to-antibody ratio or "DAR". Thus, conjugate preparations having a non-integer DAR are intended to be encompassed by formula (X).

[0477] ​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 Table 5 and Table 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.

[0478] Certain embodiments of the present disclosure relate to an ADC having formula (X), wherein D is a compound of formula (IV):

[0479]

[0480] Wherein:

[0481] R 1a is selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2;

[0482] R 2a is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3;

[0483] X is -O-, -S- or -NH-, and R 4a is selected from:

[0484] where * is the point of attachment to X, and where p is 1, 2, 3 or 4; or

[0485] X is O, and R 4a -X- is selected from:

[0486] R 5a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0487] R 8a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0488] Each R 9a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; or R 9a is absent, and X b =X;

[0489] Each R 10a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl and

[0490] Each R 10a’ is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0491] Each R 10b is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0492] R 11a is absent or is -C1-C6 alkyl;

[0493] R 12a is selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16a and

[0494] R 13a is selected from: -H and -C1-C6 alkyl;

[0495] R 14a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0496] R 14a’ is selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0497] R 16a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0498] R 21 is selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl and –(C1-C6 alkyl)-O-R 5a ;

[0499] R 22 and R 23 are each independently selected from: -H, -halogen, -C1-C6 alkyl and -C3-C8 cycloalkyl;

[0500] R 24 , R 25 and R 26 are each -C1-C6 alkyl;

[0501] X a and X b are each independently selected from: NH, O, and S;

[0502] X c is selected from: O, S, and S(O)2, and

[0503] represents the point of attachment to linker L.

[0504] In some embodiments, in the compound of formula (IV), R 1a is selected from: -CH3, -CF3, -OCH3, -OCF3, and -NH2.

[0505] In some embodiments, in the compound of formula (IV), R 1a is selected from: -CH3, -CF3, -OCH3, and -OCF3.

[0506] In some embodiments, in the compound of formula (IV), R 1a is selected from: -CH3, -OCH3, and NH2.

[0507] In some embodiments, in the compound of formula (IV), R 1a is selected from: -CH3 and -OCH3.

[0508] In some embodiments, in the compound of formula (IV), R 2a is selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0509] In some embodiments, in the compound of formula (IV), R 2a is selected from: -H, -F, and -Cl.

[0510] In some embodiments, in the compound of formula (IV), R 2a is -F.

[0511] In some embodiments, in the compound of formula (IV), X is -O-, -S-, or -NH-, and R 4a is selected from:

[0512] In some embodiments, in the compound of formula (IV), X is -O- or -NH-.

[0513] In some embodiments, in the compound of formula (IV), each R 9aIndependently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0514] In some embodiments, in the compound of formula (IV), each R 9a Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0515] In some embodiments, in the compound of formula (IV), each R 10a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and

[0516] In some embodiments, in the compound of formula (IV), each R 10a Independently selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and

[0517] In some embodiments, in the compound of formula (IV), R 12a Selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 .

[0518] In some embodiments, in the compound of formula (IV), R 13a Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0519] In some embodiments, in the compound of formula (IV), R 14a’ Selected from: H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl.

[0520] In some embodiments, in the compound of formula (IV), R 16a Selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0521] In some embodiments, in the compound of formula (IV), R 22 and R 23 Each independently selected from: -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, and -C3-C8 cycloalkyl.

[0522] In some embodiments, in the compound of formula (IV), Xa and X b are each independently selected from: NH and O.

[0523] In some embodiments, in the compound of formula (IV), X a and X b are each O.

[0524] In some embodiments, in the compound of formula (IV), X is O; R 4a is X a and X b are each O, and R 9a is -C1-C6 alkyl.

[0525] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3; X is O; R 4a is X a and X b are each O; and R 9a is -C1-C6 alkyl.

[0526] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3; R 2a is H or F; X is O; R 4a is X a and X b are each O; and R 9a is -C1-C6 alkyl.

[0527] Also contemplated are other combinations of any of the foregoing embodiments of the compound of formula (IV), and each combination forms a separate embodiment for the purposes of the present disclosure.

[0528] Certain embodiments of the present disclosure relate to an ADC having formula (X), wherein D is a compound of formula (V):

[0529]

[0530] wherein:

[0531] R 2a is selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3;

[0532] R 20a is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 、

[0533] -CO2R 8 、 - aryl, - heteroaryl, –(C1-C6 alkyl)-aryl,

[0534] R 5 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0535] R 6 and R 7 each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 、 -C3-C8 heterocycloalkyl and -C(O)R 17 ;

[0536] R 8 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl;

[0537] each R 9 independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0538] each R 10 independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl and -NR 14 R 14’ ;

[0539] each R 10’ independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl;

[0540] R 11 selected from: -H and -C1-C6 alkyl;

[0541] R 12 selected from: -H, -C1-C6 alkyl, -CO2R 8 、 -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and

[0542] R 13 selected from: -H and -C1-C6 alkyl;

[0543] R 14 and R 14’Each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0544] R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0545] R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0546] R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 ;

[0547] R 24 、R 25 and R 26 each is -C1-C6 alkyl;

[0548] X a and X b each independently selected from: NH, O, and S;

[0549] X c Selected from: O, S, and S(O)2, and

[0550] represents the point of attachment to linker L.

[0551] In some embodiments, in the compound of formula (V), R 2a Selected from: -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0552] In some embodiments, in the compound of formula (V), R 2a Selected from: -CF3, -F, -Cl, and -OCH3.

[0553] In some embodiments, in the compound of formula (V), R 2a is F.

[0554] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 、 -CO2R 8 、 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl,

[0555] In some embodiments, in the compound of formula (V), R 20a is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , -(C1-C6 alkyl)-aryl,

[0556] In some embodiments, in the compound of formula (V), R 20a is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , -(C1-C6 alkyl)-aryl, In some embodiments, in the compound of formula (V), R 20 a is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 ,

[0557] In some embodiments, in the compound of formula (V), R 20a is selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, –(C1-C6 alkyl)-aminoaryl,

[0558] In some embodiments, in the compound of formula (V), R 6 and R 7 are independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 .

[0559] In some embodiments, in the compound of formula (V), R 6 is H, and R 7 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl and -C(O)R17 .

[0560] In some embodiments, in the compound of formula (V), R 6 is H, and R 7 is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 .

[0561] In some embodiments, in the compound of formula (V), R 6 and R 7 are each independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R 17 .

[0562] 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.

[0563] 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.

[0564] In some embodiments, in the compound of formula (V), each R 9 is independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0565] In some embodiments, in the compound of formula (V), 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.

[0566] In some embodiments, in the compound of formula (V), each R 10 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0567] In some embodiments, in the compound of formula (V), each R10 Independently selected from: -C1-C6 alkyl, -NR 14 R 14’ , -aryl, and -(C1-C6 alkyl)-aryl.

[0568] 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.

[0569] In some embodiments, in the compound of formula (V), R 12 is selected from: -H, -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16 .

[0570] In some embodiments, in the compound of formula (V), R 12 is selected from: -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

[0571] 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.

[0572] 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.

[0573] In some embodiments, in the compound of formula (V), R 16 is selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0574] 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.

[0575] 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.

[0576] In some embodiments, in the compound of formula (V), R 18 and R 19 together with the N atom to which they are attached form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from: halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-O-R 5 .

[0577] In some embodiments, in the compound of formula (V), R 17 is -C1-C6 alkyl.

[0578] In some embodiments, in the compound of formula (V), X a and X b each independently selected from: NH and O.

[0579] In some embodiments, in the compound of formula (V), X a and X b are each O.

[0580] In some embodiments, in the compound of formula (V), R 20a is –(C1-C6 alkyl)-O-R 5 .

[0581] In some embodiments, in the compound of formula (V), R 20a is –(C1-C6 alkyl)-O-R 5 , and R 5 is H.

[0582] In some embodiments, in the compound of formula (V), R 2a is F; R 20a is –(C1-C6 alkyl)-O-R 5 , and R 5 is H.

[0583] Also contemplated are other combinations of any of the foregoing embodiments of the compound of formula (V), and each combination forms a separate embodiment for the purposes of the present disclosure.

[0584] Certain embodiments of the present disclosure relate to an ADC having formula (X), wherein D is a compound of formula (VI):

[0585]

[0586] Wherein:

[0587] R 2a is selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3;

[0588] X is -O-, -S-, or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a 、-CO2R 8a 、-aryl, -heteroaryl, –(C1-C6 alkyl)-aryl,

[0589]

[0590] where * is the point of attachment to X, and where p is 1, 2, 3, or 4; or

[0591] X is O, and R 25 -X- is selected from:

[0592] R 5a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0593] R 6a is selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0594] R 7a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5a 、-C3-C8 heterocycloalkyl, and -C(O)R 17a ;

[0595] R 8a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0596] Each R 9a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl; or R9a is absent, and X b = X;

[0597] Each R 10a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, and

[0598] Each R 10a’ is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0599] Each R 10b is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0600] R 11a is absent or is -C1-C6 alkyl;

[0601] R 12a is selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16a and

[0602] R 13a is selected from: -H and -C1-C6 alkyl;

[0603] R 14a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0604] R 14a’ is selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl;

[0605] R 16a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl;

[0606] 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;

[0607] R 21 is selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl, and –(C1-C6 alkyl)-O-R 5a ;

[0608] R 22 and R 23 are each independently selected from: -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl;

[0609] R 24 、R 25 and R 26 are each -C1-C6 alkyl;

[0610] X a and X b are each independently selected from: NH, O, and S;

[0611] X c is selected from: O, S, and S(O)2, and

[0612] represents the point of attachment to linker L.

[0613] In some embodiments, in the compound of formula (VI), R 2a is selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

[0614] In some embodiments, in the compound of formula (VI), R 2a is selected from: -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0615] In some embodiments, in the compound of formula (VI), R 2a is selected from: F and Cl.

[0616] In some embodiments, in the compound of formula (VI), R 2a is F.

[0617] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a 、-(C1-C6 alkyl)-aryl,

[0618]

[0619] Or X is O, and R 25 -X- is selected from:

[0620] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a , -(C1-C6 alkyl)-aryl,

[0621]

[0622] In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a ,

[0623] In some embodiments, in the compound of formula (VI), X is -O-, -S- or -NH-, and R 25 is selected from:

[0624] In some embodiments, in the compound of formula (VI), X is -O- or -NH-.

[0625] In some embodiments, in the compound of formula (VI), R 6a is H.

[0626] 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.

[0627] In some embodiments, in the compound of formula (VI), R 7a is selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17a .

[0628] 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.

[0629] In some embodiments, in the compound of formula (VI), each R 9a is independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0630] In some embodiments, in the compound of formula (VI), each R 10a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, aryl, -(C1-C6 alkyl)-aryl and

[0631] In some embodiments, in the compounds of formula (VI), each R 10a is independently selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and

[0632] In some embodiments, in the compounds of formula (VI), R 12a is selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and -S(O)2R 16a .

[0633] In some embodiments, in the compounds of formula (VI), R 13a is selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0634] In some embodiments, in the compounds 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.

[0635] In some embodiments, in the compounds of formula (VI), R 16a is selected from: -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0636] In some embodiments, in the compounds of formula (VI), R 17a is -C1-C6 alkyl.

[0637] In some embodiments, in the compounds 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.

[0638] In some embodiments, in the compounds of formula (VI), X a and X b are each independently selected from: NH and O.

[0639] In some embodiments, in the compounds of formula (VI), X a and X b are each O.

[0640] In some embodiments, in the compound of formula (VI), X is O, and R 25 is -C1-C6 alkyl.

[0641] In some embodiments, in the compound of formula (VI), R 2a is F; X is O, and R 25 is -C1-C6 alkyl.

[0642] Other combinations of any of the foregoing embodiments of the compound of formula (VI) are also contemplated, and each combination forms a separate embodiment for the purposes of the present disclosure.

