Anti-napi2b antibody-drug conjugates and methods of use

Abstract

Described herein are anti-NaPi2b 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 antibody construct of the ADC comprises an amino acid modification in the Fc that knocks out binding to Fc gamma receptors. The ADCs are useful as therapeutic agents, among others, in the treatment of cancer.

Description

Cross-referencing

[0001] This application claims priority and benefit to U.S. Provisional Application No. 63 / 638,403, filed April 24, 2024, and U.S. Provisional Application No. 63 / 544,847, filed October 19, 2023, the entire contents of each of which are incorporated herein by reference for all purposes. Technical Field

[0002] This disclosure relates to the field of immunotherapeutic agents, and more particularly to antibody-drug conjugates against human sodium-dependent phosphate transporter 2B (hNaPi2b). Background Technology

[0003] Sodium-dependent phosphate transporter 2B (NaPi2b) is a transmembrane protein encoded by the SLC34A2 gene. The NaPi2b polypeptide is 690 amino acids long and has a limited extracellular domain of 188-361 amino acids exposed on the cell surface. It is widely expressed in normal tissues and overexpressed in various cancers, including ovarian cancer, endometrial cancer, and lung cancer.

[0004] Given the overexpression of NaPi2b in certain types of cancer, NaPi2b-targeting agents have been investigated in clinical trials for cancer treatment, but the results have been mixed. Mersana Therapeutics conducted a Phase I / II clinical trial to investigate the efficacy of upifitamab rilsodotin (an antibody-drug conjugate (ADC) of NaPi2b-targeting antibody MX35 with an auristatin-F payload (Dolaflexin platform)) in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC). The NSCLC group study was discontinued due to lack of efficacy, while upifitamab rilsodotin was granted Fast Track Designation for the treatment of platinum-resistant ovarian cancer patients who have received three to four prior therapies. Mersana has also completed a Phase I / II clinical trial of XMT-1592 in ovarian cancer; XMT-1592 is a site-specific ADC consisting of antibody MX35 conjugated to an auristatin-F payload using its Dolasynthen platform. Development of this ADC has been discontinued. Lifastuzumab vedotin (an ADC of lifastuzumab with an MMAE payload) was investigated in a Genentech-sponsored clinical trial for patients with ovarian cancer or NSCLC, but the trial was later discontinued.

[0005] Camptothecin analogues 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 analogue deruxtecan (Dxd) is conjugated to the anti-HER2 antibody trastuzumab via a cleavable tetrapeptide-based linker; and sacituzumab govitecan (Trodelvy™), in which the camptothecin analogue SN-38 is conjugated to the anti-Trop-2 antibody sacituzumab via a hydrolyzable pH-sensitive linker.

[0006] Other camptothecin analogues and derivatives, as well as ADCs containing them, have been described. See, for example, international (PCT) application publication numbers WO 2019 / 195665; WO 2019 / 236954; WO 2020 / 200880 and WO 2020 / 219287.

[0007] The purpose of providing this background information is to convince the applicant that the known information available may be relevant to this disclosure. It is not necessarily intended to admit, nor should it be construed, that any of the foregoing information constitutes prior art to the claimed invention. Summary of the Invention

[0008] This article describes the anti-NaPi2b antibody-drug conjugate and its usage.

[0009] One aspect of this disclosure relates to antibody-drug conjugates having the following structure: Wherein n is between about 2 and about 6, and T is an anti-NaPi2b antibody construct as described herein, wherein T is an anti-NaPi2b antibody construct comprising two heavy chains comprising the sequence shown in SEQ ID NO:68 and two light chains comprising the sequence shown in SEQ ID NO:67, or wherein T is an anti-NaPi2b antibody construct comprising two heavy chains comprising the sequence shown in SEQ ID NO:66 and two light chains comprising the sequence shown in SEQ ID NO:67.

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

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

[0012] Another aspect of this disclosure relates to a method for killing cancer cells, which includes contacting cells with an effective amount of an antibody-drug conjugate as described herein.

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

[0014] Another aspect of this disclosure relates to antibody-drug conjugates as described herein for use in therapies.

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

[0016] Another aspect of this disclosure relates to the use of antibody-drug conjugates, as described herein, in the manufacture of medicaments for treating cancer.

[0017] Another aspect of this disclosure relates to a kit comprising an antibody-drug conjugate as described herein and a label and / or packaging instruction containing instructions for use. Attached Figure Description

[0018] Figure 1A The transplantation into the human VH germline (IGHV1-46) is shown. The sequence of the chimeric anti-NaPi2b antibody v23855 mouse heavy chain variable domain (CDR) on 03) and Figure 1B The transplantation into the human VL framework (IGKVID-39) is shown. The sequence of the mouse light chain variable domain (CDR) of the chimeric antibody v23855 on 01). The CDR is specified by AbM definition and marked in bold and underline.

[0019] Figure 2A The non-reduced (NR) SDS-PAGE spectra of all humanized variants and their parental chimeric variant 23855 are shown. Figure 2B The reduced (R) SDS-PAGE maps of all humanized variants and their parent chimeric variant 23855 are shown. Figure 2C The UPLC-SEC pattern of the parent mouse-human chimeric antibody v23855 is shown. Figure 2D The UPLC-SEC pattern of the representative humanized antibody v29456 is shown.

[0020] Figure 3A The binding of humanized antibody variants v29456, MX-35 (v18992), and lifatozumab (v18993) to human NaPi2b was depicted. Figure 3BThe binding of humanized antibody variants v29456, MX-35 (v18992), and lifatozumab (v18993) to cynomolgus monkey NaPi2b was depicted. Figure 3C The binding of humanized antibody variants v29456, MX-35 (v18992), and lifatuzumab (v18993) to mouse NaPi2b was depicted.

[0021] Figure 4A N-curve analysis was performed to depict the binding of v29814 to NaPi2b expressed in IGROWV-1 cells. Figure 4B N-curve analysis was performed to depict the binding of v36123 to NaPi2b expressed on IGROWV-1 cells. Figure 4C N-curve analysis was used to depict the binding of v36124 to NaPi2b expressed on IGROV-1 cells. For each plot, the right curve shows data for a constant binding pair at 500 pM, and the left curve shows data for a constant binding pair at 50 pM.

[0022] Figure 5A The ability of v23855 (parental chimera), v29456 (H1L2), v18992 (MX35) and v18993 (lifatozumab) to internalize in HCC-78 cells is shown as a comparison. Figure 5B The ability of v23855 (parental chimera), v29456 (H1L2), v18992 (MX35) and v18993 (lifatozumab) to internalize in NCI-H441 cells was compared.

[0023] Figure 6 The binding of parental chimeric antibody (v23855), humanized antibody variants v29452 and v29456 to IGROWV-1 cells, in addition to MX35 and lifatuzumab ADC, was depicted.

[0024] Figure 7 The ability of the v29456 ADC to act as a bystander is described.

[0025] Figure 8A The cytotoxicity of v29456 ADC in 2D monolayer cultures of HCC-78 cells was characterized. Figure 8B The cytotoxicity of v29456 ADC in 2D monolayer cultures of IGROWV-1 cells was characterized. Figure 8C The cytotoxicity of v29456 ADC in 2D monolayer cultures of HCT116 cells was characterized.

[0026] Figure 9AThe cytotoxicity of v29456 ADC in 2D monolayer cultures of IGROWV-1 cells was characterized. Figure 9B The cytotoxicity of v29456 ADC in 2D monolayer cultures of TOV-21G cells was characterized.

[0027] Figure 10A The cytotoxicity of v29456 ADC in 3D spheroids of HCC-78 cells was characterized. Figure 10B The cytotoxicity of v29456 ADC in 3D spheroids of IGROWV-1 cells was characterized.

[0028] Figure 11A The cytotoxicity of v29456 ADC in 3D spheroids of IGROWV-1 cells was characterized. Figure 11B The cytotoxicity of v29456 ADC in 3D spheroids of TOV-21G cells was characterized.

[0029] Figure 12 The efficacy of v29456 ADC in an OVCAR3 xenograft model of ovarian cancer was described.

[0030] Figure 13 The efficacy of v29456 conjugated to DXd1 in the NCI-H441 xenograft model of lung cancer was described.

[0031] Figure 14A The efficacy of v29456 ADC in an NCI-H441 xenograft model of lung cancer when administered at 0.3 mg / kg was described. Figure 14B The efficacy of v29456 ADC in an NCI-H441 xenograft model of lung cancer was described when administered at 1 mg / kg.

[0032] Figure 15A The efficacy of v29456 ADC in the patient-derived (PDX) CTG-2025 ovarian cancer model was described. Figure 15B The efficacy of v29456 ADC in the patient-derived (PDX) CTG-0958 ovarian cancer model was described.

[0033] Figure 16 The p-p-k curves of v29456 ADC in Tg32 mice are shown.

[0034] Figure 17A The ability of the ADC of v29456 (H1L2) to be internalized in OVCAR-3 cells was demonstrated compared with v18992 (MX35) and v18993 (lifatuzumab). Figure 17BThe ability of the ADC of v29456 (H1L2) to be internalized in IGROWV-1 cells was demonstrated compared with v18992 (MX35) and v18993 (lifatuzumab).

[0035] Figure 18A The cytotoxicity of v29456 ADC in 3D spheroids of IGROWV-1 cells was characterized. Figure 18B The cytotoxicity of v29456 ADC in 3D spheroids of NCI-H441 cells was characterized. Figure 18C The cytotoxicity of v29456 ADC in 3D spheroids of TOV-21G cells was characterized.

[0036] Figure 19A The results were depicted using the Membrane Proteome Array™ v38591. Figure 19B The verification data for CLDN3 is described.

[0037] Figure 20 Cellular binding of v38591 and v38591 ADC on IGROWV-1 and OVCAR-3 cells was depicted.

[0038] Figure 21 The cross-reactivity of v38591 and v38591 ADC with NaPi2b in cynomolgus monkeys and mice was depicted.

[0039] Figure 22 The specificity of v38591 and v38591 ADC for human NaPi2b, NaPi2a and NaPi2c is shown.

[0040] Figure 23 The internalization of anti-NaPi2b ADC and naked antibody is shown.

[0041] Figure 24 Bystander activity of anti-NaPi2b ADC against NaPi2b-negative EBC-1 cell lines was depicted.

[0042] Figure 25 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-0703 xenograft model.

[0043] Figure 26 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-1301 xenograft model.

[0044] Figure 27 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-3718 xenograft model.

[0045] Figure 28 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-1703 xenograft model.

[0046] Figure 29 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-2025 xenograft model.

[0047] Figure 30 The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the CTG-0958 xenograft model.

[0048] Figure 31 Pharmacokinetic curves of DAR4 and DAR8 anti-NaPi2b ADCs were plotted in cynomolgus monkeys.

[0049] Figure 32A The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU5213 lung PDX model. Figure 32B The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU5245 lung PDX model. Figure 32C The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU6802 lung PDX model. Figure 32D The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU6904 lung PDX model. Figure 32E The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU11692 lung PDX model. Figure 32F The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU11796 lung PDX model. Figure 32G The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU11870 lung PDX model. Figure 32H The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the LU11876 lung PDX model.

[0050] Figure 33A The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the UT14026 PDX model of endometrial cancer. Figure 33B The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the UT5318 PDX model of endometrial cancer. Figure 33C The effects of DAR4 and DAR8 anti-NaPi2bADC on tumor growth were shown in the UT5326 PDX model of endometrial cancer. Figure 33D The effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth were shown in the UT5321 PDX model of endometrial cancer.

[0051] Figure 34 The binding of v38591 and v40502 (LALADS) ADCs, parental antibodies, and controls to IGROWV-1, HCC-78, H441, and EBC-1 cells is shown.

[0052] Figure 35A -C depicts the effects of v38591 and v40502 (LALADS) antibodies and ADCs on NaPi2b-expressing cells, namely IGROWV-1 ( Figure 35A HCC-78 Figure 35B ) and H441 ( Figure 35C Internalization in ).

[0053] Figure 36 The cytotoxicity of v38591 and v40502 (LALADS) antibodies and ADCs in 3D spheroids of cells expressing NaPi2b was depicted.

[0054] Figure 37A The ability of compound 139 DAR 4 containing v40502 (LALADS)-MC-GGFG-AM- to resist NaPi2bADC-mediated ADCC was described. Figure 37B The ability of compound 139DAR 4 containing v40502 (LALADS)-MC-GGFG-AM- to resist NaPi2b ADC-mediated ADCP was described.

[0055] Figure 38 The ability of anti-NaPi2bADC containing v40502 (LALADS)-MC-GGFG-AM-compound 139 DAR 4 to inhibit tumor growth in an H441 xenograft model of lung cancer was demonstrated.

[0056] Figure 39 The ability of the anti-NaPi2bADC containing v40502 (LALADS)-MC-GGFG-AM-compound 139 DAR 4 to inhibit tumor growth in a LU6802 xenograft model of lung cancer was described.

[0057] Figure 40AThe cytotoxic effects of anti-NaPi2bADC containing v40502 (LALADS)-MC-GGFG-AM-compound 139 DAR 4 on erythroid progenitor cells were described. Figure 40B The cytotoxic effects of the anti-NaPi2b ADC containing v40502 (LALADS)-MC-GGFG-AM-compound 139 DAR 4 on myeloid progenitor cells were described.

[0058] Figure 41A The effects of v38591-MC-GGFG-AM-compound 139 ADC with wild-type Fc and v40502 (LALADS)-MC-GGFG-AM-compound 139 ADC with Fc silence on tumor growth in a NOD / SCID mouse UT5321 endometrial cancer PDX model are shown. Figure 41B The total antibody pharmacokinetics of v38591-MC-GGFG-AM-compound 139 ADC with wild-type Fc and v40502 (LALADS)-MC-GGFG-AM-compound 139 ADC with Fc silenced in a NOD / SCID mouse UT5321 endometrial cancer PDX model are shown. Detailed Implementation

[0059] This disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct (anti-NaPi2b antibody construct) that binds to a sodium-dependent phosphate transporter 2B (NaPi2b) conjugated to a camptothecin analog as described herein (I). The ADCs of this disclosure can be used, for example, as therapeutic agents, particularly in the treatment of cancer.

[0060] 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.

[0061] As used herein, the term "about" refers to a change of approximately + / - 10% from a given value. It should be understood that such changes are always included in any given value provided herein, unless otherwise specifically mentioned.

[0062] When used in conjunction with the term "comprising" in this text, the word "a / kind" may mean "a / kind", but it also means "a / kind or more / kinds", "at least one / kind" and "a / kind or more than one / kind".

[0063] In cases where a range of values ​​is provided in this document, such as when a value is defined as being “between” the upper and lower limits, it should be understood that the range covers the upper and lower limits as well as each intermediate value.

[0064] As used herein, the terms “comprising,” “having,” “including,” and “containing,” and their grammatical variations, are inclusive or open-ended and do not exclude additional, unlisted elements and / or method steps. When used herein in conjunction with a composition, use, or method, the term “consistently comprising” indicates that additional elements and / or method steps may be present, but these additions do not materially affect how the listed composition, method, or use functions. The term “consisting of” when used herein in conjunction with a composition, use, or method does not include the presence of additional elements and / or method steps. A composition, use, or method described herein as comprising certain elements and / or steps may also consist substantially of those elements and / or steps in some embodiments, and in other embodiments, those elements and / or steps, whether or not these embodiments are specifically mentioned.

[0065] The "complementarity-determining region" or "CDR" is an amino acid sequence that contributes to the antigen-binding specificity and affinity of the antibody constructs described herein. The "framework" region (FR) helps maintain the correct conformation of the CDR to facilitate binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, the light chain variable region (VL) and heavy chain variable region (VH) of the antibody typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs as LCDR1, LCDR2, and LCDR3. The CDRs provide most of the contact residues for the binding of the antibody to the antigen or epitope. Typically, three heavy chain CDRs and three light chain CDRs are required to bind the antigen. 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 can also occur through a combination of at least one or more CDRs selected from the VH and / or VL domains (e.g., HCDR3).

[0066] Many different definitions of CDR sequences are commonly used, including those 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 Those described in (196:901-917), as well as IMGT, AbM (University of Bath) and Contact (MacCallum et al., 1996, J Mol BiolThe definitions are as follows: , 262(5):732-745). For example, the CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Therefore, it will be apparent to those skilled in the art that the exact numbering and placement of CDRs can vary based on the numbering system employed. However, it should be understood that the disclosure herein of VH includes disclosure of the associated (inherent) heavy chain CDR (HCDR) as defined by any known numbering system. Similarly, the disclosure herein of VL includes disclosure of the associated (inherent) light chain CDR (LCDR) as defined by any known numbering system.

[0067] Table 1: General CDR Definitions 1

[0068] 1 The Kabat or Chothia numbering system can be used for all defined HCDR2, HCDR3, and light chain CDRs except for Contact, which uses Chothia numbering. 2 Kabat numbering is used. The positions in the Kabat numbering scheme that define the ends of the Chothia and IMGT CDR-H1 rings vary depending on the length of the ring, as Kabat places insertions outside the CDR definition at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 rings can be explicitly defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0069] In the context of two or more polynucleotide or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to achieve maximum similarity over a comparison window or a specified region, as measured by one of commonly used sequence comparison algorithms known to those skilled in the art or by manual alignment and visual inspection, the sequence is considered "substantially identical" if it has a certain percentage of identical amino acid residues or nucleotides (e.g., approximately 80%, 85%, 90%, 95%, or 98% identity within a specified region). For sequence comparisons, a test sequence is typically compared to a specified reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0070] A “comparison window” refers to a segment of a sequence containing consecutive amino acid or nucleotide positions, which may be, for example, about 10 to 600 consecutive amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 consecutive amino acid or nucleotide positions, at which, after optimal alignment of two sequences, the test sequence can be compared with a reference sequence of the same number of consecutive positions. Sequence alignment methods used for comparison are known to those skilled in the art. The best sequence alignment for comparison can be implemented, for example, by the following: local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; similarity retrieval method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (added in 1995), Cold Spring Harbor Laboratory Press). Examples of suitable algorithms for determining the percentage of sequence identity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0071] As used herein, the term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0072] The term "acyloxy group" refers to the group -OC(O)R, where R is an alkyl group.

[0073] As used herein, the term "alkoxy" refers to the group -OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0074] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group containing a specified number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, etc.

[0075] As used herein, the term "alkylaminoaryl" refers to an alkyl group as defined herein that is substituted with an aminoaryl group as defined herein.

[0076] As used herein, the term "alkyl heterocyclic alkyl" refers to an alkyl group as defined herein that is substituted by a heterocyclic alkyl group as defined herein.

[0077] As used herein, the term "alkylthio" refers to the group -SR, where R is an alkyl group.

[0078] As used herein, the term "amide" refers to the group -C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0079] As used herein, the term "amino" refers to the group -NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0080] As used herein, the term "aminoalkyl" refers to an alkyl group as defined herein that is substituted with one or more amino groups (e.g., one, two, or three amino groups).

[0081] As used herein, the term "aminoaryl" refers to an aryl group as defined herein that is substituted with an amino group.

[0082] As used herein, the term "aryl" refers to a 6- to 12-membered monocyclic or bicyclic hydrocarbon ring system in which at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, indanyl, etc.

[0083] As used herein, the term "carboxyl" refers to the group -C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cyclohexaalkyl.

[0084] As used in this article, the term "cyano" refers to the group -CN.

[0085] As used herein, the term "cycloalkyl" refers to a monocyclic or bicyclic saturated hydrocarbon containing a specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, etc.

[0086] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen atoms.

[0087] As used herein, the terms “halogen” and “halogenated” refer to fluorine (F), bromine (Br), chlorine (Cl), and iodine (I).

[0088] As used herein, the term "heteroaryl" refers to a 6- to 12-membered monocyclic or bicyclic cyclic system in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S, and N. Examples of heteroaryl groups include, but are not limited to, pyridinyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrroleyl, indoleyl, etc.

[0089] As used herein, the term "heterocyclic alkyl" refers to a monocyclic or bicyclic non-aromatic ring system containing a specified number of atoms, wherein at least one ring atom is a heteroatom (e.g., O, S, or N). Heterocyclic substituents may be linked via any of their available ring atoms (e.g., cyclic carbon or cyclic nitrogen). Examples of heterocyclic alkyl groups include, but are not limited to, acridine, azacyclic butyl, piperidinyl, morpholinyl, piperazine, pyrrolidinyl, etc.

[0090] As used in this article, the terms “hydroxyl” and “hydroxyl group” refer to the group -OH.

[0091] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein that is substituted with one or more hydroxyl groups.

[0092] As used in this article, the term "nitro" refers to the group -NO2.

[0093] As used herein, the term "sulfonyl" refers to the group -S(O)2R, where R is H, alkyl, or aryl.

[0094] As used herein, the term "sulfonamide" refers to the group -NH-S(O)2R, where R is H, alkyl, or aryl.

[0095] As used in this article, the terms “thio group” and “thiol” refer to the group -SH.

[0096] Unless explicitly stated as “unsubstituted,” any alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group mentioned herein shall be understood as “optionally substituted,” meaning that each such reference includes both the unsubstituted and substituted forms of these groups. For example, the 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, halogens, acyl groups, acyloxy groups, alkoxy groups, carboxyl groups, hydroxyl groups, amino groups, amide groups, nitro groups, cyano groups, azide groups, alkylthio groups, thio groups, sulfonyl groups, sulfonamide groups, alkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl, or heteroaryl groups. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group mentioned herein is optionally substituted with one or more substituents selected from the group consisting of: halogens, acyl groups, acyloxy groups, alkoxy groups, carboxyl groups, hydroxyl groups, amino groups, amide groups, nitro groups, cyano groups, azide groups, alkylthio groups, thio groups, sulfonyl groups, and sulfonamide groups.

[0097] A chemical group described herein as “substituted” may include one or more substituents up to the full valence of the substitution. For example, a methyl group may include one, two, or three substituents, and a phenyl group may include one, two, three, four, or five substituents. When a group is substituted by more than one substituent, the substituents may be the same or they may be different.

[0098] As used herein, the term "subject" refers to an animal, and in some embodiments, a mammal, which is the object of treatment, observation, or experimentation. The animal may be a human, a non-human primate, a companion animal (e.g., a dog, cat, etc.), a farm animal (e.g., a cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., a rat, mouse, guinea pig, non-human primate, etc.). In some embodiments, the subject is a human.

[0099] It is anticipated that any of the embodiments discussed herein can be implemented by any of the methods, uses, or compositions disclosed herein, and vice versa.

[0100] The specific features, structures, and / or characteristics described in connection with one embodiment disclosed herein may be combined with the features, structures, and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more other embodiments.

[0101] It should also be understood that an affirmative statement of a feature in one embodiment is the basis for excluding said feature in another embodiment. For example, when a list of options is provided for a given embodiment or claim, it should be understood that one or more options may be removed from the list, and a shortened list may form alternative embodiments, regardless of whether such alternative embodiments are specifically mentioned.

[0102] Antibody-drug conjugates This disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In some embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) in: T is the anti-NaPi2b antibody construct as described in this article; L stands for connector; D is a camptothecin analogue as described in this article; m is an integer between 1 and 4, and n is an integer between 1 and 10.

[0103] The components of formula (X) are as follows.

[0104] In one embodiment, T is an anti-NaPi2b antibody construct having a silenced Fc region. ADCs containing such Fc-silenced anti-NaPi2b antibodies are a subset of the ADCs of this disclosure and are referred to herein as anti-NaPi2b Fc-silenced ADCs. Further descriptions of anti-NaPi2b Fc-silenced ADCs are given in Examples 39 through 58. In some embodiments, the anti-NaPi2b Fc-silenced ADCs of this disclosure contain a LALADS mutation in their Fc region, as further described herein.

