Antibody-drug conjugates targeting NaPi2b and methods of use

Abstract

Described herein are antibody-drug conjugates (ADCs) comprising an antibody construct that binds to human sodium-dependent phosphate transporter 2B (NaPi2b) conjugated to a camptothecin analog of Formula (I). The ADCs are useful as therapeutic agents, particularly in the treatment of cancer.

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of immunotherapeutics, and in particular to antibody-drug conjugates that target human sodium-dependent phosphate transporter 2B (hNaPi2b). [Background technology]

[0002] background Sodium-dependent phosphate transporter 2B (NaPi2b) is a transmembrane protein encoded by the SLC34A2 gene. The NaPi2b polypeptide is 690 amino acids long, with a limited extracellular domain (amino acids 188–361) exposed on the cell surface. It is widely expressed in normal tissues and overexpressed in various cancers, including ovarian, endometrial, and lung cancers.

[0003] Given the overexpression of NaPi2b in certain types of cancer, NaPi2b-targeting agents have been tested in clinical trials for the treatment of cancer, but mixed results have been reported. Mersana Therapeutics conducted a Phase I / II clinical trial testing upifytamavrilsodutin, an antibody-drug conjugate (ADC) consisting of the NaPi2b-targeting antibody MX35 and an auristatin-F payload (Draflexin platform) in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC). The NSCLC arm of the trial was discontinued due to lack of efficacy, but upifytamavrilsodutin received fast track designation for the treatment of platinum-resistant ovarian cancer patients who had received three to four prior lines of therapy. Mersana also completed a Phase I / II clinical trial of XMT-1592 in ovarian cancer. XMT-1592 is a site-specific ADC consisting of the antibody MX35 conjugated to an auristatin-F payload using its Draflexin platform. Development of this ADC has been discontinued. Rifastuzumab vedotin, an ADC of rifastuzumab with an MMAE payload, was tested in a clinical trial sponsored by Genentech in patients with ovarian cancer or NSCLC, but the trial was subsequently discontinued.

[0004] Camptothecin analogs have been developed as payloads for ADCs. Two such ADCs have been approved for the treatment of cancer: trastuzumab deruxtecan (Enhertu™), in which the camptothecin analog deruxtecan (Dxd) is conjugated to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™), in which the camptothecin analog SN-38 is conjugated to the anti-Trop-2 antibody sacituzumab via a hydrolyzable pH-sensitive linker.

[0005] Other camptothecin analogs and derivatives, and ADCs containing them, have been described, see, for example, International (PCT) Publication Nos. WO2019 / 195665 (Patent Document 1), WO2019 / 236954 (Patent Document 2), WO2020 / 200880 (Patent Document 3), and WO2020 / 219287 (Patent Document 4).

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2019 / 195665 [Patent Document 2] WO2019 / 236954 [Patent Document 3] WO2020 / 200880 [Patent Document 4] WO2020 / 219287 Summary of the Invention

[0008] overview Described herein are antibody-drug conjugates (ADCs) that target human NaPi2b and methods of use. One aspect of the disclosure is a conjugate of formula (X): T-[L-(D) m ] n (X) and an antibody-drug conjugate comprising: During the ceremony, m is an integer between 1 and 4; n is an integer between 1 and 10, T is an anti-NaPi2b antibody construct described herein; L is a linker, D is a compound of formula I: TIFF2025535239000002.tif53165, During the ceremony, R 1 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 1 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000003.tif27165-CO2R 8 , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025535239000004.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from -H and -C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025535239000005.tif22165, R 13 is selected from -H and -C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 18 and R 19together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0009] Another aspect of the present disclosure is a compound having the structure: TIFF2025535239000006.tif68165, where n is 4 and T is an anti-NaPi2b antibody construct described herein.

[0010] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or diluent.

[0011] Another aspect of the present disclosure relates to a method of inhibiting the proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

[0012] Another aspect of the present disclosure relates to a method of killing cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate described herein.

[0013] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody-drug conjugate described herein.

[0014] Another aspect of the present disclosure relates to the antibody-drug conjugates described herein for use in therapy.

[0015] Another aspect of the present disclosure pertains to the antibody-drug conjugates described herein for use in the treatment of cancer.

[0016] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer.

[0017] Another aspect of the present disclosure relates to a kit comprising an antibody-drug conjugate described herein and a label and / or package insert containing instructions for use. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1A shows the mouse heavy chain variable domain CDR sequences of the chimeric anti-NaPi2b antibody v23855 grafted onto the human VH germline (IGHV1-46*03), and Figure 1B shows the mouse light chain variable domain CDR sequences of the chimeric antibody v23855 grafted onto the human VL framework (IGKVID-39*01). CDRs are assigned using the AbM definition and are marked in bold and underlined. [Figure 2A] Non-reducing (NR) SDS-PAGE profiles of all humanized variants and the parent chimeric variant 23855 are shown. [Figure 2B] The reducing (NR) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855 are shown. [Figure 2C] 1 shows the UPLC-SEC profile of the parental mouse-human chimeric antibody v23855. [Figure 2D] 1 shows the UPLC-SEC profile of a representative humanized antibody v29456. [Figure 3A] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to human NaPi2b. [Figure 3B] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to cynomolgus monkey NaPi2b. [Figure 3C] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to mouse NaPi2b. [Figure 4A] N-curve analysis of binding of v29814 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 4B] N-curve analysis of binding of v36123 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 4C] N-curve analysis of binding of v36124 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 5A] A comparison of the ability of v23855 (parent chimera), v29456 (H1L2), v18992 (MX35), and v18993 (rifastuzumab) to internalize into HCC-78 cells is shown. [Figure 5B] A comparison of the ability of v23855 (parent chimera), v29456 (H1L2), v18992 (MX35), and v18993 (rifastuzumab) to internalize into NCI-H441 cells is shown. [Figure 6]Binding of MX35 and rifastuzumab ADCs to IGROV-1 cells, as well as binding of the parent chimeric antibody (v23855), humanized antibody variants v29452 and v29456, are depicted. [Figure 7] Illustrates the v29456 ADC's ability to exert a bystander effect. [Figure 8A] Illustrates the cytotoxic effect of v29456 ADC in 2D monolayer cultures of HCC-78 cells. [Figure 8B] Illustrates the cytotoxic effect of v29456 ADC in 2D monolayer cultures of IGROV-1 cells. [Figure 8C] Illustrates the cytotoxic effect of v29456 ADC in 2D monolayer cultures of HCT116 cells. [Figure 9A] Illustrates the cytotoxic effect of v29456 ADC in 2D monolayer cultures of IGROV-1 cells. [Figure 9B] Illustrates the cytotoxic effect of v29456 ADC in 2D monolayer cultures of TOV-21G cells. [Figure 10A] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of HCC-78 cells. [Figure 10B] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of IGROV-1 cells. [Figure 11A] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of IGROV-1 cells. [Figure 11B] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of TOV-21G cells. [Figure 12] 1 illustrates the efficacy of the v29456 ADC in the OVCAR3 xenograft model of ovarian cancer. [Figure 13] 1 illustrates the efficacy of v29456 conjugated to DXd1 in the NCI-H441 xenograft model of lung cancer. [Figure 14A]1 illustrates the efficacy of the v29456 ADC in the NCI-H441 xenograft model of lung cancer when administered at 0.3 mg / kg. [Figure 14B] 1 illustrates the efficacy of the v29456 ADC in the NCI-H441 xenograft model of lung cancer when administered at 1 mg / kg. [Figure 15A] Illustrates efficacy of the v29456 ADC in the patient-derived (PDX) CTG-2025 ovarian cancer model. [Figure 15B] Illustrates efficacy of the v29456 ADC in the patient-derived (PDX) CTG-0958 ovarian cancer model. [Figure 16] 1 shows the PK profile of v29456 ADC in Tg32 mice. [Figure 17A] 1 shows the ability of the v29456 (H1L2) ADC to internalize into OVCAR-3 cells compared to v18992 (MX35) and v18993 (rifastuzumab). [Figure 17B] 1 shows the ability of the v29456 (H1L2) ADC to internalize into IGROV-1 cells compared to v18992 (MX35) and v18993 (rifastuzumab). [Figure 18A] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of IGROV-1 cells. [Figure 18B] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of NCI-H441 cells. [Figure 18C] Illustrates the cytotoxic effect of v29456 ADC in 3D spheroids of TOV-21G cells. [Figure 19] Figure 19A illustrates the results of a Membrane Proteome Array™ assay using v38591. Figure 19B illustrates the validation data for CLDN3. [Figure 20] Illustrates cell binding of v38591 and v38591 ADC to IGROV-1 and OVCAR-3 cells. [Figure 21]1 illustrates the cross-reactivity of v38591 and v38591 ADC to cynomolgus monkey and mouse NaPi2b. [Figure 22] 1 shows the specificity of v38591 and v38591 ADC for human NaPi2b, NaPi2a, and NaPi2c. [Figure 23] Internalization of anti-NADC2b ADC and naked antibody is shown. [Figure 24] 1 illustrates the bystander activity of anti-NaPi2b ADCs against the NaPi2b-negative EBC-1 cell line. [Figure 25] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-0703 xenograft model. [Figure 26] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-1301 xenograft model. [Figure 27] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-3718 xenograft model. [Figure 28] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-1703 xenograft model. [Figure 29] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-2025 xenograft model. [Figure 30] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in a CTG-0958 xenograft model. [Figure 31] 1 illustrates the pharmacokinetic profiles of DAR4 and DAR8 anti-NaPi2b ADCs in cynomolgus monkeys. [Figure 32A] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU5213 lung PDX model. [Figure 32B] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU5245 lung PDX model. [Figure 32C]1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU6802 lung PDX model. [Figure 32D] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU6904 lung PDX model. [Figure 32E] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11692 lung PDX model. [Figure 32F] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11796 lung PDX model. [Figure 32G] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11870 lung PDX model. [Figure 32H] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11876 lung PDX model. [Figure 33A] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT14026 PDX endometrial cancer model. [Figure 33B] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5318 PDX endometrial cancer model. [Figure 33C] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5326 PDX endometrial cancer model. [Figure 33D] 1 shows the effect of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5321 PDX endometrial cancer model. [Figure 34] Binding of v38591 and v40502 (LALADS) ADCs, parental antibodies, and controls to IGROV-1, HCC-78, H441, and EBC-1 cells is shown. [Figure 35A] Illustrates internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line IGROV-1. [Figure 35B]Illustrates internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line HCC-78. [Figure 35C] Illustrates internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line H441. [Figure 36] Illustrates the cytotoxicity of v38591 and v40502 (LALADS) antibodies and ADCs in 3D spheroids of NaPi2b-expressing cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct that binds to sodium-dependent phosphate transporter 2B (NaPi2b) (anti-NaPi2b antibody construct) conjugated to a camptothecin analog of Formula (I) described herein. The ADCs of the present disclosure may find use as therapeutic agents, for example, particularly in the treatment of cancer.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0021] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0022] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0023] Where a range of values ​​is given herein, for example, where a value is defined as from an upper limit to a lower limit, it is understood that the range includes both the upper and lower limits, as well as each value therebetween.

[0024] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. When used herein in connection with a composition, use, or method, the term "consisting essentially of" means that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. When used herein in connection with a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including particular elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not those embodiments are specifically mentioned.

[0025] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) may help maintain the proper conformation of the CDRs to promote binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required for antigen binding. However, in some cases, even a single variable domain can confer antigen-binding specificity. Furthermore, as is known in the art, in some cases, antigen binding may also occur through a combination of at least one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0026] Several different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, as will be readily apparent to one skilled in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that the disclosure of a VH herein includes disclosure of the associated (unique) heavy chain CDRs (HCDRs), as defined by any of the known numbering systems. Similarly, the disclosure of a VL herein includes disclosure of the associated (unique) light chain CDRs (LCDRs), as defined by any of the known numbering systems.

[0027] (Table 1) Common CDR definitions 1 TIFF2025535239000007.tif180165

[0028] The term "identical" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have the same percentage of amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95%, or about 98% identity over a designated region) when compared and aligned for maximum correspondence over a comparison window or over a designated region, as measured using one of the commonly used sequence comparison algorithms known to those skilled in the art or by manual alignment and visual inspection. For sequence comparison, a test sequence is typically compared to a designated reference sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0029] A "comparison window" refers to a segment of a sequence that includes contiguous amino acid or nucleotide positions, for example, about 10 to about 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, and a test sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison purposes are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI)), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0030] The term "acyl," as used herein, refers to the group --C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0031] The term "acyloxy" refers to the group --OC(O)R, where R is alkyl.

[0032] The term "alkoxy," as used herein, refers to the group --OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0033] The term "alkyl," as used herein, refers to a straight-chain or branched saturated hydrocarbon group containing the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, t-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.

[0034] The term "alkylaminoaryl," as used herein, refers to an alkyl group, as defined herein, substituted with an aminoaryl group, as defined herein.

[0035] The term "alkylheterocycloalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one heterocycloalkyl group, as defined herein.

[0036] The term "alkylthio," as used herein, refers to the group --SR, where R is an alkyl group.

[0037] The term "amide," as used herein, refers to the group --C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0038] The term "amino," as used herein, refers to the group --NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0039] The term "aminoalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more amino groups, for example, 1, 2, or 3 amino groups.

[0040] The term "aminoaryl," as used herein, refers to an aryl group, as defined herein, substituted with an amino group.

[0041] The term "aryl," as used herein, refers to a 6- to 12-membered monocyclic or bicyclic hydrocarbon ring system in which at least one ring is aromatic. Examples of aryl include, but are not limited to, phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8-tetrahydro-naphthalenyl, indanyl, and the like.

[0042] The term "carboxy," as used herein, refers to the group --C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0043] The term "cyano," as used herein, refers to the group --CN.

[0044] The term "cycloalkyl," as used herein, refers to a monocyclic or bicyclic saturated hydrocarbon containing the specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, and the like.

[0045] The term "haloalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more halogen atoms.

[0046] The terms "halogen" and "halo" as used herein refer to fluorine (F), bromine (Br), chlorine (Cl) and iodine (I).

[0047] The term "heteroaryl," as used herein, refers to a 6- to 12-membered monocyclic or bicyclic ring system in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S, and N. Examples of heteroaryls include, but are not limited to, pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

[0048] The term "heterocycloalkyl," as used herein, refers to a monocyclic or bicyclic non-aromatic ring system containing the specified number of atoms, wherein at least one ring atom is a heteroatom, such as O, S, or N. The heterocyclyl substituent can be attached via any available ring atom, such as a ring carbon or ring nitrogen. Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.

[0049] The terms "hydroxy" and "hydroxyl," as used herein, refer to the group --OH.

[0050] The term "hydroxyalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one or more hydroxy groups.

[0051] The term "nitro," as used herein, refers to the group --NO.sub.2.

[0052] The term "sulfonyl," as used herein, refers to the group --S(O)2R, where R is H, alkyl, or aryl.

[0053] The term "sulfonamide," as used herein, refers to the group --NH--S(O)2R, where R is H, alkyl, or aryl.

[0054] The terms "thio" and "thiol" as used herein refer to the group --SH.

[0055] Unless specifically stated as "unsubstituted," any alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group mentioned herein is understood to be "optionally substituted," i.e., each such reference includes both unsubstituted and substituted forms of those groups. For example, a reference to "-C1-C6 alkyl" includes both unsubstituted -C1-C6 alkyl and -C1-C6 alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group mentioned herein is optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0056] The "substituted" chemical groups described herein may contain one substituent or multiple substituents up to the maximum valence of substitution for that group. For example, a methyl group can contain one, two, or three substituents, and a phenyl group can contain one, two, three, four, or five substituents. When a group is substituted with multiple substituents, the substituents may be the same or different.

[0057] The term "subject," as used herein, refers to an animal, in some embodiments, a mammal, that is the object of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, non-human primate, etc.). In certain embodiments, the subject is a human.

[0058] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0059] Particular features, structures, and / or characteristics described in connection with an embodiment disclosed herein may be combined in any suitable manner with features, structures, and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0060] It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for the exclusion of that feature in an alternative embodiment. For example, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically mentioned.

[0061] Antibody-drug conjugates The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) and During the ceremony, T is an anti-NaPi2b antibody construct described herein; L is a linker, D is a camptothecin analog described herein; m is an integer between 1 and 4; n is an integer between 1 and 10.

[0062] The components of formula (X) are described below.

[0063] Anti-NaPi2b antibody construct The ADCs of the present disclosure include anti-NaPi2b antibody constructs. In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides comprising one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When an antibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may further comprise a scaffold, and one or more antigen-binding domains may be fused or covalently linked to the scaffold, optionally via a linker.

[0064] According to the present disclosure, the anti-NaPi2b antibody construct of the ADC comprises at least one antigen-binding domain that specifically binds to human NaPi2b (hNaPi2b). "Specifically binds" to hNaPi2b means that the antibody construct binds to hNaPi2b but does not exhibit significant binding to NaPi2a or NaPi2c. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure can bind to NaPi2b from one or more non-human species. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure can bind to cynomolgus monkey NaPi2b.

[0065] Human NaPi2b is also known as human "solute carrier family 34 member 2" or "SLC34A2." Protein F sequences of hNaPi2b from various sources are known in the art and readily available from publicly accessible databases such as GenBank or UniProtKB. Exemplary hNaPi2b sequences include those provided under NCBI reference numbers NP_006415.3, NP_001171470.2, and NP_001171469.2. An exemplary hNaPi2b protein sequence is provided in Table 2 as SEQ ID NO: 1 (UniProt ID: 095436). An exemplary cynomolgus monkey NaPi2b protein sequence is also provided in Table 2 (SEQ ID NO: 2, UniProt ID: A0A2K5UHY1), as is an exemplary mouse NaPi2b protein sequence (SEQ ID NO: 3, UniProt ID: Q9DBP0).

[0066] Table 2. Human, cynomolgus, and mouse NaPi2b protein sequences TIFF2025535239000008.tif154165TIFF2025535239000009.tif151165TIFF2025535239000010.tif157165

[0067] Specific binding of an antigen-binding domain to a target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or conventional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as binding to a non-target protein (e.g., hNaPi2a or hNaPi2c) that is about 5% to about 10% less than binding to hNaPi2b, as measured, for example, by ELISA or flow cytometry.

[0068] Dissociation constant (K Dor K d The term "K" as used herein is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, a ligand-protein interaction refers to, but is not limited to, a protein-protein interaction or an antibody-antigen interaction. D measures the tendency of two proteins (e.g., AB) complexed with each other to reversibly dissociate into their components (A+B), which is called the “off rate (k off ) and the dissociation rate constant or "on rate (k on )" Therefore, K D is k off / k on and is expressed as molar concentration (M). D The smaller the K, the stronger the binding affinity. D A decrease in K indicates an increase in affinity. D has a K of 1 nM D The affinity is expressed as K A or K a It is sometimes measured as K D or K d The K between an antibody and its antigen D can be determined using methods well established in the art. D One method for determining K is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. Isothermal titration calorimetry (ITC) can be used to determine K D The Octet™ system may also be used to measure the affinity of an antibody for a target antigen.

[0069] In certain embodiments, the specific binding of the antibody construct to NaPi2b has a dissociation constant (Kd or K) of <1 μM, e.g., <500 nM, <250 nM, <100 nM, <50 nM, or <10 nM. DIn certain embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M or less, e.g., 10 -7 M or less, or 10 -8 The dissociation constant (K D In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M~10 -9 M, e.g., 10 -7 M~10 -9 Dissociation constant of M (K D ) As is known in the art, the numerical value of the dissociation constant obtained can vary depending on the test method. For example, the expression level of NaPi2b in the cell line, the format of the antibody construct (i.e., monovalent or bivalent), and the type of assay (i.e., ELISA or flow cytometry) can affect the numerical value of the dissociation constant when measured in a cell-based assay. Data provided in the Examples illustrate this general point, as shown in Examples 10, 11, and 16.

[0070] In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure have a Kd lower than that of the reference antibody rifastuzumab and equivalent to that of the reference antibody MX35, as measured by flow cytometry in cells expressing high levels of NaPi2b. Thus, in these embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain with an affinity for human NaPi2b higher than that of the reference antibody rifastuzumab and equivalent to that of the reference antibody MX35.

[0071] In certain embodiments, the anti-NaPi2b antibody constructs exhibit comparable levels of internalization to the reference antibody MX35 and higher levels of internalization compared to the reference antibody rifastuzumab in high and medium NaPi2b-expressing cells. In some embodiments, internalization is measured 4 hours, 5 hours, or 24 hours after treatment.