[0643] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl as defined in any of formulas (IV), (V), or (VI) is optionally substituted with one or more substituents selected from the group consisting of: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl as defined in any of formulas (IV), (V), or (VI) is optionally substituted with one or more substituents selected from the group consisting of: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azide, alkylthio, thio, sulfonyl, and sulfonamido.

[0644] 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; R 2a is F; X is -O-; R 4a is R 9a is -C1-C6 alkyl, and X a and X b are each O.

[0645] In certain 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)-O-R 5 or In some embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R 2a is F; R 20ais H, -(C1-C6)-O-R 5 or R 5 is H, and R 18 and 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, in the ADC having formula (X), D is a compound of formula (V), wherein R 2a is F; R 20a is -(C1-C6)-O-R 5 , and R 5 is H.

[0646] 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-C6 alkyl.

[0647] Linker L

[0648] The conjugate of formula (X) includes 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. The bifunctional (or monovalent) linker L links a single compound D to a single site on the anti-GPC3 antibody construct T, while the polyfunctional (or multivalent) linker L links more than one compound D to a single site on the anti-GPC3 antibody construct T. A linker that links one compound D to more than one site on the anti-GPC3 antibody construct T can also be considered polyfunctional.

[0649] Linker L includes a functional group capable of reacting with one or more target groups on the anti-GPC3 antibody construct T and at least one functional group capable of reacting with a target 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 serve as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde and ketone groups.

[0650] Non-limiting examples of functional groups capable of reacting with thiol include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. In this case, the "self-stabilizing" maleimide as described by Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062 can also be used.

[0651] Non-limiting examples of functional groups capable of reacting with amines include activated esters (such as N-hydroxysuccinimide (NHS) esters and sulfo-NHS esters), imidoesters (such as Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include converting a carboxyl group to an activated ester using succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and then the activated ester can react with an amine.

[0652] Non-limiting examples of functional groups capable of reacting with electrophilic groups such as aldehyde or ketone carbonyls include hydrazides, oximes, amines, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0653] In certain embodiments, linker L may comprise a functional group that allows for the bridging of two interchain cysteines on the anti-GPC3 antibody construct, such as ThioBridge TM linkers (Badescu et al., 2014, Bioconjug. Chem. 25:1124–1136), dithio maleimide (DTM) linkers (Behrens et al., 2015, Mol. Pharm. 12:3986–3998), dithioaryl (TCEP) pyridazinone-based linkers (Lee et al., 2016, Chem. Sci., 7:799-802), or dibromopyridazinone-based linkers (Maruani et al., 2015, Nat. Commun., 6:6645).

[0654] Alternatively, the anti-GPC3 antibody construct T can be modified to include non-natural reactive groups, such as azides, which allow for conjugation to a linker via complementary reactive groups on the linker. For example, conjugation of the linker to the anti-GPC3 antibody construct can utilize click chemistry reactions (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97), such as azide-alkyne cycloaddition (AAC) reactions, which have been successfully used in the development of antibody-drug conjugates. The AAC reaction can be a copper-catalyzed AAC (CuAAC) reaction, which involves the conjugation of an azide with a linear alkyne; or a strain-promoted AAC (SPAAC) reaction, which involves the conjugation of an azide with a cyclooctyne.

[0655] The linker L can be a cleavable or non-cleavable linker. A cleavable linker is a linker that is prone to cleavage under specific conditions, such as intracellular conditions (such as in endosomes or lysosomes) or near the target cell (such as in the 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 the cell, which typically results in the release of the amino acid-linker-drug moiety.

[0656] Examples of cleavable linkers include, for example, linkers that contain an amino acid sequence that serves as 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, for example, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix near the target cell, such as a cancer cell, can be used. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteinases (MMPs), elastase, and kallikrein-related peptidases.

[0657] For conjugates that are intended to be internalized by cells, the linker L can contain an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease. Examples of such proteases include, for example, cathepsin B, C, D, H, L, and S, and legumain.

[0658] The cleavage recognition sequence can be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that can 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.

[0659] Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl 4-(2-pyridyldithio)-2-sulfonate butyrate (sulfonyl-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance near the disulfide bond to improve the extracellular stability of the linker, e.g., containing gem-dimethyls. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Linkers containing a combination of these functionalities may also be useful, e.g., linkers containing both a hydrazone and a disulfide bond are known in the art.

[0660] Another example of a cleavable linker is a linker containing β-glucuronide, which 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). β-Glucuronide can also be used to improve the hydrophilicity of linker L.

[0661] Another example of a linker that is cleaved intracellularly and improves hydrophilicity is a linker containing a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0662] In certain embodiments, linker L contained in the conjugate of formula (X) is a cleavable linker. In some embodiments, linker L contains a cleavage recognition sequence. In some embodiments, linker L may contain an amino acid sequence that is recognized and cleaved by lysosomal proteases.

[0663] The cleavable linker may optionally also contain one or more additional functional groups such as self-immolative and self-eliminating groups, spacer groups, or hydrophilic moieties.

[0664] Self-decomposing and self-eliminating groups that can be used in linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED), and hemiacetal groups. Other examples of self-decomposing groups include, but are not limited to, aromatic compounds that are electronically similar to PAB or PABC groups, such as heterocyclic derivatives, for example, 2-aminoimidazole-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that cyclize upon hydrolysis of an amide bond, such as substituted and unsubstituted 4-aminobutyramide (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionamide (Amsberry et al., 1990, J. Org. Chem. 55:5867-5877). Self-decomposing / self-eliminating groups are typically attached to an amino or hydroxyl group on compound D. Self-decomposing / self-eliminating groups are typically included, either alone or in combination, in peptide-based linkers, but can also be included in other types of linkers.

[0665] Spacer groups that can be used in linkers for drug conjugates include, for example, alkylene groups and spacer groups based on aliphatic acids, diacids, amines, or diamines, such as diglycolate, malonate, caproate, and capramide. Other spacer groups include, for example, glycine-based spacer groups and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) spacer groups.

[0666] PEG and mPEG spacer groups can also be used as hydrophilic moieties within the linker. For example, PEG or mPEG can be "directly inserted" or included as a side group in the linker to increase the hydrophilicity of the linker (see, for example, 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 linker L include, for example, β-glucuronide, sulfonic acid groups, carboxylic acid groups, and pyrophosphate diesters.

[0667] In certain embodiments, the ADC of formula (X) can comprise a cleavable linker. In some embodiments, the ADC of formula (X) can comprise a peptide-containing linker. In some embodiments, the ADC of formula (X) can comprise a protease-cleavable linker.

[0668] In some embodiments, in the ADC of formula (X), m is 1, and linker L is a cleavable linker having formula (XI):

[0669]

[0670] Wherein:

[0671] Z is a functional group capable of reacting with a target group on the anti-GPC3 antibody construct T;

[0672] Str is a spacer group;

[0673] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r forms a protease cleavage site;

[0674] X is a self-cleaving group;

[0675] q is 0 or 1;

[0676] r is 1, 2 or 3;

[0677] s is 0, 1 or 2;

[0678] # is the point of attachment to the anti-GPC3 antibody construct T, and

[0679] % is the point of attachment to the camptothecin analogue D.

[0680] In some embodiments, in the linker of formula (XI), q is 1.

[0681] In some embodiments, in the linker of formula (XI), s is 1. In some embodiments, in the ADC of formula (XI), s is 0.

[0682] In some embodiments, in the linker of formula (XI), r is 1. In some embodiments, in the ADC of formula (XI), r is 3.

[0683] In some embodiments, in the linker of formula (XI):

[0684] Z is where # is the point of attachment to T, and * is the point of attachment to the remainder of the linker.

[0685] In some embodiments, in the linker of formula (XI), Str is selected from:

[0686]

[0687] wherein:

[0688] R is H or C1-C6 alkyl;

[0689] t is an integer between 2 and 10, and

[0690] u is an integer between 1 and 10.

[0691] In some embodiments, in the linker of formula (XI), Str is selected from:

[0692]

[0693] wherein:

[0694] t is an integer between 2 and 10, and

[0695] u is an integer between 1 and 10.

[0696] 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] r has a sequence selected from: 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.

[0697] 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.

[0698] 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.

[0699] In certain embodiments, in the ADC of formula (X), m is 1 and the linker L is a cleavable linker having formula (XII):

[0700]

[0701] Wherein:

[0702] Z is a functional group capable of reacting with a target group on the anti-GPC3 antibody construct T;

[0703] Str is a spacer group;

[0704] AA1 and AA2 are each independently an amino acid, where AA1-[AA2] r forms a protease cleavage site;

[0705] Y is -NH-CH2-;

[0706] q is 0 or 1;

[0707] r is 1, 2 or 3;

[0708] v is 0 or 1;

[0709] # is the point of attachment to the anti-GPC3 antibody construct T, and

[0710] % is the point of attachment to the camptothecin analogue D.

[0711] In some embodiments, in the linker of formula (XII), q is 1.

[0712] In some embodiments, in the linker of formula (XII), v is 0. In some embodiments, in the ADC of formula (XII), s is 1.

[0713] In some embodiments, in the linker of formula (XII), r is 1. In some embodiments, in the ADC of formula (XII), r is 3.

[0714] In some embodiments, in the linker of formula (XII):

[0715] Z is where # is the point of attachment to T and * is the point of attachment to the remainder of the linker.

[0716] In some embodiments, in the linker of formula (XII), Str is selected from:

[0717]

[0718]

[0719] Wherein:

[0720] R is H or a C1-C6 alkyl group;

[0721] t is an integer between 2 and 10, and

[0722] u is an integer between 1 and 10.

[0723] In some embodiments, in the linker of formula (XII), Str is selected from:

[0724]

[0725] wherein:

[0726] t is an integer between 2 and 10, and

[0727] u is an integer between 1 and 10.

[0728] 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 has a sequence selected from: 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.

[0729] 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, Asn-Pro-Val.

[0730] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is 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.

[0731] In some embodiments, in the linker of formula (XII), Y is -NH-CH2. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH2.

[0732] 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 a cleavable linker having formula (XIII):

[0733]

[0734] Wherein:

[0735] Z is a functional group capable of reacting with a target group on the anti-GPC3 antibody construct T;

[0736] Q is -(CH2) p - or -(CH2CH2O) q -, where p and q are each independently an integer between 1 and 10;

[0737] Each R is independently H or a C1-C6 alkyl;

[0738] n is 1, 2, or 3;

[0739] # is the point of attachment to the anti-GPC3 antibody construct T, and

[0740] % is the point of attachment to the camptothecin analogue D.

[0741] In some embodiments, the ADC of formula (X) may comprise a β-glucuronide-containing linker.

[0742] A variety of non-cleavable linkers are known in the art for attaching a drug to a targeting moiety and can be used in certain embodiments in the ADCs of the present disclosure. Examples of non-cleavable linkers include linkers having an N-succinimidyl or N-sulfo-succinimidyl moiety for reacting with an anti-GPC3 antibody construct and a maleimide- or haloacetyl-based moiety for reacting with a camptothecin analogue, or vice versa. An example of such a non-cleavable linker is based on sulfo-succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via the maleimide group, which reacts with a thiol (-SH) group on the camptothecin analogue, while the sulfo-NHS ester is reactive towards primary amines (such as those found in lysine and at the N-terminus of a protein or peptide) on the anti-GPC3 antibody construct. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyl-(6-aminohexanoate) ("long-chain" SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl) isocyanate (PMPI). Other examples include those containing a haloacetyl-based functional group (such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP)).

[0743] Non-limiting examples of the drug-linkers of the camptothecin analogs of formula (I) are shown in Tables 7, 8, and 9. Non-limiting examples of the conjugates comprising these drug-linkers are shown in Tables 10, 11, and 12. In certain embodiments, the ADC of formula (X) comprises a drug-linker selected from the drug-linkers shown in Tables 7, 8, and 9. In certain embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, wherein T is an anti-GPC3 antibody construct and n is between 1 and 10. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, wherein T is an anti-GPC3 antibody construct and n is between 2 and 8. In some embodiments, the ADC of formula (X) is selected from the conjugates shown in Tables 10, 11, and 12, wherein T is an anti-FRα antibody construct and n is between 4 and 8.