[0105] Anti-NaPi2b antibody construct The ADC disclosed herein comprises an anti-NaPi2b antibody construct. In this context, the term "antibody construct" refers to a polypeptide or group of polypeptides comprising one or more antigen-binding domains, each of which specifically binds an epitope or antigen. In the case where the antibody construct comprises two or more antigen-binding domains, each antigen-binding domain may bind the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may also comprise a scaffold, and the one or more antigen-binding domains may be fused or covalently linked to the scaffold, optionally via a linker. In some embodiments, the scaffold is an Fc region, such as the human IgG Fc region.

[0106] According to this disclosure, the anti-NaPi2b antibody construct of the ADC includes at least one antigen-binding domain that specifically binds to human NaPi2b (hNaPi2b). "Specifically binding" hNaPi2b means that the antibody construct binds hNaPi2b but does not exhibit significant binding to NaPi2a or NaPi2c. In some embodiments, the anti-NaPi2b antibody construct of this disclosure is capable of binding NaPi2b from one or more non-human species. In some embodiments, the anti-NaPi2b antibody construct of this disclosure is capable of binding cynomolgus monkey NaPi2b.

[0107] Human NaPi2b is also known as human “Solute Carrier Family 34 Member 2” or “SLC34A2”. Protein sequences of hNaPi2b from various sources are known in the art and are readily available from publicly accessible databases such as GenBank or UniProtKB. Examples of hNaPi2b sequences include, for example, 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).

[0108] Table 2: NaPi2b protein sequences in humans, cynomolgus monkeys, and mice

[0109] The specific binding of the antigen-binding domain to the target antigen or epitope can be achieved, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (using instruments such as BIAcore) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry or conventional binding assay (Heeley, 2002, Endocr Res The specific binding can be measured using the method described in 28:217-229. In some embodiments, specific binding can be defined as, for example by ELISA or flow cytometry, a binding degree to a non-target protein (e.g., hNaPi2a or hNaPi2c) that is less than about 5% to less than about 10% of the binding to hNaPi2b.

[0110] As used in this article, the term "dissociation constant (K)" D or K dThe term "ligand-protein interaction" is intended to refer to the equilibrium dissociation constant of a specific ligand-protein interaction. As used herein, ligand-protein interaction refers to, but is not limited to, protein-protein interactions or antibody-antigen interactions. D The tendency of two proteins (e.g., AB) to reversibly dissociate into their constituent components (A+B) is measured and defined as the dissociation rate constant (also known as the "dissociation rate (k)"). off The association rate constant or association rate (k) on The ratio of K to "). Therefore, K D equals k off / k on And expressed as molar concentration (M). Therefore, K D The smaller the value, the stronger the binding affinity, therefore K D The decrease indicates an increase in affinity. Therefore, with 1 nM K D Compared to 1 mM K D Indicates weak binding affinity. Affinity is sometimes indicated by K. A or K a To measure, it is K D or K d The reciprocal of the number. The K-value between the antibody and its antigen. D It can be determined using methods well-established in the art. For determining this type of K... D One approach is to use surface plasmon resonance (SPR), typically employing biosensor systems such as the Biacore® system. Isothermal titration calorimetry (ITC) is another method that can be used to measure K. D Another method. The Octet™ system can also be used to measure the affinity of an antibody for a target antigen.

[0111] In some implementations, the specific binding of the antibody construct to NaPi2b can be achieved via a dissociation constant (K). d or K D ) < 1 μM, for example < 500 nM, < 250 nM < 100 nM < 50 nM or < Defined as 10 nM. In some implementations, the specific binding of the antibody construct to a particular antigen or epitope can be determined by the dissociation constant (K). D ) is 10 -6 M or smaller, such as 10 -7 M or smaller or 10 -8 M or smaller is used for definition. In some implementations, the specific binding of the antibody construct to a particular antigen or epitope can be determined by the dissociation constant (K). D Between 10-6 M and 10 -9 Between M, for example, between 10 -7 M and 10 -9 The value of the dissociation constant is defined between M and M. As is known in the art, the numerical value of the obtained dissociation constant can vary depending on the testing method. For example, the expression level of NaPi2b in the cell line, the form of the antibody construct (i.e., ， The type of assay (i.e., monovalent or bivalent) and the type of assay (i.e., ELISA or flow cytometry) can affect the value of the dissociation constant when measured in a cell-based assay. The data provided in the examples illustrate this general viewpoint, as demonstrated in Examples 10, 11, and 16.

[0112] In some embodiments, when measured by flow cytometry in cells expressing high levels of NaPi2b, the K of the anti-NaPi2b antibody construct of this disclosure... d K lower than the reference antibody lifatuzumab d And with reference antibody MX35 K d Therefore, in these embodiments, the anti-NaPi2b antibody construct of this disclosure includes an antigen-binding domain having an affinity for human NaPi2b greater than that of the reference antibody lifatuzumab and an affinity comparable to that of the reference antibody MX35.

[0113] In some embodiments, the anti-NaPi2b antibody construct exhibits internalization levels comparable to the reference antibody MX35, and higher internalization levels compared to the reference antibody lifatuzumab in cells with high and moderate NaPi2b expression. In some embodiments, internalization is measured 4 hours, 5 hours, or 24 hours after treatment.

[0114] Antibody internalization can be measured using methods known in the art, for example, by means of methods according to Schmidt, M. et al., 2008. Cancer Immunol. Immunother. The direct internalization method detailed in 57:1879-1890, or the use of commercially available fluorescent dyes such as pHAb dye (Promega Corporation, Madison, WI), pHrodo iFL, and deep red dye (ThermoFisher Scientific Corporation, Waltham, MA) and Incucyte. ® Fabfluor-pH antibody labeling reagent (Sartorius AG, Göttingen, Germany), and analytical techniques such as microscopy, FACS, high-content imaging, or other plate-based assays.

[0115] NaPi2b expression varies depending on the cell type indicated throughout this disclosure, and the level of NaPi2b expression may be referred to herein as “high,” “intermediate,” “low,” or “negative.” These terms are used for reference to describe general expression levels according to the names shown in Table 15.1 of Example 15 and are not intended to be limited to specific numerical values ​​of average NaPi2b protein per cell included. Alternatively, the expression level of NaPi2b in cells or tumors may 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 may be examined, and an H score may be calculated as known in the art and, for example, as described in Example 35 herein. A higher H score indicates higher NaPi2b expression in the tissue sample.

[0116] Antigen-binding domain The anti-NaPi2b antibody construct of the ADC disclosed herein includes at least one antigen-binding domain capable of binding hNaPi2b. The at least one antigen-binding domain capable of binding hNaPi2b is typically based on an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAb).

[0117] A “Fab fragment” contains a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain, as well as variable domains (VL and VH) of both the light and heavy chains. The Fab′ fragment differs from the Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab fragments can also be single-chain Fab molecules, where the Fab light chain and Fab heavy chain are linked by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain can be linked to the N-terminus of the Fab heavy chain in a single-chain Fab molecule.

[0118] The “scFv” contains both a heavy chain variable domain (VH) and a light chain variable domain (VL) of the antibody within a single polypeptide chain. Optionally, the scFv may further include a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form the structure required for antigen binding. For example, the scFv may include a VL connected from the C-terminus to the N-terminus of the VH via a polypeptide linker. Alternatively, the scFv may include a VH connected from its C-terminus to the N-terminus of the VL via a polypeptide linker (see [link to scFv]). The Pharmacology of Monoclonal AntibodiesIn Pluckthun's review in Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0119] The “sdAb” format refers to a single immunoglobulin domain. sdAbs can be, for example, of camel origin. Camel antibodies lack a light chain and their antigen-binding site consists of a single domain, called a “VHH”. sdAbs contain three CDR / hypervariant loops that form the antigen-binding sites: CDR1, CDR2, and CDR3. sdAbs are quite stable and readily expressed, for example, as fusions with antibody Fc chains (see, for example, Harmsen & De Haard, 2007). Appl. Microbiol Biotechnol. 77(1):13-22).

[0120] In embodiments where the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain (such as an antigen-binding antibody fragment) or a non-immunoglobulin-based domain (such as a non-immunoglobulin-based antibody mimic), or other peptides or small molecules capable of specifically binding to their targets (e.g., natural or engineered ligands). Non-immunoglobulin-based antibody mimics include, for example, anticalin, fynomer, affimer, antigen, DARPin, and avimer.

[0121] This disclosure describes the identification of a mouse antibody that specifically binds to hNaPi2b; a mouse-human chimeric variant of this antibody was identified as variant 23855. The anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain derived from the mouse antibody or a humanized antibody variant thereof. Representative humanized antibody variants of the mouse antibody (e.g., v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460) are also described. In some embodiments, the anti-NaPi2b antibody construct described herein specifically binds to human NaPi2b having the sequence shown in SEQ ID NO:1.

[0122] In some implementations, the anti-NaPi2b antibody construct of the ADC competes with any one of the humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or with the parental chimeric antibody v23855, for binding to human NaPi2b. In the competition evaluation 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.

[0123] Those skilled in the art can use competitive assays known in the art to determine whether an antibody construct competes with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460 or the parental chimeric antibody v23855 for binding to hNaPi2b. For example, the competing reference antibody is first allowed to bind to hNaPi2b under saturation conditions, and then the ability of the test antibody construct to bind to hNaPi2b is measured. If the test antibody construct can bind to hNaPi2b simultaneously with the competing reference antibody, it is considered that the test antibody construct binds to a different epitope than the competing reference antibody. Conversely, if the test antibody construct cannot bind to hNaPi2b simultaneously with the competing reference antibody, it is considered that the test antibody construct binds to the same epitope, an overlapping epitope, or a very close epitope as the competing reference antibody. Such competitive assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, and flow cytometry. "Antibody competing with a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its epitope by 50% or more in the competitive assay.

[0124] In some embodiments, the anti-NaPi2b antibody construct of this disclosure comprises at least one antigen-binding domain that specifically binds to hNaPi2b, wherein the antigen-binding domain comprises a set of CDRs based on the CDR of the parental chimeric antibody v23855 described herein. The CDR sequences of the parental chimeric antibody v23855 and representative humanized antibody variants are shown in Table 3.

[0125] Table 3: CDR sequences of anti-NaPi2b antibody constructs

[0126] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 7, 8, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 19, 20, and 18.

[0127] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1 and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 4, 5, and 6, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 17 and SEQ ID NO: 18 and the LCDR2 sequence YTS.

[0128] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 10, 11, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 19, 20, and 18.

[0129] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 12, 13, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 19, 20, and 18.

[0130] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 14, 15, and 16, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NO: 21, 22, and 23.

[0131] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure includes an antigen-binding domain having: (i) The HCDR1 amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; the HCDR2 amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29459, or v29460. 4. The HCDR2 amino acid sequence of any one of v29455, v29456, v29457, v29458, v29459, or v29460; and the 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, and (ii) An LCDR1 amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; and an LCDR2 amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29459, or v29460. 54. The LCDR2 amino acid sequence of any one of v29455, v29456, v29457, v29458, v29459, or v29460; and the LCDR3 amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. The CDR amino acid sequence is defined by any of the numbering systems such as IMGT, Chothia, Kabat, Contact, or AbM (see, for example, Table 3).

[0132] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequence is selected from variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29454, v29455, v29452, v29453, v29454, v2945 ... The heavy chain CDR amino acid sequence of any one of v29455, v29456, v29457, v29458, v29459 or v29460, wherein the light chain CDR amino acid sequence is selected from the light chain CDR amino acid sequence of any one of the 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.

[0133] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain comprising a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) as defined by any of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460 in the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0134] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure includes an antigen-binding domain having a VH sequence, the VH sequence comprising a CDR sequence of the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460. In some embodiments, the anti-NaPi2b antibody construct of this disclosure comprises an antigen-binding domain having a VL sequence comprising a CDR sequence of the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0135] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR, VH, or VL sequences of a known antibody without the antibody losing its ability 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 binding activity of the resulting variants can be tested using standard techniques. Therefore, in some embodiments, the anti-NaPi2b antibody construct of this disclosure comprises an antigen-binding domain having a set of CDRs with 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity with any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460 (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3), wherein the sequence identity % is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0136] In some embodiments, the anti-NaPi2b antibody construct of this disclosure comprises an antigen-binding domain comprising a variant of any one of the following CDR sequences: v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant contains 1 to 10 amino acid substitutions in the set of CDR sequences (i.e., the CDRs can be modified by up to 10 amino acid substitutions, wherein any combination of the six CDRs is modified), and wherein the antigen-binding domain retains the ability to bind hNaPi2b. In some embodiments, the anti-NaPi2b antibody construct of this disclosure comprises an antigen-binding domain comprising a variant of any one of the following CDR sequences: v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant comprises 1 to 7 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 2 amino acid substitutions, or 1 amino acid substitution, and wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0137] In some embodiments, the anti-NaPi2b antibody construct of this disclosure includes an antigen-binding domain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a 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 hNaPi2b. In some embodiments, the anti-NaPi2b antibody construct of this disclosure includes an antigen-binding domain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a 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 hNaPi2b.

[0138] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure includes an antigen-binding domain comprising a VH amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In some embodiments, the anti-NaPi2b antibody construct of this disclosure includes an antigen-binding domain comprising a VL amino acid sequence selected from any one of the variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0139] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain comprising a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VH sequence of v23855, and a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VL sequence of v23855, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0140] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain comprising a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VH sequence of v29456, and a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VL sequence of v29456, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0141] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises an antigen-binding domain comprising a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VH sequence of v29452, and a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the VL sequence of v29452, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0142] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this 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 of the VH and VL sequences of these variants are provided in Table 4 below. The sequences themselves are provided in Table 7.4 of the examples.

[0143] Table 4: VH and VL sequences of parental chimeric and humanized anti-NaPi2b antibodies

[0144] 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.

[0145] In some embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises: a) a VH sequence having 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 with the VH sequence of v29456; and b) a VL sequence having 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 with the VL sequence of v29456, wherein the HCDRs and LCDRs are defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0146] In some other embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises: a) a VH sequence having 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 with the VH sequence of v29452; and b) a VL sequence having 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 with the VL sequence of v29452, wherein the HCDRs and LCDRs are defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0147] In one embodiment, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 63 and two light chains having the amino acid sequence shown in SEQ ID NO: 62. In one embodiment, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 61 and two light chains having the amino acid sequence shown in SEQ ID NO: 62.

[0148] The anti-NaPi2b antibody construct of the ADC of this disclosure may include a silenced Fc region (anti-NaPi2b Fc-silenced ADC) as described elsewhere herein. In these embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 66 and two light chains having the amino acid sequence shown in SEQ ID NO: 67. In other embodiments, the anti-NaPi2b antibody construct of the ADC of this disclosure comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 68 and two light chains having the amino acid sequence shown in SEQ ID NO: 67.

[0149] In another embodiment, the anti-NaPi2b antibody construct of the anti-NaPi2b Fc silencing ADC of this disclosure comprises (a) two heavy chains having 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%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 66, and (b) two light chains having 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%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 67.

[0150] In other embodiments, the anti-NaPi2b antibody construct of the anti-NaPi2b Fc silencing ADC of this disclosure comprises (a) two heavy chains 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 with the amino acid sequence shown in SEQ ID NO: 68, and (b) two light chains having 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%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 67.

[0151] Format Anti-NaPi2b antibody constructs of ADCs can be in various formats. The minimum component of an anti-NaPi2b antibody construct is an antigen-binding domain that binds to hNaPi2b. The anti-NaPi2b antibody construct may also optionally include one or more additional antigen-binding domains and / or a scaffold. In those embodiments in which the anti-NaPi2b antibody construct includes two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within hNaPi2b, bind to a different epitope within hNaPi2b, or bind to a different antigen. Therefore, the anti-NaPi2b antibody construct can be, for example, monospecific, bicomplementary, bispecific, or multispecific.

[0152] In some embodiments, the anti-NaPi2b antibody construct comprises at least one hNaPi2b-binding antigen-binding domain and a scaffold, wherein the antigen-binding domain is operatively linked to the scaffold. As used herein, the term "operatively linked" means that the described components are in a relationship that allows them to function in their intended manner. Suitable scaffolds are described below.

[0153] In some embodiments, the anti-NaPi2b antibody construct includes two antigen-binding domains optionally operably linked to a scaffold. In some embodiments, the anti-NaPi2b antibody construct may include three or four antigen-binding domains and an optional scaffold. In these formats, when a scaffold is included, at least a first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domains may each be operably linked independently to either the scaffold or the first antigen-binding domain, or, when more than two antigen-binding domains are present, operably linked to another antigen-binding domain.

[0154] Scaffold-less 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 domains may be in the format of scFv, Fab, sdAb, or combinations thereof. For example, using scFv as the antigen-binding domain, formats such as tandem scFv ((scFv)2 or taFv) can be constructed, where the scFvs are linked together by flexible linkers. scFv can also be used to construct biantibody formats, which contain two scFvs linked by short linkers (typically about 5 amino acids in length). The restricted linker length causes the scFvs to dimerize in a head-to-tail manner. In any of the aforementioned formats, the scFvs can be further stabilized by including interdomain disulfide bonds. For example, a disulfide bond can be introduced between VL and VH by replacing non-cysteine ​​residues in each chain with cysteine ​​residues (e.g., at position 44 of VH and position 100 of VL) (see, for example, Fitzgerald et al., 1997). Protein Engineering (10:1221-1225), or a disulfide bond can be introduced between the two VHs to provide a construct with a DART format (see, for example, Johnson et al., 2010). J Mol. Biol., 399:436-449).

[0155] Similarly, in some implementations, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable adapter may be used. Other examples of anti-NaPi2b antibody constructs lacking a scaffold include those based on Fab fragments, such as the Fab2 and F(ab')2 formats, where the Fab fragments are linked by adapters or IgG hinge regions.

[0156] Combinations of antigen-binding domains of different hours can also be used to generate alternative scaffold-free formats. For example, scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of the Fab fragment to produce a bivalent (Fab-scFv / sdAb) construct.

[0157] In some embodiments, the anti-NaPi2b antibody construct may be an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as a full-size antibody format (FSA) or Mab format and includes an anti-NaPi2b antibody construct containing two Ig heavy chains and two Ig light chains. In some embodiments, the anti-NaPi2b antibody construct may be based on IgG class immunoglobulins, such as IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the anti-NaPi2b antibody construct may be based on IgG1 immunoglobulin. In the context of this disclosure, when an anti-NaPi2b antibody construct is based on a specified immunoglobulin isotype, it means that the anti-NaPi2b antibody construct contains all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-NaPi2b antibody construct based on a given Ig isotype may include at least one antigen-binding domain operatively linked to an Ig scaffold, wherein the scaffold includes an Fc region from the given isotype and optionally an Ig hinge region from the same or different isotypes. It should be understood that, in some embodiments, the anti-NaPi2b antibody construct may also comprise a heterozygote of the same type and / or subtype. It should also be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desired functional properties as known in the art. Therefore, in some embodiments, the anti-NaPi2b antibody construct comprises a VH amino acid sequence fused to an IgG1 constant domain amino acid sequence (i.e., CH1, hinge, CH2, CH3 amino acid sequences) and a VL amino acid sequence fused to a κ or λ constant amino acid sequence domain (i.e., CL amino acid sequence). Exemplary amino acid sequences are provided in the examples and sequence listing.

[0158] In some implementations, the anti-NaPi2b antibody construct can be derived from two or more immunoglobulins from different species; for example, the anti-NaPi2b antibody construct can be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from more than one species.

[0159] Chimeric antibodies typically contain at least one variable domain derived from a non-human antibody, such as a rabbit or rodent (e.g., mouse) antibody, and at least one constant domain derived from a human antibody. The human constant domain of a chimeric antibody does not need to have the same isotype as the non-human constant domain it replaces. Chimeric antibodies are exemplified, for example, by Morrison et al., 1984. Proc. Natl. Acad. Sci. USA This is discussed in 81:6851-55 and U.S. Patent No. 4,816,567.

[0160] "Humanized antibodies" are a class of chimeric antibodies containing minimal sequences derived from non-human antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced by residues from a non-human species (donor antibody) with the desired specificity and affinity for the target antigen. These non-human species include mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR transplantation."

[0161] In some cases, further modifications are made to further improve antibody performance. For example, some framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues, or humanized antibodies may contain residues not found in either the recipient or donor antibody. Generally, the variable domains in humanized antibodies will contain all or almost all hypervariable regions from non-human immunoglobulins and all or almost all FRs from the human immunoglobulin sequence. Humanized antibodies, for example, in Jones et al. , 1986, Nature 321:522-525; Riechmann et al. , 1988, Nature 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., A more detailed description can be found in 2:593-596.

[0162] Numerous methods are known in the art for selecting the most appropriate human frame in which a nonhuman CDR is transplanted. Early methods used a finite subset of well-characterized human antibodies, independent of sequence identity with the nonhuman antibody providing the CDR (“fixed frame” method). More recent methods have employed variable regions with high amino acid sequence identity to the variable region of the nonhuman antibody providing the CDR (“homology matching” or “best fit” method). Alternative methods involve selecting fragments of the frame sequence from the variable region of each light or heavy chain from several different human antibodies. In some cases, CDR transplantation may result in partial or complete loss of affinity of the transplanted molecule for its target antigen. In such cases, affinity can be restored by reverting some human-derived residues to the corresponding nonhuman residues. Methods for preparing humanized antibodies using these approaches are well known in the art (see, e.g., Tsurushita & Vasquez, 2004). Humanization of Monoclonal Antibodies , Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature , 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al. , 1997, Cancer Res, 57(20):4593-4599).

[0163] Alternatively, or in addition to these traditional methods, newer techniques can be employed to further reduce the immunogenicity of humanized antibodies with transplanted CDRs. For example, a human frame based on a germline sequence or a shared sequence can be used as the recipient human frame instead of a human frame with somatic mutations. Another technique aimed at reducing the potential immunogenicity of non-human CDRs is transplanting only specificity-determining residues (SDRs). In this approach, only the minimum number of CDR residues (“SDRs”) required for antigen-binding activity are transplanted into the human germline frame. This method improves the “humanity” (i.e., similarity to human germline sequences) of the humanized antibody, thus helping to reduce the risk of immunogenicity in the variable region. These techniques have been described in various publications (see, for example, Almagro and Fransson, 2008). Front Biosci, 13:1619-1633; Tan et al., 2002, J Immunol, 169:1119-1125; Hwang et al., 2005, Methods, 36:35-42; Pelat et al., 2008, J Mol Biol, 384:1400-1407; Tamura et al., 2000, J Immunol , 164:1432-1441; Gonzales et al., 2004, Mol Immunol, 1:863-872, and Kashmiri et al., 2005, Methods , 36:25-34).

[0164] In some embodiments, the anti-NaPi2b antibody constructs of this disclosure comprise humanized antibody sequences, such as one or more humanized variable domains. In some embodiments, the anti-NaPi2b antibody construct may be a humanized antibody. Non-limiting examples of humanized antibodies based on anti-NaPi2b antibody v23855 are described herein (see examples and sequence listings, as well as sequences of v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, v38951, and v40502).