[0072] Antibody internalization can be measured using methods known in the art, for example, by the direct internalization method 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 dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dye (ThermoFisher Scientific Corporation, Waltham, MA), and Incucyte® Fabfluor-pH antibody labeling reagent (Sartorius AG, Goettingen, Germany), and analytical techniques such as microscopy, FACS, high-content imaging, or other plate-based assays.

[0073] As shown throughout this disclosure, NaPi2b expression varies depending on the cell type, and NaPi2b expression levels are referred to herein as "high," "moderate," "low," or "negative." These terms are used for reference to generally describe expression levels, as specified in Table 15.1 of Example 15, and are not intended to be limited to the specific numerical values ​​of average NaPi2b protein per cell contained therein. Alternatively, NaPi2b expression levels in cells or tumors can be assessed by immunohistochemistry (IHC) according to methods known in the art. For example, IHC can be used to stain NaPi2b in tumor tissue samples from xenograft models, cell-derived (CDX), or patient-derived (PDX). Tissue samples can be examined, and an H-score can be calculated as known in the art and described, for example, in Example 35 herein. The higher the H-score, the higher the expression of NaPi2b in the tissue sample.

[0074] antigen-binding domain The anti-NaPi2b antibody construct of the ADC of the present disclosure comprises at least one antigen-binding domain capable of binding to hNaPi2b. The at least one antigen-binding domain capable of binding to hNaPi2b is typically an immunoglobulin-based binding domain such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fabs (scFabs), single-chain Fvs (scFvs), and single-domain antibodies (sdAbs).

[0075] A "Fab fragment" contains the light and heavy chain variable domains (VL and VH, respectively), as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab fragments may also be single-chain Fab molecules, i.e., Fab molecules in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, in a single-chain Fab molecule, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain.

[0076] An "scFv" comprises an antibody heavy chain variable domain (VH) and light chain variable domain (VL) in a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For example, an scFv may comprise a VL connected from the C-terminus of the VL to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may comprise a VH connected via the C-terminus of the VH to the N-terminus of the VL by a polypeptide linker (see review in Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0077] The "sdAb" format refers to a single immunoglobulin domain. sdAbs may, for example, be of camelid origin. Camelid antibodies lack light chains and their antigen-binding site consists of a single domain called "VHH". sdAbs form three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and are easily expressed, for example, as fusions with the Fc chain of an antibody (e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0078] In those embodiments in which the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain can independently be an immunoglobulin-based domain, e.g., an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, e.g., a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule, e.g., a natural or engineered ligand, capable of specifically binding to its target. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers.

[0079] The present disclosure describes herein the identification of a murine antibody that specifically binds to hNaPi2b. A murine-human chimeric variant of this antibody is identified as variant 23855. The anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise an antigen-binding domain derived from this murine antibody or its humanized antibody variants. Representative humanized antibody variants of the murine antibody (v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460) are also described. In certain embodiments, the anti-NaPi2b antibody constructs described herein specifically bind to human NaPi2b having the sequence set forth in SEQ ID NO: 1.

[0080] In certain embodiments, the anti-NaPi2b antibody construct of the ADC competes for binding to human NaPi2b with any one of humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or with the parent chimeric antibody v23855. When assessing competition as described below, each of variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, and v23855 is referred to as a competitive reference antibody.

[0081] Using competitive assays known in the art, it can be determined whether an antibody construct competes with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or the parent chimeric antibody v23855, for binding to hNaPi2b. For example, a competitive reference antibody is first bound to hNaPi2b under saturating conditions, and then the ability of a test antibody construct to bind to hNaPi2b is measured. If the test antibody construct can bind to hNaPi2b simultaneously with the competitive reference antibody, the test antibody construct is considered to bind to a different epitope than the competitive reference antibody. Conversely, if the test antibody construct is unable to bind to hNaPi2b simultaneously with the competing reference antibody, the test antibody construct is considered to bind to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the competing reference antibody. Such competition assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, flow cytometry, and the like. An "antibody that competes with" a reference antibody refers to an antibody that blocks binding of the competing reference antibody to its epitope by 50% or more in a competition assay.

[0082] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to hNaPi2b, wherein the antigen-binding domain comprises a set of CDRs based on the CDRs of the parent chimeric antibody v23855 described herein. The CDR sequences of the parent chimeric antibody v23855 and representative humanized antibody variants are shown in Figure 1.

[0083] Table 3. CDR sequences of anti-NaPi2b antibody constructs TIFF2025535239000011.tif165165

[0084] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 7, 8, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 19, 20, and 18.

[0085] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 4, 5, and 6, and light chain CDR amino acid sequences (LCDR1 and LCDR3) comprising the sequences set forth in SEQ ID NOs: 17 and 18, and an antigen-binding domain having the LCDR sequence YTS.

[0086] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 10, 11, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 19, 20, and 18.

[0087] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 12, 13, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 19, 20, and 18.

[0088] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 14, 15, and 16, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 21, 22, and 23.

[0089] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises: (i) an HCDR1 amino acid sequence selected from the HCDR1 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456 , v29457, v29458, v29459, or v29460, and an HCDR3 amino acid sequence selected from the HCDR3 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; (ii) an LCDR1 amino acid sequence selected from any one of the LCDR1 amino acid sequences of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; an LCDR2 amino acid sequence selected from any one of the LCDR2 amino acid sequences of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; and an LCDR3 amino acid sequence selected from any one of the LCDR3 amino acid sequences of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. and an antigen-binding domain having the formula: wherein the CDR amino acid sequences are as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Figure 11).

[0090] In certain embodiments, the anti-NaPi2b antibody construct of the ADCs of the disclosure comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. and HCDR3), and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) selected from the light chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0091] In certain embodiments, the anti-NaPi2b antibody construct of the ADCs of the disclosure comprises an antigen-binding domain comprising the heavy chain CDR amino acid sequences (HCDR1, HCDR2, HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0092] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain having a VH sequence that includes the CDR sequences of the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain having a VL sequence comprising the CDR sequences of the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0093] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without losing the ability of the antibody to bind to 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 resulting variants are tested for binding activity by standard techniques. Thus, in certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) having 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to the set of CDRs of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the % sequence identity is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0094] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant comprises 1 to 10 amino acid substitutions across the set of CDRs (i.e., the CDRs may be modified by modifying any combination of the six CDRs, resulting in up to 10 amino acid substitutions), and wherein the antigen-binding domain retains the ability to bind to hNaPi2b. In some embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant comprises 1-7 amino acid substitutions, 1-5 amino acid substitutions, 1-4 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution across the set of CDRs, and wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0095] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen-binding domain retains the ability to bind to hNaPi2b. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0096] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0097] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v23855, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v23855, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0098] In certain embodiments, the anti-NaPi2b antibody constructs of the ADCs of the disclosure comprise a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v29456, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29456, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0099] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v29452, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29452, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0100] In some embodiments, the anti-NaPi2b antibody construct of the ADCs of the disclosure comprises the VH and VL sequences of any one of v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. The SEQ ID NOs for the VH and VL sequences of these variants are set forth in Table 4 below. The sequences themselves are provided in Table 7.4 in the Examples.

[0101] Table 4. VH and VL sequences of parent chimeric and humanized anti-NaPi2b antibodies TIFF2025535239000012.tif96165

[0102] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises the VH and VL sequences of v29456. In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises the VH and VL sequences of v29452.

[0103] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises: a) a VH sequence having the three HCDRs of v29456 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29456; and b) a VL sequence having the three LCDRs of v29456 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29456, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0104] In certain other embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises: a) a VH sequence having the three HCDRs of v29452 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29452; and b) a VL sequence having the three LCDRs of v29452 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29452, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0105] In one embodiment, an anti-NaPi2b antibody construct of an ADC of the present disclosure comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO: 63 and two light chains having the amino acid sequence set forth in SEQ ID NO: 62. In one embodiment, an anti-NaPi2b antibody construct of an ADC of the present disclosure comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO: 61 and two light chains having the amino acid sequence set forth in SEQ ID NO: 62. In yet another embodiment, an anti-NaPi2b antibody construct of an ADC of the present disclosure comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO: 66 and two light chains having the amino acid sequence set forth in SEQ ID NO: 67. In yet another embodiment, an anti-NaPi2b antibody construct of an ADC of the present disclosure comprises two heavy chains having the amino acid sequence set forth in SEQ ID NO: 68 and two light chains having the amino acid sequence set forth in SEQ ID NO: 67.

[0106] format The anti-NaPi2b antibody construct of the ADC can have a variety of formats. The minimum component of the anti-NaPi2b antibody construct is an antigen-binding domain that binds to hNaPi2b. The anti-NaPi2b antibody construct can optionally further comprise one or more additional antigen-binding domains and / or scaffolds. In those embodiments in which the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within hNaPi2b, a different epitope within hNaPi2b, or a different antigen. Thus, the anti-NaPi2b antibody construct can be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0107] In certain embodiments, the anti-NaPi2b antibody construct comprises at least one antigen-binding domain that binds to hNaPi2b and a scaffold, wherein the antigen-binding domain is operably linked to the scaffold. The term "operably linked," as used herein, means that the described components are in a relationship permitting them to function in their intended manner. Suitable scaffolds are described below.

[0108] In certain embodiments, the anti-NaPi2b antibody construct comprises two antigen-binding domains operably linked to an optional scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise three or four antigen-binding domains and, optionally, a scaffold. In these formats, when a scaffold is included, at least the first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or the first antigen-binding domain, or, if more than two antigen-binding domains are present, may be linked to another antigen-binding domain.

[0109] Scaffold-free anti-NaPi2b antibody constructs may contain a single antigen-binding domain in a suitable format, such as an sdAb, or they may contain two or more antigen-binding domains, optionally operably linked by one or more linkers. In such anti-NaPi2b antibody constructs, the antigen-binding domain may be in the form of an scFv, Fab, sdAb, or a combination thereof. For example, using scFvs as antigen-binding domains allows for the construction of formats such as tandem scFvs ((scFv)2 or taFv), in which scFvs are connected together by a flexible linker. scFvs can also be used to construct diabody formats containing two scFvs connected by a short linker (usually about 5 amino acids in length). The limited length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between the VL and VH by substituting a non-cysteine ​​residue in each chain with a cysteine ​​residue (e.g., at position 44 in the VH and position 100 in the VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0110] Similarly, in some embodiments, formats comprising two sdAbs, such as VH or VHH, connected together via a suitable linker may be used. Other examples of anti-NaPi2b antibody construct formats lacking a scaffold include those based on Fab fragments, e.g., Fab2 and F(ab')2 formats, where the Fab fragments are connected via a linker or IgG hinge region.

[0111] Combinations of different forms of antigen-binding domains can also be used to create alternative scaffold-less formats, for example, an scFv or sdAb can be fused to the C-terminus of either or both the light and heavy chains of a Fab fragment, resulting in a bivalent (Fab-scFv / sdAb) construct.

[0112] In certain embodiments, the anti-NaPi2b antibody construct may be in an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as full-size antibody format (FSA) or Mab format, and includes anti-NaPi2b antibody constructs comprising two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-NaPi2b antibody construct may be based on an IgG class immunoglobulin, e.g., an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-NaPi2b antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-NaPi2b antibody construct is based on a particular immunoglobulin isotype, it means that the anti-NaPi2b antibody construct comprises all or a portion of the constant region of the particular immunoglobulin isotype. For example, an anti-NaPi2b antibody construct based on a given Ig isotype can comprise at least one antigen-binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and, optionally, an Ig hinge region from the same or a different isotype. It should be understood that in some embodiments, the anti-NaPi2b antibody construct can also comprise isotype and / or subclass hybrids. It should also be understood that the Fc region and / or hinge region can be optionally modified to confer one or more desirable functional properties known in the art. Thus, in certain embodiments, the anti-NaPi2b antibody construct comprises a VH amino acid sequence (i.e., CH1, hinge, CH2, CH3 amino acid sequences) fused to an IgG1 constant domain amino acid sequence and a VL amino acid sequence (i.e., CL amino acid sequence) fused to a kappa or lambda constant amino acid domain. Exemplary amino acid sequences are provided in the Examples and Sequence Listing.

[0113] In some embodiments, the anti-NaPi2b antibody construct may be derived from two or more immunoglobulins from different species, e.g., the anti-NaPi2b antibody construct may be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" both generally refer to antibodies that combine immunoglobulin regions or domains from multiple species.

[0114] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.

[0115] A "humanized antibody" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity and affinity for the target antigen. This technique for producing humanized antibodies is often referred to as "CDR grafting."

[0116] In some cases, additional modifications are made to further refine antibody performance. For example, some framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains of humanized antibodies contain all or substantially all of the hypervariable regions from the non-human immunoglobulin and all or substantially all of the FRs from the human immunoglobulin sequence. Humanized antibodies are described in further detail in, for example, Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0117] Several approaches for selecting the most appropriate human framework for grafting nonhuman CDRs are known in the art. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the nonhuman antibody providing the CDRs (the "fixed framework" approach). More recent approaches use variable regions with high amino acid sequence identity with the variable regions of the nonhuman antibody providing the CDRs (the "homology matching" or "best-fit" approach). An alternative approach is to select fragments of framework sequences within each light or heavy chain variable region from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding nonhuman-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0118] Alternatively or in addition to these traditional approaches, more recent techniques may be used to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on a human germline sequence or consensus sequence may be used as the acceptor human framework, rather than a human framework with somatic mutation(s). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen-binding activity are grafted onto a human germline framework. This method can help improve the "humanness" of humanized antibodies (i.e., similarity to human germline sequences), thus reducing the risk of immunogenicity of the variable regions. These techniques are described in various publications (see, e.g., Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0119] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise humanized antibody sequences, e.g., one or more humanized variable domains. In some embodiments, the anti-NaPi2b antibody constructs can be humanized antibodies. Non-limiting examples of humanized antibodies based on anti-NaPi2b antibody v23855 are described herein (see the Examples and Sequence Listing for v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460).

[0120] scaffold In certain embodiments, the anti-NaPi2b antibody construct comprises one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerization peptides (e.g., leucine zipper, heterodimer-forming "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0121] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance.When the scaffold is a polypeptide, each antigen-binding domain of the anti-NaPi2b antibody construct can be linked to either the N-terminus or C-terminus of the polypeptide scaffold.In certain embodiments, the anti-NaPi2b antibody construct also includes a polypeptide scaffold in which one or more antigen-binding polypeptide constructs are linked to a region other than the N-terminus or C-terminus, for example, via an amino acid side chain with or without a linker.

[0122] In embodiments in which the anti-NaPi2b antibody construct comprises a peptide or polypeptide scaffold, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0123] Several protein domains containing selective pairs of two different polypeptides are known in the art and can be used to form scaffolds. One example is the selectively paired leucine zipper domains, 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 barnase pair (Deyev, et al., Nat Biotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(1-2):23-26 (1999)), and split fluorescent protein pairs (International Patent Application Publication No. WO2011 / 135040).

[0124] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Mueller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154; and U.S. Patent Application Publication No. 2009 / 0285816).

[0125] For example, fusing an antigen-binding moiety, such as an scFv, diabody, or single-chain diabody, to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Mueller et al., ibid.). The antigen-binding moiety may be fused to the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0126] Derivatives of albumin have been described in the form of heteromultimers containing two transporter polypeptides obtained by segmenting the albumin protein so that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer contains four termini and can therefore be fused, optionally via linkers, to up to four different antigen-binding moieties.

[0127] In certain embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. In some embodiments, the anti-NaPi2bt antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0128] Fc area As used herein, the terms "Fc region," "Fc," or "Fc domain" refer to the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0129] In certain embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an immunoglobulin Fc region. The Fc region may be dimeric and comprised of two Fc polypeptides, or alternatively, the Fc region may be comprised of a single polypeptide.

[0130] An "Fc polypeptide" in the context of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0131] An Fc region may comprise a CH3 domain, or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0132] In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a human IgG1 Fc region.

[0133] In certain embodiments, an anti-NaPi2b antibody construct may comprise an IgG Fc region-based scaffold that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence and optionally a CH2 sequence, wherein the first and second Fc polypeptides are different. In some embodiments, an anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, an anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications.

[0134] In some embodiments, an anti-NaPi2b antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, the modified CH3 domain being 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, in which an 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 may result in only one of the two amino acids at the same respective amino acid position in each sequence being modified, or in both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences being differently modified. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may comprise one or more asymmetric amino acid modifications.

[0135] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, the modified CH3 domain comprising one or more amino acid modifications that promote the formation of a heterodimeric Fc over the formation of a homodimeric Fc, in some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0136] Amino acid modifications that can be made to the CH3 domain of an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies to produce stable asymmetrically modified Fc regions, such as those described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. In certain embodiments, anti-NaPi2b antibody constructs may comprise scaffolds based on modified Fc regions such as those described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

[0137] Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain. Table 5 also lists CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Applications WO2012 / 058768 and WO2013 / 063702.

[0138] In certain embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4 or variant 5 modifications as shown in Table 5.

[0139] Table 5: Human IgG1 Fc sequence 1, and amino acid modifications in the CH3 domain that promote heterodimer formation TIFF2025535239000013.tif124165

[0140] In some embodiments, an anti-NaPi2b antibody construct can comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications. Modifications in the CH2 domain can affect binding to Fc of Fc receptors (FcR), such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0141] In some embodiments, the anti-NaPi2b antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, where modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0142] Many amino acid modifications to the CH2 domain that selectively alter the affinity of Fc for various Fcγ receptors are known in the art. Amino acid modifications that increase binding and those that decrease binding can each be useful in specific indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased lysis of target cells. Similarly, decreasing binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations. Reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable in certain indications. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0143] Among the amino acid modifications in the CH2 domain, examples of amino acid modifications that alter Fc binding via the Fcγ receptor include S298A / E333A / K334A and S298A / E333A / K334A / K326A (improved affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (improved affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), and F243L / R292P / Y300L / L235V / P396L (improved affinity for FcγRIIIa) (Nordstrom JL, et al. al., 2011, Breast Cancer Res, 13(6):R123), F243L (improved affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8), S298A / E333A / K334A (improved affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604), S239D / I332E / A330L and S239D / I332E (improved affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (improved affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter Fc binding by FcγRIIb are described in International Publication No. WO2021 / 232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, page 283).

[0144] In certain embodiments, the anti-NaPi2b antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, which contains one or more amino acid modifications that result in reduced or eliminated binding of the entire Fc region to Fcγ receptors (i.e., a "knockout" variant).

[0145] Various publications describe strategies used to engineer antibodies to generate "knockout" variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0146] Examples of mutations that can be introduced into the hinge or CH2 domain to generate a "knockout" variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0147] In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise a scaffold based on an IgG Fc with modified native glycosylation. As is known in the art, glycosylation of Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a deglycosylated Fc lacking all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0148] Conversely, removal of fucose from the oligosaccharide attached to heavy chain N297 has been shown to enhance ADCC due to improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to the carbohydrate attached to N297 (International Publication No. WO 03 / 035835), or in other cells that produce defucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002 (ibid.); and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, WO2009 / 135181 describes the addition of fucose analogs to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody.

[0149] Other methods for producing antibodies with little or no fucose on the Fc glycosylation site (N297) are known in the art, e.g., GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0150] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which biantennary oligosaccharides attached to the Fc region of an antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include those with at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, e.g., International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0151] Preparation of anti-NaPi2b antibody constructs The anti-NaPi2b antibody constructs described herein may be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0152] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-NaPi2b antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the anti-NaPi2b antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be understood by those skilled in the art, the number of polynucleotides required for expression of an anti-NaPi2b antibody construct will depend on the format of the construct, including whether the antibody construct includes a scaffold. For example, if the anti-NaPi2b antibody construct is in a monospecific mAb format or FSA format, two polynucleotides, each encoding one polypeptide chain, will be required. If multiple polynucleotides are required, they may be incorporated into one vector or into multiple vectors.

[0153] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector(s) along with one or more control elements, such as transcriptional elements required for efficient transcription of the polynucleotide. Examples of such control elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the selection of control elements will depend on the host cell selected for expression of the antibody construct and that such control elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal or integrating vector.