[0744] In certain embodiments, the ADC of formula (X) comprises a drug-linker selected from the following (L-(D) m ): 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, and n is 4 or 8. In some embodiments, the ADC of formula (X) comprises a drug-linker selected from the following (L-(D) m ): 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, and n is 8.

[0745] Preparation of ADC

[0746] The ADCs of formula (X) can be prepared by standard methods known in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, e.g., 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)). In addition, 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 Piramal Pharma Solutions (Grangemouth, UK).

[0747] Generally, the preparation of an ADC involves first preparing a drug - linker D - L comprising one or more camptothecin analogs of formula (I) and a linker L, and then conjugating the drug - linker D - L to a suitable group on the anti - GPC3 antibody construct T. However, the ligation of the linker L to the anti - GPC3 antibody construct T and subsequent ligation of the anti - GPC3 antibody construct - linker T - L to one or more camptothecin analogs D of formula (I) are alternative methods that can be used in some embodiments.

[0748] In any of the above methods, suitable groups on the compound D of formula (I) for attaching the linker L include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of the present disclosure, the linker L is attached to the compound D of formula (I) via a hydroxyl or amine group on the compound.

[0749] In any of the above methods, suitable groups on the anti - GPC3 antibody construct T for attaching the linker L include thiol groups (e.g., on the side chain of a cysteine residue), amino groups (e.g., on the side chain of a lysine residue), carboxylic acid groups (e.g., on the side chain of an aspartic or glutamic acid residue), and carbohydrate groups.

[0750] For example, the anti-GPC3 antibody construct T may comprise one or more naturally occurring thiol groups, allowing the anti-GPC3 antibody construct T to bond to linker L through the sulfur atoms of the thiol groups. Alternatively, the anti-GPC3 antibody construct T may comprise one or more lysine residues, which may be chemically modified to introduce one or more thiol 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 reagent). Alternatively, the anti-GPC3 antibody construct T may comprise one or more carbohydrate groups, which may be chemically modified to contain one or more thiol groups.

[0751] The carbohydrate groups on the anti-GPC3 antibody construct T may also be oxidized to provide aldehyde (-CHO) groups (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which may then react with linker L, for example, via hydrazine or hydroxylamine groups on linker L.

[0752] The anti-GPC3 antibody construct T may also be modified to include additional cysteine residues (see, for example, 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, for example, 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 may be modified to include unnatural reactive groups, such as azides, which allow conjugation to the linker via complementary reactive groups on the linker, for example, by click chemistry (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). Another option is to use the GlycoConnect TM technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to allow linker attachment by metal-free click chemistry (see, for example, European Patent No. EP 2 911 699).

[0753] Other schemes for modifying proteins to link or associate with linker L are known in the art, including those described in Coligan et al., Current Protocols in Protein Science, Volume 2, John Wiley & Sons (2002).

[0754] Alternatively, ADCs can be prepared using transglutaminase, especially 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 formamide of glutamine (amine acceptor, usually on the antibody) and an alkylamino group (amine donor, usually on the drug - linker), and the alkylamino group can be, for example, the ε - amino group of lysine or a 5 - amino - n - pentyl group. Antibodies can also be modified to include a peptide or "tag" containing glutamine, which allows conjugation of the antibody to the drug - linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US2013 / 0230543 and International (PCT) Publication No. WO 2016 / 144608).

[0755] Similar conjugation methods utilize the enzyme sortase A. In this method, antibodies are typically modified to contain a sortase A recognition motif (LPXTG, where X is any natural amino acid), and the drug - linker is designed to contain an oligoglycine motif (usually GGG) to allow sortase A - mediated transpeptidation (see, e.g., Beerli et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0756] Once conjugation is complete, the average number of compounds of formula (I) conjugated to the anti - GPC3 antibody construct (i.e., the "drug - 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 - TOF MS. Additionally, the distribution of the drug - linked forms (e.g., the fraction of anti - GPC3 antibody constructs T containing zero, one, two, three, etc. compounds of formula (I)) can optionally be analyzed. Various techniques for measuring the DAR distribution are known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reversed - phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Wakankar et al., 2011, mAbs, 3:161 - 172).

[0757] Drug composition

[0758] For therapeutic use, the ADCs of the present disclosure are generally formulated as drug compositions. Accordingly, certain embodiments of the present disclosure relate to drug compositions comprising an ADC as described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such drug compositions can be prepared by known procedures using well-known and readily available ingredients.

[0759] The drug compositions can be formulated for administration to a subject by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or insufflation. As used herein, the term "parenteral" includes subcutaneous injection, as well as intradermal, intra-articular, intravenous, intramuscular, intracapsular, intrasternal, intrathecal injection or infusion. The drug compositions will generally be formulated in a form suitable for administration to a subject, for example, as syrups, elixirs, tablets, troches, lozenges, hard or soft gelatin capsules, pills, suppositories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectable solutions or solutions. The drug compositions can be provided as unit dosage formulations.

[0760] In certain embodiments, the drug composition comprising the ADC is formulated for parenteral administration, for example, in the form of a lyophilized formulation or an aqueous solution. Such drug compositions can be provided, for example, in unit dosage injectable form.

[0761] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed. Examples of such carriers include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, benzyl alcohol, alkyl parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin or 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 ion; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0762] In certain embodiments, a composition comprising an ADC can be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension can contain the ADC in a non-toxic parenterally acceptable diluent or carrier. Acceptable diluents and carriers that can be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Additionally, a sterile non-volatile oil can be used as a carrier. For this purpose, various mild non-volatile oils can be employed, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid can be used in the preparation of injectables. Adjuvants such as local anesthetics, preservatives, and / or buffering agents can also be included in the injectable solution or suspension.

[0763] In certain embodiments, a composition comprising an ADC can be formulated for intravenous administration to a human. Generally, a composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also contain solubilizing agents and / or local anesthetics such as lidocaine to alleviate the pain at the injection site. Generally, the ingredients are provided separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or an anhydrous concentrate in a sealed container (such as an ampoule or vial) indicating the amount of the active agent. When the composition is to be administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade 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 before administration.

[0764] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0765] Methods of Use

[0766] Certain embodiments of the present disclosure relate to the therapeutic use of the ADCs described herein. Some embodiments relate to the use of the ADCs as therapeutic agents.

[0767] Certain embodiments of the present disclosure relate to methods of inhibiting the growth of abnormal cancerous or tumor cells; inhibiting cancer cell or tumor cell proliferation in a subject, or treating cancer, comprising administering the ADCs described herein. In certain embodiments, the ADCs described herein can be used to treat cancer. Accordingly, some embodiments of the present disclosure relate to the use of the ADCs as anti-cancer agents.

[0768] Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells, which comprise contacting said cells with an ADC as described herein, such as an ADC of formula (X). Some embodiments relate to methods of killing cancer or tumor cells, which comprise contacting said cells with an ADC as described herein, such as an ADC of formula (X).

[0769] Some embodiments relate to methods of treating a subject having cancer by administering to the subject an ADC as described herein, such as an ADC of formula (X). In such cases, treating the subject can result in one or more of the following: reduction in tumor size, slowing or prevention of an increase in tumor size, prolonging the disease-free survival time between disappearance or removal of the tumor and its recurrence, preventing subsequent occurrence of the tumor (e.g., metastasis), prolonging the time to progression, reducing one or more adverse symptoms associated with the tumor, and / or prolonging the overall survival time of the subject having cancer.

[0770] Certain embodiments relate to the use of an ADC as described herein, such as an ADC of formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC as described herein, such as an ADC of formula (X), in a method of inhibiting cancer cell proliferation and / or killing cancer cells in vitro. Some embodiments relate to the use of an ADC as described herein, such as an ADC of formula (X), in a method of inhibiting cancer cell proliferation and / or killing cancer cells in a subject having cancer.

[0771] Examples of cancers that can be treated in certain embodiments are carcinomas, including adenocarcinoma and squamous cell carcinoma; melanoma and sarcoma. Carcinomas and sarcomas are also commonly referred to as "solid tumors". Examples of common 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, stomach cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also result in the formation of solid tumors and thus can also be considered solid tumors in certain cases. Generally, the cancer to be treated is a cancer that expresses GPC3.

[0772] Certain embodiments relate to a method of inhibiting the growth of GPC3-positive tumor cells, which method comprises contacting the cells with an ADC as described herein (such as an ADC of formula (X)). The cells can be in vitro or in vivo. In certain embodiments, the ADC can be used in a method of treating a GPC3-positive cancer or tumor in a subject.

[0773] In some embodiments, the ADCs described herein can be used to treat a subject having a cancer that overexpresses GPC3. Cancers that overexpress GPC3 are typically solid tumors. Examples include, but are not limited to, hepatocellular carcinoma (HCC), melanoma, lung cancer, and hepatoblastoma.

[0774] Drug kit

[0775] Certain embodiments relate to a drug kit comprising an ADC as described herein, such as an ADC of formula (X).

[0776] Typically, the kit will include a container housing the ADC and a label and / or package insert on or accompanying the container. The label or package insert contains instructions typically included in the commercial packaging of a therapeutic product, providing information about indications, usage, dosage, administration, contraindications, and / or warnings for using such a therapeutic product. The label or package insert may also include a notice in a form required by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, which reflects the approval of the manufacturing agency for the use or sale for administration to humans or animals. In some embodiments, the container may have a sterile inlet. For example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a subcutaneous injection needle.

[0777] In addition to the container housing 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 glucose solution), other buffers, or diluents.

[0778] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The containers can be made of various materials such as glass or plastic. In appropriate cases, one or more components of the kit may be lyophilized or provided in a dry form (such as a powder or granule), and the kit may additionally contain a suitable solvent for reconstituting the lyophilized or dry components.

[0779] The kit may also include other materials that are desirable from a commercial or user perspective, such as filters, needles, and syringes.

[0780] Tables 7 to 12

[0781] Table 7: Exemplary drug-linker (DL) structures comprising a camptothecin analogue of formula (I) having a C7 bond

[0782]

[0783]

[0784]

[0785]

[0786] Table 8: Exemplary Drug-Linker (DL) Structures Containing Camptothecin Analogs of Formula (I) with a C10 Bond

[0787]

[0788]

[0789]

[0790]

[0791] Table 9: Exemplary Drug-Linker (DL) Structures Containing Camptothecin Analogs of Formula (I) with a C7 or C10 Bond

[0792]

[0793]

[0794]

[0795] Table 10: Exemplary Conjugate (DC) Structures Containing Camptothecin Analogs of Formula (I) with a C7 Bond

[0796]

[0797]

[0798]

[0799] Table 11: Exemplary Conjugate (DC) Structures Containing Camptothecin Analogs of Formula (I) with a C10 Bond

[0800]

[0801]

[0802] Table 12: Exemplary Conjugate (DC) Structures Containing Camptothecin Analogs of Formula (I) with a C7 or C10 Bond

[0803]

[0804]

[0805]

[0806] The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.

[0807] Examples

[0808] Examples 1 to 3 below illustrate various methods for preparing camptothecin analogs of formula (I). It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known in the art. It should also be understood that those skilled in the art will be able to use the methods described below or similar methods to prepare other compounds of formula (I) not specifically described below by using appropriate starting components and modifying the synthetic parameters as needed. Generally, the starting components can be obtained from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (ThermoFisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or synthesized according to sources 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 prepared as described herein.

[0809] Abbreviations

[0810] 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; DMMTM: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc: fluorenylmethyloxycarbonyl; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; LC / MS: liquid chromatography mass spectrometry; MC: maleimidocaproyl; MT: maleimidotriethyleneglycolate; 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.