[0165] support In some embodiments, the anti-NaPi2b antibody construct includes one or more antigen-binding domains operatively linked to a scaffold. The antigen-binding domains can be one or a combination of the forms described above (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumins, albumin analogs and derivatives, heterodimerized peptides (such as leucine zippers, heterodimer-forming "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase-barstar toxins), 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, for example, Chang et al.) , 2007, Clin. Cancer Res., 13:5586s-5591s).

[0166] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical entity. When the scaffold is a polypeptide, each antigen-binding domain of the anti-NaPi2b antibody construct can be linked to the N-terminus or C-terminus of the polypeptide scaffold. In some embodiments, anti-NaPi2b antibody constructs comprising polypeptide scaffolds are also envisioned, wherein one or more antigen-binding polypeptide constructs are linked to regions other than the N-terminus or C-terminus, with or without a linker, for example via amino acid side chains.

[0167] In embodiments where the anti-NaPi2b antibody construct comprises a scaffold as a peptide or polypeptide, the antigen-binding domain is linked to the scaffold via genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain is linked to the scaffold via genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen-binding domain is linked to the scaffold via chemical conjugation.

[0168] Many protein domains are known in the art that contain selective pairings of two different polypeptides and can be used to form scaffolds. Examples are 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, barnase-barstar pairs (Deyev et al.). ,NatBiotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri et al.) , FEBS Letters, 450(1–2):23-26 (1999)) and split fluorescent protein pair (International Patent Application Publication No. WO 2011 / 135040).

[0169] Other examples of protein scaffolds include immunoglobulin Fc regions, albumins, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding portions has been described (see, for example, Müller et al.). , 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al. , 2012, Mol. Cancer Ther., 11:582-593; Vallera et al. , 2005, Clin. Cancer Res., 11:3879-3888; Song et al. , 2006, Biotech. Appl. Biochem (45:147-154 and U.S. Patent Application Publication No. 2009 / 0285816).

[0170] For example, it has been demonstrated that fusing antigen-binding moieties such as scFv, biantibodies, or single-chain biantibodies with albumin can improve the serum half-life of the antigen-binding moieties (Müller et al.). ， (Same as above). The antigen-binding portion may optionally be fused to the N-terminus and / or C-terminus of albumin via a linker.

[0171] Albumin derivatives in heteropolyform form have been described, comprising two transporter polypeptides obtained through albumin fragmentation, such that the transporter polypeptides self-assemble to form a protein resembling native albumin (see International Patent Application Publications WO 2012 / 116453 and WO 2014 / 012082). Due to albumin fragmentation, the heteropolymer includes four ends, thus allowing optional fusion via linkers to up to four different antigen-binding moieties.

[0172] In some 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 the Fc region of an immunoglobulin, albumin, or an albumin analogue or derivative. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold based on the Fc region of an immunoglobulin (e.g., the IgG Fc region).

[0173] Fc area As used herein, the terms “Fc region,” “Fc,” or “Fc domain” refer to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0174] In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the immunoglobulin Fc region. The Fc region may be dimer and composed of two Fc peptides, or alternatively, the Fc region may be composed of a single peptide.

[0175] In the case of dimer Fc, "Fc polypeptide" refers to one of the two polypeptides that form the dimer Fc domain, namely, a polypeptide containing one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to the dimer Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" are used interchangeably, provided that the Fc region contains one first Fc polypeptide and one second Fc polypeptide.

[0176] The Fc region may contain a CH3 domain or may contain both CH3 and CH2 domains. For example, in some embodiments, the Fc polypeptide of the dimer IgG Fc region may contain an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain contains two CH3 sequences, one from each of the two Fc polypeptides of the dimer Fc region, and the CH2 domain contains two CH2 sequences, one from each of the two Fc polypeptides of the dimer Fc region.

[0177] In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on the IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on the human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on the IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on the human IgG1 Fc region.

[0178] In some embodiments, the anti-NaPi2b antibody construct may comprise an IgG Fc region-based scaffold, the IgG Fc region being a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each of the first and second Fc polypeptides comprising a CH3 sequence and optionally a CH2 sequence, and wherein the first Fc polypeptide and the second Fc polypeptide are distinct. In some embodiments, the anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences, wherein at least one CH3 sequence comprises one or more amino acid modifications. In some embodiments, the anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, wherein at least one of the CH2 sequences comprises one or more amino acid modifications.

[0179] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc region containing a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification, such as substitution or insertion, in which the amino acid at a specific position on the first CH3 or CH2 sequence differs from the amino acid at the same position on the second CH3 or CH2 sequence. These asymmetric amino acid modifications may result in modification of only one of two amino acids at the same corresponding amino acid position on each sequence, or in different modifications of two amino acids at the same corresponding position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may contain one or more asymmetric amino acid modifications.

[0180] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote the formation of the heterodimeric Fc relative to the formation of the homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0181] Amino acid modifications to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art, and include, for example, International Publication No. WO 96 / 027011 (“Pepper-Mortar”), Gunasekaran et al., 2010. J Biol Chem , 285, 19637-46 (“Electrostatic Manipulation”), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (Blockchain-Switched Engineered Structure Domain (SEED) Technology) and Labrijn et al. , 2013, Proc Natl Acad Sci USA The amino acid modifications described in ,110(13):5145-50 (Fab arm exchange) are examples of such modifications. Other examples include methods that combine positive and negative design strategies to produce stable, asymmetric modified Fc regions, as described in International Publications WO 2012 / 058768 and WO 2013 / 063702. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a modified Fc region, as described in International Publications WO 2012 / 058768 or WO 2013 / 063702.

[0182] Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 60), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence contains amino acids 341-447 of the full-length human IgG1 heavy chain. Table 5 also shows the amino acid modifications of the CH3 domain that promote the formation of heterodimer Fc, as described in International Patent Application Publications WO 2012 / 058768 and WO2013 / 063702.

[0183] In some 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, as shown in Table 5.

[0184] Table 5: Promotion heterodimer formation Human IgG1 Fc sequence 1 and amino acid modification of the CH3 domain

[0185] 1 Sequence number 231-447 (EU number) In some embodiments, the anti-NaPi2b antibody construct may comprise an Fc-based scaffold containing two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences containing one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to Fc, such as FcγRI, FcγRII, and FcγRIII subclasses of receptors.

[0186] In some implementations, the anti-NaPi2b antibody construct comprises a scaffold based on IgGFc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0187] Selective alteration of the CH2 domain by various amino acid modifications that increase the affinity of Fc for different Fcγ receptors is known in the art. Amino acid modifications leading to increased binding and those leading to decreased binding can each be used for certain indications. For example, increasing the binding affinity of Fc for FcγRIIIa (an activating receptor) can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased lysis of target cells. Decreasing binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some cases. In some indications, it may be necessary to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb or decrease or eliminate binding of the Fc region to all Fcγ receptors (“knockout” variants) may be useful.

[0188] In some embodiments of the ADC described herein, anti-NaPi2b antibody constructs having amino acid modifications in the Fc region that eliminate binding to the Fcγ receptor provide benefits associated with reduced ADC toxicity compared to ADCs having anti-NaPi2b antibody constructs with a wild-type Fc region (see Kumagai et al., 2020). Cancer Science , 111:4636-4645).

[0189] Examples of amino acid modifications to the CH2 domain that alter the binding of the Fcγ receptor to Fc include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu et al., 2011). J Immunol Methods , 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al.) , 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al.) , 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al.) , 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar et al., 2006, Proc Natl Acad Sci USA , 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al. , 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter the binding of FcγRIIb to Fc are described in international publication WO 2021 / 232162. Other modifications affecting the binding of Fc to the Fcγ receptor are described in... Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, page 283).

[0190] In some embodiments, the anti-NaPi2b antibody construct comprises a scaffold based on IgG Fc having a modified CH2 domain, wherein the modified CH2 domain contains one or more amino acid modifications that cause a reduction or elimination of binding of the Fc region to all Fcγ receptors (i.e., a "knockout" variant).

[0191] Various publications describe strategies that have been used to engineer antibodies to produce “knockout” variants (see, for example, Strohl, 2009). Curr Opin Biotech 20:685-691, and Strohl & Strohl, “ Antibody Fc engineering for optimal antibody performance(See *Therapeutic Antibody Engineering*, Cambridge: Woodhead Publishing, 2012, pp. 225-249). These strategies include reducing effector function through glycosylation modifications, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al.). , 2012, J. Mol. Biol., 420:204-219).

[0192] Examples of mutations that can introduce hinge or CH2 domains to produce “knockout” variants include amino acid modifications L234A / L235A (LALA) and L234A / L235A / D265S (LALADS). LALA substitution in IgG1 antibodies has been shown to reduce antibody binding to FcγR (Wilkinson et al., PLoS One. 2021; 16(12)), thereby disrupting (but not completely knocking out) the ability of these antibodies to interact with certain cells in tissues. Generally, antibodies with Fc regions containing LALADS modifications are Fc-silenced because they do not exhibit ADCC or ADCP (antibody-dependent phagocytosis) and do not bind C1q. LALADS modifications are described in International Patent Publication No. WO 2014 / 012085.

[0193] In some embodiments, the anti-NaPi2b antibody construct described herein may comprise an IgG Fc-based scaffold in which the native glycosylation has been modified. As is known in the art, the glycosylation of the Fc can be modified to increase or decrease effector function. For example, a mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a non-glycosylated Fc lacking all effector functions (Bolt et al., 1993, ). Eur. J. Immunol., 23:403-411; Tao&Morrison, 1989, J. Immunol., 143:2595-2601).

[0194] Conversely, removal of fucose from heavy-chain N297-linked oligosaccharides has been shown to enhance ADCC based on improved binding with FcγRIIIa (see, for example, Shields et al., 2002). J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucosylation antibodies can be generated, for example, in the following cells: 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 Lec 13 (International Publication No. WO 03 / 035835), which showed a reduced ability to link fucose to N297-linked carbohydrates, or in other cells that produced antibodies against fucose (see, for example, Li et al., 2006, 87:614-622); Nat Biotechnol , 24:210-215; Shields et al. , 2002, ibid He and Shinkawa et al., 2003, J. Biol. Chem (278:3466-3473). Additionally, International Publication No. WO 2009 / 135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into carbohydrates on the antibody.

[0195] Other methods for generating antibodies containing little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology , 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0196] Other glycosylation variants include those having bimeric oligosaccharides, such as variants in which the biantennary oligosaccharide linked to the Fc region of the antibody is bimeric by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, for example, International Publication No. WO 2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US 2005 / 0123546). Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide linked to the Fc region, which may have improved CDC function (see, for example, International Publications Nos. WO 1997 / 030087, WO 1998 / 58964, and WO 1999 / 22764).

[0197] Anti-NaPi2b antibody construct with silenced Fc In one embodiment, the anti-NaPi2b antibody construct of the ADC of this disclosure is an Fc-silenced anti-NaPi2b antibody construct comprising a LALADS amino acid substitution in the Fc region. In some embodiments, the Fc-silenced anti-NaPi2b antibody construct comprising a LALADS amino acid substitution in the Fc region binds to, and / or is internalized by, NaPi2b-expressing cancer cells. In some embodiments, the target-independent binding of the Fc-silenced anti-NaPi2b ADC of this disclosure, i.e., binding to cells that do not (or only minimally) express NaPi2b, such as binding of the Fc region to one or more Fc receptors on non-target cells (e.g., macrophages), is lower compared to the binding of a comparable Fc WT anti-NaPi2b ADC. In some embodiments, anti-NaPi2b antibody constructs containing LALADS amino acid substitutions in the Fc region exhibit reduced binding to one or more Fcγ receptors compared to a reference anti-NaPi2b antibody construct containing a wild-type Fc region (e.g., an Fc region without LALADS amino acid substitutions). In some embodiments, anti-NaPi2b antibody constructs containing LALADS amino acid substitutions in the Fc region exhibit reduced ADCC and / or ADCP compared to a reference anti-NaPi2b antibody construct containing a wild-type Fc region (e.g., an Fc region without LALADS amino acid substitutions). Without being bound by any theory, reduced ADCC and / or ADCP, and decreased binding of the Fc region of the anti-NaPi2b antibody construct containing LALADS amino acid substitutions in the Fc region to NaPi2b-negative cells, may indicate reduced off-target cell binding and may potentially translate into reduced in vivo toxicity. Therefore, in some embodiments, Fc silencing of the ADC of this disclosure may have the potential to minimize toxicity driven by target-independent cellular uptake via FcγR.

[0198] Preparation of anti-NaPi2b antibody construct The anti-NaPi2b antibody constructs described herein can be generated using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and " Antibodies: A Laboratory Manual "2nd Edition, edited by Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014."

[0199] Typically, to generate antibody constructs through recombination, a polynucleotide or set of polynucleotides encoding an anti-NaPi2b antibody construct is produced and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotide encoding the anti-NaPi2b antibody construct can be generated using standard methods known in the art (see, for example, Ausubel et al.). ,Current Protocols in Molecular Biology John Wiley & Sons, New York, 1994 and updated, and " Antibodies: A Laboratory Manual "[…](2nd edition, edited by Greenfield, Cold SpringHarbor Laboratory Press, New York, 2014)". As those skilled in the art will understand, the number of polynucleotides required to express an anti-NaPi2b antibody construct will depend on the construct's format, including whether the antibody construct contains a scaffold. For example, when the anti-NaPi2b antibody construct is in a monospecific mAb or FSA format, two polynucleotides, each encoding one polypeptide chain, will be required. When multiple polynucleotides are required, they can be incorporated into one or more vectors.

[0200] Typically, for expression, a polynucleotide or a group of polynucleotides is incorporated into one or more expression vectors along with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the choice of regulatory elements depends on the selection of the host cell for expressing the antibody construct, and that such regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate the expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags, such as metal affinity tags, histidine tags, avidin / streptavitin-coding sequences, glutathione S-transferase (GST) coding sequences, and biotin-coding sequences. The expression vector can be an extrachromosomal vector or an integrative vector.

[0201] Suitable host cells for cloning or expressing anti-NaPi2b antibody constructs include a variety of prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as yeast cells). Saccharomyces ) or Pichia pastoris ( Pichia Prokaryotic host cells include, for example, *Escherichia coli* (…). E. coli Aeromonas salmonicida or Bacillus subtilis ( B. subtilis )cell.

[0202] In some embodiments, anti-NaPi2b antibody constructs can be generated in bacteria, especially when glycosylation and Fc effector function are not required, such as, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, See Volume 248, pp. 245-254, edited by BKC Lo, Humana Press, Totowa, NJ, 2003.

[0203] In some implementations, eukaryotic microorganisms such as filamentous fungi or yeast can be suitable expression host cells, especially fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in antibody constructs with partial or complete human glycosylation patterns (see, for example, Gerngross, 2004). Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0204] Suitable host cells for expressing glycosylated anti-NaPi2b antibody constructs are typically eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe PLATNIBODIES™ technology for generating antigen-binding constructs in transgenic plants. Mammalian cell lines suitable for growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed from SV40 (COS-7), human embryonic kidney (HEK) line 293, or 293 cells (see, e.g., Graham et al.). , 1977, J. Gen Virol., 36:59), juvenile hamster kidney cells (BHK), mouse Setolly™4 cells (see, for example, Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer (HeLa) cells, canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells (see, for example, Mather et al.) , 1982, Annals N.Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, and Chinese hamster ovary (CHO) cells (including DHFR) - CHO cells, see Urlaub et al. , 1980, Proc NatlAcad Sci USA , 77:4216) and myeloma cell lines (such as Y0, NS0 and Sp2 / 0). In Yazaki and Wu, Methods in Molecular Biology Exemplary mammalian host cell lines suitable for generating antibody constructs are reviewed in Volume 248, pp. 255-268 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003).

[0205] In some 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, NSO, or COS cell line, or a cell line derived from any of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for the maturation of the antibody construct.

[0206] Host cells containing an expression vector encoding an anti-NaPi2b antibody construct can be cultured using conventional methods to produce the anti-NaPi2b antibody construct. Alternatively, in some embodiments, host cells containing an expression vector encoding an anti-NaPi2b antibody construct can be used therapeutically or prophylactically to deliver the anti-NaPi2b antibody construct to a subject, or the polynucleotide or expression vector can be administered ex vivo to cells from a subject, and then the cells can be returned to the subject.

[0207] Typically, anti-NaPi2b antibody constructs are purified after expression. Proteins can be isolated or purified using a variety of methods known to those skilled in the art (see, for example...). Protein Purification: Principles and Practice (3rd edition, Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, fractionation chromatography, or gel filtration and reversed-phase chromatography, performed at atmospheric or high pressure using systems such as FPLC and HPLC. Other purification methods include electrophoresis, immunoassay, precipitation, dialysis, and focused chromatography. The combination of ultrafiltration and percolation techniques with protein concentration is also useful. As is well known in the art, many natural proteins bind to Fc regions and antibodies, and these proteins can be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to Fc regions. Similarly, bacterial protein L binds to the Fab regions of some antibodies. Purification can also be performed using specific fusion couplers. For example, if a GST fusion is used, antibodies can be purified using glutathione resin; if a His-tagged fusion is used, Ni... +2Antibodies can be purified using affinity chromatography, or, if a flag tag is used, purified using immobilized anti-flag antibodies. The required level of purification will vary depending on the anti-NaPi2b antibody construct used. In some cases, purification may not be necessary.

[0208] In some embodiments, the anti-NaPi2b antibody construct is substantially pure. When used with respect to the anti-NaPi2b antibody construct described herein, the term "substantially pure" (or "substantially purified") means that the antibody construct is substantially or essentially free of components that are typically associated with or interact with proteins, as found in their natural environment (such as native cells, or, in the case of recombinantly generated constructs, host cells). In some embodiments, the substantially pure anti-NaPi2b antibody construct is a protein formulation 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 proteins.

[0209] Some embodiments of this disclosure relate to a method for preparing an anti-NaPi2b antibody construct, the method comprising culturing a host cell having incorporated one or more polynucleotides encoding the anti-NaPi2b antibody construct or one or more expression vectors encoding the anti-NaPi2b antibody construct under conditions suitable for expression of the anti-NaPi2b antibody construct, and optionally recovering the anti-NaPi2b antibody construct from the host cell (or from the host cell culture medium).

[0210] Post-translational modification In some embodiments, the anti-NaPi2b antibody construct described herein may include one or more post-translational modifications. Such post-translational modifications may occur in vivo or in vitro after isolating the anti-NaPi2b antibody construct from host cells.

[0211] Post-translation modifications include various modifications as known in the art (see, for example) Proteins - Structure and Molecular Properties , 2nd edition, TE Creighton, WH Freeman and Company, NewYork, 1993; Post-Translational Covalent Modification of Proteins , BC 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 those embodiments in which the anti-NaPi2b antibody construct includes one or more post-translational modifications, the construct may contain the same type of modification at one or more sites, or it may contain different modifications at different sites.

[0212] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization via known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH4.

[0213] Other examples of post-translational modifications include, for example, the addition or removal of N- or O-linked carbohydrate chains, chemical modifications of N- or O-linked carbohydrate chains, N-terminal or C-terminal treatments, the linking of chemical motifs to the amino acid backbone, and the addition or deletion of N-terminal methionine residues produced by prokaryotic host cell expression. Post-translational modifications may also include modifications with detectable markers (such as enzyme markers, fluorescent markers, luminescent markers, isotope markers, or affinity markers) to allow for the detection and separation of proteins. Examples of suitable enzyme markers include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, 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.

[0214] Other examples of post-translational modifications include acetylation, ADP-ribosylation, amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, γ-carboxylation, GPI anchoring, hydroxylation, iodination, methylation, tetradecylation, polyethylene glycolation, isopentenylation, racemization, selenoylation, sulfation, and transfer-RNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

[0215] Camptothecin analogues The ADC disclosed herein contains a camptothecin analogue that is a compound having formula (I): in: R 1 Selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3, and -NH2, and R 2 Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3. And among them: When R 1 When it is -NH2, then R is R 3 or R 4 And when R 1 If it is not -NH2, then R is R 4 ; R 3 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 4 Selected from: , , , , , , , , , , , and ; R 5 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl and -(C1-C6 alkyl)-aryl; R 6 and R 7 Each is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 ; R 8 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl groups; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and ; R 13 Selected from: -H and -C1-C6 alkyl groups; R 14 and R 14’ Each is independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings with 0 to 3 substituents selected from the following: halogens, -C1-C6 alkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 ; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and X b Each is independently selected from: NH, O, and S, and X c Selected from: O, S, and S(O)2 The condition is that the compound is not ( S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14 H -pyrano[3',4':6,7]inzizo[1,2-b]quinoline-3,14(4 H )-Diketone.

[0216] In some embodiments, the camptothecin analogue is a compound of formula (I), provided that R 1 When it is NH2, R 2 Not H.

[0217] In some embodiments, in the compound of formula (I), R 1 Selected from: -CH3, -CF3, -OCH3, -OCF3 and NH2.

[0218] In some embodiments, in the compound of formula (I), R 1 It is NH2.

[0219] In some embodiments, in the compound of formula (I), R 1 Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3.

[0220] In some embodiments, in the compound of formula (I), R 1 Selected from: -CH3, -CF3, -OCH3 and -OCF3.

[0221] In some embodiments, in the compound of formula (I), R 2 Selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0222] In some embodiments, in the compound of formula (I), R 2 Selected from: -CH3, -CF3, -F, -Cl, -OCH3 and -OCF3.

[0223] In some embodiments, in the compound of formula (I), R 2 Selected from: -H, -F, -Br and -Cl.

[0224] In some embodiments, in the compound of formula (I), R 2 Selected from: -F, -Br, and -Cl.

[0225] In some embodiments, in the compound of formula (I), R 3 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR5 , -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0226] In some embodiments, in the compound of formula (I), R 4 Selected from: , , , , , , , , , and .

[0227] In some embodiments, in the compound of formula (I), R 5 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0228] In some embodiments, in the compound of formula (I), R 6 and R 7 Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 .

[0229] In some embodiments, in the compound of formula (I), R 8 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 implementations, in the compound of formula (I), each R 9 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0231] In some implementations, in the compound of formula (I), each R 9 Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0232] In some implementations, in the compound of formula (I), each R 9 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.

[0233] In some implementations, in the compound of formula (I), each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0234] In some implementations, in the compound of formula (I), each R 10 Independently selected from: -C1-C6 alkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0235] In some implementations, in the compound of formula (I), each R 10 Independently selected from: unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ Unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0236] In some embodiments, in the compound of formula (I), R 10’ 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.

[0237] In some embodiments, in the compound of formula (I), R 11 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0238] In some embodiments, in the compound of formula (I), R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0239] In some embodiments, in the compound of formula (I), R 12 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and .

[0240] In some embodiments, in the compound of formula (I), R 13 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0241] In some embodiments, in the compound of formula (I), R 14 and R 14’ Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0242] In some embodiments, in the compound of formula (I), R 16 Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0243] In some embodiments, in the compound of formula (I), R 16 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.

[0244] In some embodiments, in the compound of formula (I), R 17 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.

[0245] In some embodiments, in the compound of formula (I), R 18 and R 19Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings having 0 to 3 substituents selected from the following: halogens, unsubstituted C1-C6 alkyl groups, -C1-C6 haloalkyl groups, -C1-C6 hydroxyalkyl groups, -C1-C6 aminoalkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 .

[0246] In some embodiments, in the compound of formula (I), X a and X b Each is independently selected from: NH and O.