[0154] Suitable host cells for cloning or expressing anti-NaPi2b antibody constructs include various prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces cells or Pichia cells). Prokaryotic host cells include, for example, E. coli cells, A. salmonicida cells, or B. subtilis cells.

[0155] In certain embodiments, anti-NaPi2b antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed. Humana Press, Totowa, NJ, 2003.

[0156] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0157] Suitable host cells for the expression of glycosylated anti-NaPi2b antibody constructs are usually eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe the PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension can be particularly useful for the expression of antibody constructs. Examples include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod., 23:243-251); monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci, 383: 44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR - Exemplary mammalian host cell lines suitable for the production of antibody constructs include, but are not limited to, CHO cells; see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed. Humana Press, Totowa, NJ, 2003).

[0158] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0159] Host cells containing expression vector(s) encoding the anti-NaPi2b antibody constructs can be cultured using conventional methods to produce the anti-NaPi2b antibody constructs. Alternatively, in some embodiments, host cells containing expression vector(s) encoding the anti-NaPi2b antibody constructs can be used therapeutically or prophylactically to deliver the anti-NaPi2b antibody constructs to a subject, or the polynucleotide or expression vector can be administered ex vivo to cells from a subject, which can then be returned to the subject's body.

[0160] Typically, anti-NaPi2b antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion exchange, hydrophobic interaction, affinity, sizing, or gel filtration, and reverse-phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can be used to purify specific antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can also be enabled by specific fusion partners. For example, antibodies can be purified by binding to glutathione resins when a GST fusion is used, or Ni when a His tag is used. +2 Purification may be performed using affinity chromatography or, if a flag tag is used, using an immobilized anti-flag antibody. The degree of purification required varies depending on the use of the anti-NaPi2b antibody construct. In some cases, no purification may be required.

[0161] In certain embodiments, the anti-NaPi2b antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used with reference to the anti-NaPi2b antibody constructs described herein, means that the antibody construct is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, such as in natural cells or, in the case of recombinantly produced constructs, in host cells. In certain embodiments, a substantially pure anti-NaPi2b 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.

[0162] Certain embodiments of the present disclosure relate to methods for producing anti-NaPi2b antibody constructs, comprising culturing host cells into which one or more polynucleotides encoding the anti-NaPi2b antibody constructs or one or more expression vectors encoding the anti-NaPi2b antibody constructs have been introduced under conditions suitable for expression of the anti-NaPi2b antibody constructs, and optionally recovering the anti-NaPi2b antibody constructs from the host cells (or host cell culture medium).

[0163] Post-translational modifications In certain embodiments, the anti-NaPi2b antibody constructs described herein may contain one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-NaPi2b antibody construct from a host cell.

[0164] Post-translational modifications include various modifications known in the art (see, e.g., Proteins—Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., 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 embodiments in which an anti-NaPi2b antibody construct contains one or more post-translational modifications, the construct may contain the same type of modification at one or more sites, or may contain different modifications at different sites.

[0165] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage (by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0166] Other examples of post-translational modifications include, for example, the addition or removal of N- or O-linked glycans, chemical modification of N- or O-linked glycans, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to enable detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0167] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0168] Camptothecin analogues Camptothecin analogs encompassed by the ADCs of the present disclosure have formula (I): TIFF2025535239000014.tif53165, During the ceremony, R 1 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 1 is -NH2, R is R 3 or R 4 and R 1 is other than -NH2, R is R 4 and R 3 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000015.tif27165-CO2R 8 , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 4 teeth, Selected from TIFF2025535239000016.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from -H and -C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025535239000017.tif22165, R 13 is selected from -H and -C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 18 and R 19together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0169] In some embodiments, the camptothecin analog is a compound of formula (I), except that R 1 When is NH2, R 2 is other than H.

[0170] In some embodiments, in the compound of Formula (I), R 1 is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 and NH 2 .

[0171] In some embodiments, in the compound of Formula (I), R 1 is NH2.

[0172] In some embodiments, in the compound of Formula (I), R 1 is selected from —H, —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 , and —OCF 3 .

[0173] In some embodiments, in the compound of Formula (I), R 1 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0174] In some embodiments, in the compound of Formula (I), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0175] In some embodiments, in the compound of Formula (I), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0176] In some embodiments, in the compound of Formula (I), R 2 is selected from -H, -F, -Br and -Cl.

[0177] In some embodiments, in the compound of Formula (I), R 2 is selected from -F, -Br and -Cl.

[0178] In some embodiments, in the compound of Formula (I), R 3 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000018.tif27165-CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0179] In some embodiments, in the compound of Formula (I), R 4 teeth, Selected from TIFF2025535239000019.tif58165.

[0180] In some embodiments, in the compound of Formula (I), R 5is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0181] In some embodiments, in the compound of Formula (I), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from.

[0182] 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.

[0183] 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.

[0184] In some embodiments, in the compound of Formula (I), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0185] In some embodiments, in the compound of Formula (I), each R 9are 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.

[0186] In some embodiments, in the compound of Formula (I), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0187] In some embodiments, in the compound of Formula (I), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0188] In some embodiments, in the compound of Formula (I), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0189] In some embodiments, in the compound of Formula (I), R 10’ is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aryl.

[0190] In some embodiments, in the compound of Formula (I), R 11is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0191] In some embodiments, in the compound of Formula (I), each R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0192] In some embodiments, in the compound of Formula (I), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF2025535239000020.tif22165.

[0193] In some embodiments, in the compound of Formula (I), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0194] 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.

[0195] In some embodiments, in the compound of Formula (I), R 16 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0196] 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.

[0197] 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.

[0198] In some embodiments, in the compound of Formula (I), R 18 and R 19 taken together with the N atom to which they are attached represent halogen, unsubstituted C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the following.

[0199] In some embodiments, in the compound of formula (I), X a and X b are each independently selected from NH and O.

[0200] Combinations of any of the foregoing embodiments for compounds of formula (I) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0201] In certain embodiments, the compound of formula (I) has formula (II): TIFF2025535239000021.tif53165, During the ceremony, R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 20 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000022.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000023.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from -H and -C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025535239000024.tif22165, R 13 is selected from -H and -C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; Xc is selected from O, S and S(O)2; However, the compound is other than (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0202] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 and —OCF 3 .

[0203] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0204] In some embodiments, in the compound of Formula (II), R 2 is selected from F and Cl.

[0205] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000025.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000026.tif58165.

[0206] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000027.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000028.tif58165.

[0207] In some embodiments, in the compound of Formula (II), R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535239000029.tif58165.

[0208] In some embodiments, in the compound of Formula (II), R 20 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000030.tif27165-CO2R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, Selected from TIFF2025535239000031.tif68165.

[0209] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000032.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000033.tif58165.

[0210] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000034.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000035.tif58165.

[0211] In some embodiments, in the compound of Formula (II), R 2 is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3, and R 20 is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535239000036.tif58165.

[0212] In some embodiments, in the compound of Formula (II), R 5 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0213] In some embodiments, in the compound of Formula (II), R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0214] In some embodiments, in the compound of Formula (II), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from.

[0215] In some embodiments, in the compound of Formula (II), R 6 is H and R7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 is selected from.

[0216] In some embodiments, in the compound of Formula (II), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from.

[0217] 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.

[0218] 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.

[0219] In some embodiments, in the compound of Formula (II), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0220] In some embodiments, in the compound of Formula (II), each R 9 are 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.

[0221] In some embodiments, in the compound of Formula (II), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0222] In some embodiments, in the compound of Formula (II), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0223] In some embodiments, in the compound of Formula (II), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0224] 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.

[0225] 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.

[0226] In some embodiments, in the compound of Formula (II), R 12is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0227] In some embodiments, in the compound of Formula (II), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF2025535239000037.tif22165.

[0228] 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.

[0229] 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.

[0230] In some embodiments, in the compound of Formula (II), R 16 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0231] In some embodiments, in the compound of Formula (II), R 16is selected from unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl, and -(C1-C6 alkyl)-aminoaryl.

[0232] In some embodiments, in the compound of Formula (II), R 17 is —C1 to C6 alkyl.

[0233] 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.

[0234] In some embodiments, in the compound of Formula (II), R 18 and R 19 taken together with the N atom to which they are attached represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the following.

[0235] In some embodiments, in the compound of formula (II), X a and X b are each independently selected from NH and O.

[0236] Combinations of any of the foregoing embodiments for compounds of formula (II) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0237] In certain embodiments, the compound of formula (I) has formula (III): TIFF2025535239000038.tif53165, During the ceremony, R 2 is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 15 is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; R 4 teeth, Selected from TIFF2025535239000039.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16and Selected from TIFF2025535239000040.tif22165, R 13 is selected from -H and -C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2.

[0238] In some embodiments, in the compound of Formula (III), R 2 is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0239] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F and -Cl.

[0240] In some embodiments, in the compound of Formula (III), R 15 is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0241] In some embodiments, in the compound of Formula (III), R 15 is selected from —CH3 and —OCH3.

[0242] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F and -Cl, R 15 is selected from —CH3, —CF3, —OCH3 and —OCF3.

[0243] In some embodiments, in the compound of Formula (III), R 2 is selected from -H, -F and -Cl, R 15 is selected from —CH3 and —OCH3.

[0244] In some embodiments, in the compound of Formula (III), R 4 teeth, Selected from TIFF2025535239000041.tif58165.

[0245] 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.

[0246] 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.

[0247] In some embodiments, in the compound of Formula (III), each R 9is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0248] In some embodiments, in the compound of Formula (III), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0249] In some embodiments, in the compound of Formula (III), each R 9 are 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.

[0250] In some embodiments, in the compound of Formula (III), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0251] In some embodiments, in the compound of Formula (III), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0252] In some embodiments, in the compound of Formula (III), each R 10 are independently unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0253] 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.

[0254] 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.

[0255] In some embodiments, in the compound of Formula (III), R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -(C1-C6 alkyl)-aryl and -S(O)R 16 is selected from.

[0256] In some embodiments, in the compound of Formula (III), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and Selected from TIFF2025535239000042.tif22165.

[0257] In some embodiments, in the compound of Formula (III), R 13 is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0258] 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.

[0259] In some embodiments, in the compound of Formula (III), R 16 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0260] 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.

[0261] In some embodiments, in the compound of Formula (III), R 18 and R 19 taken together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0262] In some embodiments, in the compound of formula (III), X a and X b are each independently selected from NH and O.

[0263] Combinations of any of the foregoing embodiments for compounds of formula (III) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0264] In certain embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (I), (II), or (III) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (I), (II), or (III) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0265] In certain embodiments, the camptothecin analog included in an ADC according to the present disclosure is a compound having formula (I) and is selected from the compounds shown in Tables 6 and 7.

[0266] In certain embodiments, the camptothecin analog is a compound having formula (II): In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 ,or TIFF2025535239000043.tif27165. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 is H, -(C1~C6)-OR 5 or TIFF2025535239000044.tif27165, R 5 is H and R 18and R 19 are taken together with the N atom to which they are attached to form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 is F and R 20 Ha-(C1~C6)-OR 5 and R 5 is H. In certain embodiments, the camptothecin analog is a compound having formula (II) and is selected from the compounds shown in Table 6.

[0267] In certain embodiments, the camptothecin analog is a compound having the formula (III): In certain embodiments, the camptothecin analog is a compound having the formula (III), wherein R 2 is F and R 15 is -CH3 and R 4 teeth TIFF2025535239000045.tif32165, -R 9 is -C1-C6 hydroxyalkyl, and X a and X b are each O. In certain embodiments, the camptothecin analog is a compound having formula (III) and is selected from the compounds shown in Table 7.

[0268] In certain embodiments, the camptothecin analog included in an ADC according to the disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog included in an ADC according to the disclosure is Compound 139 or Compound 141.

[0269] Table 6: Exemplary camptothecin analogs of formula (II) TIFF2025535239000046.tif191165TIFF2025535239000047.tif212165TIFF2025535239000048.tif213165TIFF20255352390 00049.tif177165TIFF2025535239000050.tif188165TIFF2025535239000051.tif188165TIFF2025535239000052.tif201165

[0270] Table 7: Exemplary camptothecin analogs of formula (III) TIFF2025535239000053.tif172165TIFF2025535239000054.tif188165TIFF20255352390 00055.tif223165TIFF2025535239000056.tif194165TIFF2025535239000057.tif200165 TIFF2025535239000058.tif199165TIFF2025535239000059.tif193165TIFF20255352390 00060.tif193165TIFF2025535239000061.tif199165TIFF2025535239000062.tif141165

[0271] It will be understood that throughout this disclosure, references to compounds of formula (I) include, in various embodiments, compounds of formula (II) and formula (III), as well as the individual compounds set forth in Tables 6 and 7, to the same extent as if the embodiments individually reciting each of these formulas or compounds were specifically recited.

[0272] Antibody-drug conjugates The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) and During the ceremony, T is an anti-NaPi2b antibody construct described herein; L is a linker, D is a camptothecin analog having formula (I): m is an integer between 1 and 4; n is an integer between 1 and 10.

[0273] In certain embodiments, in the conjugate of formula (X), m is between 1 and 2. In some embodiments, m is 1.

[0274] 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 2 and 4.

[0275] 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.

[0276] As discussed above and reflected by the parameters m and n in formula (X), an anti-NaPi2 antibody construct "T" can be conjugated to multiple compounds "D" of formula (I). While any particular anti-NaPi2 antibody construct T is conjugated to an integer number of compounds D, those skilled in the art will understand that analysis of a conjugate preparation to determine the ratio of compound D to anti-NaPi2 antibody construct T may yield a non-integer result reflecting a statistical average. This ratio of compound D to targeting moiety T is sometimes commonly referred to as the drug-to-antibody ratio, or "DAR." Thus, conjugate preparations having a non-integer DAR are intended to be encompassed by formula (X).

[0277] In certain embodiments, in the conjugate of Formula (X), D is a compound of Formula (II) or Formula (III). In certain embodiments, in the conjugate of Formula (X), D is a compound selected from the compounds shown in Tables 6 and 7. 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.

[0278] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (IV): The compound is TIFF2025535239000063.tif63165. During the ceremony, R 1a is selected from —H, —CH3, —CHF2, —CF3, —F, —Br, —Cl, —OH, —OCH3, —OCF3, and —NH2; R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S- or -NH-, and R 4a teeth, TIFF2025535239000064.tif68165, where * is the point of attachment to X and p is 1, 2, 3 or 4; or X is O and R 4a -X- is Selected from TIFF2025535239000065.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9aare independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl, or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000066.tif22165, Each R 10a’ are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b are independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and Selected from TIFF2025535239000067.tif22165, R 13a is selected from -H and -C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R21 is -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2; TIFF2025535239000068.tif17165 shows the attachment point to the linker L.

[0279] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , —OCF 3 and —NH 2 .

[0280] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —CF 3 , —OCH 3 , and —OCF 3 .

[0281] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH 3 , —OCH 3 and NH 2 .

[0282] In some embodiments, in the compound of formula (IV), R 1a is selected from —CH3 and —OCH3.

[0283] In some embodiments, in the compound of formula (IV), R 2a is selected from —H, —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0284] In some embodiments, in the compound of formula (IV), R 2a is selected from -H, -F and -Cl.

[0285] In some embodiments, in the compound of formula (IV), R 2a is -F.

[0286] In some embodiments, in the compound of Formula (IV), X is —O—, —S—, or —NH—, and R 4a teeth, Selected from TIFF2025535239000069.tif58165.

[0287] In some embodiments, in the compound of Formula (IV), X is —O— or —NH—.

[0288] In some embodiments, in the compound of Formula (IV), each R 9a is independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, and -(C1-C6 alkyl)-aryl.

[0289] In some embodiments, in the compound of Formula (IV), each R 9a is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0290] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000070.tif22165.

[0291] In some embodiments, in the compound of Formula (IV), each R 10a are independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000071.tif22165.

[0292] In some embodiments, in the compound of formula (IV), R 12a -C1-C6 alkyl, -aryl, -(C1-C 6 -S(O)R 16 is selected from.

[0293] In some embodiments, in the compound of formula (IV), R 13a is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0294] In some embodiments, in the compound of formula (IV), R 14a’ is selected from H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl.

[0295] In some embodiments, in the compound of formula (IV), R 16a is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0296] In some embodiments, in the compound of formula (IV), R 22 and R 23 are each independently selected from -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, and -C3-C8 cycloalkyl.

[0297] In some embodiments, in the compound of formula (IV), X a and X b are each independently selected from NH and O.

[0298] In some embodiments, in the compound of formula (IV), X a and X b are O respectively.

[0299] In some embodiments, in the compound of Formula (IV), X is O and R 4a teeth TIFF2025535239000072.tif27165, X a and X b are O and R 9a is -C1 to C6 alkyl.

[0300] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, X is O, and R 4a teeth, TIFF2025535239000073.tif27165, X a and X b are O and R 9a is —C1 to C6 alkyl.

[0301] In some embodiments, in the compound of formula (IV), R 1a is -CH3 or -OCH3, and R 2a is H or F, X is O, and R 4a teeth, TIFF2025535239000074.tif27165, X a and X b are O and R, respectively. 9a is —C1 to C6 alkyl.

[0302] Other combinations of any of the foregoing embodiments relating to compounds of formula (IV) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0303] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group of formula (V): The compound is TIFF2025535239000075.tif53165. During the ceremony, R 2a is selected from —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3 and —OCF3; R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000076.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000077.tif68165, R 5 is selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from R 8 is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; Each R 9 are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and -NR 14 R 14’ is selected from Each R 10’ are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11 is selected from —H and —C1-C6 alkyl; R 12 is -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and Selected from TIFF2025535239000078.tif22165, R 13 is selected from —H and —C1-C6 alkyl; R 14 and R 14’ are each independently selected from —H, C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17 is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 18 and R 19 together with the N atom to which they are attached, represent halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2; TIFF2025535239000079.tif17165 shows the attachment point to the linker L.

[0304] In some embodiments, in the compound of Formula (V), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0305] In some embodiments, in the compound of Formula (V), R 2a is selected from —CF3, —F, —Cl and —OCH3.

[0306] In some embodiments, in the compound of Formula (V), R 2a is F.

[0307] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000080.tif27165-CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000081.tif58165.

[0308] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000082.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000083.tif58165.

[0309] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000084.tif27165-(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000085.tif58165.

[0310] In some embodiments, in the compound of Formula (V), R 20a is -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , Selected from TIFF2025535239000086.tif58165.

[0311] In some embodiments, in the compound of Formula (V), R 20a is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , TIFF2025535239000087.tif27165-CO2R 8 , unsubstituted aryl, aminoaryl, heteroaryl, (C1-C6 alkyl) aminoaryl, Selected from TIFF2025535239000088.tif68165.

[0312] In some embodiments, in the compound of Formula (V), R 6 and R 7 are each independently -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C(O)R 17 is selected from.

[0313] In some embodiments, in the compound of Formula (V), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from.

[0314] In some embodiments, in the compound of Formula (V), R 6 is H and R 7 is -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 is selected from.

[0315] In some embodiments, in the compound of Formula (V), R 6 and R 7 are each independently -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , —C3-C8 heterocycloalkyl and —C(O)R 17 is selected from.

[0316] 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.

[0317] 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.

[0318] In some embodiments, in the compound of Formula (V), each R 9 is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0319] In some embodiments, in the compound of Formula (V), each R 9are 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.

[0320] In some embodiments, in the compound of Formula (V), each R 10 are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0321] In some embodiments, in the compound of Formula (V), each R 10 are independently -C1-C6 alkyl, -NR 14 R 14’ , -aryl and -(C1-C6 alkyl)-aryl.

[0322] 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.

[0323] In some embodiments, in the compound of Formula (V), R 12 is -H, -C1-C6 alkyl, -aryl, -(C1-C 6 -S(O)R 16 is selected from.

[0324] In some embodiments, in the compound of Formula (V), R 12 is -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R16 and Selected from TIFF2025535239000089.tif22165.

[0325] 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.

[0326] 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.

[0327] In some embodiments, in the compound of Formula (V), R 16 is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0328] 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.

[0329] 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.

[0330] In some embodiments, in the compound of Formula (V), R 18 and R 19 taken together with the N atom to which they are attached, represent halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 and forming a 4-, 5-, 6-, or 7-membered ring having 0 to 3 substituents selected from:

[0331] In some embodiments, in the compound of Formula (V), R 17 is —C1 to C6 alkyl.

[0332] In some embodiments, in the compound of formula (V), X a and X b are each independently selected from NH and O.