[0811] General chemical procedures

[0812] General procedure 1: Conversion of chloride to amine

[0813] To a stirred solution of the chloride compound in dimethylformamide (0.05 M to 0.1 M), add an appropriate secondary amine (3 equivalents). After completion (determined by LC / MS, typically 1 to 3 hours), purify the reaction mixture by reverse-phase HPLC to provide the desired product after lyophilization.

[0814] General procedure 2: Conversion of amine to amide

[0815] To a stirred solution of the amine compound in dimethylformamide (0.05 - 0.1 M), add triethylamine (1.2 equivalents), a suitable carboxylic acid (1.1 equivalents), and then add a solution of DMMTM (2 equivalents) in water (1 M). After completion (determined by LC / MS, typically 16 hours), purify the reaction mixture by reverse-phase HPLC to provide the desired product after lyophilization.

[0816] General procedure 3: Conversion of amine to sulfonamide

[0817] To a stirred solution of the amine compound in dimethylformamide (0.05 M to 0.1 M), add DIPEA (3 equivalents), followed by an appropriate sulfonyl chloride. After completion (determined by LC / MS, typically 16 hours), purify the reaction mixture by reverse-phase HPLC to provide the desired product after lyophilization.

[0818] General procedure 4: Two-step conversion of amine to urea (Synthetic Scheme IV; Figure 1D)

[0819] Step 1: To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05 M to 0.1 M), add p-nitrophenyl carbonate (1 equivalent), and then add triethylamine (2 equivalents). After completion (determined by LC / MS, typically 1 to 4 hours), concentrate the reaction mixture to dryness and then purify by reverse-phase HPLC to provide the desired PNP-carbamate intermediate after lyophilization. This intermediate can be used to generate a single analogue or divided into multiple batches for generating multiple analogues in the second step. Step 2: To the PNP-carbamate intermediate (0.1 M to 0.2 M) in dimethylformamide, add an appropriate primary amine (3 equivalents). After completion (determined by LC / MS, typically 1 hour), purify the reaction mixture by reverse-phase HPLC to provide the desired product after lyophilization.

[0820] General procedure 5: Conversion of amine to carbamate

[0821] To a stirred solution of the amine compound (0.05 M to 0.1 M) in dichloromethane or dimethylformamide, add p-nitrophenyl carbonate (1 equiv), then add triethylamine (2 equiv). After completion (determined by LC / MS, typically 1 to 4 h), add the appropriate alcohol to the resulting PNP-carbamate intermediate. After completion (determined by LC / MS, typically 1 to 16 h), purify the reaction mixture by reverse-phase HPLC to afford the desired product after lyophilization.

[0822] General Procedure 6: Removal of Boc protecting group

[0823] To a stirred solution of the Boc-protected amine compound (0.1 M) in dichloromethane, add TFA (20 vol%). After completion (determined by LC / MS, typically 1 h), concentrate the reaction mixture in vacuo to afford a crude solid or purify as described in General Procedure 9.

[0824] General Procedure 7: Copper-mediated amide coupling

[0825] To a rapidly stirred solution of Boc-GGFG-OH (3 equiv) and HOAt (3 equiv) in a 10% v / v mixture of dimethylformamide in dichloromethane (0.02 M), add EDC (HCl salt, 3 equiv). After 5 min, add a solution of the amine payload (1 equiv) in a 10% v / v mixture of dimethylformamide in dichloromethane (0.02 M), then immediately add CuCl2 (4 equiv). After completion (determined by LC / MS, typically 1 to 16 h), concentrate the reaction mixture in vacuo to afford a crude solid or purify by preparative HPLC to afford the desired product after lyophilization.

[0826] General Procedure 8: MT placement

[0827] To a stirred solution of the amine compound (1 equiv) in dimethylformamide (ca. 0.02 M), add a solution of MT-OTfp (1.2 equiv to 1.5 equiv) in acetonitrile (ca. 0.02 M), then add DIPEA (10 μL, 4 equiv). After completion (determined by LC / MS, typically 1 to 16 h), concentrate the reaction mixture in vacuo to afford a crude solid, which is purified by preparative HPLC to afford the desired product after lyophilization.

[0828] General Procedure 9: Compound purification

[0829] Flash chromatography : Purify the crude reaction product with Snap Ultra column (10 g, 25 g, 50 g or 100 g) (Biotage, Charlotte, NC) in Isolera TM Elution was performed using a linear gradient of ethyl acetate / hexane or methanol / dichloromethane on an automated rapid system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was performed using a SnapUltra C18 column (12 g, 30 g, 60 g, or 120 g) with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The organic solvent was removed by rotary evaporation or the acetonitrile / water mixture was lyophilized to isolate the purified compound.

[0830] Preparative HPLC: Reverse-phase HPLC of the crude compound was performed using a 5-μm (150 × 30 mm) column (Phenomenex, Torrance, CA) on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was isolated by lyophilizing the acetonitrile / water mixture.

[0831] General Procedure 10: Compound Analysis

[0832] LC / MS: The completion of the reaction was monitored and the purified compound was analyzed using a 2.6-μm (30 × 3 mm) column (Phenomenex, Torrance, CA) on an Agilent 1290 HPLC / 6120 single quadrupole LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) with a linear gradient of 10% to 100% 0.1% formic acid / acetonitrile / 0.1% formic acid / water.

[0833] NMR: 1 1H NMR spectra were collected on a Bruker AVANCE III 300 spectrometer (300 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts were reported in parts per million (ppm).

[0834] Example 1: Preparation of Camptothecin Analogs with a Methyl Group at the C10 Position

[0835] 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)

[0836]

[0837] The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

[0838] 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)

[0839]

[0840] The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

[0841] 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)

[0842]

[0843] The title compound was prepared starting from compound 1.1 (10 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0844] LC / MS: C 26 H 26 Calculated value for FN3O5 m / z = 479.2, found value [M+H] + =480.4.

[0845] 11H 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).

[0846] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 102)

[0847]

[0848] The title compound was prepared from Compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a 20% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0849] LC / MS: C 32 H 31 Calculated for C29H32FN4O6 m / z = 618.2, found [M+H] + = 619.4.

[0850] 1 1H 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).

[0851] 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)

[0852]

[0853] The title compound was prepared starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9, eluting with a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA to afford the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0854] LC / MS: C 32 H 32 Calculated value for FN5O6 m / z = 633.2, found value [M+H] + =634.4.

[0855] 1 H NMR (300MHz, MeOD) δ8.32(d,J=8.0Hz,1H),7.85(d,J=10.5Hz,1H),7.65(s,1H),7.46(d,J=8.7Hz,2H),6.74(d,J=8.7Hz,2H),5.61(d,J= 16.5Hz,1H),5.44(s,2H),5.41(d,J=16.5Hz,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=7.3Hz,3H).

[0856] 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)

[0857]

[0858] The title compound was prepared starting from Compound 1.1 (10 mg) and N-methylpiperazine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

[0859] LC / MS: C 27 H 29 Calculated m / z for C + H

[0860] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 108)

[0861]

[0862] The title compound was prepared starting from Compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0863] LC / MS: C 32 H 32 Calculated m / z for C + H

[0864] 1 H 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).

[0865] 1.8: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 110)

[0866]

[0867] The title compound was prepared starting from Compound 1.1 (10 mg) and piperidine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with gradient elution using 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0868] LC / MS: C 27 H 28 Calculated for C25H29FN3O4 m / z = 477.2, found [M+H] + = 478.2.

[0869] 1 H 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).

[0870] 1.9: (S)-4-((4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl)piperazine-1-carboxylic acid tert-butyl ester (Compound 111)

[0871]

[0872] The title compound was prepared starting from Compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with gradient elution using 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

[0873] LC / MS: C 31 H 35 Calculated m / z for FN4O6 = 578.2, found [M+H] + = 579.4

[0874] 1.10: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 112)

[0875]

[0876] The title compound was prepared starting from Compound 111 (5.0 mg) according to General Procedure 6, and the title compound (TFA salt, 4.4 mg) was obtained as an off-white solid

[0877] LC / MS: C 26 H 27 Calculated m / z for FN4O4 = 478.2, found [M+H] + = 479.2

[0878] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 113)

[0879]

[0880] The title compound was prepared from Compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 10% to 60% CH3CN / H2O + 0.1% TFA, and the title compound (TFA salt, 4.6 mg, 32% yield) was obtained as an off-white solid

[0881] LC / MS: C 27 H 28 Calculated m / z for FN3O6 = 509.2, found [M+H] + = 510.4

[0882] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 114)

[0883]

[0884] The title compound was prepared starting from Compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0885] LC / MS: calculated m / z = 525.6 (C 27 H 28 FN3O5S), found [M+H] + = 526.5.

[0886] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 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).

[0887] 1.13: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 115)

[0888]

[0889] The title compound was prepared starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0890] LC / MS: C 28 H 28 Calculated for C + H

[0891] 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).

[0892] 1.14: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxothiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 116)

[0893]

[0894] The title compound was prepared starting from compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ 6 -thiomorpholine-1,1-dione according to General Procedure 1. Purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0895] LC / MS: Calculated m / z = 557.6 (C 27 H 28(FN3O7S), measured value [M+H] + = 558.4

[0896] 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).

[0897] 1.15: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]hept-3-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 117)

[0898]

[0899] The title compound was prepared starting from Compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with gradient elution using 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0900] LC / MS: C 28 H 28 Calculated value of m / z for C + H

[0901] 11H 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).

[0902] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 118)

[0903]

[0904] The title compound was prepared from Compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0905] LC / MS: Calcd for m / z = 497.5 (C 26 H 25 F2N3O5), found [M + H] + = 498.4.

[0906] 1 1H 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).

[0907] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 119)

[0908]

[0909] The title compound was prepared from Compound 1.1 (10 mg) and azetidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a 10% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0910] LC / MS: C 26 H 26 Calculated m / z for C24H28FN3O5 = 479.5, found [M+H] + = 480.4.

[0911] 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).

[0912] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 120)

[0913]

[0914] The title compound was prepared starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to afford the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

[0915] LC / MS: calculated m / z = 543.5 (C 28 H 28 F3N3O5), found [M+H] + = 544.4.

[0916] 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).

[0917] 1.19: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-7-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 121)

[0918]

[0919] The title compound was prepared starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to afford the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0920] LC / MS: Calculated m / z for C 29 H 30 FN3O5 is 519.6, found [M+H] + = 520.4.

[0921] 1 1H 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).

[0922] 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]indazino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 122)

[0923]

[0924] The title compound was prepared from Compound 1.2 (10 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to afford the title compound as an off-white solid (0.8 mg, 7% yield).

[0925] LC / MS: C 23 H 22 Calculated for C21H23FN3O6S m / z = 487.1, found [M+H] + = 488.2.

[0926] 1 1H 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).

[0927] 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]indazino[1,2-b]quinolin-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (Compound 124)

[0928]

[0929] The title compound was prepared starting from Compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (5.0 mg, 17% yield).

[0930] LC / MS: C 29 H 25 Calculated for C26H24FN4O8S m / z = 608.1, found [M+H] + = 609.2.

[0931] 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).

[0932] 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]indazino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 125)

[0933]

[0934] The title compound was prepared starting from Compound 1.2 (10 mg) and benzenesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (9.8 mg, 73% yield).

[0935] LC / MS: C 28 H 24 Calculated for FN3O6S m / z = 549.6, found [M+H] + = 550.6.

[0936] 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).

[0937] 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]indazino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (Compound 1.23)

[0938]

[0939] The title compound was prepared from Compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride according to General Procedure 3. Purification of the title compound was completed using a 12 g C18 column and eluting with a gradient of 5% to 75% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (37.8 mg, 47% yield).

[0940] LC / MS: C 28 H 23 Calculated for FN4O8S m / z = 594.6, found [M+H] + = 595.2.

[0941] 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]indazino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 127)

[0942]

[0943] 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 H2 and then stirred for 45 minutes under a H2 atmosphere at room temperature. 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).

