Antibody-drug conjugates targeting NAPI2B and methods of use thereof

Abstract

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

Description

Technical Field

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

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

[0003] Given the overexpression of NaPi2b in certain types of cancer, NaPi2b-targeting agents have been studied in clinical trials for the treatment of cancer, but have returned mixed results. Mersana Therapeutics conducted a Phase I / II clinical trial to study the efficacy of upifitamab rilsodotin, an antibody-drug conjugate (ADC) of the 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 arm of the study was discontinued due to lack of efficacy, and upifitamab rilsodotin was granted Fast Track Designation for the treatment of patients with platinum-resistant ovarian cancer 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 the antibody MX35 conjugated to an auristatin-F payload using its Dolasynthen platform. Development of this ADC has been discontinued. Lifastuzumab vedotin (an ADC of rifatuzumab with an MMAE payload) was being studied in a Genentech-sponsored clinical trial in patients with ovarian cancer or NSCLC, but the trial was later discontinued.

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

[0005] Other camptothecin analogs and derivatives, and ADCs comprising them, have been described. See, for example, International (PCT) Publication Nos. WO 2019 / 195665, WO 2019 / 236954, WO 2020 / 200880, and WO 2020 / 219287.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be potentially relevant to the present disclosure. It is not necessarily intended to, nor should it be construed as, an admission that any of the foregoing information constitutes prior art to the claimed invention. Summary of the invention

[0007] Antibody-drug conjugates (ADCs) targeting human NaPi2b and methods of use are described herein. One aspect of the present disclosure relates to antibody-drug conjugates having formula (X):

[0008] T-[L-(D) m ] n

[0009] (X)

[0010] in:

[0011] m is an integer between 1 and 4;

[0012] n is an integer between 1 and 10;

[0013] T is an anti-NaPi2b antibody construct as described herein;

[0014] L is a connector, and

[0015] D is a compound of formula I:

[0016]

[0017] in:

[0018] R 1 Selected from: -H, -CH 3 , -CHF 2 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 、-OCF3 and -NH 2 ,and

[0019] R 2 Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ,

[0020] And among them:

[0021] When R 1 For -NH 2 When R is R 3 or R 4 , and when R 1 No -NH 2 When R is R 4 ;

[0022] R 3 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0023] R 4 Selected from:

[0024] R 5 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl, -aryl and –(C 1 -C 6 alkyl)-aryl;

[0025] R 6 and R 7 Each independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 ;

[0026] R 8 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0027] Each R 9 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0028] Each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0029] R 10’ Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0030] R 11 Selected from: -H and -C 1 -C 6 alkyl;

[0031] R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16and

[0032] R 13 Selected from: -H and -C 1 -C 6 alkyl;

[0033] R 14 and R 14’ Each independently selected from: -H, C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0034] R 16 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0035] R 17 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, –(C 1 -C 6 Alkyl)-C 3 -C 8 Heterocycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0036] R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 ;

[0037] R 24 , R 25 and R 26 Each is -C 1 -C 6alkyl;

[0038] X a and X b are each independently selected from: NH, O and S, and

[0039] X c Selected from: O, S and S(O) 2 ,

[0040] Provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0041] Another aspect of the present disclosure relates to an antibody-drug conjugate having the following structure:

[0042]

[0043] wherein n is 4, and T is an anti-NaPi2b antibody construct as described herein.

[0044] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0045] Another aspect of the present disclosure relates to a method of inhibiting proliferation of cancer cells, comprising contacting the cells with an effective amount of an antibody-drug conjugate as described herein.

[0046] Another aspect of the present disclosure relates to a method of killing cancer cells comprising contacting the cells with an effective amount of an antibody-drug conjugate as described herein.

[0047] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody-drug conjugate as described herein.

[0048] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy.

[0049] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in the treatment of cancer.

[0050] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.

[0051] Another aspect of the present disclosure relates to a kit comprising an antibody-drug conjugate as described herein and a label and / or package insert containing instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A shows the sequence of the mouse heavy chain variable domain CDRs of the chimeric anti-NaPi2b antibody v23855 grafted onto the human VH germline (IGHV1-46*03), and Figure 1 B shows the sequence of the mouse light chain variable domain CDRs of the chimeric antibody v23855 grafted onto a human VL framework (IGKVID-39*01). The CDRs are designated according to the AbM definition and are marked in bold and underlined.

[0053] Figure 2A Non-reducing (NR) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855 are shown. Figure 2B Reducing (R) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855 are shown. Figure 2C The UPLC-SEC profile of the parental mouse-human chimeric antibody v23855 is shown. Figure 2D Shown is the UPLC-SEC profile of a representative humanized antibody v29456.

[0054] Figure 3A Binding of humanized antibody variants v29456, MX-35 (v18992), and rifatuzumab (v18993) to human NaPi2b is depicted. Figure 3B Binding of humanized antibody variants v29456, MX-35 (v18992), and rifatuzumab (v18993) to cynomolgus monkey NaPi2b is depicted. Figure 3C Binding of humanized antibody variants v29456, MX-35 (v18992), and rifatuzumab (v18993) to mouse NaPi2b is depicted.

[0055] Figure 4A N-curve analysis of v29814 binding to NaPi2b expressed on IGROV-1 cells is depicted. Figure 4B N-curve analysis of v36123 binding to NaPi2b expressed on IGROV-1 cells is depicted. Figure 4C N-curve analysis of v36124 binding to NaPi2b expressed on IGROV-1 cells is depicted. For each graph, the right curve shows data at 500 pM constant binding partner, and the left curve shows data at 50 pM constant binding partner.

[0056] Figure 5A Shown is a comparison of the ability of v23855 (parental chimeric), v29456 (H1L2), v18992 (MX35), and v18993 (rifatuzumab) to internalize in HCC-78 cells. Figure 5BShown is a comparison of the ability of v23855 (parental chimeric), v29456 (H1L2), v18992 (MX35), and v18993 (rifatuzumab) to internalize in NCI-H441 cells.

[0057] Figure 6 Depicted are the binding of the parental chimeric antibody (v23855), humanized antibody variants v29452 and v29456 to IGROV-1 cells, in addition to the binding of MX35 and rifatuzumab ADC to the cells.