[0333] In some embodiments, in the compound of formula (V), X a and X b are O respectively.

[0334] In some embodiments, in the compound of Formula (V), R 20a is -(C1-C6 alkyl)-OR 5 is.

[0335] In some embodiments, in the compound of Formula (V), R 20a is -(C1-C6 alkyl)-OR 5 and R 5 is H.

[0336] In some embodiments, in the compound of Formula (V), R 2a is F and R 20a is -(C1-C6 alkyl)-OR 5 and R 5 is H.

[0337] Other combinations of any of the foregoing embodiments for compounds of formula (V) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0338] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a group represented by formula (VI): The compound is TIFF2025535239000090.tif63165. During the ceremony, R 2a is selected from —H, —CH3, —CF3, —F, —Br, —Cl, —OH, —OCH3, and —OCF3; X is -O-, -S-, or -NH-, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, TIFF2025535239000091.tif94165, where * is the point of attachment to X and p is 1, 2, 3, or 4; or X is O and R 25 -X- is Selected from TIFF2025535239000092.tif37165, R 5a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 6a is selected from —H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 7a is -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5a , —C3-C8 heterocycloalkyl and —C(O)R 17a is selected from R 8a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9a are independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl, or R 9a does not exist and X b =X, Each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000093.tif22165, Each R 10a’ are independently selected from -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; Each R 10b are independently selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 11a is absent or is -C1-C6 alkyl, R 12a is -C1 to C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and Selected from TIFF2025535239000094.tif22165, R 13a is selected from —H and —C1-C6 alkyl; R 14a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ is selected from H, —C1-C6 alkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl; R 16ais selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, and -(C1-C6 alkyl)-aryl; R 17a is selected from -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 21 is -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5a is selected from R 22 and R 23 are each independently selected from -H, -halogen, C1-C6 alkyl, and -C3-C8 cycloalkyl; R 24 , R 25 and R 26 are each -C1 to C6 alkyl, X a and X b are each independently selected from NH, O, and S; X c is selected from O, S and S(O)2; TIFF2025535239000095.tif17165 shows the attachment point to the linker L.

[0339] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Br, —Cl, —OH, —OCH 3 and —OCF 3 .

[0340] In some embodiments, in the compound of formula (VI), R 2a is selected from —CH 3 , —CF 3 , —F, —Cl, —OCH 3 and —OCF 3 .

[0341] In some embodiments, in the compound of formula (VI), R 2a is selected from F and Cl.

[0342] In some embodiments, in the compound of formula (VI), R 2a is F.

[0343] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH— and R 25 -C1~C6 alkyl, -(C1~C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, TIFF2025535239000096.tif58165, or X is O and R 25 -X- is Selected from TIFF2025535239000097.tif37165.

[0344] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, Selected from TIFF2025535239000098.tif58165.

[0345] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 is -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , Selected from TIFF2025535239000099.tif58165.

[0346] In some embodiments, in the compound of Formula (VI), X is —O—, —S—, or —NH—, and R 25 teeth, Selected from TIFF2025535239000100.tif58165.

[0347] In some embodiments, in the compound of Formula (VI), X is —O— or —NH—.

[0348] In some embodiments, in the compound of formula (VI), R 6a is H.

[0349] 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.

[0350] In some embodiments, in the compound of formula (VI), R 7a is -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17a is selected from.

[0351] 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.

[0352] In some embodiments, in the compound of Formula (VI), each R 9a is independently selected from -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0353] In some embodiments, in the compound of Formula (VI), each R 10a are independently -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000101.tif22165.

[0354] In some embodiments, in the compound of Formula (VI), each R 10a are independently -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl, and Selected from TIFF2025535239000102.tif22165.

[0355] In some embodiments, in the compound of formula (VI), R 12a -C1-C6 alkyl, -aryl, -(C1-C 6 -S(O)R 16a is selected from.

[0356] In some embodiments, in the compound of formula (VI), R 13a is selected from -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, and -C1-C6 aminoalkyl.

[0357] In some embodiments, in the compound of formula (VI), R 14a’ is selected from H, unsubstituted —C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 hydroxyalkyl, —C1-C6 aminoalkyl, —C3-C8 cycloalkyl, and —C3-C8 heterocycloalkyl.

[0358] In some embodiments, in the compound of formula (VI), R 16a is selected from -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0359] In some embodiments, in the compound of formula (VI), R 17a is —C1 to C6 alkyl.

[0360] In some embodiments, in the compound of formula (VI), R 22 and R 23 are each independently selected from -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, and -C3-C8 cycloalkyl.

[0361] In some embodiments, in the compound of formula (VI), Xa and X b are each independently selected from NH and O.

[0362] In some embodiments, in the compound of formula (VI), X a and X b are O respectively.

[0363] In some embodiments, in the compound of Formula (VI), X is O and R 25 is —C1 to C6 alkyl.

[0364] In some embodiments, in the compound of formula (VI), R 2a is F, X is O, and R 25 is —C1 to C6 alkyl.

[0365] Other combinations of any of the foregoing embodiments relating to compounds of formula (VI) are also envisioned, with each combination forming a separate embodiment for purposes of this disclosure.

[0366] In certain embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (IV), (V), or (VI) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups as defined in any one of formulas (IV), (V), or (VI) are each optionally substituted with one or more substituents selected from halogen, acyl, acyloxy, alkoxy, carboxy, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, and sulfonamido.

[0367] In certain embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a is -CH3 and R 2a is F. In some embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a is -CH3 and R 2a is F, X is -O-, and R 4a teeth TIFF2025535239000103.tif27165, R 9a is -C1-C6 alkyl, and X a and X b are O respectively.

[0368] In certain embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R 2a is F and R 20 a is H, -(C1-C6)-OR 5 ,or TIFF2025535239000104.tif27165. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a is H, -(C1~C6)-OR 5 ,or TIFF2025535239000105.tif27165, R 5 is H and R 18 and R 19 are taken together with the N atom to which they are attached to form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, in the ADC having Formula (X), D is a compound of Formula (V), wherein R 2a is F and R 20a -(C1~C6)-OR 5 and R 5 is H.

[0369] In certain embodiments, in the ADC having formula (X), D is a compound of formula (VI), wherein R 2ais F, X is -O-, and R 25 is -C1 to C6 alkyl.

[0370] Linker L The conjugate of formula (X) comprises a linker L, which is a bifunctional or polyfunctional moiety capable of linking one or more camptothecin analogs D to the anti-NaPi2b antibody construct T. A bifunctional (or monovalent) linker L links a single compound D to a single site on the anti-NaPi2b antibody construct T, whereas a polyfunctional (or polyvalent) linker L links multiple compounds D to a single site on the anti-NaPi2b antibody construct T. A linker that links one compound D to multiple sites on the anti-NaPi2b antibody construct T may also be considered polyfunctional.

[0371] The linker L comprises a functional group capable of reacting with a targeting group(s) on the anti-NaPi2b antibody construct T and at least one functional group capable of reacting with a targeting group on the camptothecin analog D. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Groups on the anti-NaPi2b antibody construct T and the camptothecin analog D that can serve as targeting groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0372] Non-limiting examples of functional groups that can react with thiols include maleimides, haloacetamides, haloacetyls, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In this context, the "self-stabilizing" maleimides described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062, are also useful.

[0373] Non-limiting examples of functional groups that can react with amines include activated esters (e.g., N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters (e.g., Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (e.g., diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the conversion of a carboxyl group to an activated ester using succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), which can then be reacted with an amine.

[0374] Non-limiting examples of functional groups that can react with electrophilic groups such as aldehyde or ketone carbonyl groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0375] In certain embodiments, the linker L may comprise a functional group that allows for bridging of two interchain cysteines on an anti-NaPi2bt antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802), or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0376] Alternatively, the anti-NaPi2b antibody construct T may be modified to include a non-natural reactive group, such as an azide, that allows conjugation with a linker via a complementary reactive group on the linker. For example, conjugation of a linker to the anti-NaPi2b antibody construct may utilize click chemistry, such as the azide-alkyne cycloaddition (AAC) reaction, which has been successfully used in the development of antibody-drug conjugates (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling an azide with a cyclooctyne.

[0377] The linker L may be a cleavable or non-cleavable linker. A cleavable linker is a linker that is susceptible to cleavage under certain conditions, such as intracellular conditions (e.g., in endosomes or lysosomes) or in the vicinity of target cells (e.g., tumor microenvironment). Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on the degradation of the antibody in cells, which typically results in the release of the amino acid-linker-drug moiety.

[0378] Examples of cleavable linkers include linkers containing an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix surrounding target cells, such as cancer cells, may be utilized. Examples of extracellular tumor-associated proteases include plasmin, matrix metalloproteinases (MMPs), elastase, and kallikrein-related peptidases.

[0379] For conjugates intended to be internalized by cells, the linker L may comprise an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease, examples of which include cathepsins B, C, D, H, L, and S, and legumain.

[0380] The cleavage recognition sequence may be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that may be included in a cleavable linker include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys. Examples of tripeptide and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0381] Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Disulfide-containing linkers may optionally contain additional groups adjacent to the disulfide bond to provide steric hindrance (e.g., incorporation of geminal dimethyl groups) to improve the extracellular stability of the linker. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers containing a combination of these functional groups may also be useful; for example, linkers containing both hydrazones and disulfides are known in the art.

[0382] Further examples of cleavable linkers are linkers containing β-glucuronides that can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007 / 011968). The β-glucuronide can also function to increase the hydrophilicity of the linker L.

[0383] Another example of a linker that is cleaved intracellularly to increase hydrophilicity is a linker that contains a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0384] In certain embodiments, the linker L included in the conjugate of formula (X) is a cleavable linker. In some embodiments, the linker L comprises a cleavage recognition sequence. In some embodiments, the linker L may comprise an amino acid sequence that is recognized and cleaved by a lysosomal protease.

[0385] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative group, a self-leaving group, a stretcher, or a hydrophilic moiety.

[0386] Self-immolative and self-leaving groups that find use as linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED), and hemiaminal groups. Other examples of self-immolative groups include aromatic compounds electronically similar to PAB or PABC groups, such as heterocyclic derivatives, for example, but not limited to, 2-aminoimidazole-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). The self-immolative / self-leaving group is typically attached to an amino or hydroxyl group of compound D. Self-immolative / self-leaving groups, alone or in combination, are often included in peptide-based linkers, but may also be included in other types of linkers.

[0387] Stretchers used in linkers for drug conjugates include, for example, alkylene groups and fatty acid, dibasic acid, amine, or diamine-based stretchers, such as diglycolate, malonate, caproate, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers.

[0388] PEG and mPEG stretchers can also function as hydrophilic moieties within the linker. For example, PEG or mPEG can be included in the linker either "linearly" or as a pendant group to increase the hydrophilicity of the linker (see, e.g., U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd. (Plain City, OH). Other hydrophilic groups that can optionally be incorporated into the linker L include, for example, β-glucuronide, sulfonate group, carboxylate group, and pyrophosphate diester.

[0389] In certain embodiments, an ADC of Formula (X) may comprise a cleavable linker. In certain embodiments, an ADC of Formula (X) may comprise a peptide-containing linker. In certain embodiments, an ADC of Formula (X) may comprise a protease-cleavable linker.

[0390] In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XI): TIFF2025535239000106.tif32165, During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-NaPi2b antibody construct T; Str is a stretcher AA1 and AA2 are each independently an amino acid, and AA1-[AA2] r forms a protease cleavage site, X is a self-immolative group, q is 0 or 1, r is 1, 2 or 3; s is 0, 1 or 2; # is the attachment point for the anti-NaPi2b antibody construct T; % is the point of attachment to camptothecin analogue D.

[0391] In some embodiments, q is 1 in the linker of formula (XI).

[0392] In some embodiments, in the linker of formula (XI), s is 1. In some embodiments, in the ADC of formula (XI), s is 0.

[0393] In some embodiments, in the linker of Formula (XI), r is 1. In some embodiments, in the ADC of Formula (XI), r is 3.

[0394] In some embodiments, a compound of formula (XI): In the linker, Z is TIFF2025535239000107.tif33165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0395] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025535239000108.tif53165, where: R is H or C1-C6 alkyl; t is an integer between 2 and 10, u is an integer between 1 and 10.

[0396] In some embodiments, in the linker of formula (XI), Str is Selected from TIFF2025535239000109.tif22165, where: t is an integer between 2 and 10, u is an integer between 1 and 10.

[0397] In some embodiments, in the linker of formula (XI), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XI), AA1-[AA2] rare Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0398] 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.

[0399] 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.

[0400] In certain embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XII): TIFF2025535239000110.tif32165, During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-NaPi2b antibody construct T; Str is a stretcher AA1 and AA2 are each independently an amino acid, and AA1-[AA2] r forms a protease cleavage site, Y is -NH-CH-; q is 0 or 1, r is 1, 2 or 3; v is 0 or 1, # is the attachment point for the anti-NaPi2b antibody construct T; % is the point of attachment to camptothecin analogue D.

[0401] In some embodiments, q is 1 in the linker of formula (XII).

[0402] In some embodiments, in the linker of Formula (XII), v is 0. In some embodiments, in the ADC of Formula (XII), s is 1.

[0403] In some embodiments, in the linker of Formula (XII), r is 1. In some embodiments, in the ADC of Formula (XII), r is 3.

[0404] In some embodiments, a compound of formula (XII): In the linker, Z is TIFF2025535239000111.tif32165, where # is the point of attachment to T and * is the point of attachment to the rest of the linker.

[0405] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025535239000112.tif53165, where: R is H or C1-C6 alkyl; t is an integer between 2 and 10, u is an integer between 1 and 10.

[0406] In some embodiments, in the linker of formula (XII), Str is Selected from TIFF2025535239000113.tif22165, where: t is an integer between 2 and 10, u is an integer between 1 and 10.

[0407] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XII), AA1-[AA2] r are Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVa It has a sequence selected from l-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, phenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0408] In some embodiments, in the linker of formula (XII), AA1-[AA2] r is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XII), AA1-[AA2] r has a sequence selected from Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0409] 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.

[0410] In some embodiments, in the linker of formula (XII), Y is -NH-CH. In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH.

[0411] In some embodiments, the ADC of formula (X) may comprise a disulfide-containing linker. In some embodiments, in the ADC of formula (X), m is 1 and the linker L is of formula (XIII): TIFF2025535239000114.tif32165, During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-NaPi2b antibody construct T; Q is -(CH2) p -or-(CH2CH2O) q -, and p and q are each independently an integer between 1 and 10; each R is independently H or C1-C6 alkyl; n is 1, 2 or 3; # is the attachment point for the anti-NaPi2b antibody construct T; % is the point of attachment to camptothecin analogue D.

[0412] In some embodiments, an ADC of Formula (X) may comprise a b-glucuronide-containing linker.

[0413] Various non-cleavable linkers for linking drugs to targeting moieties are known in the art and may be useful in certain embodiments for the ADCs of the present disclosure. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with anti-NaPi2b antibody constructs and a maleimide or haloacetyl-based moiety for reaction with camptothecin analogs, or vice versa. An example of such a non-cleavable linker is a sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC)-based linker. Sulfo-SMCC conjugation typically occurs via a maleimide group that reacts with sulfhydryls (thiols, -SH) on camptothecin analogs, while sulfo-NHS esters are reactive with primary amines (such as those found on lysines and the N-termini of proteins or peptides) on the anti-NaPi2b antibody constructs. Other non-limiting examples of such linkers include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) ("long chain" SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid These include linkers based on N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI).Other examples include those containing haloacetyl-based functional groups such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0414] Non-limiting examples of drug-linkers comprising camptothecin analogs of Formula (I) are shown in Tables 8, 9, and 10. Non-limiting examples of conjugates comprising these drug-linkers are shown in Tables 11, 12, and 13. In certain embodiments, an ADC of Formula (X) comprises a drug-linker selected from those shown in Tables 8, 9, and 10. In certain embodiments, an ADC of Formula (X) is selected from a conjugate shown in Tables 11, 12, and 13, where T is an anti-NaPi2b antibody construct and n is between 1 and 10. In some embodiments, an ADC of Formula (X) is selected from a conjugate shown in Tables 11, 12, and 13, where T is an anti-NaPi2b 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 11, 12, and 13, where T is an anti-FRα antibody construct and n is between 4 and 8.

[0415] In certain embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m), and n is 4 or 8. In some embodiments, the ADC of Formula (X) comprises a drug-linker (L-(D)) selected from MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148, and MC-GGFG-Compound 148. m ), and n is 8.

[0416] Preparation of ADCs ADCs of formula (X) can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Additionally, various antibody-drug conjugation services are commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramel Pharma Solutions (Grangemouth, UK).

[0417] In general, preparation of an ADC involves first preparing a drug-linker DL comprising one or more camptothecin analogs of Formula (I) and a linker L, and then conjugating the drug-linker DL to an appropriate group on an anti-NaPi2b antibody construct T. However, ligation of the linker L to the anti-NaPi2b antibody construct T followed by ligation of the anti-NaPi2b antibody construct-linker TL to one or more camptothecin analogs D of Formula (I) remains an alternative approach available in some embodiments.

[0418] Suitable groups on compound D of formula (I) for attachment of linker L in any of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of the present disclosure, linker L is attached to the compound through a hydroxyl or amine group on compound D of formula (I).

[0419] Suitable groups on the anti-NaPi2b antibody construct T for attachment of the linker L in any of the above approaches include sulfhydryl groups (e.g., in the side chain of a cysteine ​​residue), amino groups (e.g., in the side chain of a lysine residue), carboxylic acid groups (e.g., in the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

[0420] For example, the anti-NaPi2b antibody construct T may contain one or more naturally occurring sulfhydryl groups that allow the anti-NaPi2b antibody construct T to be bound to the linker L via the sulfhydryl group's sulfur atom. Alternatively, the anti-NaPi2b antibody construct T may contain one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"), and 2-iminothiolane hydrochloride (Traut's reagent). Alternatively, the anti-NaPi2b antibody construct T may contain one or more carbohydrate groups that can be chemically modified to contain one or more sulfhydryl groups.

[0421] Carbohydrate groups on the anti-NaPi2b antibody construct T may also be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which may then be reacted with a linker L, for example, via a hydrazine or hydroxylamine group on the linker L.

[0422] The anti-NaPi2b antibody construct T can also be modified to include additional cysteine ​​residues (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622; and 9,000,130) or unnatural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow site-specific conjugation. Alternatively, the anti-NaPi2b antibody construct T can be modified to include a non-natural reactive group, such as an azide, that allows for conjugation with a linker via a complementary reactive group on the linker, e.g., click chemistry (e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to enable attachment of a linker via metal-free click chemistry (see, e.g., European Patent No. EP2911699).

[0423] Other protocols for modifying proteins for the attachment or association of a linker L are known in the art, including those described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002).

[0424] Alternatively, ADCs may be prepared using the enzyme transglutaminase, particularly bacterial transglutaminase (BTG) from Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of glutamine (typically an amine acceptor on an antibody) and an alkyleneamino group (typically an amine donor on a drug-linker), which may be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a glutamine-containing peptide, or "tag," that allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US2013 / 0230543 and International (PCT) Publication No. WO2016 / 144608).

[0425] A similar conjugation approach utilizes the enzyme sortase A. In this approach, an antibody is typically modified to contain a sortase A recognition motif (LPXTG, where X is any naturally occurring amino acid), and the drug-linker is designed to contain an oligoglycine motif (typically GGG) to enable sortase A-mediated transpeptidation (see, e.g., Beerli, et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0426] Once conjugation is complete, the average number of compounds of formula (I) conjugated to the anti-NaPi2b antibody construct T (i.e., the "drug-to-antibody ratio" or DAR) may 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 drug-linked forms (e.g., the proportion of anti-NaPi2b antibody construct T containing 0, 1, 2, 3, etc. compounds of formula (I) D) may also be optionally analyzed. Various techniques for assessing DAR distribution are known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Wakankar et al., 2011, mAbs, 3:161-172).

[0427] Pharmaceutical Composition For therapeutic use, the ADCs of the present disclosure are typically formulated as pharmaceutical compositions. Accordingly, certain embodiments of the present disclosure relate to pharmaceutical compositions comprising an ADC described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

[0428] Pharmaceutical compositions may be formulated for administration to a subject, for example, by oral (e.g., buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. The term "parenteral" as used herein includes subcutaneous injection, and intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to a subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard capsule, soft capsule, pill, suppository, oily suspension, aqueous suspension, dispersible powder, dispersible granule, emulsion, injection, or solution. Pharmaceutical compositions may be provided as unit-dose formulations.