[0944] LC / MS: C 28 H 24 Calculated for C + H

[0945] 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).

[0946] 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]indazino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 129)

[0947]

[0948] The title compound was prepared from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a 25% to 50% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (1.3 mg, 13% yield).

[0949] LC / MS: C 24 H 24 Calculated for C + H

[0950] 1 1H 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).

[0951] 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]indazino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 131)

[0952]

[0953] To a solution of chlorosulfonyl isocyanate (3 μL) in dichloromethane (1 mL) was added tert-butanol (3 μL). The solution was stirred for 1 h, then compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 μL). The reaction mixture was stirred for 1 h and then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was completed as described in General Procedure 9, with gradient elution using 10% to 50% CH3CN / H2O + 0.1% TFA. Trifluoroacetic acid (200 μL) was added to the purified solid in dichloromethane (1 mL). The reaction mixture was stirred for 16 h and then concentrated to dryness to give the title compound as an off-white solid (7.5 mg, 48% yield).

[0954] LC / MS: C 22 H 21 Calculated for C21H22FN4O6S m / z = 488.1, found [M + H] + = 489.0.

[0955] 11H 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).

[0956] 1.27: (S)-((4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl) 4-nitrophenyl carbamate (Compound 1.27)

[0957]

[0958] The title PNP-carbamate intermediate compound was prepared starting from Compound 1.2 (24 mg) according to the first step of General Procedure 4. As described in General Procedure 9, purification was completed using a 12 g column C18 column with a gradient elution of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (14 mg, 53% yield).

[0959] LC / MS: C 29 H 23 Calculated for C24H23FN4O8S m / z = 574.2, found [M + H] + = 575.2

[0960] 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]indazino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (Compound 132)

[0961]

[0962] The title compound was prepared according to General Procedure 4, starting from Compound 1.2 (25 mg) and aqueous methylamine (500 μL, 40 wt% in water) as the primary amine. In this case, the crude intermediate PNP-carbamate was used. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (8.9 mg, 31% yield).

[0963] LC / MS: C 24 H 23 The calculated m / z of FN4O5 = 466.2, measured value [M+H] + = 467.2.

[0964] 1 H 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).

[0965] 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]indazino[1,2-b]quinolin-11-yl)methyl)urea (Compound 134)

[0966]

[0967] According to the second step of General Procedure 4, the title compound was prepared using Compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Purification by preparative HPLC was completed as described in General Procedure 9, with gradient elution using 12% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (0.6 mg, 20% yield).

[0968] LC / MS: C 30 H 28 The calculated m / z of FN5O5 = 557.2, measured value [M+H] + = 558.4.

[0969] 11H 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).

[0970] 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]indazino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (Compound 136)

[0971]

[0972] According to the second step of General Procedure 4, the title compound was prepared using Compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 10% to 50% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (2.4 mg, 66% yield).

[0973] LC / MS: C 25 H 25 Calculated for C24H27FN4O6 m / z = 496.2, found [M+H] + = 497.2.

[0974] 1 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).

[0975] 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]indazino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 138)

[0976]

[0977] 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 completed as described in General Procedure 9, eluting with a 20% to 50% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (3.5 mg, 6% yield).

[0978] LC / MS: C 24 H 22 Calculated for C23H24FN3O6 m / z = 467.2, found [M+H] + = 468.2.

[0979] 1 H 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).

[0980] 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]indazino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 139)

[0981]

[0982] The title compound was prepared according to General Procedure 5 starting from Compound 1.2 (18 mg) and reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a 10% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (4.2 mg, 19% yield).

[0983] LC / MS: C 25 H 24 The calculated m / z for FN3O7 = 497.2, measured value [M+H] + = 498.2.

[0984] 1 H 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).

[0985] Example 2: Preparation of a camptothecin analogue having a methoxy group at the C10 position

[0986] 2.1: 1-(2-Amino-4-fluoro-5-methoxyphenyl)-2-chloroethan-1-one (Compound 2.1)

[0987]

[0988] A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0 °C. First, a DCM solution of 1 M BCl3 (71 mL, 71 mmol) was added to this solution, then a DCM solution of 1 M chloro(diethyl)alane (71 mL, 71 mmol) was added, and finally 2-chloroacetonitrile (6.4 g, 85 mmol) was added. The solution was heated to reflux for 3 hours, cooled to room temperature and quenched by adding 2 M aqueous HCl. The resulting heterogeneous mixture was heated to reflux for 1 hour, cooled to room temperature, and then the pH was adjusted to about 12 with Na2CO3. 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 purified by flash chromatography eluting with 0% to 20% EtOAc / hexane as described in General Procedure 9 to give the title compound (6 g, 28 mmol, 39% yield).

[0989] LC / MS: The calculated m / z for C9H9ClFNO2 = 217.1, measured value [M+H] + = 218.1.

[0990] 11H 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)

[0991] 2.2: (S)-11-(Chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 2.2)

[0992]

[0993] 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]indazole-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, which precipitated as a yellow solid, was collected by filtration to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0994] LC / MS: C 22 H 18 Calculated for C19H18ClFN2O5 m / z = 445.2, found [M+H] + = 445.1.

[0995] 1 1H 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)

[0996] 2.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 101)

[0997]

[0998] The title compound was prepared from Compound 2.2 (10 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 41% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (5.6 mg, 20% yield).

[0999] LC / MS: C 26 H 26 Calculated m / z for C + H

[1000] 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).

[1001] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 103)

[1002]

[1003] The title compound was prepared from Compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 20% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (2.5 mg, 14% yield).

[1004] LC / MS: C 32 H 31 Calculated m / z for C + H

[1005] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 105)

[1006]

[1007] The title compound was prepared starting from Compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with gradient elution using 20% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (4.0 mg, 23% yield).

[1008] LC / MS: C 32 H 32 Calculated m / z for C + H FN5O7S = 649.2, found [M+H] 1

[1009] H 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).

[1010] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 107)

[1011]

[1012] The title compound was prepared starting from Compound 2.2 (10 mg) and N-methylpiperazine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, with gradient elution using 20% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (2.1 mg, 19% yield).

[1013] LC / MS: C 27 H 29 The calculated m / z of FN4O5 = 508.2, measured value [M+H] + = 509.4.

[1014] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 109)

[1015]

[1016] The title compound was prepared starting from Compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (3.2 mg, 20% yield).

[1017] LC / MS: C 32 H 32 The calculated m / z of FN5O5 = 585.2, measured value [M+H] + = 586.4.

[1018] 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).

[1019] 2.8: (S)-11-(Aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 2.8)

[1020]

[1021] 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 addition of iPr2NEt (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 about 1 / 2 volume, 1 M aqueous HCl (1.5 mL) was added, stirred for 5 min and then concentrated to give a brown residue. Purification was completed as described in General Procedure 9, using a 12 g C18 flash column eluted with a gradient of 5% to 40% CH3CN / H2O + 0.1% TFA to give the title compound as a pale yellow solid (179 mg, 75% yield).

[1022] LC / MS: C 22 H 20 Calculated for C21H20FN3O5 m / z = 425.4, found [M+H] + = 426.2

[1023] 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]indazino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 123)

[1024]

[1025] The title compound was prepared from compound 2.8 (10 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 5% to 65% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (8.5 mg, 91% yield).

[1026] LC / MS: C 23 H 22 Calculated for C22H22FN3O7S m / z = 503.1, found [M+H] + = 504.2.

[1027] 11H 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).

[1028] 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]indazino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 126)

[1029]

[1030] The title compound was prepared starting from Compound 2.8 (7.5 mg) and benzenesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 5% to 70% CH3CN / H2O + 0.1% TFA gradient, to give the title compound as an off-white solid (4.6 mg, 46% yield).

[1031] LC / MS: C 28 H 24 Calculated for C25H24FN3O7S m / z = 565.6, found [M + H] + = 566.2.

[1032] 1 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).

[1033] 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]indazino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (Compound 2.11)

[1034]

[1035] The title compound was prepared from Compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride according to General Procedure 3. Purification was completed using a 12 g C18 flash column eluted with a gradient of 5% to 75% CH3CN / H2O + 0.1% TFA to give the title compound as a pale yellow solid (9.7 mg, 71% yield) as described in General Procedure 9.

[1036] LC / MS: C 28 H 23 Calculated for C24H23FN4O9S m / z = 610.6, found [M+H] + = 611.5.

[1037] 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]indazino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 128)

[1038]

[1039] 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 H2 and then stirred for 45 minutes under a H2 atmosphere at room temperature. The mixture was filtered through a pad of Celite, washed with DMF, and then the filtrate was evaporated to give the title compound as a pale yellow solid (1.5 mg, 16% yield).

[1040] LC / MS: C 28 H 25 Calculated for C24H25FN4O7S m / z = 580.6, found [M+H] + = 581.4.

[1041] 11H 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 = 7.3 Hz, 3H), 0.90 (s, 1H).

[1042] 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]indazino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 130)

[1043]

[1044] The title compound was prepared starting from Compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 15% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (2.2 mg, 22% yield).

[1045] LC / MS: C 24 H 24 Calculated for C21H24FN3O8S m / z = 533.1, found [M+H] + = 534.2.

[1046] 1 1H 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).

[1047] 2.14: (S)-((4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl) 4-nitrophenylcarbamate (Compound 2.14)

[1048]

[1049] According to the first step of General Procedure 4, starting from Compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as the solvent, the title PNP-carbamate intermediate compound was prepared. Flash purification was completed as described in General Procedure 9, using a 12 g C12 column eluted with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (61 mg, 86% yield). This intermediate was separated and used to generate the following compounds.

[1050] LC / MS: C 29 H 23 Calculated for C24H23F4N4O9 m / z = 590.1, found [M+H] + = 591.2.

[1051] 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]indazino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (Compound 133)

[1052]

[1053] 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, the title compound was prepared. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (5.8 mg, 47% yield).

[1054] LC / MS: C 24 H 23 Calculated for C23H25F4N4O6 m / z = 482.2, found [M+H] + = 483.2.

[1055] 11H 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).

[1056] 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]indazino[1,2-b]quinolin-11-yl)methyl)urea (Compound 135)

[1057]

[1058] According to the second step of General Procedure 4, the title compound was prepared using Compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 20% to 60% CH3CN / H2O + 0.1% TFA gradient to give the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

[1059] LC / MS: C 30 H 28 Calculated for C29H33FN5O6 m / z = 573.2, found [M+H] + = 574.2.

[1060] 1 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).

[1061] 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]indazino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (Compound 137)

[1062]

[1063] According to the second step of General Procedure 4, the title compound was prepared using compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 12% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (1.5 mg, 20% yield).

[1064] LC / MS: C 25 H 25 Calculated for C + H

[1065] 1 H 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).

[1066] Example 3: Preparation of a camptothecin analogue having an amino group at the C10 position

[1067] 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (Compound 3.1)

[1068]

[1069] To a stirred solution of HNO3 (121.2 mL, 67% purity, 2.0 equiv) in H2SO4 (500 mL) at 0 °C was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 equiv). After addition was complete, the ice bath was removed and the reaction mixture was stirred at 25 °C for 5 h. The mixture was poured into ice (5 L), filtered, and then dried in vacuo. The title compound was obtained as a yellow solid (219 g).

[1070] 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).

[1071] 3.2: tert-Butyl (2-fluoro-5-formyl-4-nitrophenyl)carbamate (Compound 3.2)

[1072]

[1073] A mixture of Compound 3.1 (219 g, 1.0 equiv), tert-butyl carbamate (124 g, 1.2 equiv), Cs2CO3 (575 g, 2 equiv), Pd2(dba)3 (40 g, 0.05 equiv), and XPhos (84 g, 0.2 equiv) in toluene (2000 mL) was degassed and purged with N2 for three cycles. The mixture was then stirred at 90 °C under a N2 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), then 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 afford the title compound as a yellow solid (140 g, 56% yield).