[0247] Combinations of any of the foregoing embodiments of the compound of formula (I) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0248] In some embodiments, the compound of formula (I) has formula (II): in: R 2 Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; R 20 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, , , , , , , , , , , , and ; R 5 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 6 and R 7 Each is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 ; R 8Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl groups; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and ; R 13 Selected from: -H and -C1-C6 alkyl groups; R 14 and R 14’ Each is independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings with 0 to 3 substituents selected from the following: halogens, -C1-C6 alkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 ; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and Xb Each is independently selected from: NH, O, and S, and X c Selected from: O, S, and S(O) 2， The condition is that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione.

[0249] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3.

[0250] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Cl, -OCH3 and -OCF3.

[0251] In some embodiments, in the compound of formula (II), R 2 Selected from F and Cl.

[0252] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , –(C1-C6 alkyl)-aryl , , , , , , , , , and .

[0253] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , –(C1-C6 alkyl)-aryl , , , , , , , and .

[0254] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , , , , , , , , , and .

[0255] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 Unsubstituted aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl , , , , , , , , , , , and .

[0256] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , –(C1-C6 alkyl)-aryl , , , , , , , , , , and .

[0257] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , –(C1-C6 alkyl)-aryl , , , , , , , and .

[0258] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3, and R 20 Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , , , , , , , , , and .

[0259] In some embodiments, in the compound of formula (II), R 5 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0260] In some embodiments, in the compound of formula (II), R 6 and R 7 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 .

[0261] In some embodiments, in the compound of formula (II), R 6 For H, and R 7 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5-C3-C8 heterocyclic alkyl groups and -C(O)R 17 .

[0262] In some embodiments, in the compound of formula (II), R 6 For H, and R 7 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 .

[0263] In some embodiments, in the compound of formula (II), R 6 and R 7 Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 .

[0264] In some embodiments, in the compound of formula (II), R 8 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0265] In some embodiments, in the compound of formula (II), each R 9 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0266] In some embodiments, in the compound of formula (II), each R 9 Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0267] In some embodiments, in the compound of formula (II), each R 9 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.

[0268] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0269] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: -C1-C6 alkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0270] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ Unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0271] In some embodiments, in the compound of formula (II), R 10’ 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.

[0272] In some embodiments, in the compound of formula (II), R 11 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0273] In some embodiments, in the compound of formula (II), R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0274] In some embodiments, in the compound of formula (II), R 12 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and .

[0275] In some embodiments, in the compound of formula (II), R 13Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0276] In some embodiments, in the compound of formula (II), R 14 and R 14’ Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0277] In some embodiments, in the compound of formula (II), R 16 Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0278] In some embodiments, in the compound of formula (II), R 16 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.

[0279] In some embodiments, in the compound of formula (II), R 17 It is a -C1-C6 alkyl group.

[0280] In some embodiments, in the compound of formula (II), R 17 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.

[0281] In some embodiments, in the compound of formula (II), R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings having 0 to 3 substituents selected from the following: halogens, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 .

[0282] In some embodiments, in the compound of formula (II), X a and X b Each is independently selected from: NH and O.

[0283] Combinations of any of the foregoing embodiments of the compound of formula (II) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0284] In some embodiments, the compound of formula (I) has formula (III): in: R 2 Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; R 15 Selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; R 4 Selected from: , , , , , , , , , , , and ; R 5 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 8 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 10’ Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl groups; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and ; R 13 Selected from: -H and -C1-C6 alkyl groups; R 14 and R 14’ Each is independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings with 0 to 3 substituents selected from the following: halogens, -C1-C6 alkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 ; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and X b Each is independently selected from: NH, O, and S, and X c Selected from: O, S and S(O)2.

[0285] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0286] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -F, and -Cl.

[0287] In some embodiments, in the compound of formula (III), R 15 Selected from: -CH3, -CF3, -OCH3 and -OCF3.

[0288] In some embodiments, in the compound of formula (III), R 15 Selected from: -CH3 and -OCH3.

[0289] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -F, and -Cl, and R 15Selected from: -CH3, -CF3, -OCH3 and -OCF3.

[0290] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -F, and -Cl, and R 15 Selected from: -CH3 and -OCH3.

[0291] In some embodiments, in the compound of formula (III), R 4 Selected from: , , , , , , , , , and .

[0292] In some embodiments, in the compound of formula (III), R 5 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aminoaryl.

[0293] In some embodiments, in the compound of formula (III), R 8 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0294] In some implementations, in the compound of formula (III), each R 9 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0295] In some implementations, in the compound of formula (III), each R 9 Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0296] In some implementations, in the compound of formula (III), each R 9Independently 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.

[0297] In some implementations, in the compound of formula (III), each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0298] In some implementations, in the compound of formula (III), each R 10 Independently selected from: -C1-C6 alkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0299] In some implementations, in the compound of formula (III), each R 10 Independently selected from: unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ Unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0300] In some embodiments, in the compound of formula (III), R 10' 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.

[0301] In some embodiments, in the compound of formula (III), R 11 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0302] In some embodiments, in the compound of formula (III), R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0303] In some embodiments, in the compound of formula (III), R 12 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and .

[0304] In some embodiments, in the compound of formula (III), R 13 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0305] In some embodiments, in the compound of formula (III), R 14 and R 14’ Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0306] In some embodiments, in the compound of formula (III), R 16 Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0307] In some embodiments, in the compound of formula (III), R 16 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.

[0308] In some embodiments, in the compound of formula (III), R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings having 0 to 3 substituents selected from the following: halogens, unsubstituted C1-C6 alkyl groups, -C1-C6 haloalkyl groups, -C1-C6 hydroxyalkyl groups, -C1-C6 aminoalkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 .

[0309] In some embodiments, in the compound of formula (III), X a and X b Each is independently selected from: NH and O.

[0310] Combinations of any of the foregoing embodiments of the compound of formula (III) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0311] In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group as defined in any one of formulas (I), (II), or (III) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group as defined in any one of formulas (I), (II), or (III) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, and sulfonamide.

[0312] In some embodiments, the camptothecin analog contained in the ADC according to this disclosure is a compound having formula (I) and selected from the compounds shown in Tables 6 and 7.

[0313] In some 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 Let F be the integer part of the integer part, and R be the integer part of the integer part. 20 H, -(C1-C6)-OR 5 or In some embodiments, the camptothecin analogue is a compound having formula (II), wherein R 2 For F; R 20 H, -(C1-C6)-OR 5 or ;R 5 For H, and R 18 and R 19 Together with the N atoms they are bonded to, they form unsubstituted 4-, 5-, 6-, or 7-membered rings. In some embodiments, camptothecin analogs are compounds having formula (II), wherein R 2 For F; R 20 -(C1-C6)-OR 5 And R 5 H. In some embodiments, the camptothecin analog is a compound having formula (II) and selected from the compounds shown in Table 6.

[0314] In some embodiments, the camptothecin analog is a compound having formula (III). In some embodiments, the camptothecin analog is a compound having formula (III), wherein R... 2 For F; R 15 -CH3; R 4 for ;R 9 It is a -C1-C6 hydroxyalkyl group, and X a and X b Each is O. In some embodiments, the camptothecin analog is a compound having formula (III) and selected from the compounds shown in Table 7.

[0315] In some embodiments, the ADC according to this disclosure comprises a camptothecin analogue of compound 139, compound 140, compound 141, or compound 148. In some embodiments, the ADC according to this disclosure comprises a camptothecin analogue of compound 139 or compound 141.

[0316] Table 6: Exemplary camptothecin analogs of formula (II)

[0317] Table 7: Exemplary camptothecin analogs of formula (III)

[0318] It should be understood that references to compounds of formula (I) throughout this disclosure include, in various embodiments, compounds of formulas (II) and (III) as well as the various compounds shown in Tables 6 and 7, to the same extent as embodiments in which each of these formulas or compounds is specifically listed individually.

[0319] Antibody-drug conjugates This disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In some embodiments, the ADC has formula (X): T-[L-(D) m ] n (X) in: T is the anti-NaPi2b antibody construct as described in this article; L stands for connector; D is a camptothecin analogue with formula (I); m is an integer between 1 and 4, and n is an integer between 1 and 10.

[0320] In some embodiments, m in the conjugate of formula (X) is between 1 and 2. In some embodiments, m is 1.

[0321] In some embodiments, in the conjugate of formula (X), n is between 1 and 8, for example, between 2 and 8. In some embodiments, n is between 4 and 8.

[0322] In some 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. In other embodiments, in the conjugate of formula (X), m is between 1 and 2, and n is between 2 and 6. In still other embodiments, in the conjugate of formula (X), m is 1, and n is between 2 and 6. In still other embodiments, in the conjugate of formula (X), m is 1, and n is between 3 and 5. In yet another embodiment, in the conjugate of formula (X), m is 1, and n is approximately 4.

[0323] As shown above and reflected by the parameters m and n in formula (X), the anti-NaPi2b antibody construct “T” can be conjugated to at most one compound “D” of formula (I). Those skilled in the art will understand that while any particular anti-NaPi2b antibody construct T is conjugated to an integer number of compounds D, analyzing the formulation of the conjugate to determine the ratio of compound D to anti-NaPi2b antibody construct T can yield non-integer results, thus reflecting a statistical average. This ratio of compound D to the target portion T is generally referred to as the drug-to-antibody ratio or “DAR”. Therefore, conjugate preparations with non-integer DAR are intended to be covered by formula (X). In one embodiment, the ADC of this disclosure has a DAR between 2 and 8. In other embodiments, the ADC of this disclosure has a DAR between 3 and 5. In yet another embodiment, the ADC of this disclosure has a DAR between 3.5 and 4.5.

[0324] In some embodiments, in the conjugate of formula (X), D is a compound of formula (II) or formula (III). In some embodiments, in the conjugate of formula (X), D is a compound selected from the compounds shown in Tables 6 and 7. In some 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.

[0325] Some embodiments of this disclosure relate to an ADC having formula (X), where D is a compound of formula (IV): in: R 1a Selected from: -H, -CH3, -CHF2, -CF3, -F, -Br, -Cl, -OH, -OCH3, -OCF3, and -NH2; R 2a Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; X is -O-, -S-, or -NH-, and R 4a Selected from: , , , , , , , , , and ,in It is the connection point with X, where p is 1, 2, 3, or 4; or X is O, and R 4a -X- is selected from: and ; R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and –C3-C8 heterocycloalkyl; Each R 9a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; or R 9a It does not exist, and X b =X; Each R 10a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl and ; Each R 10a’ Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10b Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11a It is absent or is a -C1-C6 alkyl group; R 12a Selected from: -C1-C6 alkyl, -CO2R 8a -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and ; R 13a Selected from: -H and -C1-C6 alkyl groups; R 14a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 21 Selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl and –(C1-C6 alkyl)-OR 5a ; R 22 and R 23 Each is independently selected from: -H, -halogen, -C1-C6 alkyl and -C3-C8 cycloalkyl; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and X b Each is independently selected from: NH, O, and S; X c Selected from: O, S, and S(O)2, and This indicates the connection point with connector L.

[0326] In some implementations, in compounds of formula (IV), R 1a Selected from: -CH3, -CF3, -OCH3, -OCF3, and -NH2.

[0327] In some implementations, in compounds of formula (IV), R 1a Selected from: -CH3, -CF3, -OCH3 and -OCF3.

[0328] In some implementations, in compounds of formula (IV), R 1a Selected from: -CH3, -OCH3 and NH2.

[0329] In some implementations, in compounds of formula (IV), R 1a Selected from: -CH3 and -OCH3.

[0330] In some implementations, in compounds of formula (IV), R 2a Selected from: -H, -CH3, -CF3, -F, -Cl, -OCH3, and -OCF3.

[0331] In some implementations, in compounds of formula (IV), R 2a Selected from: -H, -F, and -Cl.

[0332] In some implementations, in compounds of formula (IV), R 2a Yes -F.

[0333] In some embodiments, in compounds of formula (IV), X is -O-, -S-, or -NH-, and R 4aSelected from: , , , , , , and .

[0334] In some embodiments, in compounds of formula (IV), X is -O- or -NH-.

[0335] In some implementations, in the compound of formula (IV), each R 9a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0336] In some implementations, in the compound of formula (IV), each R 9a Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0337] In some implementations, in the compound of formula (IV), each R 10a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl and .

[0338] In some implementations, in the compound of formula (IV), each R 10a Independently selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl and .

[0339] In some implementations, in compounds of formula (IV), R 12a Selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0340] In some implementations, in compounds of formula (IV), R 13a Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0341] In some implementations, in compounds of formula (IV), R 14a’ Selected from: H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0342] In some implementations, in compounds of formula (IV), R 16a Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0343] In some implementations, in compounds of formula (IV), R 22 and R 23 Each is independently selected from: -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl and -C3-C8 cycloalkyl.

[0344] In some implementations, in the compound of formula (IV), X a and X b Each is independently selected from: NH and O.

[0345] In some implementations, in the compound of formula (IV), X a and X b Each is O.

[0346] In some embodiments, in the compound of formula (IV), X is O; R 4a for ;X a and X b Each is O, and R 9a It is a -C1-C6 alkyl group.

[0347] In some implementations, in compounds of formula (IV), R 1a -CH3 or -OCH3; X is O; R 4a for ;X a and X b Each is O; and R 9a It is a -C1-C6 alkyl group.

[0348] In some implementations, in compounds of formula (IV), R 1a -CH3 or -OCH3; R 2a H or F; X is O; R 4a for ;X a and X b Each is O; and R 9a It is a -C1-C6 alkyl group.

[0349] Other combinations of any of the foregoing embodiments of the compound of formula (IV) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0350] Some embodiments of this disclosure relate to an ADC having formula (X), where D is a compound of formula (V): in: R 2a Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3; R 20a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, , , , , , , , , , , , and ; R 5 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 6 and R 7 Each is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 ; R 8 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; Each R 9 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl and -NR 14 R 14’ ; Each R 10’ Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl groups; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and ; R 13 Selected from: -H and -C1-C6 alkyl groups; R 14 and R 14’ Each is independently selected from: -H, C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 17 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings with 0 to 3 substituents selected from the following: halogens, -C1-C6 alkyl groups, -C3-C8 cycloalkyl groups, and -(C1-C6 alkyl)-OR groups. 5 ; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and X b Each is independently selected from: NH, O, and S; X c Selected from: O, S, and S(O)2, and This indicates the connection point with connector L.

[0351] In some implementations, in the compound of formula (V), R 2a Selected from: -CH3, -CF3, -F, -Cl, -OCH3 and -OCF3.

[0352] In some implementations, in the compound of formula (V), R 2a Selected from: -CF3, -F, -Cl and -OCH3.

[0353] In some implementations, in the compound of formula (V), R 2a It is F.

[0354] In some implementations, in the compound of formula (V), R 20a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, , , , , , , , , , and .

[0355] In some implementations, in the compound of formula (V), R 20a Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , -(C1-C6 alkyl)-aryl , , , , , , , , , and .

[0356] In some implementations, in the compound of formula (V), R 20a Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , -(C1-C6 alkyl)-aryl , , , , , , , and .

[0357] In some implementations, in the compound of formula (V), R 20a Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR5 , , , , , , , , , and .

[0358] In some implementations, in the compound of formula (V), R 20a Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl , , , , , , , , , , , and .

[0359] In some implementations, in the compound of formula (V), R 6 and R 7 Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 .

[0360] In some implementations, in the compound of formula (V), R 6 For H, and R 7 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 .

[0361] In some implementations, in the compound of formula (V), R 6 For H, and R 7 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17 .

[0362] In some implementations, in the compound of formula (V), R6 and R 7 Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 -C3-C8 heterocyclic alkyl groups and -C(O)R 17 .

[0363] In some implementations, in the compound of formula (V), R 8 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0364] In some implementations, in the compound of formula (V), each R 9 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0365] In some implementations, in the compound of formula (V), each R 9 Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0366] In some implementations, in the compound of formula (V), each R 9 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.

[0367] In some implementations, in the compound of formula (V), each R 10 Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0368] In some implementations, in the compound of formula (V), each R 10 Independently selected from: -C1-C6 alkyl, -NR 14 R 14’ -aryl and -(C1-C6 alkyl)-aryl.

[0369] In some implementations, in the compound of formula (V), R 11Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0370] In some implementations, in the compound of formula (V), R 12 Selected from: -H, -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16 .

[0371] In some implementations, in the compound of formula (V), R 12 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -CO2R 8 Unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C1-C6 alkyl)-aminoaryl, -S(O)2R 16 and .

[0372] In some implementations, in the compound of formula (V), R 13 Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0373] In some implementations, in the compound of formula (V), R 14 and R 14’ Each is independently selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0374] In some implementations, in the compound of formula (V), R 16 Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0375] In some implementations, in the compound of formula (V), R 16 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.

[0376] In some implementations, in the compound of formula (V), R 17Selected 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.

[0377] In some implementations, in the compound of formula (V), R 18 and R 19 Together with the N atoms they are bonded to, they form 4-, 5-, 6-, or 7-membered rings having 0 to 3 substituents selected from the following: halogens, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 aminoalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl, and -(C1-C6 alkyl)-OR 5 .

[0378] In some implementations, in the compound of formula (V), R 17 It is a -C1-C6 alkyl group.

[0379] In some implementations, in the compound of formula (V), X a and X b Each is independently selected from: NH and O.

[0380] In some implementations, in the compound of formula (V), X a and X b Each is O.

[0381] In some implementations, in the compound of formula (V), R 20a –(C1-C6 alkyl)-OR 5 .

[0382] In some implementations, in the compound of formula (V), R 20a –(C1-C6 alkyl)-OR 5 And R 5 For H.

[0383] In some implementations, in the compound of formula (V), R 2a For F; R 20a –(C1-C6 alkyl)-OR 5 And R 5 For H.

[0384] Other combinations of any of the foregoing embodiments of the compound of formula (V) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0385] Some embodiments of this disclosure relate to an ADC having formula (X), where D is a compound of formula (VI): in: R 2a Selected from: -H, -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3, and -OCF3; X is -O-, -S-, or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a -CO2R 8a -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, , , , , , , , , , , , and ,in It is the connection point with X, where p is 1, 2, 3, or 4; or X is O, and R 25 -X- is selected from: and ; R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 6a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 7a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5a -C3-C8 heterocyclic alkyl groups and -C(O)R 17a ; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and –C3-C8 heterocycloalkyl; Each R 9a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; or R 9a It does not exist, and X b =X; Each R 10aIndependently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl and ; Each R 10a’ Independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; Each R 10b Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11a It is absent or is a -C1-C6 alkyl group; R 12a Selected from: -C1-C6 alkyl, -CO2R 8a -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and ; R 13a Selected from: -H and -C1-C6 alkyl groups; R 14a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl, and -C3-C8 heterocycloalkyl; R 16a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 17a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, –(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and –(C1-C6 alkyl)-aryl; R 21 Selected from: -C1-C6 alkyl, –C3-C8 cycloalkyl and –(C1-C6 alkyl)-OR 5a ; R 22 and R 23 Each is independently selected from: -H, -halogen, -C1-C6 alkyl and -C3-C8 cycloalkyl; R 24 R 25 and R 26 Each is a -C1-C6 alkyl group; X a and X b Each is independently selected from: NH, O, and S; X c Selected from: O, S, and S(O)2, and This indicates the connection point with connector L.

[0386] In some embodiments, in the compound of formula (VI), R 2a Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3.

[0387] In some embodiments, in the compound of formula (VI), R 2a Selected from: -CH3, -CF3, -F, -Cl, -OCH3 and -OCF3.

[0388] In some embodiments, in the compound of formula (VI), R 2a Selected from: F and Cl.

[0389] In some embodiments, in the compound of formula (VI), R 2a It is F.

[0390] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a –(C1-C6 alkyl)-aryl , , , , , , , and Or X is O, and R 25 -X- is selected from: and .

[0391] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a -(C1-C6 alkyl)-aryl , , , , , , , and .

[0392] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , , , , , , , , and .

[0393] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: , , , , , , , and .

[0394] In some embodiments, in compounds of formula (VI), X is -O- or -NH-.

[0395] In some embodiments, in the compound of formula (VI), R 6a It is H.

[0396] In some embodiments, in the compound of formula (VI), R 6a Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0397] In some embodiments, in the compound of formula (VI), R 7a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C(O)R 17a .

[0398] In some implementations, in the compound of formula (VI), each R 9a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl and -(C1-C6 alkyl)-aryl.

[0399] In some implementations, in the compound of formula (VI), each R 9a Independently selected from: -C1-C6 alkyl and -(C1-C6 alkyl)-aryl.

[0400] In some implementations, in the compound of formula (VI), each R 10a Independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -(C1-C6 alkyl)-aryl and .

[0401] In some implementations, in the compound of formula (VI), each R 10a Independently selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl and .

[0402] In some embodiments, in the compound of formula (VI), R 12a Selected from: -C1-C6 alkyl, -aryl, -(C1-C6 alkyl)-aryl and -S(O)2R 16a .

[0403] In some embodiments, in the compound of formula (VI), R 13a Selected from: -H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl and -C1-C6 aminoalkyl.

[0404] In some embodiments, in the compound of formula (VI), R 14a’ Selected from: H, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl.

[0405] In some embodiments, in the compound of formula (VI), R 16a Selected from: -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl.

[0406] In some embodiments, in the compound of formula (VI), R 17a It is a -C1-C6 alkyl group.

[0407] In some embodiments, in the compound of formula (VI), R 22 and R 23 Each is independently selected from: -H, -halogen, unsubstituted -C1-C6 alkyl, -C1-C6 haloalkyl, -C1-C6 hydroxyalkyl, -C1-C6 aminoalkyl and -C3-C8 cycloalkyl.

[0408] In some embodiments, in the compound of formula (VI), X a and X b Each is independently selected from: NH and O.

[0409] In some embodiments, in the compound of formula (VI), X a and X b Each is O.

[0410] In some embodiments, in the compound of formula (VI), X is O, and R 25 It is a -C1-C6 alkyl group.

[0411] In some embodiments, in the compound of formula (VI), R 2a F is F; X is O, and R is O. 25 It is a -C1-C6 alkyl group.

[0412] Other combinations of any of the foregoing embodiments of the compound of formula (VI) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0413] In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group as defined in any one of formulas (IV), (V), or (VI) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group as defined in any one of formulas (IV), (V), or (VI) is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amide, nitro, cyano, azide, alkylthio, thio, sulfonyl, and sulfonamide.

[0414] In some embodiments, in an ADC having formula (X), D is a compound of formula (IV), wherein R 1a It is -CH3, and R 2a For F. In some embodiments, in an ADC having formula (X), D is a compound of formula (IV), wherein R 1a -CH3; R 2a F; X is -O-; R 4a for ;R 9a It is a -C1-C6 alkyl group, and X a and X b Each is O.

[0415] In some embodiments, in an ADC having formula (X), D is a compound of formula (V), wherein R 2a Let F be the integer part of the integer part, and R be the integer part of the integer part. 20a H, -(C1-C6)-OR 5 or In some embodiments, in an ADC having formula (X), D is a compound of formula (V), where R... 2a For F; R 20a H, -(C1-C6)-OR 5 or ;R 5 For H, and R 18 and R 19 Together with the N atoms they are bonded to, they form unsubstituted 4-, 5-, 6-, or 7-membered rings. In some embodiments, in an ADC having formula (X), D is a compound of formula (V), wherein R 2a For F; R 20a -(C1-C6)-OR 5 And R 5 For H.

[0416] In some embodiments, in an ADC having formula (X), D is a compound of formula (VI), wherein R 2a F is F; X is -O-, and R is -O-. 25 It is a -C1-C6 alkyl group.