[0429] In certain embodiments, a pharmaceutical composition comprising an ADC is formulated for parenteral administration, e.g., as a lyophilized preparation or an aqueous solution. Such a pharmaceutical composition may be provided, for example, in a unit dosage injectable form.

[0430] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or These include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0431] In certain embodiments, compositions containing the ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension may contain the ADC in a non-toxic parenterally acceptable diluent or carrier. Acceptable diluents and carriers that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Additionally, sterile fixed oils may be used as carriers. For this purpose, various bland fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions. Auxiliaries such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0432] In certain embodiments, a composition comprising an ADC may be formulated for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0433] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"; Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000)).

[0434] How to use Certain embodiments of the present disclosure relate to therapeutic uses of the ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents.

[0435] Certain embodiments of the present disclosure relate to methods of inhibiting abnormal cancer or tumor cell proliferation, methods of inhibiting cancer or tumor cell proliferation, or methods of treating cancer in a subject, comprising administering an ADC described herein. In certain embodiments, the ADC described herein can be used to treat cancer. Accordingly, some embodiments of the present disclosure relate to the use of an ADC as an anticancer agent.

[0436] Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X). Some embodiments relate to methods of killing cancer or tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X).

[0437] Some embodiments relate to methods of treating a subject with cancer by administering to the subject an ADC described herein, e.g., an ADC of Formula (X). In this context, treating the subject can result in one or more of: a reduction in tumor size; a delay or prevention of an increase in tumor size; an increase in disease-free survival between the disappearance or removal and reappearance of a tumor; prevention of subsequent development of a tumor (e.g., metastasis); an increase in time to progression; a reduction in one or more adverse symptoms associated with the tumor; and / or an increase in overall survival of the subject with cancer.

[0438] Certain embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vitro. Some embodiments relate to the use of an ADC described herein, e.g., an ADC of Formula (X), in a method of inhibiting cancer cell growth and / or killing cancer cells in vivo in a subject with cancer.

[0439] Examples of cancers that may be treated in certain embodiments include carcinomas, melanomas, and sarcomas, including adenocarcinomas and squamous cell carcinomas. Carcinomas and sarcomas are also often referred to as "solid tumors." Examples of commonly occurring solid tumors that may be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, renal cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors and therefore may also be considered solid tumors in certain circumstances. Typically, the cancer to be treated is a NaPi2b-expressing cancer.

[0440] Certain embodiments relate to methods of inhibiting the growth of NaPi2b-positive tumor cells, comprising contacting the cells with an ADC described herein, e.g., an ADC of Formula (X). The cells may be in vitro or in vivo. In certain embodiments, the ADC may be used in methods of treating a NaPi2b-positive cancer or tumor in a subject.

[0441] Cancers that overexpress NaPi2b are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, and lung cancer (such as non-small cell lung cancer (NSCLC)). In one embodiment, the ADCs described herein may be used in a method for treating ovarian cancer or lung cancer. In one embodiment, the ADCs described herein may be used in a method for treating NSCLC.

[0442] Medicine Kit Certain embodiments relate to pharmaceutical kits that include an ADC described herein, e.g., an ADC of Formula (X).

[0443] The kit typically includes a container holding the ADC and a label and / or package insert on or associated with the container. The label or package insert includes instructions customarily included in commercial packaging of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic product. The label or package insert may further include a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, such notice reflecting approval by that agency for manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle.

[0444] In addition to the container holding the ADC, the kit may optionally include one or more additional containers containing other components of the kit, for example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0445] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dried form, such as a powder or granules, and the kit may additionally include a suitable solvent for the reconstitution of the lyophilized or dried component(s).

[0446] Kits may further include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0447] Tables 8~13

[0448] Table 8: Exemplary Drug-Linker (DL) Structures, Including Camptothecin Analogs of Formula (I) with a C7 Linkage TIFF2025535239000115.tif185165TIFF2025535239000116.tif191165TIFF2025535239000117.tif232165

[0449] Table 9: Exemplary Drug-Linker (DL) Structures, Including Camptothecin Analogs of Formula (I) with a C10 Linkage TIFF2025535239000118.tif196165TIFF2025535239000119.tif194165TIFF2025535239000120.tif205165TIFF2025535239000121.tif216165

[0450] Table 10: Exemplary Drug-Linker (DL) Structures, Including Camptothecin Analogs of Formula (I) with Either a C7 or C10 Bond TIFF2025535239000122.tif143165TIFF2025535239000123.tif173165TIFF2025535239000124.tif138165

[0451] Table 11: Exemplary conjugate (DC) structures containing camptothecin analogs of formula (I) with a C7 linkage TIFF2025535239000125.tif181165TIFF2025535239000126.tif210165TIFF2025535239000127.tif210165TIFF2025535239000128.tif228165

[0452] Table 12: Exemplary conjugate (DC) structures containing camptothecin analogs of formula (I) with a C10 bond TIFF2025535239000129.tif202165TIFF2025535239000130.tif231165TIFF2025535239000131.tif103165

[0453] Table 13: Exemplary conjugate (DC) structures containing camptothecin analogs of formula (I) with either a C7 or C10 bond TIFF2025535239000132.tif189165TIFF2025535239000133.tif202165TIFF2025535239000134.tif213165

[0454] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0455] Examples 1-3 below illustrate various methods for preparing camptothecin analogs of Formula (I). It is understood that those skilled in the art can produce these compounds by similar methods or by combining other methods known in the art. It is also understood that those skilled in the art can produce other compounds of Formula (I) not specifically shown below using the methods described below or similar methods by using appropriate starting materials and modifying the synthetic parameters as needed. In general, the starting materials can be obtained from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (Thermo Fisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or can be synthesized according to materials known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013), or can be prepared as described herein.

[0456] Abbreviation The following abbreviations are used throughout the Examples section: BCA: bicinchoninic acid, Boc: di-tert-butyl dicarbonate, CE-SDS: capillary electrophoresis sodium dodecyl sulfate, DCM: dichloromethane, DTPA: diethylenetriaminepentaacetic acid, DIPEA: N,N-diisopropylethylamine, DMF: dimethylformamide, DMM™: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride, EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Fmoc: fluorenylmethyloxycarbonyl, HATU: azabenzotriazole tetramethylwt. tetrafluorophenylalanine, HCl: hydrophobic interaction chromatography, HOAt: 1-hydroxy-7-azabenzotriazole, HPLC: high performance liquid chromatography, LC / MS: liquid chromatography mass spectrometry, MC: maleimidocaproyl, MT: maleimidotriethylene glycolate, NMM: N-methylmorpholine, PNP: p-nitrophenol, RP-UPLC-MS: reversed-phase ultra-performance liquid chromatography mass spectrometry, SEC: size exclusion chromatography, TCEP: tris(2-carboxyethyl)phosphine, Tfp: tetrafluorophenyl, TLC: thin layer chromatography, TFA: trifluoroacetic acid.

[0457] General Chemistry Procedures General Procedure 1: Conversion of Chlorides to Amine To a stirred solution of the chloride compound in dimethylformamide (0.05-0.1 M) was added the appropriate secondary amine (3 equiv.). Upon completion (as determined by LC / MS, typically 1-3 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0458] General Procedure 2: Conversion of amines to amides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added triethylamine (1.2 equiv.), the appropriate carboxylic acid (1.1 equiv.), followed by a solution of DMMTM (2 equiv.) in water (1 M). Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0459] General Procedure 3: Conversion of amines to sulfonamides To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added DIPEA (3 equiv.), followed by the appropriate sulfonyl chloride. Upon completion (as determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse-phase HPLC to give the desired product after lyophilization.

[0460] General Procedure 4: Two-Step Conversion of Amines to Ureas (Synthetic Scheme IV) Step 1: To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 equivalent), followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the reaction mixture was concentrated to dryness and then purified by reverse-phase HPLC to yield the desired PNP-carbamate intermediate after lyophilization. This intermediate may be used to generate a single analog or may be split into multiple batches to generate multiple analogs in a second step. Step 2: To the PNP-carbamate intermediate in dimethylformamide (0.1-0.2 M) was added the appropriate primary amine (3 equivalents). Upon completion (as determined by LC / MS, typically 1 hour), the reaction mixture was purified by reverse-phase HPLC to yield the desired product after lyophilization.

[0461] General Procedure 5: Conversion of amines to carbamates To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05-0.1 M) was added p-nitrophenyl carbonate (1 equivalent), followed by triethylamine (2 equivalents). Upon completion (as determined by LC / MS, typically 1-4 hours), the appropriate alcohol was added to the resulting PNP-carbamate intermediate. Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was purified by reverse-phase HPLC to yield the desired product after lyophilization.

[0462] General Procedure 6: Removal of the Boc protecting group To a stirred solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20% by volume). Upon completion (as determined by LC / MS, typically 1 h), the reaction mixture was concentrated in vacuo to give a crude solid, or purified as described in General Procedure 9.

[0463] General Procedure 7: Copper-Mediated Amide Coupling To a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 equiv.) and HOAt (3 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added EDC (HCl salt, 3 equiv.). After 5 min, a solution (0.02 M) of the amine-containing payload (1 equiv.) in a 10% v / v mixture of dimethylformamide in dichloromethane was added, followed immediately by CuCl (4 equiv.). Upon completion (as determined by LC / MS, typically 1-16 h), the reaction mixture was concentrated in vacuo to give a crude solid or purified by preparative HPLC to give the desired product after lyophilization.

[0464] General Procedure 8: MT Installation To a stirred solution (approximately 0.02 M) of the amine compound (1 equivalent) in dimethylformamide was added a solution (approximately 0.02 M) of MT-OTfp (1.2-1.5 equivalents) in acetonitrile, followed by DIPEA (10 μL, 4 equivalents). Upon completion (as determined by LC / MS, typically 1-16 hours), the reaction mixture was concentrated in vacuo to give a crude solid, which was purified by preparative HPLC to give the desired product after lyophilization.

[0465] General Procedure 9: Compound Purification Flash chromatography Crude reaction products were purified using Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC) eluting with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane on a Biotage® Isolera™ automated flash system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was performed using Biotage® Snap Ultra C18 columns (12, 30, 60, or 120 g) eluting with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. Purified compounds were isolated either by removal of organic solvents by rotary evaporation or by lyophilization of the acetonitrile / water mixture.

[0466] Preparative HPLC Reverse-phase HPLC of the crude compound was performed on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) using a Luna® 5-μm C18 100 Å (150 × 30 mm) column (Phenomenex, Torrance, CA) eluted with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was isolated by lyophilization of the acetonitrile / water mixture.

[0467] General Procedure 10: Compound Analysis LC / MSReaction completion was monitored, and purified compounds were analyzed on an Agilent 1290 HPLC / 6120 Single Quad LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) using a Kinetex® 2.6-μm C18 100 Å (30 × 3 mm) column (Phenomenex, Torrance, CA) eluted with a 10–100% linear gradient of 0.1% formic acid in acetonitrile / 0.1% formic acid in water.

[0468] NMR : 1 H NMR spectra were collected using a Bruker AVANCE III 300 Spectrometer (300 MHz) (Bruker Corporation, Billerica, Mass.) Chemical shifts are reported in parts per million (ppm).

[0469] Example 1: Preparation of camptothecin analogs bearing methyl at C10 position 1.1: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 1.1) TIFF2025535239000135.tif42165 The title compound was prepared according to the procedure set forth in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0470] 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) TIFF2025535239000136.tif47165 The title compound was prepared according to the procedure set forth in Li, et al., 2019, ACS Med. Chem. Lett., 10(10):1386-1392.

[0471] 1.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 100) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0472] LC / MS:C 26 H 26 Calculated m / z for FN3O5 = 479.2, observed [M+H] + =480.4.

[0473] 1 H NMR (300 MHz, CDCl3) δ 8.20 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 10.4 Hz, 1H), 7.67 (s, 1H), 5.77 (d, J = 16.4 Hz, 1H), 5.42 (s, 2H), 5.33 (d, J = 16.4 Hz, 1H), 4.26 (s, 2H), 3.81 (t, J = 4.7 Hz, 4H), 2.82 - 2.76 (m, 4H), 2.57 (d, J = 1.7 Hz, 3H), 1.99 - 1.82 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H).

[0474] 1.4: ((S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 102) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0475] LC / MS:C 32 H 31 Calculated m / z for FN4O6 = 618.2, observed [M+H] + =619.4.

[0476] 1 H NMR (300 MHz, CDCl3) δ 8.07 (d, J = 7.9 Hz, 1H), 7.88 - 7.44 (m, 7H), 5.73 (d, J = 16.4 Hz, 1H), 5.33 (s, 2H), 5.33 - 5.26 (m, 1H), 4.19 (s, 2H), 3.12 (s, 4H), 2.80 (s, 4H), 2.54 (s, 3H), 1.90 (dt, J = 11.6, 7.0 Hz, 2H), 1.04 (t, J = 7.3 Hz, 3H).

[0477] 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) TIFF2025535239000139.tif63165 The title compound was prepared according to general procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was performed as described in general procedure 9 eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0478] LC / MS:C 32 H 32 Calculated m / z for FN5O6 = 633.2, found [M+H] + =634.4.

[0479] 1 H NMR (300 MHz, MeOD) δ 8.32 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 10.5 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 5.61 (d, J = 16.5 Hz, 1H), 5.44 (s, 2H), 5.41 (d, J = 16.5 Hz, 1H), 4.51 (s, 2H), 3.22 - 3.07 (m, 8H), 2.58 (s, 3H), 2.03 - 1.93 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H).

[0480] 1.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 106) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

[0481] LC / MS:C 27 H 29 Calculated m / z for FN4O4 = 492.2, found [M+H] + =493.4.

[0482] 1.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 108) TIFF2025535239000141.tif58165 The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0483] LC / MS:C 32 H 32 Calculated m / z for FN5O4 = 569.2, observed [M+H] + =570.4.

[0484] 1H NMR (300 MHz, MeOD) δ 8.39 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 10.6 Hz, 1H), 7.21 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 5.62 (d, J = 16.4 Hz, 1H), 5.49 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.45 (s, 2H), 3.44 - 3.38 (m, 4H), 3.06 - 3.00 (m, 4H), 2.58 (d, J = 1.8 Hz, 3H), 2.00 - 1.89 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0485] 1.8: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 110) The title compound was prepared according to General Procedure 1, starting from compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0486] LC / MS:C 27 H 28 Calculated m / z for FN3O4 = 477.2, observed [M+H] + =478.2.

[0487] 1H NMR (300 MHz, MeOD) δ 8.34 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 10.3 Hz, 1H), 7.70 (s, 1H), 5.63 (d, J = 16.4 Hz, 1H), 5.52 (s, 2H), 5.44 (d, J = 16.5 Hz, 1H), 4.99 (s, 2H), 3.73 - 3.46 (m, 4H), 2.64 (s, 3H), 2.03 - 1.90 (m, 2H), 1.90 - 1.84 (m, 6H), 1.03 (t, J = 7.4 Hz, 3H).

[0488] 1.9: tert-Butyl (S)-4-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazine-1-carboxylate (compound 111) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

[0489] LC / MS:C 31 H 35 Calculated m / z for FN4O6 = 578.2, observed [M+H] + =579.4.

[0490] 1.10: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 112) TIFF2025535239000144.tif53165 The title compound was prepared according to general procedure 6 starting from compound 111 (5.0 mg) to afford the title compound as an off-white solid (TFA salt, 4.4 mg).

[0491] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [M+H] + =479.2.

[0492] 1.11: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(((R)-2-(hydroxymethyl)morpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 113) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).

[0493] LC / MS:C 27 H 28 Calculated m / z for FN3O6 = 509.2, found [M+H] + =510.4.

[0494] 1.12: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)thiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 114) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0495] LC / MS:C 27 H 28 Calculated m / z for FN3O5S = 525.6, observed [M+H] + =526.5.

[0496] 1 H NMR (300 MHz, 10%D2O / CD3CN) 8.36 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J = 16.4 Hz, 1H), 5.52 - 5.29 (m, 3H), 5.02 (d, J = 14.6 Hz, 1H), 4.71 - 4.54 (m, 1H), 4.27 (dd, J = 12.4, 5.0 Hz, 1H), 3.98 (dd, J = 12.3, 3.4 Hz, 1H), 3.55 (s, 1H), 3.30-3.03 (m, 4H) 2.97 - 2.72 (m, 3H), 2.62 (s, 1H), 2.55 (s, 3H), 0.95 (t, J = 7.4 Hz, 3H).

[0497] 1.13: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 115) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0498] LC / MS:C 28 H 28 Calculated m / z for FN3O6 = 521.5, observed [M+H] + =522.5.

[0499] 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).

[0500] 1.14: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxidethiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 116) The title compound was prepared according to general procedure 1 starting from compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ-thiomorpholine-1,1-dione. Purification was carried out as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0501] LC / MS:C 27 H 28 Calculated m / z for FN3O7S = 557.6, found [M+H] + =558.4.

[0502] 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).

[0503] 1.15: (4S)-4-Ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]heptan-3-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 117) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0504] LC / MS:C 28 H 28 Calculated m / z for FN3O5 = 505.5, observed [M+H] + =506.6.

[0505] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.25 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 10.6 Hz, 1H), 7.50 (s, 1H), 5.65 - 5.27 (m, 4H), 4.98 (s, 2H), 4.24 (s, 1H), 3.83 - 3.57 (m, 4H), 2.54 (s, 5H), 2.01-1.86 (m, 2H), 1.70 (s, 2H), 0.95 (t, J = 7.3 Hz, 3H).

[0506] 1.16: (S)-4-Ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 118) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0507] LC / MS:C 26 H 25 Calculated m / z for F2N3O5 = 497.5, observed [M+H] + =498.4.

[0508] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.24 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 10.7 Hz, 1H), 7.50 (s, 1H), 5.57 (d, J = 16.5 Hz, 1H), 5.48 - 5.28 (m, 3H), 4.98 (s, 2H), 4.44 - 4.14 (m, 4H), 3.78 (d, J = 14.9 Hz, 2H), 2.01-1.86 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).

[0509] 1.17: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)azetidin-1-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 119) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and azetidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0510] LC / MS:C 26 H 26 Calculated m / z for FN3O5 = 479.5, observed [M+H] + =480.4.

[0511] 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).

[0512] 1.18: (4S)-11-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 120) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

[0513] LC / MS:C 28 H 28 Calculated m / z for F3N3O5 = 543.5, observed [M+H] + =544.4.

[0514] 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).

[0515] 1.19: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]heptan-7-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 121) The title compound was prepared according to general procedure 1, starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]heptan-1-ylmethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0516] LC / MS:C 29 H 30 Calculated m / z for FN3O5 = 519.6, observed [M+H] + =520.4.

[0517] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.22 (s, 1H), 7.92 (d, J = 10.7 Hz, 1H), 7.54 (s, 1H), 5.59 (dd, J = 17.6, 7.6 Hz, 2H), 5.33 (t, J = 17.4 Hz, 2H), 4.98 - 4.81 (m, 1H), 4.67 - 4.44 (m, 2H), 4.28 - 3.93 (m, 4H), 2.73 (s, 2H), 2.34 - 2.03 (m, 4H), 1.91 (d, J = 14.0 Hz, 5H), 0.96 (t, J = 7.4 Hz, 3H).

[0518] 1.20: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 122) The title compound was prepared according to general procedure 3, starting from compound 1.2 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (0.8 mg, 7% yield).

[0519] LC / MS:C 23 H 22 Calculated m / z for FN3O6S = 487.1, observed [M+H] + =488.2.

[0520] 1 H NMR (300 MHz, MeOD) δ 8.33 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.8 Hz, 1H), 7.68 (s, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.52 (s, 2H), 5.42 (d, J = 16.4 Hz, 1H), 4.87 (s, 2H), 3.06 (s, 3H), 2.59 (s, 3H), 2.06-1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0521] 1.21: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (compound 124) The title compound was prepared according to general procedure 3, starting from compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.0 mg, 17% yield).

[0522] LC / MS:C 29 H 25 Calculated m / z for FN4O8S = 608.1, observed [M+H] + =609.2.

[0523] 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).

[0524] 1.22: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 125) The title compound was prepared according to general procedure 3, starting from compound 1.2 (10 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (9.8 mg, 73% yield).

[0525] LC / MS:C 28 H 24 Calculated m / z for FN3O6S = 549.6, observed [M+H] + =550.6.