[0058] Figure 7 Depicted is the ability of the v29456 ADC to play a bystander role.

[0059] Fig. 8A The cytotoxicity of v29456 ADC in 2D monolayer culture of HCC-78 cells is depicted. Figure 8B The cytotoxicity of v29456 ADC in 2D monolayer culture of IGROV-1 cells is depicted. Figure 8C The cytotoxicity of v29456 ADC in 2D monolayer culture of HCT116 cells is depicted.

[0060] Fig.9A The cytotoxicity of v29456 ADC in 2D monolayer culture of IGROV-1 cells is depicted. Fig. 9B The cytotoxicity of v29456 ADC in 2D monolayer culture of TOV-21G cells is depicted.

[0061] Fig. 10A Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of HCC-78 cells. Fig. 10B Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of IGROV-1 cells.

[0062] Fig.11A Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of IGROV-1 cells. Fig. 11B Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of TOV-21G cells.

[0063] Fig.12 The efficacy of v29456 ADC in the OVCAR3 xenograft model of ovarian cancer is depicted.

[0064] Fig.13 The efficacy of v29456 conjugated to DXd1 in the NCI-H441 xenograft model of lung cancer is depicted.

[0065] Fig.14AThe efficacy of v29456 ADC in the NCI-H441 xenograft model of lung cancer is depicted when dosed at 0.3 mg / kg. Fig. 14B The efficacy of v29456 ADC in the NCI-H441 xenograft model of lung cancer is depicted when dosed at 1 mg / kg.

[0066] Fig.15A The efficacy of v29456 ADC in the patient-derived (PDX) CTG-2025 ovarian cancer model is depicted. Fig. 15B The efficacy of v29456 ADC in the patient-derived (PDX) CTG-0958 ovarian cancer model is depicted.

[0067] Fig.16 Shown is the PK profile of v29456 ADC in Tg32 mice.

[0068] Fig.17A The ability of the ADC v29456 (H1L2) to internalize in OVCAR-3 cells is shown compared to v18992 (MX35) and v18993 (rifatuzumab). Fig. 17B The ability of the ADC of v29456 (H1L2) to be internalized in IGROV-1 cells is shown compared to v18992 (MX35) and v18993 (rifatuzumab).

[0069] Fig.18A Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of IGROV-1 cells. Fig.18B Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of NCI-H441 cells. Fig. 18C Depicted is the cytotoxicity of v29456 ADC in 3D spheroids of TOV-21G cells.

[0070] Fig.19A Depicts the Membrane Proteome Array using v38591 TM The results of the measurement. Fig.19B Validation data for CLDN3 are depicted.

[0071] Fig. 20 Depicted are cellular binding of v38591 and v38591 ADC on IGROV-1 and OVCAR-3 cells.

[0072] Fig.21 Cross-reactivity of v38591 and v38591 ADC with cynomolgus monkey and mouse NaPi2b is depicted.

[0073] Fig. 22Specificity of v38591 and v38591 ADC for human NaPi2b, NaPi2a, and NaPi2c is shown.

[0074] Fig.23 Internalization of anti-NADC2b ADC and naked antibody is shown.

[0075] Fig.24 Depicted is the bystander activity of anti-NaPi2b ADCs against the NaPi2b-negative EBC-1 cell line.

[0076] Fig.25 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-0703 xenograft model.

[0077] Fig.26 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-1301 xenograft model.

[0078] Fig. 27 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-3718 xenograft model.

[0079] Fig.28 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-1703 xenograft model.

[0080] Fig.29 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-2025 xenograft model.

[0081] Fig.30 Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the CTG-0958 xenograft model.

[0082] Fig.31 Pharmacokinetic profiles of DAR4 and DAR8 anti-NaPi2b ADCs in cynomolgus monkeys are depicted.

[0083] Fig.32A Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU5213 lung PDX model. Fig.32B Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU5245 lung PDX model. Fig.32C Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU6802 lung PDX model. Fig.32DShown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU6904 lung PDX model. Fig.32E Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11692 lung PDX model. Fig.32F Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11796 lung PDX model. Figure 32G Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11870 lung PDX model. Fig.32H Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the LU11876 lung PDX model.

[0084] Fig.33A Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT14026 PDX model of endometrial cancer. Fig.33B Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5318 PDX model of endometrial cancer. Fig.33C Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5326 PDX model of endometrial cancer. Fig.33D Shown are the effects of DAR4 and DAR8 anti-NaPi2b ADCs on tumor growth in the UT5321 PDX model of endometrial cancer.

[0085] Fig.34 Binding of v38591 and v40502 (LALADS) ADCs, parental antibodies, and controls to IGROV-1, HCC-78, H441, and EBC-1 cells is shown.

[0086] Fig.35A Depicted are the internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line IGROV-1. Fig.35B Depicted are the internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line HCC-78. Fig.35C Depicted are the internalization of v38591 and v40502 (LALADS) antibodies and ADCs in the NaPi2b-expressing cell line H441.

[0087] Fig.36 Depicted are the cytotoxicity of v38591 and v40502 (LALADS) antibodies and ADCs in 3D spheroids of cells expressing NaPi2b. DETAILED DESCRIPTION

[0088] The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct (anti-NaPi2b antibody construct) bound to a sodium-dependent phosphate transporter 2B (NaPi2b) conjugated to a camptothecin analog of formula (I) as described herein. The ADCs of the present disclosure can be used, for example, as therapeutic agents, especially in the treatment of cancer.

[0089] definition

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0091] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

[0092] When used in conjunction with the term "comprising" in this document, the use of the word "a / kind" may mean "one / kind", but it also has the meaning of "one / kind or more / kinds", "at least one / kind" and "one / kind or more than one / kind".

[0093] Where a range of values ​​is provided herein, for example, where a value is defined as being "between" an upper value and a lower value, it should be understood that the range encompasses the upper and lower values ​​and every intervening value.