[0417] Connector L The conjugate of formula (X) includes a linker L, which is a bifunctional or multifunctional portion capable of linking one or more camptothecin analogs D to an 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, while a multifunctional (or multivalent) linker L links more than one compound D to a single site on the anti-NaPi2b antibody construct T. Linkers that link a single compound D to more than one site on the anti-NaPi2b antibody construct T can also be considered multifunctional.

[0418] The linker L includes a functional group capable of reacting with one or more target groups on the anti-NaPi2b antibody construct T and at least one functional group capable of reacting with a target group on the camptothecin analog D. Suitable functional groups are known in the art and include, for example... Bioconjugate Techniques Those described in (GT Hermanson, 2013, Academic Press). The target groups that can be used as linkers on the anti-NaPi2b antibody construct T and camptothecin analog D include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0419] Non-limiting examples of functional groups capable of reacting with thiols include maleimides, haloacetamides, haloacetyl groups, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, and tetrafluorophenyl esters), acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In this case, alternatives such as those used by Lyon et al. may also be used. , 2014, Nat. Biotechnol., The “self-stabilizing” maleimide described in 32:1059-1062.

[0420] Non-limiting examples of functional groups capable of reacting with amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfonyl-NHS esters), imine esters (such as Traut reagents), isothiocyanates, aldehydes, and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include converting the carboxyl group to an activated ester using succinimide-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), which can then react with amines.

[0421] Non-limiting examples of functional groups capable of reacting with electrophilic groups such as aldehyde or ketone carbonyl groups include acyl hydrazides, oximes, amino groups, hydrazines, thioureas, hydrazide carboxylates, and aryl acyl hydrazides.

[0422] In some embodiments, the linker L may include a functional group that allows interchain cysteine ​​bridging on the anti-NaPi2b antibody construct, such as the ThioBridge™ linker (Badescu et al., 2014). Bioconjug. Chem. 25:1124–1136), dithiomaleimide (DTM) connector (Behrens et al., 2015, Mol. Pharm. 12:3986–3998), and linkers based on dithioaryl (TCEP) pyridazinide (Lee et al., 2016, Chem. Sci., 7:799-802) or a linker based on dibromopyridazinedione (Maruani et al., 2015, Nat. Commun., 6:6645).

[0423] Alternatively, the anti-NaPi2b antibody construct T can be modified to include non-natural reactive groups, such as azides, which allow conjugation to the linker via complementary reactive groups on the linker. For example, conjugation of the linker to the anti-NaPi2b antibody construct can be achieved using click chemistry (see, for example, Chio & Bane, 2020). Methods Mol. Biol.(2078:83-97), such as azide-alkyne cycloaddition (AAC) reactions, which have been successfully used to develop antibody-drug conjugates. AAC reactions can be copper-catalyzed AAC (CuAAC) reactions, which involve the conjugation of azides with straight-chain alkynes; or strain-promoted AAC (SPAAC) reactions, which involve the conjugation of azides with cyclooctyne.

[0424] Linker L can be either cleavable or cleavable. A cleavable linker is one that is readily cleaved under specific conditions, such as intracellular conditions (e.g., in endosomes or lysosomes) or near target cells (e.g., in the tumor microenvironment). Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers. In contrast, cleavable linkers depend on the degradation of antibodies within the cell, which typically results in the release of the amino acid-linker-drug moiety.

[0425] Examples of cleavable linkers include, for example, linkers containing amino acid sequences that serve as cleavage recognition sequences for proteases. Many such cleavage recognition sequences are known in the art. For conjugates not intended for internalization, amino acid sequences that are recognized and cleaved by proteases present in the extracellular matrix near target cells, such as cancer cells, can be used. Examples of extracellular tumor-associated proteases include, for example, plasminogen activators, matrix metalloproteinases (MMPs), elastases, and kallikrein-related peptidases.

[0426] For conjugates intended for internalization, the linker L may contain an amino acid sequence that is recognized and cleaved by endosomal or lysosomal proteases. Examples of such proteases include, for example, cathepsins B, C, D, H, L, and S, as well as pod proteins.

[0427] The cleavage recognition sequence can be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that may be included in the 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, phenyl Gly-(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.

[0428] Other examples of cleavable linkers include disulfide-containing linkers, such as N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfonylbutyrate (sulfon-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance in the vicinity of the disulfide bond to improve the extracellular stability of the linker, for example, containing gem-dimethyl groups. Other cleavable linkers include those that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers containing combinations of these functionalities may also be useful; for example, linkers containing both hydrazones and disulfide bonds are known in the art.

[0429] Another example of a cleavable linker is a linker containing a β-glucuronide, which can be cleaved by β-glucuronidases, enzymes found in lysosomes and tumor stroma (see, for example, De Graaf et al., 2002). Curr. Pharm. Des. 8:1391–1403, and International Patent Publication No. WO 2007 / 011968). β-glucuronide can also be used to improve the hydrophilicity of the linker L.

[0430] Another example of a linker that is internally cleaved and improves hydrophilicity within the cell is a linker containing a pyrophosphate diester moiety (see, for example, Kern et al., 2016). J Am Chem Soc., 138:2430-1445).

[0431] In some embodiments, the linker L included in the conjugate of formula (X) is a cleavable linker. In some embodiments, linker L includes a cleavage recognition sequence. In some embodiments, linker L may include an amino acid sequence that is recognized and cleaved by lysosomal proteases.

[0432] The cleavable linker may optionally also include one or more additional functional groups, such as self-immolative and self-eliminating groups, extension groups, or hydrophilic portions.

[0433] Self-decomposing and self-eliminating groups that can be used in connectors include, for example For Aminobenzyl (PAB) and For Aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED), and hemiacetal groups. Other examples of self-decomposing groups include, but are not limited to, aromatic compounds with electron similarities to PAB or PABC groups, such as heterocyclic derivatives, for example, the 2-aminoimidazolium-5-methanol derivative described in U.S. Patent No. 7,375,078. Other examples include groups that cyclize upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995). Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. (55:5867-5877). Self-decomposing / self-eliminating groups are typically attached to an amino or hydroxyl group on compound D. Self-decomposing / self-eliminating groups are usually included alone or in combination in peptide-based linkers, but may also be included in other types of linkers.

[0434] Extension groups that can be used in the linkers of pharmaceutical conjugates include, for example, alkylene groups and extension groups based on aliphatic acids, diacids, amines, or diamines, such as diethylene glycol esters, malonic esters, hexanoates, and hexamethylene amides. Other extension groups include, for example, glycine-based extension groups and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) extension groups.

[0435] PEG and mPEG extension groups can also be used as hydrophilic portions within the linker. For example, PEG or mPEG can be “directly inserted” or included as a side group in the linker to increase its hydrophilicity (see, for example, U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that may optionally be incorporated into the linker L include, for example, β-glucuronic acid, sulfonic acid groups, carboxylic acid groups, and pyrophosphate diesters.

[0436] In some embodiments, the ADC of formula (X) may include a cleavable linker. In some embodiments, the ADC of formula (X) may include a peptide-containing linker. In some embodiments, the ADC of formula (X) may include a protease-cleavable linker.

[0437] In some implementations, in the ADC of formula (X), m is 1, and the connector L is a detachable connector of formula (XI): in: Z is a functional group capable of reacting with the target group on the anti-NaPi2b antibody construct T; Str is an extension group; AA1 and AA2 are each an amino acid independently, with AA1-[AA2] being the most abundant. r Formation of protease cleavage sites; X is a self-decomposing group; q is 0 or 1; r is 1, 2, or 3; s is 0, 1, or 2; # is the connection point with the anti-NaPi2b antibody construct T, and % represents the connection point with the camptothecin analogue D.

[0438] In some implementations, q is 1 in the connector of formula (XI).

[0439] In some implementations, s is 1 in the connector of formula (XI). In some implementations, s is 0 in the ADC of formula (XI).

[0440] In some implementations, r is 1 in the connector of formula (XI). In some implementations, r is 3 in the ADC of formula (XI).

[0441] In some implementations, in the connector of formula (XI): Z is , where # is the connection point with T, and It is the connection point with the rest of the connector.

[0442] In some implementations, in the connector of formula (XI), Str is selected from: ; ; ; ; ; and , in: R is H or a C1-C6 alkyl group; t is an integer between 2 and 10, and u is an integer between 1 and 10.

[0443] In some implementations, in the connector of formula (XI), Str is selected from: and , in: t is an integer between 2 and 10, and u is an integer between 1 and 10.

[0444] In some implementations, in the connector of formula (XI), AA1-[AA2] r It is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XI), AA1-[AA2] r It has sequences selected from the following: 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, phenyl Gly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys.

[0445] In some implementations, in the connector of formula (XI), AA1-[AA2] r It is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XI), AA1-[AA2] rIt has sequences selected from the following: 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.

[0446] In some implementations, in the connector of formula (XI), AA1-[AA2] r It is a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XI), AA1-[AA2] r It has sequences selected from the following: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0447] In some implementations, in the ADC of formula (X), m is 1, and the connector L is a detachable connector having formula (XII): in: Z is a functional group capable of reacting with the target group on the anti-NaPi2b antibody construct T; Str is an extension group; AA1 and AA2 are each an amino acid independently, with AA1-[AA2] being the most abundant. r Formation of protease cleavage sites; Y is -NH-CH2-; q is 0 or 1; r is 1, 2, or 3; v is 0 or 1; # is the connection point with the anti-NaPi2b antibody construct T, and % represents the connection point with the camptothecin analogue D.

[0448] In some implementations, q is 1 in the connector of formula (XII).

[0449] In some implementations, v is 0 in the connector of formula (XII). In some implementations, s is 1 in the ADC of formula (XII).

[0450] In some implementations, r is 1 in the connector of formula (XII). In some implementations, r is 3 in the ADC of formula (XII).

[0451] In some implementations, in the connector of formula (XII): Z is , where # is the connection point with T, and It is the connection point with the rest of the connector.

[0452] In some implementations, in the connector of formula (XII), Str is selected from: ; ; ; ; and , in: R is H or a C1-C6 alkyl group; t is an integer between 2 and 10, and u is an integer between 1 and 10.

[0453] In some implementations, in the connector of formula (XII), Str is selected from: and , in: t is an integer between 2 and 10, and u is an integer between 1 and 10.

[0454] In some implementations, in the connector of formula (XII), AA1-[AA2] r It is a dipeptide (i.e., r=1). In some embodiments, in the linker of formula (XII), AA1-[AA2] r It has sequences selected from the following: 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, phenyl Gly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys.

[0455] In some implementations, in the connector of formula (XII), AA1-[AA2] r It is a tripeptide (i.e., r=2). In some embodiments, in the linker of formula (XII), AA1-[AA2] rIt has sequences selected from the following: 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.

[0456] In some implementations, in the connector of formula (XII), AA1-[AA2] r It is a tetrapeptide (i.e., r=3). In some embodiments, in the linker of formula (XII), AA1-[AA2] r It has sequences selected from the following: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0457] In some embodiments, in the connector of formula (XII), Y is -NH-CH2. In some embodiments, in the connector of formula (XII), v is 1 and Y is -NH-CH2.

[0458] In some embodiments, the ADC of formula (X) may include a disulfide bond-containing junction. In some embodiments, in the ADC of formula (X), m is 1, and the junction L is a cleavable junction of formula (XIII): in: Z is a functional group capable of reacting with the target group on the anti-NaPi2b antibody construct T; Q is -(CH2) p -or-(CH2CH2O) q - where p and q are each independent integers between 1 and 10; Each R is independently H or a C1-C6 alkyl group; n is 1, 2, or 3; # is the connection point with the anti-NaPi2b antibody construct T, and % represents the connection point with the camptothecin analogue D.

[0459] In some implementations, the ADC of formula (X) may include a β-glucuronide-containing linker.

[0460] Various non-cleavable linkers are known in the art for attaching drugs to a target moiety and are available in certain embodiments of the ADCs disclosed herein. Examples of non-cleavable linkers include linkers having an N-succinimide or N-sulfosuccinimide moiety for reaction with an anti-NaPi2b antibody construct and a maleimide- or haloacetyl-based moiety for reaction with camptothecin analogs, or... vice versa Examples of such non-cleavable linkers include sulfosuccinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylate (sulfosuccinimide-SMCC). Sulfosuccinimide-SMCC conjugation typically occurs via a maleimide group that reacts with a thiol (-SH) on a camptothecin analog, while the sulfosuccinimide-NHS ester is reactive against primary amines on the NaPi2b antibody construct (such as those found in lysine residues and at the N-terminus of proteins or peptides). Other non-limiting examples of such linkers include N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC) and N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxyl-(6-aminohexanoate). (“Long-chain” SMCC or LC-SMCC), κ-maleimide undecanoic acid N-succinimide ester (KMUA), γ-maleimide butyric acid N-succinimide ester (GMBS), ε-maleimide hexanoic acid N-hydroxysuccinimide ester (EMCS), m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimide acetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimide propionylamino)hexanoate (SMPH), N-succinimide-4-(p-maleimide phenyl)-butyrate ester (SMPB) and N-(p-maleimide phenyl) isocyanate (PMPI). Other examples include those containing halogenated acetyl functional groups, such as N-succinimide-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimide iodoacetate (SIA), N-succinimide bromoacetate (SBA), and N-succinimide-3-(bromoacetamyl)propionate (SBAP)).

[0461] Non-limiting examples of drug-connectors comprising camptothecin analogs of formula (I) are shown in Tables 8, 9, and 10. Non-limiting examples of conjugates comprising these drug-connectors are shown in Tables 11, 12, and 13. In some embodiments, the ADC of formula (X) comprises a drug-connector selected from those shown in Tables 8, 9, and 10. In some embodiments, the ADC of formula (X) is selected from those shown in Tables 11, 12, and 13, wherein T is an anti-NaPi2b antibody construct and n is between 1 and 10. In some embodiments, the ADC of formula (X) is selected from those shown in Tables 11, 12, and 13, wherein 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 those shown in Tables 11, 12, and 13, wherein T is an anti-FRα antibody construct and n is between 4 and 8.

[0462] In some embodiments, the ADC of formula (X) comprises a drug-to-connector (L-(D)) selected from the following: m The compounds are: 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, where n is 4 or 8. In some embodiments, the ADC of formula (X) comprises a drug-connector (L-(D)) selected from the following. m ): MT-GGFG-AM-compound 139, MC-GGFG-AM-compound 139, MT-GGFG-compound 140, MC-GGFG-compound 140, MT-GGFG-AM-compound 141, MC-GGFG-AM-compound 141, MT-GGFG-compound 141, MC-GGFG-compound 141, MT-GGFG-compound 148, and MC-GGFG-compound 148, and n is 8.

[0463] ADC fabrication The ADC of formula (X) can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques(GT Hermanson, 2013, Academic Press). Various linkers and linker components are commercially available or can 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 (GT Hermanson, 2013, Academic Press). In addition, various antibody drug conjugation services are available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).

[0464] Typically, the preparation of an ADC involves first preparing a drug-connector DL ​​comprising one or more camptothecin analogs of formula (I) and a connector L, and then conjugating the drug-connector DL ​​to a suitable group on an anti-NaPi2b antibody construct T. However, the conjugation of the connector L to the anti-NaPi2b antibody construct T, and the subsequent conjugation of the anti-NaPi2b antibody construct-connector TL to one or more camptothecin analogs D of formula (I), remain alternative methods that can be used in some implementations.

[0465] In any of the methods described above, suitable groups on the compound D of formula (I) used to connect connector L include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of this disclosure, connector L is connected to compound D of formula (I) via a hydroxyl or amine group on the compound.

[0466] In any of the above methods, suitable groups on the anti-NaPi2b antibody construct T used to attach the adapter L include thiol groups (e.g., on the side chain of a cysteine ​​residue), amino groups (e.g., on the side chain of a lysine residue), carboxylic acid groups (e.g., on the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

[0467] For example, the anti-NaPi2b antibody construct T may contain one or more naturally occurring thiol groups, thereby allowing the anti-NaPi2b antibody construct T to bond to the linker L via the sulfur atom of the thiol group. Alternatively, the anti-NaPi2b antibody construct T may contain one or more lysine residues, which may be chemically modified to introduce one or more thiol groups. Reagents that can be used to modify lysine residues include, but are not limited to, those mentioned above. N - Succinimide-S-acetylthioacetate (SATA), N-succinimide-3-(2-pyridyldithio)propionate (“SPDP”), and 2-iminothiacyclopentane hydrochloride (Traut reagent). Alternatively, the anti-NaPi2b antibody construct T may contain one or more carbohydrate groups, which may be chemically modified to contain one or more thiol groups.

[0468] The carbohydrate group on the anti-NaPi2b antibody construct T can also be oxidized to provide an aldehyde (-CHO) group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which can then react with the linker L, for example, via a hydrazine or hydroxylamine group on the linker L.

[0469] The anti-NaPi2b antibody construct T can also be modified to include additional cysteine ​​residues (see, for example, U.S. Patent Nos. 7,521,541, 8,455,622, and 9,000,130) or non-natural amino acids, such as selenomethionine, to provide a reactive stem. right Acetylphenylalanine, formylglycine or right Azide-methyl-L-phenylalanine (see, for example, Hofer et al., 2009, Biochemistry , 48:12047-12057; Axup et al., 2012, PNAS , 109:16101-16106; Wu et al., 2009, PNAS , 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow site-specific conjugation. Alternatively, the anti-NaPi2b antibody construct T can be modified to include non-natural reactive groups, such as azides, which allow conjugation to the linker via complementary reactive groups on the linker, for example, through click chemistry (see, for example, Chio & Bane, 2020). Methods Mol. Biol.(2078:83-97). Another option is to use GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves the enzymatic remodeling of antibody-glycans to allow the linkers to be chemically linked via metal-free clicks (see, for example, European Patent No. EP 2 911 699).

[0470] Other methods for modifying proteins to connect or associate linkers L are known in the art, including those described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002).

[0471] Alternatively, ADCs can use transglutaminase, especially those derived from *Streptomyces mobara* (…). Streptomyces mobaraensis The preparation is made from bacterial transglutaminase (BTG) (see, for example, Jeger et al., 2010). Angew. Chem. Int. Ed. (49:9995-9997). BTG forms an amide bond between the formamide (amine acceptor, typically on antibodies) on the side chain of glutamine and an alkylene amino group (amine donor, typically on drug-linker), said alkylene amino group may be, for example, an ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a peptide or "tag" containing glutamine, which allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, for example, U.S. Patent Application Publication No. US 2013 / 0230543 and International (PCT) Publication No. WO 2016 / 144608).

[0472] Similar conjugation methods utilize the enzyme transpeptidase A. In this approach, antibodies are typically modified to include a transpeptidase A recognition motif (LPXTG, where X is any native amino acid), and the drug-linker is engineered to include an oligoglycine motif (typically GGG) to allow transpeptidase A-mediated transpeptidation (see, for example, Beerli et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, ...). Nature: Scientific Reports , 6:31899).

[0473] Once conjugation is complete, the average number of compounds of formula (I) conjugated to the anti-NaPi2b antibody construct (i.e., the "drug-antibody ratio" or DAR) can be determined using standard techniques such as UV / VIS spectroscopy, ELISA-based techniques, and 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 fraction of anti-NaPi2b antibody construct T containing zero, one, two, three, etc., compounds of formula (I)) can be optionally analyzed. Various techniques for measuring the DAR distribution are known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reversed-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al., 2011). mAbs, 3:161-172).

[0474] ADCs that resist NaPi2b Fc silence Target-independent uptake of ADCs, followed by treatment and release of cytotoxic payloads in normal tissues, can enhance the toxicity profile of ADCs. One mechanism of target-independent uptake is through binding of the ADC's Fc region to FcγR. Silencing Fc-FcγR binding via specific amino acid mutations can reduce binding to FcγR, decrease uptake into FcγR-expressing cells, and potentially alter the toxicity profile. As demonstrated and described herein, the target-independent binding of the Fc-silenced anti-NaPi2b ADC of this disclosure, i.e., binding to cells that do not express NaPi2b, such as binding of the Fc region to one or more Fc receptors, is lower compared to the binding of the FcWT anti-NaPi2b ADC. In some embodiments, the Fc-silenced anti-NaPi2b antibody construct containing LALADS amino acid substitutions in the Fc region and the corresponding ADC exhibit reduced ADCC and / or ADCP when compared to a reference anti-NaPi2b antibody construct containing a wild-type Fc region (e.g., an Fc region without LALADS amino acid substitutions). Unbound by any theory, the reduced ADCC and / or ADCP, as well as the decreased binding of the anti-NaPi2b antibody construct with Fc silence containing LALADS amino acid substitutions in the Fc region, to NaPi2b-negative cells demonstrate reduced off-target cell binding and may indicate the possibility of reduced in vivo toxicity.

[0475] In one implementation, the ADC resistant to NaPi2b Fc silencing comprises the following structure: Where n is between 2 and 6; and T is an anti-NaPi2b (sodium-dependent phosphate transporter 2B) antibody construct comprising an antigen-binding domain binding to human NaPi2b, wherein the anti-NaPi2b antibody construct comprises two heavy chains comprising the sequence shown in SEQ ID NO:68 and two light chains comprising the sequence shown in SEQ ID NO:67, or wherein the anti-NaPi2b antibody construct comprises two heavy chains comprising the sequence shown in SEQ ID NO:66 and two light chains comprising the sequence shown in SEQ ID NO:67. In a related embodiment, the anti-NaPi2b Fc-silencing ADC comprises the above structure, but wherein n is between 3.5 and 4.5. In other related embodiments, the anti-NaPi2b Fc-silencing ADC comprises the above structure, but wherein n is about 4. In various embodiments, such an anti-NaPi2b Fc-silencing ADC may also be described herein as “v40502 (LALADS)-MC-GGFG-AM-Compound 139 ADC”.

[0476] The anti-NaPi2b Fc-silencing ADCs described herein, containing the LALADS mutation in Fc, bind to and / or are internalized by NaPi2b-expressing cancer cells, and / or exhibit cytotoxicity against NaPi2b-expressing cancer cells, and / or possess antitumor activity in xenograft models of NaPi2b-expressing cancers. In some embodiments, these anti-NaPi2b Fc-silencing ADCs also exhibit target-independent binding to a reduction in normal cells expressing one or more FcγRs compared to equivalent ADCs containing the WT Fc region. In some embodiments, the anti-NaPi2b Fc-silencing ADCs of this disclosure also exhibit one or more in vivo properties that differ from equivalent ADCs containing the WT Fc region, such as one or more different PK characteristics (e.g., longer clearance time) and different antitumor activities in various tumor models.

[0477] Pharmaceutical Composition For therapeutic use, the ADCs of this disclosure are typically formulated as pharmaceutical compositions. Therefore, certain embodiments of this disclosure relate to pharmaceutical compositions comprising an ADC as described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions can be prepared using well-known and readily available ingredients through known procedures.

[0478] Pharmaceutical compositions may be formulated for administration to a subject via, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. As used herein, the term "parenteral" includes subcutaneous injection, as well as intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal injection or infusion. Pharmaceutical compositions are typically formulated in a form suitable for administration to a subject, such as syrups, elixirs, tablets, sugar tablets, lozenges, hard or soft capsules, pills, suppositories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injections, or solutions. Pharmaceutical compositions may be provided as unit-dose formulations.

[0479] In some embodiments, the pharmaceutical composition containing the ADC is formulated for parenteral administration, for example, in the form of a lyophilized formulation or an aqueous solution. Such pharmaceutical compositions may be provided, for example, in a unit-dose injectable form.