[0526] 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).

[0527] 1.23: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 1.23) The title compound was prepared according to general procedure 3, starting from compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. Purification of the title compound was carried out as described in general procedure 9 using a 12 g C18 column eluted with a 5 to 75% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (37.8 mg, 47% yield).

[0528] LC / MS:C 28 H 23 Calculated m / z for FN4O8S = 594.6, observed [M+H] + =595.2.

[0529] 1.24: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 127) TIFF2025535239000158.tif63165 To a solution of compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL) was added platinum 1% vanadium 2% carbon (75 mg). The flask was purged with H and then stirred under an H atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (30 mg, 84% yield).

[0530] LC / MS:C 28 H 24 Calculated m / z for FN4O6S = 564.6, observed [M+H] + =565.2.

[0531] 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).

[0532] 1.25: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 129) The title compound was prepared according to general procedure 3, starting from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 25 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (1.3 mg, 13% yield).

[0533] LC / MS:C 24 H 24 Calculated m / z for FN3O7S = 517.1, observed [M+H] + =518.2.

[0534] 1 H NMR (300 MHz, DMSO-d6) δ 8.30 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 10.9 Hz, 1H), 7.84 (t, J = 6.3 Hz, 1H), 7.33 (s, 1H), 5.50-5.33 (m, 4H), 5.07 (t, J = 5.4 Hz, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.07 (s, 3H), 3.80 (dt, J = 6.3 Hz, J = 5.8 Hz, 2H), 1.86 (m, 2H), 0.87 (d, J = 7.3 Hz, 3H).

[0535] 1.26: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfamide (Compound 131) TIFF2025535239000160.tif53165 To a solution of chlorosulfonyl isocyanate (3 μL) in dichloromethane (1 mL) was added tert-butanol (3 μL). This solution was stirred for 1 hour, after which compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 μL). The reaction was stirred for 1 hour and then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was performed as described in General Procedure 9, eluting with a 10 to 50% CH3CN / HO + 0.1% TFA gradient. To the purified solid in dichloromethane (1 mL) was added trifluoroacetic acid (200 μL). The reaction was stirred for 16 hours and then concentrated to dryness to give the title compound as an off-white solid (7.5 mg, 48% yield).

[0536] LC / MS:C 22 H 21 Calculated m / z for FN4O6S = 488.1, observed [M+H] + =489.0.

[0537] 1 H NMR (300 MHz, MeOD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 10.7 Hz, 1H), 7.62 (s, 1H), 5.59 (d, J = 16.4 Hz, 1H), 5.45 (s, 2H), 5.39 (d, J = 16.4 Hz, 1H), 4.81 (s, 2H), 2.55 (d, J = 1.7 Hz, 3H), 2.07 - 1.89 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0538] 1.27: 4-Nitrophenyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 1.27) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting from compound 1.2 (24 mg). Purification was carried out as described in General Procedure 9 using a 12 g C18 column eluted with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (14 mg, 53% yield).

[0539] LC / MS:C 29 H 23 Calculated m / z for FN4O8S = 574.2, observed [M+H] + =575.2.

[0540] 1.28: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 132) The title compound was prepared according to general procedure 4, starting from compound 1.2 (25 mg) and aqueous methylamine (500 μL, 40 wt % in water) as the primary amine. In this case, the intermediate PNP-carbamate was used crude. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (8.9 mg, 31% yield).

[0541] LC / MS:C 24 H 23 Calculated m / z for FN4O5 = 466.2, observed [M+H] + =467.2.

[0542] 1H NMR (300 MHz, MeOD) δ 8.26 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 10.7 Hz, 1H), 7.66 (s, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.97 (s, 2H), 2.73 (s, 3H), 2.57 (s, 3H), 2.08 - 1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0543] 1.29: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 134) The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (0.6 mg, 12% yield).

[0544] LC / MS:C 30 H 28 Calculated m / z for FN5O5 = 557.2, found [M+H] + =558.4.

[0545] 1H NMR (300 MHz, MeOD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.67 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 5.63 (d, J = 16.4 Hz, 1H), 5.48 (s, 2H), 5.43 (d, J = 16.4 Hz, 1H), 5.01 (s, 2H), 4.37 (s, 2H), 2.56 (d, J = 1.7 Hz, 3H), 2.05 - 1.94 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0546] 1.30: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 136) The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (2.4 mg, 66% yield).

[0547] LC / MS:C 25 H 25 Calculated m / z for FN4O6 = 496.2, observed [M+H] + =497.2.

[0548] 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).

[0549] 1.31: Methyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 138) The title compound was prepared according to general procedure 5, starting from compound 1.2 (50 mg) and reacting methanol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (3.5 mg, 6% yield).

[0550] LC / MS:C 24 H 22 Calculated m / z for FN3O6 = 467.2, observed [M+H] + =468.2.

[0551] 1H NMR (300 MHz, MeOD) δ 8.17 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 10.5 Hz, 1H), 7.69 (s, 1H), 5.65 (d, J = 16.5 Hz, 1H), 5.48 (s, 2H), 5.33 (d, J = 16.4 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.65 (s, 3H), 2.56 (s, 3H), 2.02 - 1.89 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0552] 1.32: 2-Hydroxyethyl (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 139) The title compound was prepared according to general procedure 5, starting from compound 1.2 (18 mg) by reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.2 mg, 19% yield).

[0553] LC / MS:C 25 H 24 Calculated m / z for FN3O7 = 497.2, observed [M+H] + =498.2.

[0554] 1H NMR (300 MHz, DMSO) δ 8.23 ​​(d, J = 8.2 Hz, 1H), 7.78 (d, J = 10.7 Hz, 1H), 7.40 (s, 1H), 5.47 (d, J = 16.5 Hz, 1H), 5.42 (s, 2H), 5.34 (d, J = 16.4 Hz, 1H), 4.77 (s, 2H), 3.99 (t, J = 4.9 Hz, 2H), 3.64 - 3.38 (m, 2H), 2.48 (s, 3H), 2.02 - 1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0555] Example 2: Preparation of camptothecin analogs with methoxy at C10 position 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethan-1-one (compound 2.1) A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0 °C. To this solution was added first 1 M BCl in DCM (71 mL, 71 mmol), followed by 1 M chloro(diethyl)almane in DCM (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated at reflux for 3 h, cooled to room temperature, and quenched by the addition of 2 M aqueous HCl. The resulting heterogeneous mixture was heated at reflux for 1 h, cooled to room temperature, and then the pH was adjusted to approximately 12 with NaCO. The layers were separated, and the aqueous layer was extracted with DCM (3 × 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and flash purified as described in general procedure 9, eluting with 0 to 20% EtOAc / hexanes, to give the title compound (6 g, 28 mmol, 39% yield).

[0556] LC / MS: m / z calculated for C9H9ClFNO2 = 217.1, found [M+H] + =218.1.

[0557] 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 9.2 Hz, 1H), 6.44 (d, J = 12.8 Hz, 1H), 4.59 (s, 2H), 3.86 (s, 3H).

[0558] 2.2: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 2.2) To a solution of compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene (200 mL) was added toluene-4-sulfonic acid (157 mg, 0.9 mmol). The solution was heated at 140° C. for 3 hours and then cooled to room temperature. The product was collected by filtration as a yellow precipitate to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0559] LC / MS:C 22 H 18 Calculated m / z for ClFN2O5 = 445.2, found [M+H] + =445.1.

[0560] 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J =12.0 Hz, 1H) 7.80 (d, J = 9.2 Hz, 1H) 7.27 (s, 1H), 6.50 (s, 1H), 5.45 (s, 2H), 5.41 (s, 2H), 5.33 (s, 2H) 4.08 (s, 3H), 1.87 - 1.83 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0561] 2.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 101) The title compound was prepared according to General Procedure 1, starting from compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (5.6 mg, 41% yield).

[0562] LC / MS:C 26 H 26 Calculated m / z for FN3O6 = 495.2, found [M+H] + =496.4.

[0563] 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).

[0564] 2.4: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 103) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.5 mg, 14% yield).

[0565] LC / MS:C 32 H 31 Calculated m / z for FN4O7S = 634.2, observed [M+H] + =635.4.

[0566] 2.5: (S)-11-((4-((4-aminophenyl)sulfonyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 105) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (4.0 mg, 23% yield).

[0567] LC / MS:C 32 H 32 Calculated m / z for FN5O7S = 649.2, found [M+H] + =650.4.

[0568] 1H NMR (300 MHz, DMSO) δ 8.08 (s, 2H), 7.90 - 7.67 (m, 2H), 7.35 (s, 1H), 7.32 - 7.26 (m, 2H), 6.67 - 6.57 (m, 2H), 5.46 (d, J = 16.5 Hz, 1H), 5.33 -5.22 (m, 3H), 3.92 (s, 3H), 3.02 - 2.72 (m, 4H), 2.75 - 2.58 (m, 4H), 1.97 - 1.70 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H).

[0569] 2.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 107) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.1 mg, 19% yield).

[0570] LC / MS:C 27 H 29 Calculated m / z for FN4O5 = 508.2, observed [M+H] + =509.4.

[0571] 2.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 109) The title compound was prepared according to general procedure 1, starting from compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 20 to 60% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (3.2 mg, 20% yield).

[0572] LC / MS:C 32 H 32 Calculated m / z for FN5O5 = 585.2, observed [M+H] + =586.4.

[0573] 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).

[0574] 2.8: (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 2.8) To a solution of compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL) was added hexamethylenetetramine (236 mg, 1.7 mmol), followed by iPrNEt (100 μL, 0.56 mmol). The solution was heated to reflux for 5 h, cooled to room temperature, and quenched with 12 M aqueous HCl (60 μL). The solution was concentrated to approximately half its volume, and 1 M aqueous HCl (1.5 mL) was added. The mixture was stirred for 5 min and then concentrated to give a brown residue. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the title compound as a pale yellow solid (179 mg, 75% yield).

[0575] LC / MS:C 22 H 20 Calculated m / z for FN3O5 = 425.4, observed [M+H] + =426.2.

[0576] 2.9: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 123) The title compound was prepared according to general procedure 3, starting from compound 2.8 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 65% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (8.5 mg, 91% yield).

[0577] LC / MS:C 23 H 22 Calculated m / z for FN3O7S = 503.1, observed [M+H] + =504.2.

[0578] 1 H NMR (300 MHz, DMSO-d6) δ 7.98 (d, J = 12.1 Hz, 1H), 7.89 (t, J = 6.4 Hz, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.28 (s, 1H), 5.42 (s, 2H), 5.39 (s, 2H), 4.77 (d, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.06 (s, 3H), 1.95-1.73 (m, 2H), 0.88 (d, J = 7.3 Hz, 3H).

[0579] 2.10: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 126) The title compound was prepared according to general procedure 3, starting from compound 2.8 (7.5 mg) and benzenesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 5 to 70% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (4.6 mg, 46% yield).

[0580] LC / MS:C 28 H 24 Calculated m / z for FN3O7S = 565.6, observed [M+H] + =566.2.

[0581] 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).

[0582] 2.11: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 2.11) The title compound was prepared according to general procedure 3 starting from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride. Purification was carried out as described in general procedure 9 using a 12 g C18 flash column eluted with a 5 to 75% CH3CN / HO + 0.1% TFA gradient to afford the title compound as a pale yellow solid (9.7 mg, 71% yield).

[0583] LC / MS:C 28 H 23 Calculated m / z for FN4O9S = 610.6, observed [M+H] + =611.5.

[0584] 2.12: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (compound 128) TIFF2025535239000178.tif63165 To a solution of compound 2.11 (9.7 mg, 0.016 mmol) in methanol (1.6 mL) was added platinum 1% vanadium 2% carbon (15 mg). The flask was purged with H2 and then stirred under an H2 atmosphere at room temperature for 45 minutes. The mixture was filtered through a pad of Celite, washed with DMF, and the filtrate was evaporated to give the title compound as a pale yellow solid (1.5 mg, 16% yield).

[0585] LC / MS:C 28 H 25 Calculated m / z for FN4O7S = 580.6, observed [M+H] + =581.4.

[0586] 1 H NMR (300 MHz, MeOD) δ 7.77 (d, J = 11.0 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.39 (d, J = 16.4 Hz, 1H), 5.30 (s, 1H), 4.56 (s, 1H), 4.10 (d, J = 3.7 Hz, 3H), 2.04 - 1.91 (m, 2H), 1.31 (s, 1H), 1.02 (t, J = 7.3Hz, 3H), 0.90 (s, 1H).

[0587] 2.13: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 130) The title compound was prepared according to general procedure 3, starting from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 15 to 50% CHCN / HO+0.1% TFA gradient to afford the title compound as an off-white solid (2.2 mg, 22% yield).

[0588] LC / MS:C 24 H 24 Calculated m / z for FN3O8S = 533.1, observed [M+H] + =534.2.

[0589] 1 H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J = 12.2 Hz, 1H), 7.89-7.79 (m, 2H), 7.29 (s, 1H), 5.43 (s, 2H), 5.40 (s, 2H), 4.76 (d, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.81 (t, J = 6.3 Hz, 2H), 3.34 (t, J = 6.3 Hz, 2H), 1.94-1.75 (m, 2H), 0.87 (d, J = 7.4 Hz, 3H).

[0590] 2.14: 4-Nitrophenyl-(S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 2.14) The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4, starting with compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as the solvent. Flash purification was performed as described in General Procedure 9 using a 12 g C12 column eluted with a 10 to 50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (61 mg, 86% yield). This intermediate was resolved and used to generate the next compound.

[0591] LC / MS:C 29 H 23 Calculated m / z for FN4O9 = 590.1, observed [M+H] + =591.2.

[0592] 2.15: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 133) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and aqueous methylamine (500 uL, 40 wt % in water) as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (5.8 mg, 47% yield).

[0593] LC / MS:C 24 H 23 Calculated m / z for FN4O6 = 482.2, observed [M+H] + =483.2.

[0594] 1H NMR (300 MHz, DMSO-d6) δ 8.00 - 7.87 (m, 2H), 7.31 (s, 1H), 5.48 - 5.39 (m, 3H), 4.81 (s, 3H), 2.56 (s, 3H), 1.93 - 1.81 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0595] 2.16: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (compound 135) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

[0596] LC / MS:C 30 H 28 Calculated m / z for FN5O6 = 573.2, found [M+H] + =574.2.

[0597] 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).

[0598] 2.17: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 137) The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (1.5 mg, 12% yield).

[0599] LC / MS:C 25 H 25 Calculated m / z for FN4O7 = 512.2, observed [M+H] + =513.2.

[0600] 1H NMR (300 MHz, MeOD) δ 7.93 (d, J = 12.1 Hz, 1H), 7.88 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 5.62 (d, J = 16.2 Hz, 1H), 5.52 (s, 2H), 5.45 (d, J = 16.3 Hz, 1H), 4.98 (s, 2H), 4.17 (s, 3H), 3.59 (t, J = 5.6 Hz, 2H), 3.28 (t, J = 5.6 Hz, 2H), 2.10 - 1.91 (m, 2H), 1.05 (t, J = 7.3 Hz, 3H).

[0601] Example 3: Preparation of camptothecin analogs with an amino at the C10 position 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (compound 3.1) TIFF2025535239000184.tif2716To a stirred solution of HNO3 (121.2 mL, 67% purity, 2.0 equiv.) in H2SO4 (500 mL) at 50 °C was added 3-bromo-4-fluorobenzaldehyde (180 g, 1.0 equiv.). After the addition was complete, the ice bath was removed and the reaction was stirred at 25 °C for 5 h. The mixture was poured onto ice (5 L), filtered, and then dried under reduced pressure. The title compound was obtained as a yellow solid (219 g).

[0602] 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).

[0603] 3.2: tert-Butyl (2-fluoro-5-formyl-4-nitrophenyl)carbamate (compound 3.2) A mixture of compound 3.1 (219 g, 1.0 equiv.), tert-butyl carbamate (124 g, 1.2 equiv.), CsCO (575 g, 2.0 equiv.), Pd(dba) (40 g, 0.05 equiv.), and XPhos (84 g, 0.2 equiv.) in toluene (2000 mL) was degassed and purged with N for three cycles. The mixture was then stirred at 90 °C under a N atmosphere for 15 h. The reaction mixture was diluted with H2O (800 mL) and extracted with EtOAc (300 mL × 2). The combined organic layers were washed with brine (200 mL × 2), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 20:1) to give the title compound as a yellow solid (140 g, 56% yield).

[0604] 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.94 (s, 1H), 8.42 (d, J=7.6 Hz, 1H), 8.16 (d, J=10.8 Hz, 1H), 1.50 (s, 9H).

[0605] 3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (compound 3.3) TIFF2025535239000186.tif27165 To a solution of compound 3.2 (100 g, 1.0 equiv.) in HO (300 mL) and EtOH (1200 mL) was added NH4Cl (30.5 g, 1.62 equiv.). Iron (78.6 g, 4.0 equiv.) was added portionwise at 80 °C. The mixture was stirred at 80 °C for 6 h. The mixture was filtered, water was added to the filtrate, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1) and TLC (petroleum ether) to give the title compound as a yellow solid (19.0 g, 21% yield).

[0606] LC / MS:C 12 H 15 Calculated m / z for FN2O3 = 254.1, observed [M+H] + =255.0.

[0607] 1 H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1 H), 8.57 (s, 1 H), 7.58 (d, J = 4.8 Hz, 1 H), 7.21 (s, 2 H), 6.53 (d, J = 12.8 Hz, 1 H), 1.43 (s, 9 H).

[0608] 3.4: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.4) A mixture of compound 3.3 (4.20 g, 1.2 equiv.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 equiv.), and TsOH (monohydrate, 253 mg, 0.1 equiv.) in toluene (350 mL) was stirred at 110 °C for 2 h. The reaction solution was cooled to 25 °C, and the solid was washed with methyl t-butyl ether (30 mL) and then dried under reduced pressure. The title compound was obtained as a yellow solid (4.5 g, 62% yield).

[0609] LC / MS:C 25 H 24 Calculated m / z for FN3O6 = 481.2, observed [M+H] + =482.1.

[0610] 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).

[0611] 3.5: tert-Butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.5) TIFF2025535239000188.tif47165 To a mixture of compound 3.4 (4.00 g) in MeOH (360 mL) was added a solution of FeSO (heptahydrate, 1.2 g) and HSO (280 μL) in H2O (4 mL). The reaction mixture was heated to 65 °C while H2O (24 mL, 30% purity) was added dropwise over 30 min and then stirred for 0.5 h. The reaction solution was cooled to 25 °C and filtered to give the title compound as a yellow solid (1.53 g, 33.2% yield). The filtrate was added with H2O (400 mL) and then quenched with saturated aqueous Na2SO3. The pH was adjusted to 7-8 with saturated aqueous Na2CO3, and the solution was concentrated and filtered. The solid was triturated with MeOH (30 mL) at 55° C. for 1 h and then filtered to give a second batch of the title compound as a brown solid (1.09 g, 26% yield).

[0612] LC / MS:C 26 H 26 Calculated m / z for FN3O7 = 511.2, observed [M+H] + =512.2.

[0613] 1H NMR (300 MHz, d6-DMSO) δ 9.47 (s, 1H), 8.47 (d, J =7.6 Hz, 1H), 7.94 (d, J =12.0 Hz, 1H), 7.29 (d, J =1.6 Hz, 1H), 6.49 (s, 1H), 5.86 - 5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J =4.4 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 6.4 Hz, 3H).

[0614] 3.6: tert-Butyl (S)-(4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.6) To a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), DCM (2.9 mL) was added, followed by Dess-Martin periodinane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). The solution was stirred at room temperature for 18 h, then diluted with DCM and washed with saturated aqueous NaHCO3 and brine. The layers were separated, and the combined organic layer was evaporated onto Celite. Flash purification was performed as described in General Procedure 9 using a 10 g silica column eluting with 0 to 10% DCM / MeOH to afford the title product as an orange powder (42.5 mg, 28%).

[0615] LC / MS:C 26 H 24 Calculated m / z for FN3O7 = 509.2, found [M+H] + =510.4.