[1074] 1 1H 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)

[1075] 3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (Compound 3.3)

[1076]

[1077] To a solution of Compound 3.2 (100 g, 1.0 equiv) in H2O (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 in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1), TLC (petroleum ether) to afford the title compound as a yellow solid (19.0 g, 21% yield).

[1078] LC / MS: C 12 H 15 Calculated for C12H15FN2O3 m / z = 254.1, found [M + H] + = 255.0.

[1079] 1 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.57 (s, 1H), 7.58 (d, J = 4.8 Hz, 1H), 7.21 (s, 2H), 6.53 (d, J = 12.8 Hz, 1H), 1.43 (s, 9H).

[1080] 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]indazino[1,2-b]quinolin-9-yl)carbamate (Compound 3.4)

[1081]

[1082] 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]indazine-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, filtered, and the solid was washed with methyl tert-butyl ether (30 mL) and then dried in vacuo. The title compound as a yellow solid was obtained (4.5 g, 62% yield).

[1083] LC / MS: C 25 H 24 Calculated for C22H25FN3O6 m / z = 481.2, found [M + H] + = 482.1.

[1084] 1 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)

[1085] 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]indazino[1,2-b]quinolin-9-yl)carbamate (Compound 3.5)

[1086]

[1087] To the mixture of compound 3.4 (4.00g) in MeOH (360mL), FeSO4 (heptahydrate, 1.2g), H2SO4 (280mL) in H2O (4mL) were added. The reaction mixture was heated at 65°C, while H2O2 (24mL, 30% purity) was added dropwise within 30 minutes, and then stirred for 0.5 hours. The reaction solution was cooled to 25°C, then filtered to obtain the title compound (1.53g, 33.2% yield) as a yellow solid. H2O (400mL) was added to the filtrate, and then quenched with a saturated Na2S2O3 aqueous solution. The pH was adjusted to 7 to 8 with a saturated Na2CO3 aqueous solution, and the solution was then concentrated and filtered. The solid was ground with MeOH (30mL) at 55°C for 1 hour, and then filtered to obtain the second batch of brown solid title compound (1.09g, 26% yield).

[1088] LC / MS: C 26 H 26 Calculated value for FN3O7 m / z = 511.2, found value [M+H] + =512.2.

[1089] 1 H NMR(300MHz,d6-DMSO)δ9.47(s,1H),8.47(d,J=7.6Hz,1H),7.94(d,J=12.0Hz,1H),7.29(d,J=1.6Hz,1H),6.49(s,1H),5 .86-5.76(m,1H),5.42(s,2H),5.38(s,2H),5.16(d,J=4.4Hz,2H),1.90-1.83(m,2H),1.52(s,9H),0.88(t,J=6.4Hz,3H).

[1090] 3.6: (S)-tert-butyl (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)

[1091]

[1092] To a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol) was added DCM (2.9 mL), 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 silica gel. Flash purification was completed as described in General Procedure 9, using a 10 g silica gel column eluted with 0% to 10% DCM / MeOH to give the title product as an orange powder (42.5 mg, 28%).

[1093] LC / MS: C 26 H 24 Calculated for C + H

[1094] 1 H 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).

[1095] 3.7: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 140)

[1096]

[1097] The title compound was prepared starting from compound 3.4 (40 mg) according to General Procedure 6 to give the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[1098] LC / MS: C 20 H16 The calculated m / z of FN3O4 = 381.1, the measured value [M+H] + = 382.2.

[1099] 1 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).

[1100] 3.8: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 141)

[1101]

[1102] The title compound was prepared starting from Compound 3.5 (5 mg) according to General Procedure 6 to give the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).

[1103] LC / MS: C 21 H 18 The calculated m / z of FN3O5 = 411.2, the measured value [M+H] + = 412.2.

[1104] 1 1H 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).

[1105] 3.9: (S)-(11-(Chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-9-yl)carbamic acid tert-butyl ester (Compound 3.9)

[1106]

[1107] 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, a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added again. After another 1 h, the reaction mixture 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 subsequent reaction without further purification.

[1108] LC / MS: C 26 H 25 Calculated for C + H

[1109] 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]indazino[1,2-b]quinolin-9-yl)carbamate (Compound 3.10)

[1110]

[1111] To a solution of compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg), and then DIPEA (99 μL). The solution was heated at 60 °C for 16 h and then concentrated to dryness in vacuo. Flash purification was completed as described in General Procedure 9 using a 12 g C18 column eluted with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

[1112] LC / MS: C 26 H 27 Calculated for C + H

[1113] 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).

[1114] 3.11: (S)-9-Amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 145)

[1115]

[1116] The title compound was prepared starting from Compound 3.10 (2.1 mg) according to General Procedure 6, giving the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[1117] LC / MS: C 21 H 19 Calculated m / z for C + H

[1118] 1 H 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).

[1119] Example 3.12: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 3.12)

[1120]

[1121] The title compound was prepared from Compound 3.9 (150 mg) and morpholine according to General Procedure 1. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, giving the title compound as a red solid (TFA salt, 103 mg, 52% yield).

[1122] LC / MS: C 30 H 33 Calculated m / z for C + H

[1123] 1 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).

[1124] 3.13: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 142)

[1125]

[1126] The title compound was prepared starting from Compound 3.12 (45 mg) according to General Procedure 6 to give the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[1127] LC / MS: C 25 H 25 Calculated for C + H

[1128] 1 1H 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).

[1129] 3.14: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 148)

[1130]

[1131] To a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol) was added dichloromethane (1.45 mL), 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 adding water + 0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was completed as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5% to 40% CH3CN / H2O + 0.1% TFA gradient to afford the Boc-protected intermediate as a yellow powder. The 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).

[1132] LC / MS: C 26 H 27 Calculated for C24H28FN4O4 m / z = 478.2, found [M+H] + = 479.4.

[1133] 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).

[1134] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 149)

[1135]

[1136] To a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol), dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and N-methylpiperazine (4.90 μL, 0.044 mmol) were added. The solution was stirred at room temperature for 4 h, then sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) was added and stirring was continued for an additional 45 min. Excess hydride was quenched by addition of 0.1% aqueous TFA (0.5 mL). Purification was completed as described in General Procedure 9, using a 12 g C18 flash column and eluting with a 5% to 40% CH3CN / H2O + 0.1% TFA gradient to afford the Boc-protected intermediate as a yellow powder. Deprotection of this intermediate according to General Procedure 6 gave the title product as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

[1137] LC / MS: C 26 H 28 Calculated for C25H27FN5O4 m / z = 493.2, found [M+H] + = 494.4.

[1138] 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).

[1139] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 153)

[1140]

[1141] According to General Procedure 1, the Boc-protected precursor of the title compound was prepared starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC was completed as described in General Procedure 9, eluting with a gradient of 35% to 44% CH3CN / H2O + 0.1% TFA 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 (TFA salt, 2.4 mg, 17% yield, over 2 steps).

[1142] LC / MS: C 31 H 30 Calculated for C + H

[1143] 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).

[1144] 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]indazino[1,2-b]quinolin-11-yl)methyl)acetamide (Compound 147)

[1145]

[1146] According to General Procedure 2, and then according to General Procedure 6, the title compound was prepared starting from compound 3.10 (8 mg) and acetic acid. Preparative HPLC purification of the Boc-protected intermediate compound was completed as described in General Procedure 9, eluting with a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a red solid (4.0 mg, 56% yield).

[1147] LC / MS: C 23 H 21 Calculated for C + H

[1148] 11H 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).

[1149] 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]indazino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 146)

[1150]

[1151] According to General Procedure 3, followed by General Procedure 6, the title compound was prepared starting from Compound 3.10 (8 mg) and methanesulfonyl chloride. The preparative HPLC purification of the intermediate Boc-protected compound was completed as described in General Procedure 9, with a gradient elution of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a red solid (4.4 mg, 57% yield).

[1152] LC / MS: C 22 H 21 Calculated for C21H24FN4O6S m / z = 488.1, found [M+H] + = 489.2.

[1153] 1 1H 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).

[1154] 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]indazino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 150)

[1155]

[1156] According to General Procedure 3, and then according to General Procedure 6, the title compound was prepared starting from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride. The preparative HPLC purification of the intermediate Boc-protected compound was completed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a red solid (1 mg, 16% yield).

[1157] LC / MS: C 23 H 23 Calculated m / z for C + H

[1158] 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, 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).

[1159] 3.20: (S)-((9-Amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl) 4-nitrophenylcarbamate (Compound 3.20)

[1160]

[1161] 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 about 30 minutes and then used directly in the subsequent reaction.

[1162] 3.21: (S)-((9-Amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl) methylcarbamate (Compound 143)

[1163]

[1164] The title compound was prepared by adding MeOH (100 μL) to a 200 μL solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was completed as described in General Procedure 9, using a 10% to 60% CH3CN / H2O+0.1% TFA gradient elution. The title compound (2.1 mg, 47% yield) was obtained as a red solid according to General Procedure 6.

[1165] LC / MS: C 23 H 21 Calculated value for FN4O6 m / z = 468.4, found value [M+H] + =468.3.

[1166] 1 H NMR(300MHz,10% D2O / CD3CN) δ7.72(d,J=12.2Hz,1H),7.41(d,J=18.1Hz,1H),6.96(s,1H),5.52(d,J=3.6Hz,1H),5.39–5.2 3(m,3H),4.82(s,1H),4.73(s,1H),3.63(d,J=1.2Hz,3H),1.56(s,3H),1.27(s,2H),0.94(t,J=7.4Hz,3H).

[1167] 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)

[1168]

[1169] The title compound was prepared by adding methylamine hydrochloride (10 mg) to a solution of 200 μL of compound 3.20 followed by iPr2NEt (5 μL). The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was completed as described in General Procedure 9, using a 10% to 60% CH3CN / H2O+0.1% TFA gradient elution. The title compound (2.9 mg, 64.5% yield) was obtained as a red solid according to General Procedure 6.

[1170] LC / MS: C 23 H 21 Calculated value for FN5O5 m / z = 467.5, found value [M+H]+ =468.5.

[1171] 1 H NMR(300MHz,10% D2O / CD3CN) δ8.13(d,J=9.2Hz,1H),7.92(s,1H),7.73(d,J=12.3Hz,1H),7.52–7.35(m,2H),6.94(d,J=9.2Hz,2H),5.55(d,J =16.5Hz,2H),5.44–5.27(m,4H),4.85(s,2H),4.78(s,1H),1.56(d,J=2.5Hz,3H),1.27(s,2H),0.93(q,J=11.7,9.5Hz,3H).

[1172] 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)

[1173]

[1174] The title compound was prepared by adding ethanolamine (100 μL) to a 200 μL solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was completed as described in General Procedure 9, using a 10% to 60% CH3CN / H2O+0.1% TFA gradient elution. The title compound (0.5 mg, 8.5% yield) was obtained as a red solid according to General Procedure 6.

[1175] LC / MS: C 24 H 24 Calculated value for FN5O6 m / z = 497.5, found value [M+H] + =498.5.

[1176] 1 H NMR(300MHz,10% D2O / CD3CN)δ7.77–7.61(m,1H),7.48–7.30(m,2H),5.53(d,J=16.3Hz,1H),5.31(d,J=15.4Hz,1H),5.19(s,2H),4.6 9(s,2H),3.97(dd,J=6.6,4.9Hz,2H),3.39(t,J=5.8Hz,2H),2.93(s,1H),2.01-1.83(m,2H),0.94(t,J=7.3Hz,3H).

[1177] 3.24: (S)-9-Amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 152)

[1178]

[1179] To a stirred solution of Compound 3.5 (100 mg) in 2 mL of dichloromethane was added thionyl chloride (35 mL, 2.5 equiv). The solution was stirred at room temperature for 20 minutes, and then thionyl chloride (35 mL, 2.5 equiv) was added again. After 20 minutes, toluene (1 mL) was added, and then 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 completed as described in General Procedure 9, with elution using a 5% to 50% CH3CN / H2O + 0.1% TFA gradient, to afford the title compound as an off-white solid (20 mg, 23% yield).