[0094] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended, and do not exclude additional, unlisted elements and / or method steps. When used in conjunction with compositions, uses, or methods herein, the term "consisting essentially of means that additional elements and / or method steps may be present, but these additions do not substantially affect the manner in which the enumerated compositions, methods, or uses function. The term "consisting of" does not include the presence of additional elements and / or method steps when used in combination with compositions, uses, or methods herein. Compositions, uses, or methods described herein as comprising certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and consist of those elements and / or steps in other embodiments, whether or not these embodiments are specifically mentioned.

[0095] "Complementarity determining region" or "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" region (FR) can help maintain the correct conformation of CDR to promote the binding between antigen binding region and antigen. From N-terminus to C-terminus, the light chain variable region (VL) and heavy chain variable region (VH) of an antibody generally include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3 in this article, and the three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3. CDR provides most of the contact residues for the binding of an antibody to an antigen or epitope. Generally, three heavy chain CDRs and three light chain CDRs are required to bind antigen. However, in some cases, even a single variable domain can also confer antigen binding specificity. In addition, as known in the art, in some cases, antigen binding can also occur by a combination of at least one or more CDRs (e.g., HCDR3) selected from VH and / or VL domains.

[0096] Many different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196: 901-917), and IMGT, AbM (University of Bath) and Contact (MacCallum et al., 1996, J Mol Biol, 262 (5): 732-745). For example, the CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Therefore, it is obvious to those skilled in the art that the exact numbering and placement of the CDRs may differ based on the numbering system used. However, it should be understood that the disclosure of VH herein includes the disclosure of the associated (intrinsic) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, disclosure of a VL herein includes disclosure of the associated (intrinsic) light chain CDRs (LCDRs) as defined by any known numbering system.

[0097] Table 1: Common CDR definitions 1

[0098]

[0099]

[0100] 1Kabat or Chothia numbering systems may be used for all defined HCDR2, HCDR3 and light chain CDRs except for Contact which use Chothia numbering.

[0101] 2 Kabat numbering is used. The positions in the Kabat numbering scheme that delineate the ends of the Chothia and IMGT CDR-H1 loops vary depending on the length of the loop, as Kabat places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be clearly 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.

[0102] In the context of two or more polynucleotides or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to obtain the maximum consistency measured as one of the conventional sequence comparison algorithms known to those of ordinary skill in the art or by manual alignment and visual inspection on a comparison window or in a specified region, if the sequence has a certain percentage of identical amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95% or about 98% identity in a specified region), the sequence is "substantially identical". For sequence comparison, a test sequence is usually compared with a specified reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, 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 sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0103] A "comparison window" refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions, which can be, for example, about 10 to 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, over which a test sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining sequence identity percentages are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0104] As used herein, the term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0105] The term "acyloxy" refers to the group -OC(O)R, where R is alkyl.

[0106] As used herein, the term "alkoxy" refers to the group -OR, where R is alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0107] As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group containing a specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.

[0108] As used herein, the term "alkylaminoaryl" refers to an alkyl group, as defined herein, substituted with an aminoaryl group, as defined herein.

[0109] As used herein, the term "alkylheterocycloalkyl" refers to an alkyl group, as defined herein, substituted with a heterocycloalkyl group, as defined herein.

[0110] As used herein, the term "alkylthio" refers to the group -SR, where R is an alkyl group.

[0111] As used herein, the term "amido" refers to the group -C(O)NRR', where R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0112] As used herein, the term "amino" refers to the group -NRR', wherein R and R' are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.

[0113] As used herein, the term "aminoalkyl" refers to an alkyl group, as defined herein, substituted with one or more amino groups (eg, one, two or three amino groups).

[0114] As used herein, the term "aminoaryl" refers to an aryl group, as defined herein, substituted with an amino group.

[0115] As used herein, the term "aryl" refers to a 6- to 12-membered monocyclic or bicyclic hydrocarbon ring system in which at least one ring is aromatic. Examples of aryl include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, indanyl, and the like.

[0116] As used herein, the term "carboxy" refers to the group -C(O)OR, where R is H, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.

[0117] As used herein, the term "cyano" refers to the group -CN.

[0118] As used herein, the term "cycloalkyl" refers to a monocyclic or bicyclic saturated hydrocarbon containing the specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, and the like.

[0119] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more halogen atoms.

[0120] As used herein, the terms "halogen" and "halo" refer to fluorine (F), bromine (Br), chlorine (Cl), and iodine (I).

[0121] As used herein, the term "heteroaryl" refers to a 6- to 12-membered monocyclic or bicyclic ring system in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S, and N. Examples of heteroaryl include, but are not limited to, pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolyl, benzoxazolyl, benzothiazolyl, isoquinolyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.

[0122] As used herein, the term "heterocycloalkyl" refers to a monocyclic or bicyclic non-aromatic ring system containing a specified number of atoms and at least one of the ring atoms being a heteroatom (e.g., O, S, or N). The heterocyclyl substituent may be attached via any of its available ring atoms (e.g., ring carbon or ring nitrogen). Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, etc.

[0123] As used herein, the terms "hydroxy" and "hydroxyl" refer to the group -OH.

[0124] As used herein, the term "hydroxyalkyl" refers to an alkyl group, as defined herein, substituted with one or more hydroxy groups.

[0125] As used herein, the term "nitro" refers to the group -NO2.

[0126] As used herein, the term "sulfonyl" refers to the group -S(O)2R, where R is H, alkyl, or aryl.

[0127] As used herein, the term "sulfonylamino" refers to the group -NH-S(O)2R, where R is H, alkyl, or aryl.

[0128] As used herein, the terms "sulfenyl" and "thiol" refer to the group -SH.

[0129] Unless explicitly stated as "unsubstituted", any alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups mentioned herein should be understood as "optionally substituted", i.e., each such reference includes unsubstituted and substituted forms of these groups. For example, reference to "-C1-C6 alkyl" includes unsubstituted-C1-C6 alkyl and-C1-C6 alkyl substituted with one or more substituents. Examples of substituents include, but are not limited to, halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group mentioned herein is optionally substituted with one or more substituents selected from the following: halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfonyl, sulfonamido.

[0130] A chemical group described as "substituted" herein may include one substituent or multiple substituents, up to the full valence of the substitution of the group. For example, a methyl group may include 1, 2, or 3 substituents, and a phenyl group may include 1, 2, 3, 4, or 5 substituents. When a group is substituted with more than one substituent, the substituents may be the same or they may be different.