[0616] 1H NMR (300 MHz, acetone-d6) δ 11.10 (s, 1H), 9.68 (d, J =8.6 Hz, 1H), 8.81 (s, 1H), 8.04 (d, J =11.9 Hz, 1H), 7.63 (s, 1H), 5.73 (s, 2H), 5.69 (d, J =16.2 Hz, 1H), 5.42 (d, J =16.2 Hz, 1H), 2.02-1.95 (m, 2H), 8.47 (d, J =7.6 Hz, 1H), 7.94 (d, J =12.0 Hz, 1H), 7.29 (d, J =1.6 Hz, 1H), 6.49 (s, 1H), 5.86 - 5.76 (m, 1H), 5.42 (s, 2H), 5.38 (s, 2H), 5.16 (d, J =4.4 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.52 (s, 9H), 0.88 (t, J = 6.4 Hz, 3H).

[0617] 3.7: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 140) TIFF2025535239000190.tif37165 The title compound was prepared according to general procedure 6 starting from compound 3.4 (40 mg) to afford the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[0618] LC / MS:C 20 H 16 Calculated m / z for FN3O4 = 381.1, observed [M+H] + =382.2.

[0619] 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).

[0620] 3.8: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 141) TIFF2025535239000191.tif47165 The title compound was prepared according to general procedure 6 starting from compound 3.5 (5 mg) to afford the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).

[0621] LC / MS:C 21 H 18 Calculated m / z for FN3O5 = 411.2, observed [M+H] + =412.2.

[0622] 1 H NMR (300 MHz, MeOD) δ 7.71 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.29 (d, J = 9.5 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.47 (s, 2H), 5.40 (d, J = 16.3 Hz, 1H), 5.25 (s, 2H), 2.03 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0623] 3.9: tert-Butyl (S)-(11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.9) To a stirred solution of compound 3.5 (100 mg) in dichloromethane (5 mL) was added a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL). After 1 h, additional thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added. After an additional 1 h, the reaction was diluted with dichloromethane (10 mL) and toluene (1 mL) and then concentrated in vacuo to give the title compound as a red solid, which was used in the next reaction without further purification.

[0624] LC / MS:C 26 H 25 Calculated m / z for ClFN3O6 = 529.1, found [M+H] + =530.2.

[0625] 3.10: tert-Butyl (S)-(11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.10) TIFF2025535239000193.tif47165 To compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg) followed by DIPEA (99 μL). The solution was heated at 60° C. for 16 hours and then concentrated to dryness in vacuo. Flash purification was performed as described in General Procedure 9 using a 12 g C18 column eluted with a 10 to 50% CH3CN / HO + 0.1% TFA gradient to afford the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

[0626] LC / MS:C 26 H 27Calculated m / z for FN4O6 = 510.2, observed [M+H] + =511.4.

[0627] 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).

[0628] 3.11: (S)-9-Amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 145) TIFF2025535239000194.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.10 (2.1 mg) to afford the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[0629] LC / MS:C 21 H 19 Calculated m / z for FN4O4 = 410.1, found [M+H] + =411.2.

[0630] 1H NMR (300 MHz, MeOD) δ 7.82 (d, J = 12.1 Hz, 1H), 7.60 (s, 1H), 7.37 (d, J = 9.1 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.42 (s, 2H), 5.41 (d, J = 16.3 Hz, 1H), 4.69 (s, 2H), 2.08 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0631] Example 3.12: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 3.12) The title compound was prepared according to General Procedure 1, starting from compound 3.9 (150 mg) and morpholine. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a red solid (TFA salt, 103 mg, 52% yield).

[0632] LC / MS:C 30 H 33 Calculated m / z for FN4O7 = 580.2, observed [M+H] + =581.4.

[0633] 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).

[0634] 3.13: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 142) TIFF2025535239000196.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.12 (45 mg) to afford the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[0635] LC / MS:C 25 H 25 Calculated m / z for FN4O5 = 480.2, observed [M+H] + =481.4.

[0636] 1 H NMR (300 MHz, MeOD) δ 7.73 (d, J = 12.0 Hz, 1H), 7.54 (s, 1H), 7.48 (d, J = 9.2 Hz, 1H), 5.60 (d, J = 16.3 Hz, 1H), 5.47 - 5.34 (m, 3H), 4.65 (s, 2H), 3.91 - 3.85 (m, 4H), 3.30 - 3.24 (m, 4H), 2.08 - 1.91 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H).

[0637] 3.14: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 148) To a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol), dichloromethane (1.45 mL) was added, followed by acetic acid (18.69 μL, 0.327 mmol), piperidine (21.52 μL, 0.218 mmol), and sodium triacetoxyborohydride (23.0 mg, 0.109 mmol). The solution was then stirred at room temperature for 2 h, quenched by the addition of water + 0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to general procedure 6 to give the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).

[0638] LC / MS:C 26 H 27 Calculated m / z for FN4O4 = 478.2, observed [M+H] + =479.4.

[0639] 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).

[0640] 3.15: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 149) To a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol) was added dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and N-methylpiperazine (4.90 μL, 0.044 mmol). The solution was stirred at room temperature for 4 h, followed by the addition of sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) and stirring for an additional 45 min. The excess hydride was then quenched by the addition of 0.1% aqueous TFA (0.5 mL). Purification was carried out as described in General Procedure 9 using a 12 g C18 flash column eluted with a 5 to 40% CH3CN / HO + 0.1% TFA gradient to give the Boc-protected intermediate as a yellow powder. This intermediate was deprotected according to general procedure 6 to give the title compound as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

[0641] LC / MS:C 26 H 28 Calculated m / z for FN5O4 = 493.2, observed [M+H] + =494.4.

[0642] 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).

[0643] 3.16: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 153) The Boc-protected precursor of the title compound was prepared according to General Procedure 1, starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC was performed as described in General Procedure 9, eluting with a 35 to 44% CHCN / HO + 0.1% TFA gradient, to afford the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to afford the title compound (TFA salt, 2.4 mg, 17% yield over two steps).

[0644] LC / MS: m / z calculated for C31H30FN5O6S = 619.2, found [M+H]+ = 520.4.

[0645] 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).

[0646] 3.17: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 147) The title compound was prepared according to General Procedure 2 followed by General Procedure 6, starting from compound 3.10 (8 mg) and acetic acid. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.0 mg, 56% yield).

[0647] LC / MS:C 23 H 21 Calculated m / z for FN4O5 = 452.2, observed [M+H] + =453.2.

[0648] 1 H NMR (300 MHz, MeOD) δ 7.69 (d, J = 12.1 Hz, 1H), 7.56 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.44 - 5.33 (m, 3H), 4.85 (s, 3H), 2.03 (s, 3H), 2.00 - 1.84 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0649] 3.18: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (compound 146) The title compound was prepared according to general procedure 3 followed by general procedure 6, starting from compound 3.10 (8 mg) and methanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (4.4 mg, 57% yield).

[0650] LC / MS:C 22 H 21 Calculated m / z for FN4O6S = 488.1, observed [M+H] + =489.2.

[0651] 1 H NMR (300 MHz, MeOD) δ 7.74 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.49 (d, J = 9.3 Hz, 1H), 5.61 (d, J = 16.2 Hz, 1H), 5.45 (s, 2H), 5.40 (d, J = 16.2 Hz, 1H), 4.78 (s, 2H), 3.05 (s, 3H), 2.08 - 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0652] 3.19: (S)—N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 150) The title compound was prepared according to general procedure 3 followed by general procedure 6, starting from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO+0.1% TFA gradient. The title compound was obtained as a red solid (1 mg, 16% yield).

[0653] LC / MS:C 23 H 23 Calculated m / z for FN4O7S = 518.5, observed [M+H] + =519.5.

[0654] 1H NMR (300 MHz, 10% D2O / CD3CN) δ 7.77 - 7.61 (m, 1H), 7.48 - 7.30 (m, 2H), 5.53 (d, J = 16.3 Hz, 1H), 5.31 (d, J = 15.4 Hz, 3H), 4.69 (s, 2H), 3.97 (dd, J = 6.6, 4.9 Hz, 2H), 3.39 (t, J = 5.8 Hz, 2H), 2.93 (s, 1H), 1.99-1.83 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0655] 3.20: 4-Nitrophenyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 3.20) To a solution of compound 3.10 (10 mg, 0.02 mmol) in DMF (400 μL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 μL, 0.04 mmol). The solution was stirred at room temperature for approximately 30 minutes and then used directly in the next reaction.

[0656] 3.21: Methyl (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (compound 143) The title compound was prepared by adding MeOH (100 μL) to a solution of 200 μL of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.1 mg, 47% yield) following General Procedure 6.

[0657] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.4, observed [M+H] + =468.3.

[0658] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 7.72 (d, J = 12.2 Hz, 1H), 7.41 (d, J = 18.1 Hz, 1H), 6.96 (s, 1H), 5.52 (d, J = 3.6 Hz, 1H), 5.39 - 5.23 (m, 3H), 4.82 (s, 1H), 4.73 (s, 1H), 3.63 (d, J = 1.2 Hz, 3H), 1.56 (s, 3H), 1.27 (s, 2H), 0.94 (t, J = 7.4 Hz, 3H).

[0659] 3.22: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (compound 144) The title compound was prepared by adding methylamine hydrochloride (10 mg) to 200 μL of a solution of compound 3.20, followed by the addition of iPrNEt (5 μL). The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (2.9 mg, 64.5% yield) following General Procedure 6.

[0660] LC / MS:C 23 H 21 Calculated m / z for FN5O5 = 467.5, observed [M+H] + =468.5.

[0661] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 8.13 (d, J = 9.2 Hz, 1H), 7.92 (s, 1H), 7.73 (d, J = 12.3 Hz, 1H), 7.52 - 7.35 (m, 2H), 6.94 (d, J = 9.2 Hz, 2H), 5.55 (d, J = 16.5 Hz, 2H), 5.44 - 5.27 (m, 4H), 4.85 (s, 2H), 4.78 (s, 1H), 1.56 (d, J = 2.5 Hz, 3H), 1.27 (s, 2H), 0.93 (q, J = 11.7, 9.5 Hz, 3H).

[0662] 3.23: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 151) The title compound was prepared by adding ethanolamine (100 μL) to a 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 performed as described in General Procedure 9, eluting with a 10-60% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a red solid (0.5 mg, 8.5% yield) following General Procedure 6.

[0663] LC / MS:C 24 H 24 Calculated m / z for FN5O6 = 497.5, observed [M+H] + =498.5.

[0664] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 7.77 - 7.61 (m, 1H), 7.48 - 7.30 (m, 2H), 5.53 (d, J = 16.3 Hz, 1H), 5.31 (d, J = 15.4 Hz, 1H), 5.19 (s, 2H), 4.69 (s, 2H), 3.97 (dd, J = 6.6, 4.9 Hz, 2H), 3.39 (t, J = 5.8 Hz, 2H), 2.93 (s, 1H), 2.01-1.83 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0665] 3.24: (S)-9-Amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 152) To a stirred solution of compound 3.5 (100 mg) in 2 mL of dichloromethane was added thionyl chloride (35 μL, 2.5 equiv.). The solution was stirred at room temperature for 20 minutes, after which additional thionyl chloride (35 μL, 2.5 equiv.) was added. After 20 minutes, toluene (1 mL) was added, and the reaction mixture was concentrated in vacuo. The crude solid was suspended in DMSO (1 mL), and sodium azide (19 mg, 1.5 equiv.) was added. The solution was stirred at room temperature for 16 hours. Purification was carried out as described in General Procedure 9, eluting with a 5-50% CH3CN / HO + 0.1% TFA gradient, to afford the title compound as an off-white solid (20 mg, 23% yield).

[0666] LC / MS:C 21 H 17 Calculated m / z for FN6O4 = 436.1, observed [M+H] + =437.2.

[0667] 1 H NMR (300 MHz, MeOD) δ 7.75 (d, J = 12.2 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.46 - 5.35 (m, 3H), 5.07 (s, 2H), 2.03 - 1.97 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).

[0668] 3.25: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (compound 164) The title compound was prepared according to general procedure 2, starting from compound 145 (10 mg) and glycolic acid. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (6.9 mg, 60% yield).

[0669] LC / MS:C 23 H 21 Calculated m / z for FN4O6 = 468.1, observed [M+H] + =469.2.

[0670] 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).

[0671] 3.26: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylthiourea (compound 161) To a solution of compound 145 (9 mg, 1.0 equiv.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 equiv.), followed by DIPEA (8 μL, 2.0 equiv.). The resulting solution was stirred at 25° C. for 2 h, after which complete conversion to the isothiocyanate intermediate was confirmed. Methylammonium chloride (3 mg, 2.0 equiv.) was then added, and the reaction mixture was heated at 60° C. for 30 min. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10-45% CH CN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (2.3 mg, 22% yield).

[0672] LC / MS:C 23 H 22 Calculated m / z for FN5O4S = 483.1, observed [M+H] + =484.2.

[0673] 1 H NMR (300 MHz, MeOD) δ 7.70 (d, J = 12.0 Hz, 1H), 7.60 (s, 1H), 7.38 (d, J = 9.3 Hz, 1H), 5.62 (d, J = 16.2 Hz, 1H), 5.36 (s, 2H), 5.31 (d, J = 16.2 Hz, 1H), 5.30 (s, 2H), 3.04 (s, 3H), 1.99 - 1.90 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).

[0674] 3.27: S-(2-Hydroxyethyl)-(S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamothioate (compound 160) The title compound was prepared according to general procedure 5, starting from compound 145 (10 mg) and 2-mercaptoethanol. Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 45% CHCN / HO + 0.1% TFA gradient. The title compound was obtained as a yellow solid (4.2 mg, 43% yield).

[0675] LC / MS:C 24 H 23 Calculated m / z for FN4O6S = 514.1, observed [M+H] + =515.2.

[0676] 1 H NMR (300 MHz, MeOD) δ 7.71 (d, J = 12.1 Hz, 1H), 7.60 (s, 1H), 7.36 (d, J = 9.4 Hz, 1H), 5.62 (d, J = 16.3 Hz, 1H), 5.42 (s, 2H), 5.35 (d, J = 16.2 Hz, 1H), 4.88 (d, J = 4.6 Hz, 2H), 3.68 (t, J = 6.4 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.04 - 1.92 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0677] 3.28: (S)-9-Amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 154) TIFF2025535239000211.tif47165 To a 5 mL flask containing compound 140 (50 mg), water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg), and propionaldehyde (74 μL) were added. The resulting suspension was cooled to −15 °C using an ice-salt water bath, and then sulfuric acid (0.40 mL) was added dropwise. Hydrogen peroxide (95 μL) was then added dropwise. The mixture was stirred at −15 °C for 10 minutes, then warmed to room temperature, and stirred for 2 hours. The reaction mixture was diluted with water (30 mL), and the resulting suspension was extracted with DCM (3 × 30 mL). The organic phase was then evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25 to 70% CH3CN / H2O+0.1% TFA gradient to afford the title compound as a deep orange solid (2.4 mg, 4.4% yield).

[0678] LC / MS:C 22 H 20 Calculated m / z for FN3O4 = 410.1, found [M+H] + =410.2.

[0679] 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).

[0680] 3.29: tert-Butyl-(S)-(11-((carbamoyloxy)methyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.29) Compound 3.5 (15 mg) was added to a 5 mL Erlenmeyer flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL) at −20° C. The resulting suspension was stirred at −20° C. for 5 minutes. Water (59 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 2 hours, then heated at 70° C. for 1 hour. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 40-55% CH3CN / HO + 0.1% TFA gradient, to afford the title compound as a dark orange solid (5.1 mg, 31% yield).

[0681] LC / MS:C 27 H 27 Calculated m / z for FN4O8 = 555.2, observed [M+H] + =555.2.

[0682] 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).

[0683] 3.30: (S)-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methylcarbamate (compound 169) TIFF2025535239000213.tif53165 The title compound was prepared according to general procedure 6 starting from compound 3.29 (5.1 mg) to afford the title compound as a yellow powder (TFA salt, 3.8 mg, 73% yield).

[0684] LC / MS:C 22 H 19 Calculated m / z for FN4O6 = 455.1, observed [M+H] + =455.2.

[0685] 1 H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J = 12.4 Hz, 1H), 7.29 (d, J = 9.7 Hz, 1H), 7.21 (s, 1H), 7.0-6.50 (m, 2H), 5.45 (s, 2H), 5.40 (s, 2H), 5.33 (s, 2H), 1.95-1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0686] 3.31: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 155) TIFF2025535239000214.tif47165 To a 50 mL flask containing compound 3.5 (30 mg) was added MeOH / dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL). The reaction mixture was then stirred at reflux for 24 h. The reaction mixture was concentrated, poured into water (30 mL), and extracted with DCM (3 × 50 mL). The organic phases were combined and dried over MgSO. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25-40% CHCN / H2O + 0.1% TFA gradient, to afford the title compound as a dark orange solid (5.1 mg, 16% yield).

[0687] LC / MS:C 22 H 20 FN3 Calculated m / z for O5 = 426.1, observed [M+H]+ = 426.2.

[0688] 1 H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J = 12.3 Hz, 1H), 7.24 (d, J = 9.9 Hz, 1H), 7.20 (s, 1H), 6.47 (s, 1H), 6.30-5.92 (brs, 2H), 5.40 (s, 2H), 5.24 (s, 2H), 4.93 (s, 2H), 3.43 (s, 3H), 1.95-1.75 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0689] 3.32: (4S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(((1R,5S)-6-hydroxy-3-azabicyclo[3.1.1]heptan-3-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 158) To a 5 mL Erlenmeyer flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL), followed by 3-azabicyclo[3.1.1]heptan-6-ol (10 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature, and sodium triacetoxyborohydride (9.4 mg) was added. After 1 h at room temperature, the reaction was quenched by the addition of water + 0.1% TFA and diluted with DMF. The reaction mixture was then partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient, to afford the Boc-protected title compound as a yellow powder. Deprotection was carried out according to general procedure 6, and the resulting residue was purified by preparative HPLC purification as described in general procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as a yellow powder (TFA salt, 7.1 mg, 39% yield).

[0690] LC / MS:C 27 H 27 Calculated m / z for FN4O5 = 507.2, [M+H] + =507.4.

[0691] 1 H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J = 12.1 Hz, 1H), 7.46 (d, J = 9.4 Hz, 1H), 7.23 (s, 1H), 6.64-5.85 (m, 3H), 5.60-5.25 (m, 4H), 4.85 (s, 1H), 4.10-3.95 (m, 1H), 3.68 (s, 2H), 2.45-2.33 (m, 2H), 1.96-1.72 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0692] 3.33: (S)-9-Amino-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 159) To a 5 mL Erlenmeyer flask containing compound 3.6 (15 mg) was added dichloromethane (0.6 mL), followed by (3-fluoroazetidin-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction was stirred at room temperature, and sodium triacetoxyborohydride (9.4 mg) was added. After 1 h at room temperature, the reaction was quenched by the addition of water + 0.1% TFA, diluted with DMF, and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20-50% CH3CN / HO + 0.1% TFA gradient, to afford the Boc-protected title compound as a yellow powder. Deprotection was then carried out according to General Procedure 6. The resulting residue was purified by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 50% CHCN / H0+0.1% TFA gradient to afford the title compound as a yellow powder (TFA salt, 1.8 mg, 10% yield).

[0693] LC / MS:C 25 H 24 Calculated m / z for F2N4O5 = 499.2, found [M+H] + =499.4.

[0694] 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).

[0695] 3.34: tert-Butyl-(S)-(4-ethyl-8-fluoro-4-hydroxy-11-((methylamino)methyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (compound 3.34) To a stirred solution of compound 3.9 (210 mg) in DMF (5 mL) was added sodium iodide (5.9 mg), followed by methylammonium chloride (107 mg). The reaction mixture was then stirred at room temperature overnight. Reverse-phase purification was performed as described in General Procedure 9 using a 30 g C18 column eluted with a 10 to 65% CH3CN / HO + 0.1% TFA gradient to afford the title compound as a yellow solid (15.0 mg, 7.2% yield).

[0696] LC / MS:C 27 H 29 Calculated m / z for FN4O6 = 524.2, observed [M+H] + =525.4.

[0697] 3.35: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-N-methylacetamide (compound 165) The Boc-protected form of the title compound was prepared according to General Procedure 2, starting from compound 3.34 (6.4 mg) and glycolic acid. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 20 to 50% CHCN / HO + 0.1% TFA gradient. Deprotection was then carried out according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.0 mg, 28% yield).

[0698] LC / MS:C 24 H 23 Calculated m / z for FN4O6 = 482.2, observed [M+H] + =483.2.