[1180] LC / MS: C 21 H 17 Calculated for C20H19FN6O4 m / z = 436.1, found [M+H] + = 437.2.

[1181] 1 1H 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).

[1182] 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]indazino[1,2-b]quinolin-11-yl)methyl)acetamide (Compound 164)

[1183]

[1184] The title compound was prepared starting from compound 145 (10 mg) and glycolic acid according to General Procedure 2. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).

[1185] LC / MS: C 23 H 21 Calculated m / z for C + H

[1186] 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).

[1187] 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]indazino[1,2-b]quinolin-11-yl)methyl)-3-methylthiourea (Compound 161)

[1188]

[1189] To a solution of compound 145 (9 mg, 1.0 equiv) in DMF (1 mL) was added thiocarbonyl diimidazole (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 observed. Then methylammonium chloride (3 mg, 2.0 equiv) was added and the reaction mixture was heated at 60 °C for 30 min. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a yellow solid (2.3 mg, 22% yield).

[1190] LC / MS: C 23 H 22 Calculated m / z for C + H

[1191] 1 1H 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).

[1192] 3.27: (S)-((9-Amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)methyl)thiocarbamic acid S-(2-hydroxyethyl) ester (Compound 160)

[1193]

[1194] The title compound was prepared starting from Compound 145 (10 mg) and 2-mercaptoethanol according to General Procedure 5. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).

[1195] LC / MS: C 24 H 23 Calculated for C24H25FN4O6S m / z = 514.1, found [M+H] + = 515.2.

[1196] 1 1H 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).

[1197] 3.28: (S)-9-Amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 154)

[1198]

[1199] To a 5 mL flask containing compound 140 (50 mg) was added water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg), and propionaldehyde (74 μL). The resulting suspension was cooled to -15 °C using an ice-salt bath, and then sulfuric acid (0.40 mL) was added dropwise. Then hydrogen peroxide (95 μL) was 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). Then the organic phase was evaporated to dryness. Preparative HPLC purification was completed as described in General Procedure 9, where elution was carried out with a 25% to 70% CH3CN / H2O + 0.1% TFA gradient to give the title compound as a dark orange solid (2.4 mg, 4.4% yield).

[1200] LC / MS: C 22 H 20 Calculated for C21H22FN3O4 m / z = 410.1, found [M+H] + = 410.2.

[1201] 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).

[1202] 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]indazino[1,2-b]quinolin-9-yl)carbamate (Compound 3.29)

[1203]

[1204] At -20 °C, compound 3.5 (15 mg) was added to a 5 mL conical flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL). 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, then heated at 70 °C for 1 hour. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was completed as described in General Procedure 9, with elution using a 40% to 55% CH3CN / H2O + 0.1% TFA gradient, to give the title compound as a dark orange solid (5.1 mg, 31% yield).

[1205] LC / MS: C 27 H 27 Calculated for C + H

[1206] 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).

[1207] 3.30: Methyl (S)-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-11-yl)carbamate (Compound 169)

[1208]

[1209] The title compound was prepared starting from compound 3.29 (5.1 mg) according to General Procedure 6, to give the title compound as a yellow powder (TFA salt, 3.8 mg, 73% yield).

[1210] LC / MS: C 22 H 19 Calculated for C + H

[1211] 11H 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).

[1212] 3.31: ((S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 155)

[1213]

[1214] 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 refluxed and stirred 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 MgSO4. Preparative HPLC purification was completed as described in General Procedure 9, with a gradient elution of 25% to 40% CH3CN / H2O + 0.1% TFA, to give the title compound as a dark orange solid (5.1 mg, 16% yield).

[1215] LC / MS: C 22 H 20 Calculated for C19H18FN3O5 m / z = 426.1, found [M + H] + = 426.2.

[1216] 1 1H 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).

[1217] 3.32: (4S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(((1R,5S)-6-hydroxy-3-azabicyclo[3.1.1]hept-3-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 158)

[1218]

[1219] To a 5 mL conical flask containing Compound 3.6 (15 mg), dichloromethane (0.6 mL) was added, followed by 3-azabicyclo[3.1.1]heptan-6-ol (10 mg) and acetic acid (7.6 μL). The reaction mixture was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by adding water + 0.1% TFA and diluted with DMF. Then the reaction mixture was partially evaporated. Purification by preparative HPLC was completed as described in General Procedure 9, with gradient elution using 20% to 50% CH3CN / H2O + 0.1% TFA, to give 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, with gradient elution using 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound as a yellow powder (TFA salt, 7.1 mg, 39% yield).

[1220] LC / MS: C 27 H 27 Calculated for C24H25FN4O5 m / z = 507.2, found [M+H] + = 507.4.

[1221] 1 1H 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).

[1222] 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]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 159)

[1223]

[1224] To a 5 mL conical flask containing Compound 3.6 (15 mg), dichloromethane (0.6 mL) was added, followed by (3-fluoroazetidin-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction mixture was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by adding water + 0.1% TFA, diluted with DMF, and then partially evaporated. Purification by preparative HPLC was completed as described in General Procedure 9, with gradient elution using 20% to 50% CH3CN / H2O + 0.1% TFA to give 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, with gradient elution using 20% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as a yellow powder (TFA salt, 1.8 mg, 10% yield).

[1225] LC / MS: C 25 H 24 Calculated for F2N4O5 m / z = 499.2, found [M+H] + = 499.4.

[1226] 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).

[1227] 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]indazino[1,2-b]quinoline-9-yl)carbamate (Compound 3.34)

[1228]

[1229] 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 completed as described in General Procedure 9, using a 30 g C18 column and eluting with a gradient of 10% to 65% CH3CN / H2O + 0.1% TFA to give the title compound as a yellow solid (15.0 mg, 7.2% yield).

[1230] LC / MS: C 27 H 29 Calculated for C + H

[1231] 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]indazino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-N-methylacetamide (Compound 165)

[1232]

[1233] The Boc-protected form of the title compound was prepared from Compound 3.34 (6.4 mg) and glycolic acid according to General Procedure 2. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA. Deprotection was then carried out according to General Procedure 6 to give the title compound as a yellow powder (TFA salt, 2.0 mg, 28% yield).

[1234] LC / MS: C 24 H 23 Calculated for C + H

[1235] 1 H 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).

[1236] 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]indazino[1,2-b]quinolin-11-yl)methyl)-N-methylmethanesulfonamide (Compound 166)

[1237]

[1238] According to General Procedure 3, the Boc-protected form of the title compound was prepared starting from Compound 3.34 (8.0 mg) and methanesulfonyl chloride. Preparative HPLC purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA. Then deprotection was carried out according to General Procedure 6 to give the title compound as a yellow powder (TFA salt, 2.6 mg, 34% yield).

[1239] LC / MS: C 23 H 23 Calculated m / z for C21H24FN4O6S = 502.1, found [M + H] + = 503.2.

[1240] 1 H 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).

[1241] 3.37: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 170)

[1242]

[1243] 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. While stirring in an ice-salt bath at -15 °C, sulfuric acid (0.495 mL) was added dropwise to the resulting suspension. Then, hydrogen peroxide (0.118 mL) was added dropwise. The mixture was stirred at -15 °C for 10 minutes, then allowed to warm to room temperature and stirred for 1 hour. Then the reaction mixture was 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 completed as described in General Procedure 9, where elution was performed with a 25% to 45% CH3CN / H2O + 0.1% TFA gradient to give the title compound as a dark orange solid (TFA salt, 3.1 mg, 4.4% yield).

[1244] LC / MS: C 23 H 22 Calculated m / z for C + H

[1245] 1 H 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).

[1246] 3.38: (S)-N-(4-Ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-9-yl)acetamide (Compound 171)

[1247]

[1248] 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 for 5 minutes at room temperature, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). The solution was stirred at room temperature for 24 hours and then purified directly by preparative HPLC as described in General Procedure 9, eluting with a 25% to 45% CH3CN / H2O + 0.1% TFA gradient to give the title compound as a bright yellow powder (43.0 mg, 38% yield).

[1249] LC / MS: C 22 H 18 Calculated for C19H16FN3O5 m / z = 424.1, found [M+H] + = 424.2.

[1250] 1 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 = 9.12 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).

[1251] tert-Butyl (5-formyl-2-methoxy-4-nitrophenyl)carbamate (Compound 3.39)

[1252]

[1253] To a solution of compound 3.2 (1.3 g, 1.0 equiv) in MeOH (12 mL) at 0 °C was added sodium methoxide (0.74 g, 3.0 equiv). After addition was complete, the ice bath was removed and the resulting solution was stirred at room temperature for 72 hours. The reaction was then quenched with ice water (50 mL) and extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give the title compound as an orange solid (1.2 g, 89% yield).

[1254] LC / MS: C 13 H 16 Calculated for C12H12N2O6 m / z = 296.10, found [M+H] + = 297.1.

[1255] 1 1H 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)

[1256] 3.40: tert-Butyl (4-amino-5-formyl-2-methoxyphenyl)carbamate (Compound 3.40)

[1257]

[1258] To a solution of Compound 3.39 (500 mg, 1 equiv) in MeOH (10 mL) and H2O (1 mL) was added B2(OH)4 (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 minutes. The reaction mixture was stirred for an additional 5 minutes and then quenched by pouring the solution into 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 completed as described in General Procedure 9, using a 25 g silica gel column and eluting with 10% to 50% hexane / EtOAc to afford the title compound as an orange solid (386 mg, 86%).

[1259] LC / MS: C 13 H 18 Calculated for C13H15N2O4 m / z = 266.1, found [M + H] + = 297.2.

[1260] 3.41: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 168)

[1261]

[1262] At 110 °C, a mixture of compound 3.40 (385 mg, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazole-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 for 2 h. The reaction mixture was then cooled to 25 °C and concentrated in vacuo. Purification was completed as described in General Procedure 9, where a 25 g silica gel column was used and 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, where elution was carried out with a 20% to 65% CH3CN / H2O + 0.1% TFA gradient to give the title compound as a red solid (TFA salt, 300 mg, 53% yield).

[1263] LC / MS: C 21 H 19 Calculated for C19H16N3O5 m / z = 393.2, found [M+H] + = 393.2.

[1264] 1 1H 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).

[1265] 3.42: 5-Bromo-2-nitro-4-(trifluoromethyl)benzaldehyde (Compound 3.42)

[1266]

[1267] To a stirred solution of HNO3 (2.0 g, 1.4 mL, 67% purity, 2 equiv) in H2SO4 (8 mL) at 0 °C was added 3-bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 equiv). After addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 5 h. The mixture was poured into 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 give the title compound as a yellow solid (4.4 g, 93% yield).

[1268] LC / MS: Calculated m / z for C8H3BrF3NO3 = 296.90, found [M+H] + = 298.0.

[1269] 1 H NMR (300 MHz, MeOD) δ 10.35 (s, 1H), 8.29 (s, 1H), 8.23 (s, 1H).

[1270] 3.43: tert-Butyl (5-formyl-4-nitro-2-(trifluoromethyl)phenyl)carbamate (Compound 3.43)

[1271]

[1272] A mixture of Compound 3.42 (800 mg, 1 equiv), tert-butyl carbamate (378 mg, 1.2 equiv), Cs2CO3 (1.7 g, 2 equiv), Pd2(dba)3 (122 mg, 0.05 equiv), and dicyclohexyl[2',4',6'-tris(prop-2-yl)[1,1'-biphenyl]-2-yl]phosphane ( XPhos) (256 mg, 0.2 equiv) in toluene (5 mL) was degassed and purged with N2 for three cycles. The mixture was then stirred at 90 °C under a N2 atmosphere for 15 h. The reaction mixture was diluted with H2O (25 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (2 × 25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was completed according to General Procedure 9, using a 25 g silica gel column and eluting with 0% to 25% DCM / MeOH to afford the title compound as an orange solid (750 mg, 84% yield).