[0131] As used herein, the term "subject" refers to an animal, in some embodiments a mammal, that is the subject of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., a dog, a cat, etc.), a farm animal (e.g., a cow, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, a non-human primate, etc.). In certain embodiments, the subject is a human.

[0132] It is contemplated that any embodiment discussed herein can be implemented by any method, use, or composition disclosed herein, and vice versa.

[0133] Certain features, structures, and / or characteristics described in connection with one embodiment disclosed herein may be combined with features, structures, and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0134] It should also be understood that the positive recitation of a feature in one embodiment is a basis for excluding that feature in another embodiment. For example, where a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and the shortened list may form an alternative embodiment, regardless of whether such an alternative embodiment is specifically mentioned.

[0135] Antibody-drug conjugates

[0136] The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X):

[0137] T-[L-(D) m ] n

[0138] (X)

[0139] in:

[0140] T is an anti-NaPi2b antibody construct as described herein;

[0141] L is the connector;

[0142] D is a camptothecin analog as described herein;

[0143] m is an integer between 1 and 4, and

[0144] n is an integer between 1 and 10.

[0145] The components of formula (X) are described below.

[0146] Anti-NaPi2b antibody constructs

[0147] ADC of the present disclosure comprises anti-NaPi2b antibody construct.In this context, the term "antibody construct" refers to a polypeptide or polypeptide group comprising one or more antigen binding domains, wherein each of the one or more antigen binding domains specifically binds to an epitope or antigen. In the case where the antibody construct comprises two or more antigen binding domains, each antigen binding domain can bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they can bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may also include a support, and the one or more antigen binding domains may be fused or covalently attached to the support, optionally connected via a joint.

[0148] According to the present disclosure, the anti-NaPi2b antibody construct of ADC comprises at least one antigen binding domain that specifically binds to human NaPi2b (hNaPi2b). "Specific binding" hNaPi2b means that the antibody construct binds hNaPi2b, but does not show significant binding to NaPi2a or NaPi2c. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure is capable of binding to NaPi2b from one or more non-human species. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure is capable of binding to cynomolgus monkey NaPi2b.

[0149] Human NaPi2b is also known as human "solute carrier family 34 member 2" or "SLC34A2". Protein F sequences of hNaPi2b from various sources are known in the art and can be readily obtained 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).

[0150] Table 2: Human, cynomolgus monkey and mouse NaPi2b protein sequences

[0151]

[0152]

[0153] Specific binding of the antigen binding domain to the target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (using, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17: 323-329), flow cytometry or traditional binding assays (Heeley, 2002, Endocr Res, 28: 217-229). In certain embodiments, specific binding can be defined as, for example, less than about 5% to less than about 10% of the binding to a non-target protein (e.g., hNaPi2a or hNaPi2c) as measured by ELISA or flow cytometry compared to the binding to hNaPi2b.

[0154] As used herein, the term "dissociation constant (K D or K d )” is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, ligand-protein interaction refers to, but is not limited to, protein-protein interaction or antibody-antigen interaction. D The dissociation rate constant (k) measures the tendency of two proteins in a complex (e.g., AB) to reversibly dissociate into their constituent components (A+B). off )”) and the association rate constant or “association rate (kon )”. Therefore, K D Equal to k off / k on , and is expressed as molar concentration (M). It can be seen that K D The smaller the K, the stronger the binding affinity. D A decrease in K indicates an increase in affinity. D In comparison, 1mM K D Indicates weak binding affinity. Affinity is sometimes expressed as K A or K a To measure, it is K D or K d The K between an antibody and its antigen D This can be determined using methods well established in the art. D One method is to use surface plasmon resonance (SPR), commonly used in biosensor systems such as Isothermal titration calorimetry (ITC) is a method for measuring K D Another way to do this. Octet TM The system can also be used to measure the affinity of antibodies for target antigens.

[0155] In certain embodiments, the specific binding of an antibody construct to NaPi2b can be determined by a dissociation constant (Kd or K D )≤1 μM, for example, ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM or ≤10 nM. In certain embodiments, the specific binding of an antibody construct to a particular antigen or epitope can be measured by the dissociation constant (K D ) is 10 -6 M or smaller, e.g. 10 -7 M or less or 10 -8 In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope can be measured by the dissociation constant (K D ) between 10 -6 M and 10 -9 M, for example, between 10 -7 M and 10 -9 The dissociation constant is defined between M. As is known in the art, the numerical value of the dissociation constant obtained may vary depending on how it is tested. For example, the expression level of NaPi2b in the cell line, the form of the antibody construct (i.e., monovalent or divalent) and the assay type (i.e., ELISA or flow cytometry) may affect the numerical value of the dissociation constant when measured in a cell-based assay. The data provided in the examples illustrate this general view, as demonstrated in Examples 10, 11 and 16.

[0156] In some embodiments, when measured by flow cytometry in cells expressing NaPi2b at high levels, the Kd of the anti-NaPi2b antibody constructs of the present disclosure is lower than the Kd of the reference antibody rifatuzumab and is comparable to the Kd of the reference antibody MX35. Therefore, in these embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen binding domain having an affinity for human NaPi2b greater than the affinity of the reference antibody rifatuzumab and comparable to the affinity of the reference antibody MX35.

[0157] In certain embodiments, the anti-NaPi2b antibody construct exhibits an internalization level comparable to that of the reference antibody MX35, and exhibits a higher internalization level than the reference antibody rifatuzumab in cells expressing high and medium NaPi2b. In some embodiments, internalization is measured after 4 hours, 5 hours, or 24 hours of treatment.

[0158] Antibody internalization can be measured using methods known in the art, for example, by direct internalization according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as pHAb dye (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red dye (ThermoFisher Scientific Corporation, Waltham, MA) and Fabfluor-pH antibody labeling reagent (Sartorius AG, Germany), and analytical techniques such as microscopy, FACS, high-content imaging or other plate-based assays.