[0480] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used. Examples of such carriers include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol, benzyl alcohol, alkyl esters of p-hydroxybenzoate (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; and low molecular weight (less than about 10 residues) peptides. Proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium ions; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0481] In some embodiments, the composition containing the ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be formulated using suitable dispersants 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, parenteral-acceptable diluent or carrier. Acceptable diluents and carriers that may be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Alternatively, sterile, non-volatile oils may be used as carriers. Various mild, non-volatile oils may be used for this purpose, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid may be used in the preparation of the injection. Adjuvants, such as local anesthetics, preservatives, and / or buffers, may also be included in the injectable solution or suspension.

[0482] In some embodiments, the composition containing the ADC can be formulated for intravenous administration to a human vein. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer and / or a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in a closed container (such as an ampoule or capsule) indicating the amount of active agent. When the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a single ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0483] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art, and for example in " Remington: The Science and Practice of Pharmacy "(Original name " Remingtons Pharmaceutical Sciences It is described in Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0484] How to use Some embodiments of this disclosure relate to the therapeutic use of the ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents.

[0485] Some embodiments of this disclosure relate to methods of inhibiting the growth of abnormal cancer cells or tumor cells; inhibiting the proliferation of cancer cells or tumor cells in a subject; or treating cancer, including administering the ADCs described herein. In some embodiments, the ADCs described herein can be used to treat cancer. Therefore, some embodiments of this disclosure relate to the use of ADCs as anticancer agents.

[0486] Some embodiments of this disclosure relate to methods for inhibiting the proliferation of cancer or tumor cells, comprising contacting the cells with an ADC as described herein, such as an ADC of formula (X). Some embodiments relate to methods for killing cancer or tumor cells, comprising contacting the cells with an ADC as described herein, such as an ADC of formula (X).

[0487] Some implementation methods involve treating a subject with cancer by administering an ADC as described herein, such as an ADC of formula (X). In this case, treating the subject may result in one or more of the following: tumor size reduction, slowing or preventing tumor size increase, prolonging disease-free survival between tumor disappearance or removal and its recurrence, preventing subsequent tumor development (e.g., metastasis), prolonging the time to progression, reducing one or more adverse symptoms associated with the tumor, and / or prolonging the overall survival of the subject with cancer.

[0488] Some embodiments relate to the use of ADCs as described herein, such as an ADC of formula (X), in methods for inhibiting tumor growth in a subject. Some embodiments relate to the use of ADCs as described herein, such as an ADC of formula (X), in methods for inhibiting cancer cell proliferation and / or killing cancer cells in vitro. Some embodiments relate to the use of ADCs as described herein, such as an ADC of formula (X), in methods for inhibiting cancer cell proliferation and / or killing cancer cells in a subject with cancer.

[0489] Examples of cancers that can be treated in some implementations are carcinomas, including adenocarcinoma and squamous cell carcinoma; melanoma and sarcoma. Carcinomas and sarcomas are often also referred to as “solid tumors.” Examples of common solid tumors that can be treated in some implementations include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors and therefore may be considered solid tumors in some cases. Typically, the cancer to be treated is a cancer that expresses NaPi2b.

[0490] Some embodiments involve methods for inhibiting the growth of NaPi2b-positive tumor cells, which include contacting the cells with an ADC as described herein (e.g., an ADC of formula (X)). The cells may be present in vitro or in vivo. In some embodiments, the ADC may be used in methods for treating a subject with NaPi2b-positive cancer or tumor.

[0491] 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 ADC described herein can be used in a method for treating ovarian cancer or lung cancer. In one embodiment, the ADC described herein can be used in a method for treating NSCLC.

[0492] Drug kit Some implementations involve drug kits containing ADCs as described herein, such as an ADC of formula (X).

[0493] This kit typically includes a container holding the ADC and a label and / or package insert on or attached to the container. The label or package insert contains instructions commonly included in the commercial packaging of therapeutic products, providing information on the indications, usage, dosage, administration, contraindications, and / or warnings for using such therapeutic products. The label or package insert may also include a notification, in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug or biological product, reflecting the manufacturer's approval for use or sale in humans or animals. In some embodiments, the container may have a sterile inlet. For example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle.

[0494] In addition to the container holding the ADC, the kit may optionally contain one or more additional containers containing other components of the kit. These may include, for example, pharmaceutically acceptable buffers (such as water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or glucose solution), other buffers, or diluents.

[0495] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags. These containers can be made of various materials such as glass or plastic. Where appropriate, one or more components of the kit may be provided in lyophilized or dry form (such as powder or granules), and the kit may additionally contain a suitable solvent for reconstituted lyophilized or dry components.

[0496] The kit may also include other materials that are commercially or user-required, such as filters, needles, and syringes.

[0497] Tables 8 to 13 Table 8: Exemplary drug-linker (DL) structures of camptothecin analogues of formula (I) containing a C7 bond

[0498] Table 9: Exemplary drug-linker (DL) structures of camptothecin analogues containing formula (I) with C10 bonds

[0499] Table 10: Exemplary drug-linker (DL) structures of camptothecin analogues of formula (I) containing C7 or C10 bonds

[0500] Table 11: Exemplary conjugate (DC) structures of camptothecin analogues of formula (I) containing a C7 bond

[0501] Table 12: Exemplary conjugate (DC) structures of camptothecin analogues of formula (I) containing C10 bonds

[0502] Table 13: Exemplary conjugate (DC) structures of camptothecin analogues of formula (I) containing C7 or C10 bonds

[0503] The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.

[0504] Some embodiments of this disclosure Implementation Scheme 1. An antibody-drug conjugate having the following structure: Where n is between 2 and 6; and T is an anti-NaPi2b (sodium-dependent phosphate transporter 2B) antibody construct containing an antigen-binding domain that binds to human NaPi2b, wherein the anti-NaPi2b antibody construct comprises two heavy chains containing the sequence shown in SEQ ID NO:68 and two light chains containing the sequence shown in SEQ ID NO:67, or wherein the anti-NaPi2b antibody construct comprises two heavy chains containing the sequence shown in SEQ ID NO:66 and two light chains containing the sequence shown in SEQ ID NO:67.

[0505] Implementation Scheme 2. The antibody-drug conjugate according to Implementation Scheme 1, wherein n is between 3.5 and 4.5.

[0506] Implementation Scheme 3. The antibody-drug conjugate according to Implementation Scheme 1, wherein n is about 4.

[0507] Implementation Scheme 4. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of Implementation Schemes 1 to 3 and a pharmaceutically acceptable carrier or diluent.

[0508] Implementation Scheme 5. A method for inhibiting cancer cell proliferation, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any one of Implementation Schemes 1 to 3.

[0509] Implementation Scheme 6. A method for killing cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any one of Implementation Schemes 1 to 3.

[0510] Implementation Scheme 7. A method for treating cancer in a subject in need, comprising administering to the subject an effective amount of an antibody-drug conjugate according to any one of Implementation Schemes 1 to 3.

[0511] Implementation Scheme 8. Use of an effective amount of the antibody-drug conjugate according to any one of Implementation Schemes 1 to 3 for the treatment of cancer in a subject of need.

[0512] Implementation Scheme 9. An antibody-drug conjugate according to any one of Implementation Schemes 1 to 3, used in a therapy.

[0513] Implementation Scheme 10. An antibody-drug conjugate according to any one of Implementation Schemes 1 to 3, which is used to treat cancer.

[0514] Implementation Scheme 11. Use of the antibody-drug conjugate according to any one of Implementation Schemes 1 to 3 in the manufacture of a medicament for treating cancer.

[0515] Implementation Scheme 12. A kit comprising an antibody-drug conjugate according to any one of Implementation Schemes 1 to 3 and a label and / or packaging instructions containing instructions for use.

[0516] Example Examples 1 to 3 below illustrate various methods for preparing camptothecin analogues of formula (I). It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known in the art. It should also be understood that those skilled in the art will be able to prepare other compounds of formula (I) not specifically described below using the methods described below or similar methods, by using appropriate starting components and modifying the synthetic parameters as needed. Typically, starting components are available from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (ThermoFisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI), and Fluorochem Ltd., or synthesized from sources known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (7th edition, John Wiley & Sons, Inc., 2013) or prepared as described herein.

[0517] abbreviation The following abbreviations are used throughout the Examples section: BCA: diquinoline carboxylic acid; Boc: dicarbonate. Uncle Butyl acetate; CE-SDS: Sodium dodecyl sulfate by capillary electrophoresis; DCM: Dichloromethane; DTPA: Diethylenetriaminepentaacetic acid; DIPEA: -Diisopropylethylamine; DMF: Dimethylformamide; DMMTM: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc: fluorenylmethoxycarbonyl; HATU: hexafluorophosphate azibazide tetramethylureonium; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azibazide triazole; HPLC: high performance liquid chromatography; LC / MS: liquid chromatography-mass spectrometry; MC: maleiminohexanoyl; MT: maleiminotriethylene glycol ester; NMM: N -Methylmorpholine; PNP: right Nitrophenol; RP-UPLC-MS: reversed-phase ultra-high performance chromatography-mass spectrometry; SEC: size exclusion chromatography; TCEP: tris(2-carboxyethyl)phosphine; Tfp: tetrafluorophenyl; TLC: thin-layer chromatography; TFA: trifluoroacetic acid.

[0518] General Chemical Procedures General procedure 1: Convert chloride to amine Add an appropriate secondary amine (3 equivalents) to a stirred solution of the chloride compound in dimethylformamide (0.05 M to 0.1 M). After completion (as determined by LC / MS, typically 1 to 3 hours), purify the reaction mixture by reversed-phase HPLC to provide the desired product upon lyophilization.

[0519] General Procedure 2: Converting amines to amides Triethylamine (1.2 equivalents), a suitable carboxylic acid (1.1 equivalents), and a solution of DMMTM (2 equivalents) in water (1 M) were added to a stirred solution of the amine compound in dimethylformamide (0.05–0.1 M). After completion (as determined by LC / MS, typically 16 hours), the reaction mixture was purified by reversed-phase HPLC to provide the desired product upon lyophilization.

[0520] General procedure 3: Converting amines to sulfonamides DIPEA (3 equivalents) was added to a stirred solution of the amine compound in dimethylformamide (0.05 M to 0.1 M), followed by the addition of an appropriate amount of sulfonyl chloride. After completion (as determined by LC / MS, typically 16 hours), the reaction mixture was purified by reversed-phase HPLC to provide the desired product upon lyophilization.

[0521] General Procedure 4: Two-step conversion of amine to urea (Synthetic Scheme IV) Step 1: Add the amine compound to a stirred solution of dichloromethane or dimethylformamide (0.05M to 0.1M). right Nitrophenyl carbonate (1 equivalent) is added, followed by triethylamine (2 equivalents). After completion (determined by LC / MS, typically 1 to 4 hours), the reaction mixture is concentrated to dryness and then purified by reversed-phase HPLC to provide the desired PNP-carbamate intermediate upon lyophilization. This intermediate can be used to generate a single analog or in multiple batches to generate multiple analogs in a second step. Step 2: An appropriate primary amine (3 equivalents) is added to the PNP-carbamate intermediate (0.1 M to 0.2 M) in dimethylformamide. After completion (determined by LC / MS, typically 1 hour), the reaction mixture is purified by reversed-phase HPLC to provide the desired product upon lyophilization.

[0522] General procedure 5: Converting amines to carbamates Add to a stirred solution (0.05 M to 0.1 M) of the amine compound in dichloromethane or dimethylformamide. right Nitrophenyl carbonate (1 equivalent) is added, followed by triethylamine (2 equivalents). After completion (as determined by LC / MS, typically 1 to 4 hours), an appropriate alcohol is added to the resulting PNP-carbamate intermediate. After completion (as determined by LC / MS, typically 1 to 16 hours), the reaction mixture is purified by reversed-phase HPLC to provide the desired product upon lyophilization.

[0523] General procedure 6: Removal of Boc protecting groups TFA (20 vol%) was added to a stirred solution (0.1 M) of the Boc-protected amine compound in dichloromethane. After completion (as determined by LC / MS, typically 1 hour), the reaction mixture was concentrated under vacuum to provide crude solids or purified as described in General Procedure 9.

[0524] General Procedure 7: Copper-Mediated Amide Coupling Add EDC (HCl salt, 3 equivalents) to a rapidly stirred solution (0.02 M) of a 10% v / v mixture of Boc-GGFG-OH (3 equivalents) and HOAt (3 equivalents) in dimethylformamide in dichloromethane. After 5 minutes, add a solution (0.02 M) of an amine-containing effective load (1 equivalent) in a 10% v / v mixture of dimethylformamide in dichloromethane, followed immediately by the addition of CuCl2 (4 equivalents). Once complete (as determined by LC / MS, typically 1 to 16 hours), concentrate the reaction mixture under vacuum to provide crude solids or purify by preparative HPLC to provide the desired product upon lyophilization.

[0525] General Procedure 8: MT Placement A solution of MT-OTfp (1.2 to 1.5 equivalents) in acetonitrile (about 0.02 M) was added to a stirred solution of the amine compound (1 equivalent) in dimethylformamide (about 0.02 M), followed by the addition of DIPEA (10 µL, 4 equivalents). After completion (as determined by LC / MS, typically 1 to 16 hours), the reaction mixture was concentrated under vacuum to provide a crude solid, which was purified by preparative HPLC to provide the desired product upon lyophilization.

[0526] General Procedure 9: Compound Purification Rapid chromatography The crude reaction product was purified using a Biotage® Snap Ultra column (10 g, 25 g, 50 g, or 100 g) (Biotage, Charlotte, NC) eluted with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane on a Biotage® Isolera™ automated rapid system (Biotage, Charlotte, NC). Alternatively, reverse-phase rapid purification was performed using a Biotage® Snap Ultra C18 column (12 g, 30 g, 60 g, or 120 g) eluted with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was separated by removing the organic solvent via rotary evaporation or by lyophilizing the acetonitrile / water mixture.

[0527] Preparative HPLC: Reversed-phase HPLC of the crude compound was performed using a Luna® 5-μm C18 100 Å (150 × 30 mm) column (Phenomenex, Torrance, CA) on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA), eluted with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was separated by lyophilization of the acetonitrile / water mixture.

[0528] General Procedure 10: Compound Analysis LC / MS:The reaction was monitored for completion and the purified compound was analyzed using a Kinetex® 2.6-μm C18 100 Å (30×3 mm) column (Phenomenex, Torrance, CA) on an Agilent 1290 HPLC / 6120 single quadrupole LC / MS system (Agilent Technologies, Inc., Santa Clara, CA), eluted with a 10% to 100% linear gradient of 0.1% formic acid / acetonitrile / 0.1% formic acid / water.

[0529] NMR: 1 1H NMR spectra were collected using a Bruker AVANCE III 300 spectrometer (300 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are reported in parts per million (ppm).

[0530] Example 1: Preparation of camptothecin analogue with a methyl group 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]Indazano[1,2-b]quinoline-3,14(4H)-dione (Compound 1.1) According to Li et al., 2019, ACS Med. Chem. Lett. The procedure for preparing the title compound is provided in , 10(10): 1386-1392.

[0531] 1.2: (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano [3',4':6,7]Indazano[1,2-b]quinoline-3,14(4H)-dione (compound 1.2) According to Li et al., 2019, ACS Med. Chem. Lett. The procedure for preparing the title compound is provided in , 10(10): 1386-1392.

[0532] 1.3: (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(morpholinomethyl)-1,12-dihydro-14H-pyran [3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 100) The title compound was prepared from compound 1.1 (10 mg) and morpholine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 3.6 mg, 26% yield) as a grayish-white solid.

[0533] LC / MS: C 26 H 26 The calculated value of FN3O5 is m / z = 479.2, and the measured value is [M+H]. + =480.4.

[0534] 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).

[0535] 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]inzazido[1,2-b]quinoline-3,14(4H)-dione (compound 102) The title compound was prepared from compound 1.1 (10 mg) and 1-(benzenesulfonyl)piperazine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 3.6 mg, 21% yield) as a grayish-white solid.

[0536] LC / MS: C 32 H 31 The calculated value of FN4O6 is m / z = 618.2, and the measured value is [M+H]. + =619.4.

[0537] 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).

[0538] 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]inzazido[1,2-b]quinoline-3,14(4H)-dione (compound) Object 104) The title compound was prepared from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 4.7 mg, 27% yield) as a grayish-white solid.

[0539] LC / MS: C 32 H 32 The calculated value of FN5O6 is m / z = 633.2, and the measured value is [M+H]. + =634.4.

[0540] 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).

[0541] 1.6: (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-methylpiperazin-1-yl)methyl)-1,12-di Hydrogen-14H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 106) According to general procedure 1, from compound 1.1 (10 mg) and NThe preparation of the title compound began with methylpiperazine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 3.6 mg, 25% yield) as a grayish-white solid.

[0542] LC / MS: C 27 H 29 The calculated value of FN4O4 is m / z = 492.2, and the measured value is [M+H]. + =493.4.

[0543] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 108) The title compound was prepared from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 3.7 mg, 23% yield) as a grayish-white solid.

[0544] LC / MS: C 32 H 32 The calculated value of FN5O4 is m / z = 569.2, and the measured value is [M+H]. + =570.4.

[0545] 1 H NMR (300 MHz, MeOD) δ 8.39 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 10.6 Hz, 1H), 7.21 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 5.62 (d, J = 16.4 Hz, 1H), 5.49 (s, 2H), 5.41 (d, J = 16.4 Hz, 1H), 4.45 (s, 2H), 3.44 – 3.38 (m, 4H), 3.06 – 3.00 (m, 4H), 2.58 (d, J = 1.8 Hz, 3H), 2.00 – 1.89 (m, 2H), 1.03 (t, J =7.3 Hz, 3H).

[0546] 1.8: (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyridine Brno[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 110) The title compound was prepared from compound 1.1 (10 mg) and piperidine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 1.5 mg, 11% yield) as a grayish-white solid.

[0547] LC / MS: C 27 H 28 The calculated value of FN3O4 is m / z = 477.2, and the measured value is [M+H]. + =478.2.

[0548] 1 H NMR (300 MHz, MeOD) δ 8.34 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 10.3 Hz,1H), 7.70 (s, 1H), 5.63 (d, J = 16.4 Hz, 1H), 5.52 (s, 2H), 5.44 (d, J = 16.5 Hz,1H), 4.99 (s, 2H), 3.73 – 3.46 (m, 4H), 2.64 (s, 3H), 2.03 – 1.90 (m, 2H),1.90 – 1.84 (m, 6H), 1.03 (t, J = 7.4 Hz, 3H).

[0549] 1.9: (S)-4-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-) Pyrano[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)methyl)piperazine-1-carboxylic acid tert-butyl ester (compound 111) The title compound was prepared from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 6.6 mg, 40% yield) as a grayish-white solid.

[0550] LC / MS: C 31 H 35 The calculated value of FN4O6 is m / z = 578.2, and the measured value is [M+H]. + =579.4.

[0551] 1.10: (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H- Pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 112) The title compound was prepared from compound 111 (5.0 mg) according to general procedure 6, yielding a grayish-white solid title compound (TFA salt, 4.4 mg).

[0552] LC / MS: C 26 H 27 The calculated value of FN4O4 is m / z = 478.2, and the measured value is [M+H]. + =479.2.

[0553] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 113) According to general procedure 1, from compound 1.1 (10 mg) and ( R The title compound was prepared by starting with 2-morpholino-2-ylmethanol. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound as a grayish-white solid (TFA salt, 4.6 mg, 32% yield).

[0554] LC / MS: C 27 H 28 The calculated value of FN3O6 is m / z = 509.2, and the measured value is [M+H]. + =510.4.

[0555] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 114) The title compound was prepared from compound 1.1 (10 mg) and thiomorpholino-3-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 1.5 mg, 12% yield) as a grayish-white solid.

[0556] LC / MS: (C 27 H 28 The calculated value of FN3O5S is m / z = 525.6, and the measured value is [M+H]. + = 526.5.

[0557] 1H NMR (300 MHz, 10%D2O / CD3CN) 8.36 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 10.7Hz, 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).

[0558] 1.13: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((4-(hydroxymethyl)-2-oxa-5-azabicyclo) [2.2.1]Hept-5-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazano[1,2-b]quinoline- 3,14(4H)-diketone (compound 115) The title compound was prepared from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptane-4-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (TFA salt, 3.5 mg, 29% yield) as a grayish-white solid.

[0559] LC / MS: C 28 H 28 The calculated value of FN3O6 is m / z = 521.5, and the measured value is [M+H]. + =522.5.

[0560] 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).

[0561] 1.14: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)-1,1-dioxothiomorpholino)methyl 9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione (Cyclo) Compound 116) According to general procedure 1, compounds 1.1 (10 mg) and 3-(hydroxymethyl)-1λ were extracted. 6 The title compound was prepared by starting with thiomorpholine-1,1-dione. Purification was performed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as a grayish-white solid (TFA salt, 0.2 mg, 2% yield).

[0562] LC / MS: (C 27 H 28 The calculated value of FN3O7S is m / z = 557.6, and the measured value is [M+H]. + = 558.4.

[0563] 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).

[0564] 1.15: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((6-hydroxy-3-azabicyclo[3.1.1]hept-3-yl)methyl 9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione (Cyclo) Compound 117) The title compound was prepared from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]hepta-6-ol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 1.3 mg, 11% yield) as a grayish-white solid.

[0565] LC / MS: C 28 H 28 The calculated value of FN3O5 is m / z = 505.5, and the measured value is [M+H]. + =506.6.

[0566] 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).

[0567] 1.16: (S)-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azacyclobutane-1-yl)methyl)-4-hydroxy 9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione (compound) Object 118) The title compound was prepared from compound 1.1 (10 mg) and 3-fluorozacyclobutane-3-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 1.4 mg, 12% yield) as a grayish-white solid.

[0568] LC / MS: (C 26 H 25 The calculated value of F2N3O5 is m / z = 497.5, and the measured value is [M+H]. + = 498.4.

[0569] 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).

[0570] 1.17: (S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)azacyclobutane-1-yl)methyl)-9- Methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indazano[1,2-b]quinoline-3,14(4H)-dione (compound) 119) The title compound was prepared from compound 1.1 (10 mg) and aziridine-3-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as a grayish-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0571] LC / MS: C 26 H 26 The calculated value of FN3O5 is m / z = 479.5, and the measured value is [M+H]. + =480.4.

[0572] 1H 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).

[0573] 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]inzazido[1,2-b]quinoline-3,14(4H)-dione (compound) 120) The title compound was prepared from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as a grayish-white solid (TFA salt, 4 mg, 32% yield).

[0574] LC / MS: Calculated value m / z = 543.5 (C 28 H 28 F3N3O5), measured value [M+H] + = 544.4.

[0575] 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).

[0576] 1.19: (S)-4-ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-7- 9-Methyl-1,12-dihydro-14H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-di Ketone (compound 121) The title compound was prepared from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]hept-1-ylmethanol according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound as a grayish-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0577] LC / MS: C 29 H 30 The calculated value of FN3O5 is m / z = 519.6, and the measured value is [M+H]. + =520.4.

[0578] 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).

[0579] 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]inzizo[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 122) The title compound was prepared from compounds 1.2 (10 mg) and methanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (0.8 mg, 7% yield) as a grayish-white solid.

[0580] LC / MS: C 23 H 22 The calculated value of FN3O6S is m / z = 487.1, and the measured value is [M+H]. + =488.2.

[0581] 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).

[0582] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (compound) 124) The title compound was prepared from compounds 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (5.0 mg, 17% yield) as a grayish-white solid.

[0583] LC / MS: C 29 H 25 The calculated value of FN4O8S is m / z = 608.1, and the measured value is [M+H]. + =609.2.