[1273] LC / MS: C 13 H 13 FN2O5 calculated m / z = 334.1, found [M-H] - = 333.1.

[1274] 3.44: tert-Butyl (4-amino-5-formyl-2-(trifluoromethyl)phenyl)carbamate (Compound 3.44)

[1275]

[1276] To a solution of Compound 3.43 (750 mg, 1 equiv) in MeOH (16 mL) and H2O (1.6 mL) was added B2(OH)4 (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 and then quenched by pouring the solution into 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 completed as described in General Procedure 9 using a 25 g silica gel column eluting with 10% to 50% hexanes / EtOAc to afford the title compound as an orange solid (460 mg, 67%).

[1277] LC / MS: C 13 H 15 Calculated for C + H

[1278] 3.45: (S)-9-Amino-4-ethyl-4-hydroxy-8-(trifluoromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione (Compound 167)

[1279]

[1280] At 110 °C, a mixture of Compound 3.44 (460 mg, 1 equiv) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazole-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 for 2 h. The reaction mixture was then cooled to 25 °C and concentrated in vacuo. Purification was completed as described in General Procedure 9 using a 25 g silica gel column eluting with a 0% to 50% DCM / MeOH gradient to afford the Boc-protected intermediate as a red solid. This material was then deprotected according to General Procedure 6 and subsequently purified by preparative HPLC as described in General Procedure 9 eluting with a 20% to 65% CH3CN / H2O + 0.1% TFA gradient to afford the title compound as a yellow solid (6.2 mg, 48%).

[1281] LC / MS: C 21 H 16 Calculated for C + H

[1282] 1 1H 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).

[1283] Example 4: Preparation of drug-linker

[1284] 4.1: ((9H-Fluoren-9-yl)methoxy)carbonylglycylglycine 2,5-dioxopyrrolidin-1-yl ester (Compound 4.1)

[1285]

[1286] The title compound was prepared according to the procedure described in Chinese Patent Publication No. CN105218644.

[1287] 4.2: ((9H-Fluoren-9-yl)methoxy)carbonylglycylglycyl-L-phenylalanine (Fmoc-GGF-OH; Compound 4.2)

[1288]

[1289] To a solution of L-phenylalanine (965 mg) in acetonitrile (10 mL) and dimethylformamide (0.5 mL) was added DIPEA (1.51 mL), and then Compound 4.1 (1.3 g) was added. After 1 hour, the reaction mixture was concentrated to dryness. Flash purification was completed as described in General Procedure 9, eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound as a white solid (430 mg, 30% yield).

[1290] LC / MS: C 28 H 71 Calculated for C25H24N3O6S m / z = 501.2, found [M+H] + = 502.4.

[1291] 11H NMR (300 MHz, DMSO) δ 8.16 (d, J = 8.1 Hz, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.59 (t, J = 6.0 Hz, 1H), 7.54–7.39 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.28–7.13 (m, 5H), 4.44 (td, J = 8.5, 5.1 Hz, 1H), 4.33–4.13 (m, 3H), 3.83–3.59 (m, 4H), 3.06 (dd, J = 13.7, 5.1 Hz, 1H), 2.88 (dd, J = 13.8, 9.0 Hz, 1H).

[1292] 4.3: 2,3,5,6-Tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propionate (MT-OTfp; Compound 4.3)

[1293]

[1294] The title compound was prepared according to the procedure described in International Patent Publication No. WO 2017 / 054080.

[1295] 4.4: (3-(2-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoyl)glycylglycyl-L-phenylalanine (Compound 4.4)

[1296]

[1297] To a solution of Compound 4.3 (1.61 g, 3.58 mmol) in DMF (35 mL) was added...

Claims

1. An antibody-drug conjugate having the formula (X): T-[L-(D) m n ​ (X) Wherein: m is an integer between 1 and 4; n is an integer between 1 and 10; T is an anti-GPC3 (glypican-3) antibody construct comprising an antigen-binding domain that binds to human GPC3, and the antigen-binding domain comprises: c) a heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO: 6, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO: 7, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO: 8, and d) a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO: 18, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO: 19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO: 17; L is a linker, and D is a compound of formula I: Wherein: R 1 selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2, and R 2 selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3, And wherein: When R 1 is -NH2, then R is R 3 or R 4 , and when R 1 is not -NH2, then R is R 4 ; R 3 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 4 Selected from: R 5 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl and –(C1-C6 alkyl)-aryl; R 6 and R 7 each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl and -C(O)R 17 ; R 8 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 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 10’ selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and R 13 Selected from: -H and -C1-C6 alkyl; R 14 and R 14’ each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 17 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 bonded form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the following: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-O-R 5 ; R 24 、R 25 and R 26 each is -C1-C6 alkyl; X a and X b each independently selected from: NH, O, and S, and X c Selected from: O, S, and S(O)2, Provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-3,14(4H)-dione.

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

28.

3. The antibody-drug conjugate according to claim 1 or 2, wherein D is a compound of formula (IV): Wherein: R 1a Selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3 and -NH2; R 2a Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3; X is -O-, -S- or -NH-, and R 4a is selected from: 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: R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and –(C1-C6 alkyl)-aryl; or R 9a is absent, and X b = X; Each R 10a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl and 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 selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16a and R 13a selected from: -H and -C1-C6 alkyl; R 14a selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16a selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 21 selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl and –(C1-C6 alkyl)-O-R 5a ; R 22 and R 23 each independently selected from: -H, -halogen, -C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 、R 25 and R 26 each is -C1-C6 alkyl; X a and X b each independently selected from: NH, O, and S; X c selected from: O, S, and S(O)2, and Indicates the connection point to the joint L.

4. The antibody-drug conjugate according to claim 3, wherein R 1a is selected from: -CH3, -CF3, -OCH3, -OCF3, and -NH2.

5. The antibody-drug conjugate according to claim 3, wherein R 1a is selected from: -CH3, -OCH3, and NH2.

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

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

8. The antibody-drug conjugate according to claim 1 or 2, wherein D is a compound of formula (V): Wherein: R 2a Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; R 20a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, R 5 selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5 , -C3-C8 heterocycloalkyl, and -C(O)R 17 ; R 8 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 is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, and -NR 14 R 14’ ; Each R 10’ is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16 and R 13 selected from: -H and -C1-C6 alkyl; R 14 and R 14’ each independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 17 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 bonded form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the following: halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-O-R 5 ; R 24 、R 25 and R 26 each is -C1-C6 alkyl; X a and X b each independently selected from: NH, O, and S; X c selected from: O, S, and S(O)2, and Indicates the connection point to the joint L.

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

10. The antibody-drug conjugate according to claim 8 or 9, wherein R 20a is selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5 , -(C1-C6 alkyl)-aryl, 11. The antibody-drug conjugate according to claim 1 or 2, wherein D is a compound of formula (VI): Wherein: R 2a selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3; X is -O-, -S- or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a , -CO2R 8a , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, where * is the connection point with X, and where p is 1, 2, 3, or 4; or X is O, and R 25 -X- is selected from: R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 6a selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 7a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-O-R 5a , -C3-C8 heterocycloalkyl and -C(O)R 17a ; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; or R 9a is absent, and X b = X; Each R 10a is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl and 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 selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, –(C1-C6 alkyl)-aryl, -S(O)2R 16a and R 13a Selected from: -H and -C1-C6 alkyl; R 14a selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 17a 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 Selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl and –(C1-C6 alkyl)-O-R 5a ; 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 each is -C1-C6 alkyl; X a and X b each independently selected from: NH, O, and S; X c selected from: O, S and S(O)2, and Indicates the connection point to the joint L.

12. The antibody-drug conjugate according to claim 11, wherein R 2a is selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3.

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

14. The conjugate according to any one of claims 11 to 13, wherein X is -O-, -S- or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a , -(C1-C6 alkyl)-aryl, or X is O, and R 25 -X- is selected from:

15. The conjugate according to any one of claims 11 to 13, wherein X is -O-, -S- or -NH-, and R 25 is selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-O-R 5a , -(C1-C6 alkyl)-aryl, 16. The antibody-drug conjugate according to any one of claims 1 to 15, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one or more substituents selected from: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, acylamino, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

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

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

19. The antibody-drug conjugate according to claim 1 or 2, wherein D is Compound 139 or Compound 141.

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

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

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

23. The antibody-drug conjugate according to any one of claims 20 to 22, wherein L has: (a) Formula (XI) Wherein: Z is a functional group capable of reacting with a target group on the anti-GPC3 antibody construct T; Str is a spacer group; AA1 and AA2 are each independently an amino acid, where AA1-[AA2] r forms a protease cleavage site; X is a self-degrading 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 the camptothecin analogue D, or (b) Formula (XII) Wherein: Z is a functional group capable of reacting with a target group on the anti-GPC3 antibody construct T; Str is a spacer group; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r forms a protease cleavage site; Y is -NH-CH2-; 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 the camptothecin analogue D.

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

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

26. The antibody-drug conjugate according to claim 1 or 2, wherein L-(D) in formula (X) is: MC-GGFG-AM-Compound 139 MT-GGFG-AM-Compound 139 MT-GGFG-AM-Compound 141 MC-GGFG-AM-Compound 141 MT-GGFG-Compound 141 MC-GGFG-Compound 141 27. The antibody-drug conjugate according to any one of claims 1 to 26, wherein m is between 1 and 2.

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

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

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

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

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

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

38.

34. The antibody-drug conjugate according to claim 33, wherein L-(D) in formula (X) is: MC-GGFG-AM-Compound 139 35. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier or diluent.

36. A method of inhibiting cancer cell proliferation, which comprises contacting the cells with an effective amount of the antibody-drug conjugate according to any one of claims 1 to 34.

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

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

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

40. The antibody-drug conjugate according to any one of claims 1 to 34, wherein the antibody-drug conjugate is used in the treatment of cancer.

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

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

43. An antibody-drug conjugate having the following structure: Wherein: n is between 4 and 8, and T is an anti-GPC3 (glypican-3) antibody construct comprising an antigen-binding domain that binds to human GPC3, and the antigen-binding domain comprises: a) a heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:8, and b) a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:18, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:

17.

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

30.

45. An antibody-drug conjugate having the following structure: Wherein: n is between 1 and 10, and T is an anti-GPC3 (glypican-3) antibody construct comprising an antigen-binding domain that binds to human GPC3, and the antigen-binding domain comprises: c) a heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:6, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:7, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:8, and d) i) a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:71, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:17; or ii) a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:74, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:17; or iii) a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:77, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:19, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:

17.

46. The antibody-drug conjugate according to claim 45, wherein the antigen-binding domain comprises: a) A VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:29 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:68; or b) A VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:29 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:64, or c) A VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:29 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO:

60.

47. The antibody-drug conjugate according to claim 46, wherein the antigen-binding domain comprises: a) A VH domain containing the sequence shown in SEQ ID NO:29 and a VL domain containing the sequence shown in SEQ ID NO:68, or b) A VH domain containing the sequence shown in SEQ ID NO:29 and a VL domain containing the sequence shown in SEQ ID NO:64, or c) A VH domain containing the sequence shown in SEQ ID NO:29 and a VL domain containing the sequence shown in SEQ ID NO:

60.

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

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

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

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

58.

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

53. A method of inhibiting cancer cell proliferation, which comprises contacting the cells with an effective amount of the antibody-drug conjugate according to any one of claims 43 to 51.

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

55. A method of treating cancer in a subject in need thereof, which comprises administering to said subject an effective amount of an antibody-drug conjugate according to any one of claims 43 to 51.

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

57. An antibody-drug conjugate according to any one of claims 43 to 51, said antibody-drug conjugate being used in the treatment of cancer.

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

59. A kit, which comprises an antibody-drug conjugate according to any one of claims 43 to 51 and a label and / or package insert containing instructions for use.

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