[0159] NaPi2b expression varies according to the cell type indicated throughout this disclosure, and the level of NaPi2b expression is referred to herein as "high", "medium", "low" or "negative". These terms are used for reference to describe the general expression level according to the name shown in Table 15.1 in Example 15, and are not intended to be limited to the specific numerical value of the average NaPi2b protein per cell included therein. Alternatively, the expression level of NaPi2b in a cell or tumor can be assessed by immunohistochemistry (IHC) according to methods known in the art. For example, IHC can be used to stain NaPi2b in a tumor tissue sample from a xenograft model, a cell source (CDX) or a patient source (PDX). Tissue samples can be examined and H scores calculated as known in the art and, for example, as described in Example 35 herein. The higher the H score, the higher the expression of NaPi2b in the tissue sample.

[0160] Antigen binding domain

[0161] The anti-NaPi2b antibody construct of the ADC of the present disclosure comprises at least one antigen binding domain capable of binding to hNaPi2b. The at least one antigen binding domain capable of binding to hNaPi2b is typically an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fab (scFab), single-chain Fv (scFv) and single-domain antibodies (sdAb).

[0162] A "Fab fragment" contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab' fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of a Fab light chain can be connected to the N-terminus of a Fab heavy chain in a single-chain Fab molecule.

[0163] "scFv" comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody in a single polypeptide chain. ScFv may optionally further comprise a polypeptide linker between the VH and VL domains so that the scFv can form a structure required for antigen binding. For example, scFv may include a VL connected to the N-terminus of VH from the C-terminus via a polypeptide linker. Alternatively, scFv may comprise a VH connected to the N-terminus of VL via its C-terminus via a polypeptide linker (see the review in Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0164] The "sdAb" format refers to a single immunoglobulin domain. sdAb can be, for example, of camel origin. Camel antibodies lack light chains, and their antigen binding sites consist of a single domain called "VHH". sdAb contains three CDR / hypervariable loops CDR1, CDR2, and CDR3 that form the antigen binding site. sdAb is quite stable and easy to express, for example, expressed as a fusion with an antibody Fc chain (see, e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77 (1): 13-22).

[0165] In those embodiments in which 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 an antibody mimetic that is not based on an immunoglobulin), or other polypeptides or small molecules that are capable of specifically binding to their targets (e.g., natural or engineered ligands). Non-immunoglobulin-based antibody mimetic forms include, for example, anticalins, fynomers, affimers, alphabodies, DARPins, and avimers.

[0166] The disclosure describes herein the identification of mouse antibodies that specifically bind hNaPi2b; The mouse-human chimeric variant of the antibody is identified as variant 23855. The anti-NaPi2b antibody construct of the ADC disclosed herein comprises an antigen binding domain derived from the mouse antibody or its humanized antibody variant. Representative humanized antibody variants (v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 and v29460) of mouse antibodies are also described. In certain embodiments, the anti-NaPi2b antibody construct described herein specifically binds to the human NaPi2b with a sequence as shown in SEQ ID NO: 1.

[0167] In certain embodiments, the anti-NaPi2b antibody construct of the ADC competes for binding to human NaPi2b with any of the humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or with the parent chimeric antibody v23855. In evaluating competition as described below, each of the variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, and v23855 is referred to as a competing reference antibody.

[0168] Competition assays known in the art can be used to determine whether antibody constructs compete with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 and v29460 or parent chimeric antibody v23855 for binding to hNaPi2b. For example, first allow the competing reference antibody to bind to hNaPi2b under saturation conditions, and then measure the ability of the test antibody construct to bind to hNaPi2b. 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 different epitopes from the competing reference antibody. On the contrary, 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, overlapping epitopes or very close epitopes as the epitope bound to the competing reference antibody. Such competition assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, flow cytometry, etc. An "antibody that competes with a competing reference antibody" refers to an antibody that blocks 50% or more of the binding of the reference antibody to its epitope in a competition assay.

[0169] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen binding domain that specifically binds to hNaPi2b, wherein the antigen binding domain comprises a set of CDRs based on the CDRs of the parent chimeric antibody v23855 described herein. The CDR sequences of the parent chimeric antibody v23855 and representative humanized antibody variants are shown in Table 3.

[0170] Table 3: CDR sequences of anti-NaPi2b antibody constructs

[0171]

[0172] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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 NOs: 7, 8, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NOs: 19, 20, and 18.

[0173] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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 LCDR sequence YTS.

[0174] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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 NOs: 10, 11, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NOs: 19, 20, and 18.

[0175] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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 NOs: 12, 13, and 9, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NOs: 19, 20, and 18.

[0176] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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 NOs: 14, 15, and 16, and the light chain CDR amino acid sequence comprises the sequence shown in SEQ ID NOs: 21, 22, and 23.

[0177] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having:

[0178] (i) a HCDR1 amino acid sequence selected from the HCDR1 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460; a HCDR2 amino acid sequence selected from the HCDR3 amino acid sequence of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460. a HCDR2 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460; and a 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

[0179] (ii) a LCDR1 amino acid sequence selected from the LCDR1 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; a LCDR2 amino acid sequence selected from the LCDR3 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. v29454, v29455, v29456, v29457, v29458, v29459 or v29460; and a LCDR3 amino acid sequence selected from the LCDR3 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460,

[0180] The CDR amino acid sequences are defined in any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Table 3).

[0181] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a heavy chain CDR amino acid sequence (HCDR1, HCDR2, and HCDR3) 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, v29455, v29456, v29457, v29458, v29459, v29460, v29461, v29462, v29463, v29464, v29465, v29466, v29467, v29468, v29469, v29470, v29471, v29472, v29473, v29474, v29475, v29476, v29477, v29478, v29479, v29470, v29471, v29472, v29473, v29474, v29475 The heavy chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460 are selected from the light chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460 as defined by any of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0182] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present 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) of any of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460 as defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0183] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain having a VH sequence comprising the CDR sequences of the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain having a VL sequence comprising the CDR sequences of the VL sequence of any of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0184] Those skilled in the art will appreciate that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without the antibody losing its ability to bind its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art such as alanine scanning, and the resulting variants tested for binding activity by standard techniques. Thus, in certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) having 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs of any of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the % sequence identity is calculated across all six CDRs, and wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0185] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a variant of a set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460, wherein the variant comprises 1 to 10 amino acid substitutions 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 to hNaPi2b. In some embodiments, the anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a variant of a set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460, wherein the variant comprises 1 to 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 in the set of CDRs, and wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0186] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen binding domain retains the ability to bind to hNaPi2b. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0187] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain comprising a VH amino acid sequence selected from the VH amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure comprises an antigen binding domain comprising a VL amino acid sequence selected from the VL amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460.