[0584] 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).

[0585] 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]inzizo[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 125) The title compound was prepared from compounds 1.2 (10 mg) and benzenesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (9.8 mg, 73% yield) as a grayish-white solid.

[0586] LC / MS: C 28 H 24 The calculated value of FN3O6S is m / z = 549.6, and the measured value is [M+H]. + =550.6.

[0587] 1 H NMR (300 MHz, DMSO- d 6) δ 8.60 (t, J = 6.2 Hz, 1H), 8.17 (d, J = 8.1Hz, 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).

[0588] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 1.23) The title compound was prepared from compounds 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride according to general procedure 3. Purification of the title compound was performed using a 12 g C18 column and eluted with a gradient of 5% to 75% CH3CN / H2O + 0.1% TFA as described in general procedure 9, yielding the title compound as a grayish-white solid (37.8 mg, 47% yield).

[0589] LC / MS: C 28 H 23 The calculated value of FN4O8S is m / z = 594.6, and the measured value is [M+H]. + =595.2.

[0590] 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]inzizo[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 127) To a solution (6.4 mL) of compound 1.23 (37.8 mg, 0.064 mmol) in methanol, add 1% platinum, 2% vanadium, and 75 mg carbon. Purge the flask with H2 and then stir at room temperature under an H2 atmosphere for 45 minutes. Filter the mixture through a diatomaceous earth pad, wash with DMF, and evaporate the filtrate to give the title compound (30 mg, 84% yield) as a pale yellow solid.

[0591] LC / MS: C 28 H 24 The calculated value of FN4O6S is m / z = 564.6, and the measured value is [M+H]. + =565.2.

[0592] 1 H NMR (300 MHz, DMSO- d 6) δ 8.13 (d, J = 8.2 Hz, 1H), 8.02 (t, J = 6.2Hz, 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).

[0593] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 129) The title compound was prepared from compounds 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 25% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (1.3 mg, 13% yield) as a grayish-white solid.

[0594] LC / MS: C 24 H 24 The calculated value of FN3O7S is m / z = 517.1, and the measured value is [M+H]. + =518.2.

[0595] 1 H NMR (300 MHz, DMSO- d 6) δ 8.30 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 10.9Hz, 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 = 5.8Hz, 2H), 1.86 (m, 2H), 0.87 (d, J = 7.3 Hz, 3H).

[0596] 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]inzazo[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 131) Add chlorosulfonyl isocyanate (3 μL) to a solution of dichloromethane (1 mL) Uncle Butanol (3 μL). The solution was stirred for 1 hour, then compound 1.2 (13 mg) dissolved in dichloromethane (1 mL) was added, followed by triethylamine (13 μL). The reaction mixture was stirred for 1 hour and then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was performed as described in General Procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA. Trifluoroacetic acid (200 μL) was added to the purified solid in dichloromethane (1 mL). The reaction mixture was stirred for 16 hours and then concentrated to dryness to give the title compound (7.5 mg, 48% yield) as a grayish-white solid.

[0597] LC / MS: C 22 H 21 The calculated value of FN4O6S is m / z = 488.1, and the measured value is [M+H]. + =489.0.

[0598] 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).

[0599] 1.27: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) 4-Nitrophenyl benzo[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)methyl)carbamate (compound 1.27) The title PNP-carbamate intermediate compound was prepared from compound 1.2 (24 mg) according to the first step of General Procedure 4. Purification was completed using a 12 g C18 column with a gradient elution of 10% to 50% CH3CN / H2O + 0.1% TFA as described in General Procedure 9, yielding the title compound as a grayish-white solid (14 mg, 53% yield).

[0600] LC / MS: C 29 H23 The calculated value of FN4O8S is m / z = 574.2, and the measured value is [M+H]. + =575.2 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-methylurea (compound 132) The title compound was prepared from compound 1.2 (25 mg) and an aqueous solution of methylamine (500 µL, 40 wt% in water) as a primary amine, according to general procedure 4. In this case, the crude intermediate PNP-carbamate was used. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (8.9 mg, 31% yield) as a grayish-white solid.

[0601] LC / MS: C 24 H 23 The calculated value of FN4O5 is m / z = 466.2, and the measured value is [M+H]. + =467.2.

[0602] 1 H NMR (300 MHz, MeOD) δ 8.26 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 10.7 Hz,1H), 7.66 (s, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.48 (s, 2H), 5.41 (d, J = 16.4 Hz,1H), 4.97 (s, 2H), 2.73 (s, 3H), 2.57 (s, 3H), 2.08 – 1.93 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0603] 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]inzazo[1,2-b]quinoline-11-yl)methyl)urea (compound 134) Following step 2 of general procedure 4, the title compound was prepared using compound 1.27 (4 mg) as a PNP-carbamate and 4-(aminomethyl)aniline as a primary amine. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 12% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (0.6 mg, 20% yield) as a grayish-white solid.

[0604] LC / MS: C 30 H 28 The calculated value of FN5O5 is m / z = 557.2, and the measured value is [M+H]. + =558.4.

[0605] 1 H 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).

[0606] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 136) Following step 2 of General Procedure 4, the title compound was prepared using compound 1.27 (4 mg) as a PNP-carbamate and hydroxyethylamine as a primary amine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA, yielding the title compound (2.4 mg, 66% yield) as a grayish-white solid.

[0607] LC / MS: C 25 H 25 The calculated value of FN4O6 is m / z = 496.2, and the measured value is [M+H]. + =497.2.

[0608] 1 H 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).

[0609] 1.31: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) Brønsted[3',4':6,7]inzizo[1,2-b]quinoline-11-yl)methyl)carbamate (compound 138) The title compound was prepared by reacting methanol with the intermediate PNP-carbamate, starting with compound 1.2 (50 mg), according to general procedure 5. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (3.5 mg, 6% yield) as a grayish-white solid.

[0610] LC / MS: C 24 H 22 The calculated value of FN3O6 is m / z = 467.2, and the measured value is [M+H]. + =468.2.

[0611] 1 H NMR (300 MHz, MeOD) δ 8.17 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 10.5 Hz,1H), 7.69 (s, 1H), 5.65 (d, J = 16.5 Hz, 1H), 5.48 (s, 2H), 5.33 (d, J = 16.4 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.65 (s, 3H), 2.56 (s, 3H), 2.02 – 1.89 (m, 2H),1.02 (t, J = 7.4 Hz, 3H).

[0612] 1.32: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) 2-Hydroxyethyl benzo[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)methyl)carbamate (compound 139) The title compound was prepared by reacting compound 1.2 (18 mg) with 1,2-ethylene glycol and the intermediate PNP-carbamate according to general procedure 5. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (4.2 mg, 19% yield) as a grayish-white solid.

[0613] LC / MS: C 25 H 24 The calculated value of FN3O7 is m / z = 497.2, and the measured value is [M+H]. + =498.2.

[0614] 1 H NMR (300 MHz, DMSO) δ 8.23 ​​(d, J = 8.2 Hz, 1H), 7.78 (d, J = 10.7 Hz,1H), 7.40 (s, 1H), 5.47 (d, J = 16.5 Hz, 1H), 5.42 (s, 2H), 5.34 (d, J = 16.4 Hz,1H), 4.77 (s, 2H), 3.99 (t, J = 4.9 Hz, 2H), 3.64 – 3.38 (m, 2H), 2.48 (s, 3H), 2.02 – 1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0615] Example 2: Preparation of camptothecin analogues with a methoxy group at the C10 position 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethane-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. First, a DCM solution of 1 M BCl3 (71 mL, 71 mmol) was added to this solution, followed by a DCM solution of 1 M diethylaluminane (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was refluxed for 3 hours, cooled to room temperature, and quenched by adding 2 M HCl aqueous solution. The resulting heterogeneous mixture was refluxed for 1 hour, cooled to room temperature, and the pH was adjusted to approximately 12 with Na2CO3. The layers were separated, and the aqueous layer was extracted with DCM (3 × 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and rapidly purified by elution with 0% to 20% EtOAc / hexane as described in General Procedure 9, to give the title compound (6 g, 28 mmol, 39% yield).

[0616] LC / MS: Calculated value of C9H9ClFNO2 m / z = 217.1, measured value [M+H] + =218.1.

[0617] 1 H 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) 2.2: (S)-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano [3',4':6,7]Indazano[1,2-b]quinoline-3,14(4H)-dione (Compound 2.2) Toluene-4-sulfonic acid (157 mg, 0.9 mmol) was added to a solution (200 mL) of compound 2.1 (1.65 g, 7.6 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazine-3,6,10(4H)-trione (2 g, 7.6 mmol) in toluene. The solution was heated at 140 °C for 3 h and then cooled to room temperature. The product, as a yellow precipitate, was collected by filtration to give the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0618] LC / MS: C 22 H 18 The calculated value of ClFN2O5 is m / z = 445.2, and the measured value is [M+H]. + =445.1.

[0619] 1 H NMR (400 MHz, DMSO- d 6) 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) 2.3: (S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyridine Brno[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 101) The title compound was prepared from compound 2.2 (10 mg) and morpholine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 41% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (5.6 mg, 20% yield) as a grayish-white solid.

[0620] LC / MS: C 26 H 26 The calculated value of FN3O6 is m / z = 495.2, and the measured value is [M+H]. + =496.4.

[0621] 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).

[0622] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 103) The title compound was prepared from compounds 2.2 (10 mg) and 1-(benzenesulfonyl)piperazine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (2.5 mg, 14% yield) as a grayish-white solid.

[0623] LC / MS: C 32 H 31 The calculated value of FN4O7S is m / z = 634.2, and the measured value is [M+H]. + =635.4.

[0624] 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]inzazido[1,2-b]quinoline-3,14(4H)-dione (Chemical) Compound 105) The title compound was prepared from compounds 2.2 (10 mg) and 4-(piperazine-1-ylsulfonyl)aniline according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (4.0 mg, 23% yield) as a grayish-white solid.

[0625] LC / MS: C 32 H 32 The calculated value of FN5O7S is m / z = 649.2, and the measured value is [M+H]. + =650.4.

[0626] 1 H NMR (300 MHz, DMSO) δ 8.08 (s, 2H), 7.90 – 7.67 (m, 2H), 7.35 (s,1H), 7.32 – 7.26 (m, 2H), 6.67 – 6.57 (m, 2H), 5.46 (d, J = 16.5 Hz, 1H), 5.33–5.22 (m, 3H), 3.92 (s, 3H), 3.02 – 2.72 (m, 4H), 2.75 – 2.58 (m, 4H), 1.97 –1.70 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H).

[0627] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 107) According to general procedure 1, from compound 2.2 (10 mg) and N The preparation of the title compound began with methylpiperazine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound as a grayish-white solid (2.1 mg, 19% yield).

[0628] LC / MS: C 27 H 29 The calculated value of FN4O5 is m / z = 508.2, and the measured value is [M+H]. + =509.4.

[0629] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 109) The title compound was prepared from compounds 2.2 (10 mg) and 4-(piperazin-1-yl)aniline according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, to give the title compound (3.2 mg, 20% yield) as a grayish-white solid.

[0630] LC / MS: C 32 H 32 The calculated value of FN5O5 is m / z = 585.2, and the measured value is [M+H]. + =586.4.

[0631] 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).

[0632] 2.8: (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyran [3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 2.8) Hexamethylenetetramine (236 mg, 1.7 mmol) was added to a solution of compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL), followed by the addition of... i Pr2NEt (100 µL, 0.56 mmol). The solution was heated under reflux for 5 hours, cooled to room temperature, and quenched with 12 M HCl aqueous solution (60 µL). The solution was concentrated to approximately half its volume, and 1 M HCl aqueous solution (1.5 mL) was added. The mixture was stirred for 5 minutes and then concentrated to give a brown residue. Purification was performed as described in General Procedure 9, using a 12 g C18 fast column with a gradient elution of 5% to 40% CH3CN / H2O + 0.1% TFA to give the title compound as a pale yellow solid (179 mg, 75% yield).

[0633] LC / MS: C 22 H 20 The calculated value of FN3O5 is m / z = 425.4, and the measured value is [M+H]. + =426.2 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]inzizo[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 123) The title compound was prepared from compound 2.8 (10 mg) and methanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 5% to 65% CH3CN / H2O + 0.1% TFA, to give the title compound (8.5 mg, 91% yield) as a grayish-white solid.

[0634] LC / MS: C 23 H 22 The calculated value of FN3O7S is m / z = 503.1, and the measured value is [M+H]. + =504.2.

[0635] 1 H NMR (300 MHz, DMSO- d 6) δ 7.98 (d, J = 12.1 Hz, 1H), 7.89 (t, J = 6.4Hz, 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).

[0636] 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]inzizo[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 126) The title compound was prepared from compound 2.8 (7.5 mg) and benzenesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 5% to 70% CH3CN / H2O + 0.1% TFA, to give the title compound (4.6 mg, 46% yield) as a grayish-white solid.

[0637] LC / MS: C 28 H 24 The calculated value of FN3O7S is m / z = 565.6, and the measured value is [M+H]. + =566.2.

[0638] 1 H NMR (300 MHz, DMSO- d 6) δ 8.59 (t, J = 6.3 Hz, 1H), 7.94 (d, J = 12.2Hz, 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 = 6.3Hz, 1H), 4.09 (s, 2H), 1.95 – 1.81 (m, 1H), 0.89 (t, J = 7.3 Hz, 2H).

[0639] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-4-nitrobenzenesulfonamide (compound 2.11) The title compound was prepared from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride according to general procedure 3. Purification was performed using a 12 g C18 fast column and eluted with a gradient of 5% to 75% CH3CN / H2O + 0.1% TFA as described in general procedure 9, to give the title compound (9.7 mg, 71% yield) as a pale yellow solid.

[0640] LC / MS: C 28 H 23 The calculated value of FN4O9S is m / z = 610.6, and the measured value is [M+H]. + =611.5.

[0641] 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]inzizo[1,2-b]quinoline-11-yl)methyl)benzenesulfonamide (compound 128) To a solution (1.6 mL) of compound 2.11 (9.7 mg, 0.016 mmol) in methanol, add 1% platinum, 2% vanadium, and 15 mg carbon. Purge the flask with H2 and then stir at room temperature under an H2 atmosphere for 45 minutes. Filter the mixture through a diatomaceous earth pad, wash with DMF, and then evaporate the filtrate to give the title compound (1.5 mg, 16% yield) as a pale yellow solid.

[0642] LC / MS: C 28 H 25 The calculated value of FN4O7S is m / z = 580.6, and the measured value is [M+H]. + =581.4.

[0643] 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 = 7.3 Hz, 3H), 0.90 (s, 1H).

[0644] 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]inzazido[1,2-b]quinoline-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound) 130) The title compound was prepared from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 15% to 50% CH3CN / H2O + 0.1% TFA, to give the title compound (2.2 mg, 22% yield) as a grayish-white solid.

[0645] LC / MS: C 24 H 24 The calculated value of FN3O8S is m / z = 533.1, and the measured value is [M+H]. + =534.2.

[0646] 1 H NMR (300 MHz, DMSO- d 6) 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).

[0647] 2.14: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) 4-Nitrophenyl benzo[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)methyl)carbamate (compound 2.14) Following the first step of General Procedure 4, the title PNP-carbamate intermediate compound was prepared starting with compound 2.8 (65 mg) using a 1:1 mixture of dimethylformamide and dichloromethane as a solvent. Rapid purification was performed as described in General Procedure 9, using a 12 g C12 column and eluting with a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound (61 mg, 86% yield) as a grayish-white solid. This intermediate was separated and used to generate the following compounds.

[0648] LC / MS: C 29 H23 The calculated value of FN4O9 is m / z = 590.1, and the measured value is [M+H]. + =591.2.

[0649] 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]inzizo[1,2-b]quinoline-11-yl)methyl)-3-methylurea (compound 133) Following step 2 of General Procedure 4, the title compound was prepared using compound 2.14 (15 mg) as a PNP-carbamate and aqueous methylamine (500 μL, 40 wt% in water) as a primary amine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 47% to 60% CH3CN / H2O + 0.1% TFA, yielding the title compound (5.8 mg, 20% yield) as a grayish-white solid.

[0650] LC / MS: C 24 H 23 The calculated value of FN4O6 is m / z = 482.2, and the measured value is [M+H]. + =483.2.

[0651] 1 H NMR (300 MHz, DMSO- d 6) 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 = 7.3Hz, 3H).

[0652] 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]inzazo[1,2-b]quinoline-11-yl)methyl)urea (compound 135) Following step 2 of General Procedure 4, the title compound was prepared using compound 2.14 (15 mg) as a PNP-carbamate and 4-(aminomethyl)aniline as a primary amine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 20% to 60% CH3CN / H2O + 0.1% TFA, yielding the title compound as a grayish-white solid (TFA salt, 2.1 mg, 12% yield).

[0653] LC / MS: C 30 H 28 The calculated value of FN5O6 is m / z = 573.2, and the measured value is [M+H]. + =574.2.

[0654] 1 H 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).

[0655] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 137) Following step 2 of General Procedure 4, the title compound was prepared using compound 2.14 (15 mg) as a PNP-carbamate and hydroxyethylamine as a primary amine. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 12% to 60% CH3CN / H2O + 0.1% TFA, yielding the title compound (1.5 mg, 20% yield) as a grayish-white solid.

[0656] LC / MS: C 25 H 25 The calculated value of FN4O7 is m / z = 512.2, and the measured value is [M+H]. + =513.2.

[0657] 1 H NMR (300 MHz, MeOD) δ 7.93 (d, J = 12.1 Hz, 1H), 7.88 (d, J = 9.2 Hz,1H), 7.56 (s, 1H), 5.62 (d, J = 16.2 Hz, 1H), 5.52 (s, 2H), 5.45 (d, J = 16.3 Hz,1H), 4.98 (s, 2H), 4.17 (s, 3H), 3.59 (t, J= 5.6 Hz, 2H), 3.28 (t, J = 5.6 Hz,2H), 2.10 – 1.91 (m, 2H), 1.05 (t, J = 7.3 Hz, 3H).

[0658] Example 3: Preparation of camptothecin analogues with an amino group at the C10 position 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (Compound 3.1) 3-Bromo-4-fluorobenzaldehyde (180 g, 1.0 equivalent) was added to a stirred solution (500 mL) of HNO3 (121.2 mL, 67% purity, 2.0 equivalent) in H2SO4 at 0 °C. After the addition was complete, the ice bath was removed, and the reaction mixture was stirred at 25 °C for 5 hours. The mixture was poured into ice (5 L), filtered, and then dried under vacuum. The title compound (219 g) was obtained as a yellow solid.

[0659] 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).

[0660] 3.2: (2-Fluoro-5-formyl-4-nitrophenyl)tert-butyl carbamate (compound 3.2) A mixture of compound 3.1 (219 g, 1.0 equivalent), tert-butyl carbamate (124 g, 1.2 equivalent), Cs₂CO₃ (575 g, 2.0 equivalent), Pd₂(dba)₃ (40 g, 0.05 equivalent), and XPhos (84 g, 0.2 equivalent) in toluene (2000 mL) was degassed and purged with N₂ for three cycles. The mixture was then stirred at 90 °C under N₂ atmosphere for 15 h. The reaction mixture was diluted with H₂O (800 mL) and extracted with EtOAc (300 mL × 2). The combined organic layers were washed with brine (200 mL × 2), 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 (140 g, 56% yield) as a yellow solid.

[0661] 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) 3.3: (4-Amino-2-fluoro-5-formylphenyl) tert-butyl carbamate (compound 3.3) NH4Cl (30.5 g, 1.62 equivalents) was added to a solution of compound 3.2 (100 g, 1.0 equivalents) in H2O (300 mL) and EtOH (1200 mL). Iron (78.6 g, 4.0 equivalents) was added in portions at 80 °C. The mixture was stirred at 80 °C for 6 hours. 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 vacuum. 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 (19.0 g, 21% yield) as a yellow solid.

[0662] LC / MS: C 12 H 15 The calculated value of FN2O3 is m / z = 254.1, and the measured value is [M+H]. + =255.0.

[0663] 1 H NMR (400 MHz, DMSO- d 6) 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).

[0664] 3.4: (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3', [4':6,7]Indazino[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound 3.4) Compound 3.3 (4.20 g, 1.2 equivalents) was subjected to treatment at 110 °C. S )-4-ethyl-4-hydroxy-7,8-dihydro-1 H -pyrano[3,4- f Indazine-3,6,10(4) H )-Triketone (3.5 g, 1 equivalent) and TsOH (monohydrate, 253 mg, 0.1 when quantity The mixture was stirred in toluene (350 mL) for 2 hours. The reaction solution was cooled to 25 °C, filtered, and the solid was washed with methyl tert-butyl ether (30 mL) and then dried under vacuum. The title compound (4.5 g, 62% yield) was given as a yellow solid.

[0665] LC / MS: C 25 H 24 The calculated value of FN3O6 is m / z = 481.2, and the measured value is [M+H]. + =482.1.

[0666] 1 H NMR (400 MHz, DMSO- d 6) δ 9.49 (s, 1H), 8.65 (s, 1H), 8.43 (d, J =8.4Hz, 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) 3.5: (S)-(4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound 3.5) A solution of FeSO4 (heptahydrate, 1.2 g) and H2SO4 (280 µL) in H2O (4 mL) was added to a mixture of compound 3.4 (4.00 g) in MeOH (360 mL). The reaction mixture was heated at 65 °C while H2O2 (24 mL, 30% purity) was added dropwise over 30 minutes, followed by stirring for 0.5 h. The reaction solution was cooled to 25 °C and then filtered to give the title compound as a yellow solid (1.53 g, 33.2% yield). H2O (400 mL) was added to the filtrate, followed by quenching with a saturated aqueous solution of Na2S2O3. The pH was adjusted to 7 to 8 with a saturated aqueous solution of Na2CO3, and the solution was concentrated and filtered. The solid was ground 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).

[0667] LC / MS: C 26 H 26The calculated value of FN3O7 is m / z = 511.2, and the measured value is [M+H]. + =512.2.

[0668] 1 H 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).

[0669] 3.6: (S)-(4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-) Pyrano[3',4':6,7]inzazo[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound 3.6) DCM (2.9 mL) was added to a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), followed by the addition of Desmond-Martin periodane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). The solution was stirred at room temperature for 18 hours, then diluted with DCM and washed with saturated NaHCO3 aqueous solution and brine. The layers were separated, and the combined organic layer was evaporated onto diatomaceous earth. Rapid purification was performed as described in General Procedure 9, using a 10 g silica gel column and eluting with 0% to 10% DCM / MeOH to give the title product (42.5 mg, 28%) as an orange powder.

[0670] LC / MS: C 26 H 24 The calculated value of FN3O7 is m / z = 509.2, and the measured value is [M+H]. + =510.4.

[0671] 1 H NMR (300 MHz, acetone- d 6) 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 =16.2Hz, 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).

[0672] 3.7: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazine [1,2-b]quinoline-3,14(4H)-dione (compound 140) The title compound was prepared from compound 3.4 (40 mg) according to general procedure 6, yielding the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[0673] LC / MS: C 20 H 16 The calculated value of FN3O4 is m / z = 381.1, and the measured value is [M+H]. + =382.2.

[0674] 1 H 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).

[0675] 3.8: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano [3',4':6,7]Indazano[1,2-b]quinoline-3,14(4H)-dione (compound 141) The title compound was prepared from compound 3.5 (5 mg) according to general procedure 6, yielding a red solid title compound (TFA salt, 4.1 mg, 78% yield).