[0699] 1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J = 12.3 Hz, 1H), 7.27 (d, J = 9.5 Hz, 1H), 7.22 (s, 1H), 6.48 (s, 1H), 6.28-6.02 (m, 2H), 5.40 (s, 2H), 5.21 (s, 2H), 5.06-4.93 (m, 2H), 4.18 (s, 2H), 2.80 (s, 3H), 1.92-1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0700] 3.36: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-N-methylmethanesulfonamide (compound 166) The Boc-protected form of the title compound was prepared according to General Procedure 3, starting from compound 3.34 (8.0 mg) and methanesulfonyl chloride. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient. Deprotection was then carried out according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.6 mg, 34% yield).

[0701] LC / MS:C 23 H 23 Calculated m / z for FN4O6S = 502.1, observed [M+H] + =503.2.

[0702] 1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J = 12.3 Hz, 1H), 7.41 (d, J = 9.4 Hz, 1H), 7.23 (s, 1H), 6.63-5.84 (m, 2H), 5.42 (s, 2H), 5.29 (s, 2H), 4.81-4.64 (m, 2H), 3.14 (s, 3H), 2.67 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0703] 3.37: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 170) TIFF2025535239000220.tif53165 To a 10 mL round-bottom flask containing compound 3.4 (62.0 mg), water (0.89 mL), FeSO4 (heptahydrate, 18.0 mg), and 3-methoxypropanal (113.0 mg) were added. To the resulting suspension, sulfuric acid (0.495 mL) was added dropwise while stirring in an ice-salt bath at −15°C. Hydrogen peroxide (0.118 mL) was then added dropwise. The mixture was stirred at −15°C for 10 minutes, then warmed to room temperature, and stirred for 1 hour. The reaction mixture was then diluted with water (30 mL), and the resulting suspension was extracted with DCM (3 × 30 mL). The organic phase was evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 25 to 45% CHCN / H0+0.1% TFA gradient to afford the title compound as a deep orange solid (TFA salt, 3.1 mg, 4.4% yield).

[0704] LC / MS:C 23 H 22 Calculated m / z for FN3O5 = 440.2, observed [M+H] + =440.2.

[0705] 1H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J = 12.4 Hz, 1H), 7.33 (d, J = 9.4 Hz, 1H), 7.20 (s, 1H), 6.60-6.42 (m, 2H), 5.40 (s, 2H), 5.25 (s, 2H), 3.69 (t, J = 6.5 Hz, 2H), 3.24 (s, 3H), 3.23 (t, J = 6.5 Hz, 2H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0706] 3.38: (S)-N-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)acetamide (Compound 171) To a 25 mL round-bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL) was added N-methylmorpholine (0.343 mL), HOAt (0.142 g), and HATU (0.435 g). After stirring at room temperature for 5 minutes, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). Immediately after stirring at room temperature for 24 hours, the solution was purified by preparative HPLC as described in General Procedure 9, eluting with a 25-45% CHCN / HO + 0.1% TFA gradient to afford the title compound as a bright yellow powder (43.0 mg, 38% yield).

[0707] LC / MS:C 22 H 18 Calculated m / z for FN3O5 = 424.1, observed [M+H] + =424.2.

[0708] 1H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.61 (s, 1H), 7.96 (d, J = 912.1 Hz, 1H), 7.29 (s, 1H), 6.60-6.42 (m, 2H), 5.41 (s, 2H), 5.21 (s, 2H), 2.20 (s, 3H), 1.96-1.76 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0709] 3.39: tert-Butyl (5-formyl-2-methoxy-4-nitrophenyl)carbamate (compound 3.39) To a solution of compound 3.2 (1.3 g, 1.0 equiv.) in MeOH (12 mL) at 650° C. was added sodium methoxide (0.74 g, 3.0 equiv.). After the addition was complete, the ice bath was removed and the resulting solution was stirred at room temperature for 72 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 afford the title compound as an orange solid (1.2 g, 89% yield).

[0710] LC / MS:C 13 H 16 Calculated m / z for N2O6 = 296.10, observed [M+H] + =297.1.

[0711] 1 H NMR (300 MHz, MeOD) δ 10.29 (s, 1H), 8.61 (s, 1H), 7.73 (s, 1H), 4.08 (s, 3H), 1.57 (s, 9H)

[0712] 3.40: tert-Butyl (4-amino-5-formyl-2-methoxyphenyl)carbamate (compound 3.40) To a solution of compound 3.39 (500 mg, 1 equiv.) in MeOH (10 mL) and HO (1 mL) was added B(OH) (454 mg, 3 equiv.). The resulting mixture was cooled to 0 °C, and 5 M aqueous NaOH (2.75 mL) was added with stirring over 10 min. The reaction mixture was stirred for an additional 5 min before being quenched by pouring the solution onto ice (40 mL). The resulting mixture was extracted with DCM (3 × 50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was performed as described in General Procedure 9 using a 25 g silica column eluting with 10 to 50% hexanes / EtOAc to afford the title compound as an orange solid (386 mg, 86%).

[0713] LC / MS:C 13 H 18 Calculated m / z for N2O4 = 266.1, observed [M+H] + =297.2.

[0714] 3.41: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 168) A mixture of compound 3.40 (385 mg, 1.0 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (362 mg, 0.95 equiv.), TsOH (monohydrate, 25 mg, 0.1 equiv.), and toluene (30 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 hours. The reaction mixture was then cooled to 25° C. and concentrated under reduced pressure. Purification was carried out as described in General Procedure 9 using a 25 g silica column eluted with a 0 to 50% DCM / MeOH gradient to give the Boc-protected intermediate as a red solid. This material was then deprotected according to General Procedure 6, followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CHCN / H0+0.1% TFA gradient, to afford the title compound as a red solid (TFA salt, 300 mg, 53% yield).

[0715] LC / MS:C 21 H 19 Calculated m / z for N3O5 = 393.2, observed [M+H] + =393.2.

[0716] 1 H NMR (300 MHz, MeOD) δ 8.27 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 7.11 (s, 1H), 5.61 (d, J = 16.2 Hz, 1H), 5.38 (d, J = 16.2 Hz, 1H), 5.24 (s, 2H), 4.11 (s, 3H), 2.06 - 1.91 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).

[0717] 3.42: 5-Bromo-2-nitro-4-(trifluoromethyl)benzaldehyde (compound 3.42) TIFF2025535239000225.tif3216 To a stirred solution of HNO3 (2.0 g, 1.4 mL, 67% purity, 2 equiv.) in H2SO4 (8 mL) at 50 °C was added 3-bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 equiv.). After the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 5 h. The mixture was poured 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 afford the title compound as a yellow solid (4.4 g, 93% yield).

[0718] LC / MS: m / z calculated for C8H3BrF3NO3 = 296.90, found [M+H] + =298.0.

[0719] 1 H NMR (300 MHz, MeOD) δ 10.35 (s, 1H), 8.29 (s, 1H), 8.23 ​​(s, 1H).

[0720] 3.43: tert-Butyl (5-formyl-4-nitro-2-(trifluoromethyl)phenyl)carbamate (compound 3.43) TIFF2025535239000226.tif32165 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(propan-2-yl)[1,1'-biphenyl]-2-yl]phosphane in toluene (5 mL). (A mixture of HCl (256 mg, 0.2 equiv.) and HCl (256 mg, 0.2 equiv.) was degassed and purged with N2 three times. The mixture was then stirred at 90 °C under a N2 atmosphere for 15 h. The reaction mixture was diluted with HO (25 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (2 × 25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was performed using a 25 g silica column according to General Procedure 9, eluting with 0 to 25% DCM / MeOH to afford the title compound as an orange solid (750 mg, 84% yield).

[0721] LC / MS:C 13 H 13 Calculated m / z for FN2O5 = 334.1, measured [MH] - =333.1.

[0722] 3.44: tert-Butyl (4-amino-5-formyl-2-(trifluoromethyl)phenyl)carbamate (compound 3.44) To a solution of compound 3.43 (750 mg, 1 equiv.) in MeOH (16 mL) and HO (1.6 mL) was added B(OH) (603 mg, 3 equiv.). The resulting mixture was cooled to 0 °C, and 5 M aqueous NaOH (2.75 mL) was added with stirring over 10 min. The reaction mixture was stirred for an additional 5 min before being quenched by pouring the solution onto ice (50 mL). The resulting mixture was extracted with DCM (3 × 75 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Flash purification was performed as described in General Procedure 9 using a 25 g silica column eluting with 10 to 50% hexanes / EtOAc to afford the title compound as an orange solid (460 mg, 67%).

[0723] LC / MS:C 13 H 15 Calculated m / z for F3N2O3 = 304.1, observed [M+H] + =305.2.

[0724] 3.45: (S)-9-Amino-4-ethyl-4-hydroxy-8-(trifluoromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (compound 167) A mixture of compound 3.44 (460 mg, 1 equiv.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (378 mg, 0.95 equiv.), TsOH (monohydrate, 26 mg, 0.1 equiv.), and toluene (35 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred at 110° C. for 2 hours. The reaction mixture was then cooled to 25° C. and concentrated under reduced pressure. Purification was carried out as described in General Procedure 9 using a 25 g silica column eluted with a 0 to 50% DCM / MeOH gradient to give the Boc-protected intermediate as a red solid. This material was then deprotected according to General Procedure 6, followed by preparative HPLC purification as described in General Procedure 9, eluting with a 20 to 65% CHCN / H0+0.1% TFA gradient, to afford the title compound as a yellow solid (6.2 mg, 48%).

[0725] LC / MS:C 21 H 16 Calculated m / z for F3N3O4 = 431.1, found [M+H] + =432.2.

[0726] 1 H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.27 (s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 5.59 (d, J = 16.3 Hz, 1H), 5.39 (d, J = 16.3 Hz, 1H), 5.28 (s, 2H), 2.00 - 1.89 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0727] Example 4: Preparation of Drug-Linker 4.1: 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycinate (compound 4.1) TIFF2025535239000229.tif32165 The title compound was prepared according to the procedure described in Chinese Patent Publication No. CN105218644.

[0728] 4.2: (((9H-Fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (Fmoc-GGF-OH; compound 4.2) TIFF2025535239000230.tif32165 To L-phenylalanine (965 mg) in acetonitrile (10 mL) and dimethylformamide (0.5 mL) was added DIPEA (1.51 mL), followed by compound 4.1 (1.3 g). After 1 h, the reaction was concentrated to dryness. Flash purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient, to afford the title compound as a white solid (430 mg, 30% yield).

[0729] LC / MS:C 28 H 71 Calculated m / z for N3O6S = 501.2, observed [M+H] + =502.4.

[0730] 1H 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).

[0731] 4.3: 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (MT-OTfp; compound 4.3) The title compound of TIFF2025535239000231.tif32165 was prepared according to the procedures described in International Patent Publication No. WO2017 / 054080.

[0732] 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) To a solution of compound 4.3 (1.61 g, 3.58 mmol) in DMF (35 mL) was added Gly-Gly-Phe (1 g, 3.58 mmol) in one portion, followed by iPrNEt (1.25 mL, 7.2 mmol). The solution was stirred at room temperature for 1 h and then evaporated to dryness. Purification was carried out as described in General Procedure 9 using a 30 g C flash column eluted with a 10 to 90% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (400 mg, 20% yield).

[0733] LC / MS:C 26 H 34 N4O 10 Calculated value for m / z=562.6, measured value [MH] - =561.5.

[0734] 1 H NMR (300 MHz, CDCl3) δ 7.60 (t, J = 5.6 Hz, 2H), 7.41 (d, J = 7.7 Hz, 1H), 7.32 - 7.07 (m, 5H), 6.70 (s, 2H), 6.33 - 6.07 (m, 3H), 4.72 (td, J = 7.6, 5.3 Hz, 1H), 4.12 - 3.78 (m, 4H), 3.72 (ddd, J = 15.2, 6.9, 4.8 Hz, 5H), 3.60 (dd, J = 11.6, 6.1 Hz, 10H), 3.12 (ddd, J = 48.2, 14.0, 6.5 Hz, 2H), 2.52 (d, J = 11.7 Hz, 2H).

[0735] 4.5: (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (compound 4.5) The title compound of TIFF2025535239000233.tif27165 was prepared according to the procedure described in U.S. Patent Publication No. US2017 / 021031.

[0736] 4.6: (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (compound 4.6) The title compound TIFF2025535239000234.tif42165 was prepared according to the procedure described in U.S. Patent Publication No. US2017 / 021031 using Fmoc-GGFGG-OH as the starting peptide.

[0737] 4.7: tert-Butyl (2-((2-(((S)-1-((2-((4-((4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (compound 4.7) The title compound was prepared according to general procedure 7, starting from compound 104 (20 mg). Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (14 mg, 42% yield).

[0738] LC / MS:C 52 H 58 N9O 12 Calculated m / z for S = 1051.4, observed [M+H] + =1052.6.

[0739] 4.8: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-amide)-N-(2-((4-((4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 104) The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from compound 4.7 (14 mg). Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (9.1 mg, 56% yield).

[0740] LC / MS:C 60 H 67 FN 10 O 16 Calculated m / z for S = 1234.4, observed [M+H] + =1235.8.

[0741] 4.9: tert-Butyl (2-((2-(((S)-1-((2-((4-(4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (compound 4.9) The title compound was prepared according to general procedure 7, starting from compound 108 (12 mg). Preparative HPLC purification was performed as described in general procedure 9, eluting with a 10 to 60% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (13 mg, 62% yield).

[0742] LC / MS:C 52 H 58 N9O 10 Calculated value m / z=987.4, observed value [M+H] + =988.6.

[0743] 4.10: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-amide)-N-(2-((4-(4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 108) The title compound was prepared according to Procedure 6 followed by Procedure 8 starting from compound 4.9 (13 mg). Preparative HPLC purification was performed as described in General Procedure 9, eluting with a 10 to 50% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (3.1 mg, 20% yield).

[0744] LC / MS:C 60 H 67 FN 10 O 14 Calculated value for m / z=1170.5, observed value [M+H] + =1171.6.

[0745] 4.11: (9H-Fluoren-9-yl)methyl (S)-(1-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-3,10-dioxo-7-oxa-2,4,9-triazaundecan-11-yl)carbamate (compound 4.11) To a solution of compound 1.2 (31 mg, 0.076 mmol) in DMF (750 μL) was added (9H-fluoren-9-yl)methyl (2-(((2-(((4-nitrophenoxy)carbonyl)amino)ethoxy)methyl)amino)-2-oxoethyl)carbamate (41 mg, 0.076 mmol), followed by iPrNEt (26 μL, 0.15 mmol). The solution was stirred at room temperature for 2 h and then applied directly to a 12 g C column. Purification was carried out as described in General Procedure 9, eluting with a 10 to 100% CHCN / HO + 0.1% TFA gradient to afford the title compound as a white solid (21 mg, 35% yield).

[0746] LC / MS:C 43 H 41 Calculated m / z for FN6O9 = 804.87, found [M+H] + =805.6.

[0747] 4.12: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-amide)-N-(1-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-3,10-dioxo-7-oxa-2,4,9-triazaundecan-11-yl)-3-phenylpropanamide (MT-GGFG-AM-Compound 136) Compound 4.11 (21 mg, 0.026 mmol) was dissolved in a 10% solution of piperidine in DMF (1 mL) and stirred for 10 min. The piperidine solution was evaporated, and the resulting residue was redissolved in DMF (5 mL) and evaporated to dryness again. To this residue was added DMF (50 μL) and DCM (450 μL), followed by compound 4.4 (15 mg, 0.026 mmol), NMM (10 μL), and ...

Claims

1. Formula (X): T-[L-(D) ｍ ] ｎ (X) 1. An antibody-drug conjugate having the formula: During the ceremony, m is an integer between 1 and 4; n is an integer between 1 and 10; T is an anti-NaPi2b (sodium-dependent phosphate transporter protein 2B) antibody construct comprising an antigen-binding domain that binds to human NaPi2b, said antigen-binding domain comprising: (a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 9; and (b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:

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

2. the antigen-binding domain (a) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (b) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (c) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (d) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (e) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (f) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (g) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (h) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (i) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (j) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (k) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (l) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; or (m) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 31, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

32.

2. The antibody-drug construct of claim 1, comprising:

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

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

5. R １ａ But -CH ３ , -OCH ３ , and N.H. ２ The antibody-drug conjugate of claim 3, wherein the antibody-drug conjugate is selected from the group consisting of:

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

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

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

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

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

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

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

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

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

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

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

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

18. 3. The antibody-drug conjugate of claim 1 or 2, wherein D has the structure of any one of the compounds shown in Table 6 or Table 7.

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

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

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

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

23. L, (a) Formula (XI) And, During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-NaPi2b antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, and AA １ - [AA ２ ] ｒ forms a protease cleavage site, X is a self-immolative group; q is 0 or 1; r is 1, 2 or 3; s is 0, 1 or 2; # is the point of attachment to the anti-NaPi2b antibody construct T; % is the point of attachment to camptothecin analog D; Formula (XI), or (b) Formula (XII) And, During the ceremony, Z is a functional group capable of reacting with a targeting group on the anti-NaPi2b antibody construct T; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, and AA １ - [AA ２ ] ｒ forms a protease cleavage site, Y is -NH-CH ２ - and q is 0 or 1; r is 1, 2 or 3; v is 0 or 1, # is the point of attachment to the anti-NaPi2b antibody construct T; % is the point of attachment to camptothecin analog D; The formula (XII) The antibody-drug conjugate of any one of claims 20 to 22, having the formula:

24. The antibody-drug conjugate of claim 1 or 2, wherein L-(D) in formula (X) has the structure of any one of the drug linkers (DL) shown in Tables 8 to 10.

25. The antibody-drug conjugate of claim 1 or 2, wherein L-(D) in formula (X) has the structure of any one of the drug linkers (DL) shown in Table 8 or Table 9.

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

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

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

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

30. The antibody-drug conjugate of any one of claims 1 to 29, wherein n is 4 to 8.

31. The antibody-drug conjugate of any one of claims 1 to 30, wherein the anti-NaPi2b antibody construct further comprises a scaffold, and the antigen-binding domain is operably linked to the scaffold.

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

33. 3. The antibody-drug conjugate of claim 1 or 2, wherein the anti-NaPi2b antigen-binding construct comprises two heavy chains each having the sequence set forth in SEQ ID NO: 46 and two light chains each having the sequence set forth in SEQ ID NO:

51.

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

35. structure:

1. An antibody-drug conjugate having the formula: n is 4, T is an anti-NaPi2b (sodium-dependent phosphate transporter protein 2B) antibody construct comprising an antigen-binding domain that binds to human NaPi2b, said antigen-binding domain comprising: (a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 9; and (b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:

18. Including, The antibody-drug conjugate.

36. 36. The antibody-drug construct of claim 35, wherein the antigen binding domain comprises a VH domain having the sequence set forth in SEQ ID NO:24 and a VL domain having the sequence set forth in SEQ ID NO:

29.

37. 37. The antibody-drug construct of claim 36, wherein the anti-NaPi2b antibody construct comprises two heavy chains comprising the sequence set forth in SEQ ID NO: 46 and two light chains comprising the sequence set forth in SEQ ID NO:

51.

38. 37. The antibody-drug construct of claim 36, wherein the anti-NaPi2b antibody construct comprises two heavy chains comprising the sequence set forth in SEQ ID NO:68 and two light chains comprising the sequence set forth in SEQ ID NO:

67.

39. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 38 and a pharmaceutically acceptable carrier or diluent.

40. A method for inhibiting the proliferation of cancer cells, the method comprising contacting the cells with an effective amount of the antibody-drug conjugate of any one of claims 1 to 38.

41. 39. A method of killing cancer cells, comprising contacting said cells with an effective amount of the antibody-drug conjugate of any one of claims 1 to 38.

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

43. 40. Use of an effective amount of the antibody-drug conjugate of any one of claims 1 to 38 for the treatment of cancer in a subject in need thereof.

44. An antibody-drug conjugate according to any one of claims 1 to 38 for use in therapy.

45. 39. The antibody-drug conjugate of any one of claims 1 to 38 for use in the treatment of cancer.

46. Use of the antibody-drug conjugate of any one of claims 1 to 38 in the manufacture of a medicament for the treatment of cancer.

47. A kit comprising the antibody-drug conjugate of any one of claims 1 to 38 and a label and / or package insert containing instructions for use.

48. 3. The antibody-drug conjugate of claim 1 or 2, wherein the anti-NaPi2b antigen-binding construct comprises two heavy chains, each having the heavy chain sequence of v29456, and two light chains, each having the light chain sequence of v29456.

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