[0188] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v23855, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v23855, wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0189] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v29456, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29456, wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0190] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises an antigen binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of v29452, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29452, wherein the antigen binding domain retains the ability to bind to hNaPi2b.

[0191] In some embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise the VH and VL sequences of any one of v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. The SEQ ID NOs for the VH and VL sequences of these variants are provided in Table 4 below. The sequences themselves are provided in Table 7.4 of the Examples.

[0192] Table 4: VH and VL sequences of parental chimeric and humanized anti-NaPi2b antibodies

[0193]

[0194]

[0195] In some embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises the VH sequence and VL sequence of v29456. In some embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises the VH sequence and VL sequence of v29452.

[0196] In certain embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises: a) a VH sequence having 3 HCDRs of v29456 and being 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% identical to the VH sequence of v29456, and b) a VL sequence having 3 LCDRs of v29456 and being 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% identical to the VL sequence of v29456, wherein the HCDRs and LCDRs are defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0197] In certain other embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises: a) a VH sequence having 3 HCDRs of v29452 and being 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% identical to the VH sequence of v29452, and b) a VL sequence having 3 LCDRs of v29452 and being 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% identical to the VL sequence of v29452, wherein the HCDRs and LCDRs are defined by any of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0198] In one embodiment, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises two heavy chains having an amino acid sequence as shown in SEQ ID NO:63 and two light chains having an amino acid sequence as shown in SEQ ID NO:62. In one embodiment, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises two heavy chains having an amino acid sequence as shown in SEQ ID NO:61 and two light chains having an amino acid sequence as shown in SEQ ID NO:62. In other embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises two heavy chains having an amino acid sequence as shown in SEQ ID NO:66 and two light chains having an amino acid sequence as shown in SEQ ID NO:67. In other embodiments, the anti-NaPi2b antibody construct of the ADC of the present disclosure comprises two heavy chains having an amino acid sequence as shown in SEQ ID NO:68 and two light chains having an amino acid sequence as shown in SEQ ID NO:67.

[0199] Format

[0200] The anti-NaPi2b antibody construct of ADC can have various formats. The minimum component of the anti-NaPi2b antibody construct is the antigen binding domain that binds hNaPi2b. The anti-NaPi2b antibody construct may also optionally include one or more additional antigen binding domains and / or scaffolds. 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 in hNaPi2b, may bind to different epitopes in hNaPi2b, or may bind to different antigens. Therefore, the anti-NaPi2b antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.

[0201] In certain embodiments, the anti-NaPi2b antibody construct comprises at least one antigen binding domain that binds hNaPi2b and a scaffold, wherein the antigen binding domain is operably connected to the scaffold. As used herein, the term "operably connected" means that the described components are in a relationship that allows them to function in their intended manner. Suitable scaffolds are described below.

[0202] In certain embodiments, the anti-NaPi2b antibody construct comprises two antigen binding domains that are optionally operably connected to a scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise three or four antigen binding domains and optionally a scaffold. In these formats, when a scaffold is included, at least the first antigen binding domain is operably connected to the scaffold, and the remaining antigen binding domains can each be independently operably connected to the scaffold or the first antigen binding domain, or when there are more than two antigen binding domains, operably connected to another antigen binding domain.

[0203] Anti-NaPi2b antibody constructs lacking a scaffold may comprise a single antigen binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen binding domains optionally operably connected via one or more linkers. In such anti-NaPi2b antibody constructs, the antigen binding domain may be in the format of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as the antigen binding domain, a format such as a tandem scFv ((scFv) 2 In some embodiments, the scFv is a double antibody, which is a double antibody, wherein the scFv is connected together by a flexible joint. ScFv can also be used to construct a double antibody format, which includes two scFvs connected by a short joint (usually about 5 amino acids in length). The restricted joint length causes scFv to dimerize in a head-to-tail manner. In any of the aforementioned formats, scFv can be further stabilized by comprising an interdomain disulfide bond. For example, disulfide bonds can be introduced between VL and VH by replacing the non-cysteine ​​residues in each chain with cysteine ​​residues (for example, at position 44 of VH and position 100 of VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering, 10: 1221-1225), or disulfide bonds can be introduced between two VH to provide a construct with a DART format (see, for example, Johnson et al., 2010, J Mol.Biol., 399: 436-449).

[0204] Similarly, in some embodiments, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable linker may be employed. Other examples of anti-NaPi2b antibody construct formats lacking a scaffold include those based on Fab fragments, such as Fab 2 and F(ab') 2 format, in which the Fab fragments are connected by a linker or IgG hinge region.

[0205] Combinations of antigen binding domains of varying sizes can also be employed to generate alternative scaffold-free formats. For example, a scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of a Fab fragment, thereby generating a bivalent (Fab-scFv / sdAb) construct.

[0206] In certain embodiments, the anti-NaPi2b antibody construct can be an antibody format based on immunoglobulin (Ig). This type of format is referred to herein as 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 certain embodiments, the anti-NaPi2b antibody construct can be based on IgG class immunoglobulins, such as IgG1, IgG2, IgG3 or IgG4 immunoglobulins. In some embodiments, the anti-NaPi2b antibody construct can be based on IgG1 immunoglobulins. In the context of the present disclosure, when the anti-NaPi2b antibody construct is based on a specified immunoglobulin isotype, it means that the anti-NaPi2b antibody construct includes all or part of the constant region of the specified immunoglobulin isotype. For example, the anti-NaPi2b antibody construct based on a given Ig isotype may include at least one antigen-binding domain operably connected to an Ig scaffold, wherein the scaffold includes an Fc region from a 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 include a hybrid of an isotype and / or subclass. It should also be understood that the Fc region and / or hinge region may be optionally modified to confer one or more desired functional properties as known in the art. Therefore, in certain 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 sequence) 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 Table.