[0676] LC / MS: C 21 H 18 The calculated value of FN3O5 is m / z = 411.2, and the measured value is [M+H]. + =412.2.

[0677] 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).

[0678] 3.9: (S)-(11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound 3.9) A solution of thionyl chloride (14 µL) in dichloromethane (0.1 mL) was added to a stirred solution of compound 3.5 (100 mg) in dichloromethane (5 mL). After 1 hour, another solution of thionyl chloride (14 µL) in dichloromethane (0.1 mL) was added. After another 1 hour, the reactants were diluted with dichloromethane (10 mL) and toluene (1 mL) and then concentrated under vacuum to give the title compound as a red solid, which was used in subsequent reactions without further purification.

[0679] LC / MS: C 26 H 25 The calculated value of ClFN3O6 is m / z = 529.1, and the measured value is [M+H]. + =530.2.

[0680] 3.10: (S)-(11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetra- Hydrogen-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound 3.10) Hexamethylenetetramine (79 mg) was added to an ethanol (500 µL) solution of compound 3.9 (100 mg), followed by the addition of DIPEA (99 µL). The solution was heated at 60 °C for 16 hours and then concentrated to dryness under vacuum. Rapid purification was performed as described in General Procedure 9, using a 12 g C18 column with a gradient elution of 10% to 50% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 29 mg, 24% yield) as a grayish-white solid.

[0681] LC / MS: C 26 H 27 The calculated value of FN4O6 is m / z = 510.2, and the measured value is [M+H]. + =511.4.

[0682] 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).

[0683] 3.11: (S)-9-amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano [3',4':6,7]Indazano[1,2-b]quinoline-3,14(4H)-dione (compound 145) The title compound was prepared from compound 3.10 (2.1 mg) according to general procedure 6, yielding the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[0684] LC / MS: C 21 H 19 The calculated value of FN4O4 is m / z = 410.1, and the measured value is [M+H]. + =411.2.

[0685] 1 H NMR (300 MHz, MeOD) δ 7.82 (d, J = 12.1 Hz, 1H), 7.60 (s, 1H), 7.37(d,J = 9.1 Hz, 1H), 5.61 (d, J = 16.3 Hz, 1H), 5.42 (s, 2H), 5.41 (d, J = 16.3 Hz,1H), 4.69 (s, 2H), 2.08 – 1.94 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).

[0686] Example 3.12: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro- 14H-pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 3.12) The title compound was prepared from compound 3.9 (150 mg) and morpholine according to general procedure 1. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 103 mg, 52% yield) as a red solid.

[0687] LC / MS: C 30 H 33 The calculated value of FN4O7 is m / z = 580.2, and the measured value is [M+H]. + =581.4.

[0688] 1 H 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).

[0689] 3.13: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyran [3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 142) The title compound was prepared from compound 3.12 (45 mg) according to general procedure 6, yielding the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[0690] LC / MS: C 25 H 25 The calculated value of FN4O5 is m / z = 480.2, and the measured value is [M+H]. + =481.4.

[0691] 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).

[0692] 3.14: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H- Pyrano[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 148) Dichloromethane (1.45 mL) was added to a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol), 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 hours, quenched by adding water + 0.1% TFA and DMF (1:1, 1.0 mL), and partially evaporated. Purification was completed as described in General Procedure 9, using a 12 g C18 fast column and eluting with a gradient of 5% to 40% CH3CN / H2O + 0.1% TFA to give a Boc-protected intermediate as a yellow powder. The intermediate was then deprotected according to General Procedure 6 to give the title compound (TFA salt, 32.5 mg, 98% yield) as a yellow solid.

[0693] LC / MS: C 26 H 27 The calculated value of FN4O4 is m / z = 478.2, and the measured value is [M+H].+ =479.4.

[0694] 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).

[0695] 3.15: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-di Hydrogen-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione (compound 149) Dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol), and [other components] were added to a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol). N -Methylpiperazine (4.90 μL, 0.044 mmol). The solution was stirred at room temperature for 4 hours, then sodium triacetoxyborohydride (7.8 mg, 0.037 mmol) was added and stirred for another 45 minutes. Excess hydride was then quenched by adding 0.1% TFA aqueous solution (0.5 mL). Purification was completed as described in General Procedure 9, using a 12 g C18 fast column and eluting with a gradient of 5% to 40% CH3CN / H2O + 0.1% TFA to give a Boc-protected intermediate as a yellow powder. The intermediate was deprotected according to General Procedure 6 to give the title product (TFA salt, 1.5 mg, 7.1% yield) as a yellow solid.

[0696] LC / MS: C 26 H 28 The calculated value of FN5O4 is m / z = 493.2, and the measured value is [M+H]. + =494.4.

[0697] 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).

[0698] 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]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 153) The Boc-protected precursor of the title compound was prepared from compound 3.9 (10 mg) and 1-(benzenesulfonyl)piperazine according to general procedure 1. Preparative HPLC was performed as described in general procedure 9, eluting with a gradient of 35% to 44% CH3CN / H2O + 0.1% TFA to give a yellow powdery Boc-protected intermediate. This intermediate was then deprotected according to general procedure 6 to give the title compound (TFA salt, 2.4 mg, 17% yield, in two steps).

[0699] LC / MS: C 31 H 30 The calculated value of FN5O6S is m / z = 619.2, and the measured value is [M+H]. + =520.4.

[0700] 1H 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).

[0701] 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]inzazo[1,2-b]quinoline-11-yl)methyl)acetamide (compound 147) The title compound was prepared from compound 3.10 (8 mg) and acetic acid according to general procedure 2, followed by general procedure 6. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (4.0 mg, 56% yield) was obtained as a red solid.

[0702] LC / MS: C 23 H 21 The calculated value of FN4O5 is m / z = 452.2, and the measured value is [M+H]. + =453.2.

[0703] 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).

[0704] 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]inzazo[1,2-b]quinoline-11-yl)methyl)methanesulfonamide (compound 146) The title compound was prepared from compound 3.10 (8 mg) and methanesulfonyl chloride according to general procedure 3, followed by general procedure 6. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (4.4 mg, 57% yield) was obtained as a red solid.

[0705] LC / MS: C 22 H 21 The calculated value of FN4O6S is m / z = 488.1, and the measured value is [M+H]. + =489.2.

[0706] 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).

[0707] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (compound 150) The title compound was prepared from compound 3.10 (6 mg) and 2-hydroxyethanesulfonyl chloride according to general procedure 3, followed by general procedure 6. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in general procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (1 mg, 16% yield) was obtained as a red solid.

[0708] LC / MS: C 23 H 23 The calculated value of FN4O7S is m / z = 518.5, and the measured value is [M+H]. + =519.5.

[0709] 1 H NMR (300 MHz, 10% D2O / CD3CN) δ 7.77 – 7.61 (m, 1H), 7.48 – 7.30 (m,2H), 5.53 (d, J = 16.3 Hz, 1H), 5.31 (d, J = 15.4 Hz, 3H), 4.69 (s, 2H), 3.97(dd, J = 6.6, 4.9 Hz, 2H), 3.39 (t, J = 5.8 Hz, 2H), 2.93 (s, 1H), 1.99-1.83 (m,2H), 0.94 (t, J = 7.3 Hz, 3H).

[0710] 3.20: (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) 4-Nitrophenyl benzo[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)methyl)carbamate (compound 3.20) Add 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 µL, 0.04 mmol) to a solution of compound 3.10 (10 mg, 0.02 mmol) in DMF (400 µL, 0.05 M). Stir the solution at room temperature for about 30 minutes and then use it directly in subsequent reactions.

[0711] 3.21: (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) Brønno[3',4':6,7]inzizo[1,2-b]quinoline-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 min. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (2.1 mg, 47% yield) was obtained as a red solid according to General Procedure 6.

[0712] LC / MS: C 23 H 21 The calculated value of FN4O6 is m / z = 468.4, and the measured value is [M+H]. + =468.3.

[0713] 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).

[0714] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-methylurea (compound 144) The solution of compound 3.20 was prepared by adding methylamine hydrochloride (10 mg) to a 200 μL solution, followed by the addition of... iThe title compound was prepared using Pr2NEt (5 µL). The solution was stirred at room temperature for 30 min. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (2.9 mg, 64.5% yield) was obtained as a red solid according to General Procedure 6.

[0715] LC / MS: C 23 H 21 The calculated value of FN5O5 is m / z = 467.5, and the measured value is [M+H]. + =468.5.

[0716] 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).

[0717] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-(2-hydroxyethyl)urea (compound 151) The title compound was prepared by adding ethanolamine (100 μL) to a solution of 200 μL of compound 3.20. The solution was stirred at room temperature for 30 min. Preparative HPLC purification of the compound protected by the intermediate Boc was performed as described in General Procedure 9, with elution using a gradient of 10% to 60% CH3CN / H2O + 0.1% TFA. The title compound (0.5 mg, 8.5% yield) was obtained as a red solid according to General Procedure 6.

[0718] LC / MS: C 24 H 24 The calculated value of FN5O6 is m / z = 497.5, and the measured value is [M+H]. + =498.5.

[0719] 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).

[0720] 3.24: (S)-9-amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyran [3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 152) Thionyl chloride (35 µL, 2.5 equivalents) was added to a stirred solution of compound 3.5 (100 mg) in 2 mL of dichloromethane. The solution was stirred at room temperature for 20 min, and then thionyl chloride (35 µL, 2.5 equivalents) was added again. After 20 min, toluene (1 mL) was added, and the reaction mixture was concentrated under vacuum. The crude solid was suspended in DMSO (1 mL) and sodium azide (19 mg, 1.5 equivalents) was added. The solution was stirred at room temperature for 16 h. Purification was completed as described in General Procedure 9, with elution using a gradient of 5% to 50% CH3CN / H2O + 0.1% TFA to give the title compound (20 mg, 23% yield) as a grayish-white solid.

[0721] LC / MS: C 21 H 17 The calculated value of FN6O4 is m / z = 436.1, and the measured value is [M+H]. + =437.2.

[0722] 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).

[0723] 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]inzazo[1,2-b]quinoline-11-yl)methyl)acetamide (compound 164) The title compound was prepared from compound 145 (10 mg) and glycolic acid according to general procedure 2. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound (6.9 mg, 60% yield) was obtained as a yellow solid.

[0724] LC / MS: C 23 H 21 The calculated value of FN4O6 is m / z = 468.1, and the measured value is [M+H]. + =469.2.

[0725] 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).

[0726] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-3-methylthiourea (compound 161) A solution of compound 145 (9 mg, 1.0 equivalent) in DMF (1 mL) was treated with thiocarbonyl diimidazole (6 mg, 1.5 equivalent), followed by DIPEA (8 μL, 2.0 equivalent). The resulting solution was stirred at 25 °C for 2 h, after which complete conversion to the isothiocyanate intermediate was observed. Methylammonium chloride (3 mg, 2.0 equivalent) 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, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound (2.3 mg, 22% yield) was given as a yellow solid.

[0727] LC / MS: C 23 H 22 The calculated value of FN5O4S is m / z = 483.1, and the measured value is [M+H]. + =484.2.

[0728] 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).

[0729] 3.27: (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) [3',4':6,7]Indazazino[1,2-b]quinoline-11-yl)methyl)thiocarbamate S-(2-hydroxyethyl) ester (compound) Object 160) The title compound was prepared from compound 145 (10 mg) and 2-mercaptoethanol according to general procedure 5. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 45% CH3CN / H2O + 0.1% TFA. The title compound (4.2 mg, 43% yield) was obtained as a yellow solid.

[0730] LC / MS: C 24 H 23 The calculated value of FN4O6S is m / z = 514.1, and the measured value is [M+H]. + =515.2.

[0731] 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).

[0732] 3.28: (S)-9-amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6, 7] Indazano[1,2-b]quinoline-3,14(4H)-dione (compound 154) Water (0.72 mL), FeSO4 (heptahydrate, 11.0 mg), and propionaldehyde (74 μL) were added to a 5 mL flask containing compound 140 (50 mg). The resulting suspension was cooled to -15 °C using an ice-salt 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 min, then heated to room temperature and stirred for 2 h. 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, with elution using a gradient of 25% to 70% CH3CN / H2O + 0.1% TFA, to give the title compound (2.4 mg, 4.4% yield) as a deep orange solid.

[0733] LC / MS: C 22 H 20 The calculated value of FN3O4 is m / z = 410.1, and the measured value is [M+H]. + =410.2.

[0734] 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).

[0735] 3.29: (S)-(11-((carbamoyloxy)methyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4, 12,14-Tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)tert-butyl carbamate (compound) 3.29) At -20°C, 3.5 g (15 mg) of the compound was added to a 5 mL Erlenmeyer flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL). The resulting suspension was stirred at -20°C for 5 min. Water (59 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 2 h, then heated at 70°C for 1 h. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 40% to 55% CH3CN / H2O + 0.1% TFA, to give the title compound (5.1 mg, 31% yield) as a deep orange solid.

[0736] LC / MS: C 27 H 27 The calculated value of FN4O8 is m / z = 555.2, and the measured value is [M+H]. + =555.2.

[0737] 1 H NMR (300 MHz, DMSO- d 6) 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 = 7.7Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[0738] 3.30: (S)-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridine) Methyl carbamate (compound 169) of benzo[3',4':6,7]inzizo[1,2-b]quinoline-11-yl) The title compound was prepared from compound 3.29 (5.1 mg) according to general procedure 6, yielding a yellow powder of the title compound (TFA salt, 3.8 mg, 73% yield).

[0739] LC / MS: C 22 H 19 The calculated value of FN4O6 is m / z = 455.1, and the measured value is [M+H]. + =455.2.

[0740] 1 H NMR (300 MHz, DMSO- d 6) δ 7.79 (d, J = 12.4 Hz, 1H), 7.29 (d, J = 9.7Hz, 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).

[0741] 3.31: ((S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyridine) Brno[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 155) MeOH / dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL) were added to a 50 mL flask containing 3.5 (30 mg) of compound. The reaction mixture was then refluxed and stirred for 24 hours. The reaction mixture was concentrated, poured into water (30 mL), and extracted with DCM (3 × 50 mL). The organic phases were combined and dried over MgSO4. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 25% to 40% CH3CN / H2O + 0.1% TFA, to give the title compound (5.1 mg, 16% yield) as a deep orange solid.

[0742] LC / MS: C 22 H 20 The calculated value of FN3O5 is m / z = 426.1, and the measured value is [M+H]. + =426.2.

[0743] 1 H NMR (300 MHz, DMSO- d 6) δ 7.75 (d, J = 12.3 Hz, 1H), 7.24 (d, J = 9.9Hz, 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).

[0744] 3.32: (4S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(((1R,5S)-6-hydroxy-3-azabicyclo) [3.1.1]Hept-3-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indazano[1,2-b]quinoline-3,14 (4H)-Diketone (Compound 158) Dichloromethane (0.6 mL) was added to a 5 mL Erlenmeyer flask containing 15 mg of compound 3.6, followed by 10 mg of 3-azabicyclo[3.1.1]hepta-6-ol and 7.6 μL of acetic acid. The reaction mixture was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by adding water + 0.1% TFA and diluted with DMF. The reaction mixture was then partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give a yellow powder containing the Boc-protected title compound. Deprotection was performed according to General Procedure 6, and the resulting residue was purified by preparative HPLC as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give a yellow powder containing the title compound (TFA salt, 7.1 mg, 39% yield).

[0745] LC / MS: C 27 H 27 The calculated value of FN4O5 is m / z = 507.2, and the measured value is [M+H]. + =507.4.

[0746] 1 H NMR (300 MHz, DMSO- d 6) δ 7.85 (d, J = 12.1 Hz, 1H), 7.46 (d, J = 9.4Hz, 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).

[0747] 3.33: (S)-9-amino-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azacyclobutane-1-yl)methyl )-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-3,14(4H)-dione ( Compound 159) Dichloromethane (0.6 mL) was added to a 5 mL Erlenmeyer flask containing 3.6 (15 mg) of compound, followed by (3-fluorozacyclobutane-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reaction mixture was stirred at room temperature and sodium triacetoxyborohydride (9.4 mg) was added. After 1 hour at room temperature, the reaction was quenched by adding water + 0.1% TFA, diluted with DMF, and then partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give a yellow powder containing the Boc-protected title compound. Deprotection was then performed according to General Procedure 6. The resulting residue was purified by preparative HPLC as described in General Procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA, to give a yellow powder containing the title compound (TFA salt, 1.8 mg, 10% yield).

[0748] LC / MS: C 25 H 24 The calculated value of F2N4O5 is m / z = 499.2, and the measured value is [M+H]. + =499.4.

[0749] 1 H NMR (300 MHz, DMSO- d 6) δ 7.82 (d, J = 12.4 Hz, 1H), 7.45 (d, J = 9.5Hz, 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).

[0750] 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]inzazo[1,2-b]quinoline-9-yl)carbamate (compound) 3.34) Sodium iodide (5.9 mg) was added to a stirred solution of compound 3.9 (210 mg) in DMF (5 mL), followed by methylammonium chloride (107 mg). The reaction mixture was then stirred overnight at room temperature. Reversed-phase purification was performed as described in General Procedure 9, using a 30 g C18 column with a gradient elution of 10% to 65% CH3CN / H2O + 0.1% TFA to give the title compound as a yellow solid (15.0 mg, 7.2% yield).

[0751] LC / MS: C 27 H 29 The calculated value of FN4O6 is m / z = 524.2, and the measured value is [M+H]. + =525.4.

[0752] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-2-hydroxy-N-methylacetamide (compound 165) The Boc-protected form of the title compound was prepared starting with compound 3.34 (6.4 mg) and glycolic acid according to general procedure 2. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 20% to 50% CH3CN / H2O + 0.1% TFA. Deprotection was then performed according to general procedure 6 to give the title compound (TFA salt, 2.0 mg, 28% yield) as a yellow powder.

[0753] LC / MS: C 24 H 23 The calculated value of FN4O6 is m / z = 482.2, and the measured value is [M+H]. + =483.2.

[0754] 1 H NMR (300 MHz, DMSO- d 6) δ 7.79 (d, J = 12.3 Hz, 1H), 7.27 (d, J = 9.5Hz, 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).

[0755] 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]inzazo[1,2-b]quinoline-11-yl)methyl)-N-methylmethanesulfonamide (compound 166) The Boc-protected form of the title compound was prepared from compound 3.34 (8.0 mg) and methanesulfonyl chloride according to general procedure 3. Preparative HPLC purification was performed as described in general procedure 9, with elution using a gradient of 10% to 50% CH3CN / H2O + 0.1% TFA. Deprotection was then performed according to general procedure 6 to give the title compound (TFA salt, 2.6 mg, 34% yield) as a yellow powder.

[0756] LC / MS: C 23 H 23 The calculated value of FN4O6S is m / z = 502.1, and the measured value is [M+H]. + =503.2.

[0757] 1 H NMR (300 MHz, DMSO- d 6) δ 7.81 (d, J = 12.3 Hz, 1H), 7.41 (d, J = 9.4Hz, 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 = 7.3Hz, 3H).

[0758] 3.37: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyridine Brno[3',4':6,7]inzazo[1,2-b]quinoline-3,14(4H)-dione (compound 170) Water (0.89 mL), FeSO4 (heptahydrate, 18.0 mg), and 3-methoxypropionaldehyde (113.0 mg) were added to a 10 mL round-bottom flask containing compound 3.4 (62.0 mg). Sulfuric acid (0.495 mL) was added dropwise to the resulting suspension 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 min, then warmed to room temperature and stirred for 1 h. 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 gradient of 25% to 45% CH3CN / H2O + 0.1% TFA to give the title compound (TFA salt, 3.1 mg, 4.4% yield) as a deep orange solid.

[0759] LC / MS: C 23 H 22 The calculated value of FN3O5 is m / z = 440.2, and the measured value is [M+H]. + =440.2.

[0760] 1 H NMR (300 MHz, DMSO- d 6) δ 7.75 (d, J = 12.4 Hz, 1H), 7.33 (d, J = 9.4Hz, 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).

[0761] 3.38: (S)-N-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano) [3',4':6,7]Indazazino[1,2-b]quinoline-9-yl)acetamide (compound 171) N-methylmorpholine (0.343 mL), HOAt (0.142 g), and HATU (0.435 g) were added to a 25 mL round-bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL). After stirring at room temperature for 5 minutes, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). The solution was stirred at room temperature for 24 hours, and then purified directly by preparative HPLC as described in General Procedure 9, eluting with a gradient of 25% to 45% CH3CN / H2O + 0.1% TFA, to give the title compound as a bright yellow powder (43.0 mg, 38% yield).

[0762] LC / MS: C 22 H 18 The calculated value of FN3O5 is m / z = 424.1, and the measured value is [M+H]. + =424.2.

[0763] 1 H NMR (300 MHz, DMSO- d 6) 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).

[0764] 3.39: (5-Formyl-2-methoxy-4-nitrophenyl)tert-butyl carbamate (compound 3.39) Sodium methoxide (0.74 g, 3.0 equivalent) was added to a solution of compound 3.2 (1.3 g, 1.0 equivalent) in MeOH (12 mL) at 0 °C. 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 under vacuum to give the title compound (1.2 g, 89% yield) as an orange solid.

[0765] LC / MS: C 13 H 16 The calculated value of N₂O₆ is m / z = 296.10, and the measured value is [M+H]. + =297.1.

[0766] 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) 3.40: (4-Amino-5-formyl-2-methoxyphenyl) tert-butyl carbamate (compound 3.40) Add B2(OH)4 (454 mg, 3 equivalents) to a solution of compound 3.39 (500 mg, 1 equivalent) in MeOH (10 mL) and H2O (1 mL). Cool the resulting mixture to 0 °C and add 5 M NaOH aqueous solution (2.75 mL) over 10 min with stirring. Stir the reaction mixture for another 5 min and then quench the solution by pouring it into ice (40 mL). Extract the resulting mixt...

Claims

1. An antibody-drug conjugate having the following structure: Where n is between 2 and 6; and T is an anti-NaPi2b (sodium-dependent phosphate transporter 2B) antibody construct containing an antigen-binding domain that binds to human NaPi2b, wherein the anti-NaPi2b antibody construct comprises two heavy chains containing the sequence shown in SEQ ID NO:68 and two light chains containing the sequence shown in SEQ ID NO:67, or wherein the anti-NaPi2b antibody construct comprises two heavy chains containing the sequence shown in SEQ ID NO:66 and two light chains containing the sequence shown in SEQ ID NO:

67.

2. The antibody-drug conjugate according to claim 1, wherein n is between 3.5 and 4.

5.

3. The antibody-drug conjugate according to claim 1, wherein n is about 4.

4. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.

5. A method for inhibiting the proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any one of claims 1 to 3.

6. A method for killing cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate according to any one of claims 1 to 3.

7. A method of treating cancer in a subject in need, comprising administering to the subject an effective amount of an antibody-drug conjugate according to any one of claims 1 to 3.

8. Use of an effective amount of the antibody-drug conjugate according to any one of claims 1 to 3 for the treatment of cancer in a subject of need.

9. The antibody-drug conjugate according to any one of claims 1 to 3, used in a therapy.

10. The antibody-drug conjugate according to any one of claims 1 to 3, for the treatment of cancer.

11. Use of the antibody-drug conjugate according to any one of claims 1 to 3 in the manufacture of a medicament for treating cancer.

12. A kit comprising an antibody-drug conjugate according to any one of claims 1 to 3 and a label and / or packaging instructions containing instructions for use.

Images