[0207] In some embodiments, 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.

[0208] "Chimeric antibodies" typically comprise at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not have the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851-55 and U.S. Pat. No. 4,816,567.

[0209] "Humanized antibodies" are a class of chimeric antibodies that contain minimal sequence derived from non-human antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions of the recipient are replaced with residues from hypervariable regions of a non-human species (donor antibody) having the desired specificity and affinity for the target antigen, such as mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR grafting."

[0210] In some cases, additional modifications are made to further improve antibody performance. For example, some framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues, or humanized antibodies may include residues that are not found in either the recipient antibody or the donor antibody. In general, the variable domains in humanized antibodies will include all or nearly all of the hypervariable regions from non-human immunoglobulins and all or nearly all of the FRs from human immunoglobulin sequences. Humanized antibodies are described in more detail in, for example, Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2: 593-596.

[0211] Many methods are known in the art for selecting the most appropriate human framework for transplanting non-human CDR therein. Early methods use a limited subset of fully characterized human antibodies, independent of the sequence identity of the non-human antibodies providing CDR ("fixed framework" method). The most recent method has adopted a variable region with high amino acid sequence identity to the variable region of the non-human antibodies providing CDR ("homologous matching" or "best fit" method). An alternative method is to select a fragment of framework sequence from each light chain or heavy chain variable region from several different human antibodies. In some cases, CDR transplantation may cause the affinity of the transplanted molecule to its target antigen to be partially or completely lost. In such cases, affinity can be restored by backmutating some of the residues in some human sources to corresponding non-human residues. Methods for preparing humanized antibodies by these methods are well known in the art (see, e.g., Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-329; Presta et al., 1997, Cancer Res, 57(20): 4593-4599).

[0212] Alternatively, or in addition to these traditional methods, newer technology can be used to further reduce the immunogenicity of the humanized antibody of CDR transplantation.For example, a framework based on human germline sequence or consensus sequence can be used as a receptor human framework rather than a human framework with somatic mutations.Another technology intended to reduce the potential immunogenicity of non-human CDR is to transplant specificity determining residues (SDR) only.In this method, only the minimum CDR residues (" SDR ") required for antigen binding activity are transplanted into the human germline framework.This method improves the "humanity" (i.e., similarity to human germline sequence) of humanized antibodies, and therefore can contribute to reducing the immunogenicity risk of variable regions. These techniques have been described in various publications (see, e.g., Almagro and Fransson, 2008, Front Biosci, 13: 1619-1633; Tan, et al., 2002, J Immunol, 169: 1119-1125; Hwang, et al., 2005, Methods, 36: 35-42; Pelat et al., 2008, J Mol Biol, 384: 1400-1407; Tamura et al., 2000, J Immunol, 164: 1432-1441; Gonzales et al., 2004, Mol Immunol, 1: 863-872, and Kashmiri et al., 2005, Methods, 36: 25-34).

[0213] In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure comprises a humanized antibody sequence, such as one or more humanized variable domains. In some embodiments, the anti-NaPi2b antibody construct can be a humanized antibody. Non-limiting examples of humanized antibodies based on anti-NaPi2b antibody v23855 are described herein (see Examples and Sequence Table, and sequences of v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 and v29460).

[0214] Bracket

[0215] In certain embodiments, the anti-NaPi2b antibody construct comprises one or more antigen binding domains operably connected to a support. The antigen binding domain can be one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable supports are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerization peptides (such as leucine zippers, "zipper" peptides derived from Jun and Fos forming heterodimers, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include DOCK-AND-LOCK based on IBC Pharmaceuticals, Inc. and Immunomedics, Inc. development. TM (DNL TM ) technology (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0216] 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 connected to the N-terminus or C-terminus of the polypeptide scaffold. In certain embodiments, an anti-NaPi2b antibody construct comprising a polypeptide scaffold is also envisioned, wherein one or more antigen-binding polypeptide constructs are connected to a region other than the N-terminus or C-terminus, for example, via the side chain of an amino acid, with or without a joint.

[0217] In embodiments where the anti-NaPi2b antibody construct comprises a scaffold as a peptide or polypeptide, the antigen binding domain can be connected to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen binding domain is connected to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen binding domain can be connected to the scaffold by chemical conjugation.

[0218] Many protein domains are known in the art that contain selective pairing of two different polypeptides and can be used to form a scaffold. Examples are leucine zipper domains that are selectively paired together, 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., Nat Biotechnol, 21: 1486-1492 (2003)), DNA strand pairs (Chaudri et al., FEBS Letters, 450 (1–2): 23-26 (1999)) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011 / 135040).

[0219] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen binding moieties has been described (see, e.g., 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).

[0220] For example, it has been demonstrated that fusing an antigen-binding moiety such as scFv, diabody or single-chain diabody to albumin can improve the serum half-life of the antigen-binding moiety (Müller et al., supra). The antigen-binding moiety may be fused to the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0221] Albumin derivatives in the form of heteromultimers have been described, which comprise two transporter polypeptides obtained by fragmenting albumin, such that the transporter polypeptides self-assemble to form a natural albumin-like structure (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). Due to the fragmentation of albumin, the heteromultimer comprises four termini and can therefore optionally be fused to up to four different antigen-binding moieties via a linker.

[0222] In certain embodiments, the anti-NaPi2b antibody construct may include a protein scaffold. In some embodiments, the anti-NaPi2b antibody construct may include a protein scaffold based on an immunoglobulin Fc region, albumin or an albumin analog or derivative. In some embodiments, the anti-NaPi2b antibody construct may include a protein scaffold based on an immunoglobulin Fc region (e.g., an IgG Fc region).

[0223] Fc region

[0224] As used herein, the term "Fc region," "Fc" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0225] In certain embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the immunoglobulin Fc region. The Fc region may be dimeric and consist of two Fc polypeptides, or alternatively, the Fc region may consist of a single polypeptide.

[0226] In the case of a dimeric Fc, "Fc polypeptide" refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" can be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0227] The Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0228] In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on an IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on a human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on an IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on a human IgG1 Fc region.

[0229] In certain embodiments, the anti-NaPi2b antibody construct may include a scaffold based on an IgG Fc region, the IgG Fc region being a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each of which comprises a CH3 sequence and optionally a CH2 sequence, and wherein the first Fc polypeptide and the second Fc polypeptide are different. In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on an Fc region 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 include a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.

[0230] In some embodiments, the anti-NaPi2b antibody construct may include 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 such a modification, such as substitution or insertion, in which the amino acid at a specific position on the first CH3 or CH2 sequence is different from the amino acid at the same position on the second CH3 or CH2 sequence. These asymmetric amino acid modifications can be the result of modification of only one of the two amino acids at the same corresponding amino acid position on each sequence, or the result of different modifications of two amino acids on each sequence 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 include one or more asymmetric amino acid modifications.

[0231] In some embodiments, the anti-NaPi2b antibody construct may include a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain includes one or more amino acid modifications that promote heterodimeric Fc formation relative to homodimeric Fc formation. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0232] Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("knob-mortise"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic manipulation"), Davis et al., 2010, Prot Eng Des Sel, 23(4): 195-202 (chain exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13): 5145-50 (Fab arm exchange). Other examples include methods of combining positive and negative design strategies to generate stable asymmetric modified Fc regions, such as those described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, an anti-NaPi2b antibody construct may comprise a modified Fc region based scaffold as described in International Publication Nos. WO 2012 / 058768 or WO 2013 / 063702.

[0233] Table 5 provides the amino acid sequence of a human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of a full-length human IgG1 heavy chain. The CH3 sequence comprises amino acids 341-447 of a full-length human IgG1 heavy chain. Also shown in Table 5 are CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0234] In certain embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising modifications of any one of variant 1, variant 2, variant 3, variant 4, or variant 5, as shown in Table 5.

[0235] Table 5: Human IgG1 Fc sequences that promote heterodimer formation 1 and CH3 domain amino acid modifications

[0236]

[0237] 1 Sequence from position 231 to 447 (EU numbering)

[0238] In some embodiments, the anti-NaPi2b antibody construct may include a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcR) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0239] In some embodiments, the anti-NaPi2b antibody construct comprises an IgGFc based scaffold with 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.

[0240] A variety of amino acid modifications that selectively change the affinity of Fc for different Fcγ receptors to the CH2 domain are known in the art. Amino acid modifications that lead to increased binding and amino acid modifications that lead to reduced binding can each be used for certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn causes an increase in the lysis of target cells. Reducing the binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some cases. In certain indications, it may be necessary to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, a modified CH2 domain ("knockout" variant) comprising an amino acid modification that leads to an increase in binding to FcγRIIb or reduces or eliminates the binding of the Fc region to all Fcγ receptors may be useful.

[0241] Examples of amino acid modifications to the CH2 domain that alter the binding of Fcγ receptors to Fc include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenha gen, 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 change the binding of FcγRIIb to Fc are described in International Publication No. WO 2021 / 232162. Other modifications that affect the binding of Fc to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).

[0242] In certain embodiments, the anti-NaPi2b antibody construct comprises an IgGFc based scaffold with a modified CH2 domain comprising one or more amino acid modifications that result in reduced or eliminated binding of the Fc region to all Fcγ receptors (ie, a “knockout” variant).

[0243] 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" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp. 225-249). These strategies include reducing effector function by glycosylation modification, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 domains of 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).

[0244] Examples of mutations that can be introduced into the hinge or CH2 domain to generate "knockout" variants include amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0245] In certain embodiments, the anti-NaPi2b antibody constructs described herein may include a scaffold based on IgG Fc, wherein the native glycosylation has been modified. As known in the art, the glycosylation of Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine (i.e., N297A, Q, K or H) results in the production of 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).

[0246] In contrast, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC based on improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells that lack fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in a variant CHO cell line Lec 13 with reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or other cells that produce afucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, International Publication No. WO 2009 / 135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into carbohydrates on the antibody.

[0247] Other methods for producing antibodies containing little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, Technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12): 1607-1618 and US Pat. No. 8,409,572).

[0248] Other glycosylation variants include those with bisected oligosaccharide variants, for example, wherein the biantennary oligosaccharide connected to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc) variants. Such glycosylation variants can 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 variants having at least one galactose residue in the oligosaccharide connected to the Fc region, which can have improved CDC function (see, for example, International Publication No. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).

[0249] Preparation of anti-NaPi2b antibody constructs

[0250] The anti-NaPi2b antibody constructs described herein can be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2nd edition, ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0251] Typically, in order to recombinantly produce an antibody construct, a polynucleotide or a group of polynucleotides encoding an anti-NaPi2b antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. The polynucleotides encoding an anti-NaPi2b antibody construct can be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and updated, and "Antibodies: A Laboratory Manual", 2nd edition, Greenfield edited, Cold Spring Harbor Laboratory Press, New York, 2014). As will be appreciated by those skilled in the art, the number of polynucleotides required for expressing an anti-NaPi2b antibody construct will depend on the format of the construct, including whether the antibody construct includes a support. For example, when an anti-NaPi2b antibody construct is in a monospecific mAb format or a FSA format, two polynucleotides encoding a polypeptide chain will be required. When multiple polynucleotides are needed, they can be incorporated into a vector or more than one vector.

[0252] Typically, for expression, a polynucleotide or a group of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcription elements, which are required for the effective transcription of the polynucleotides. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. It will be appreciated by those skilled in the art that the selection of regulatory elements depends on the host cell selected for expressing the antibody construct, and such regulatory elements may be derived from a variety of sources, including bacteria, fungi, viruses, mammals, or insect genes. The expression vector may optionally further contain a heterologous nucleic acid sequence, which facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags, such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal vector or an integration vector.

[0253] Suitable host cells for cloning or expressing anti-NaPi2b antibody constructs include various prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, Escherichia coli (E. coli), A. salmonicida, or Bacillus subtilis (B. subtilis) cells.

[0254] In certain embodiments, anti-NaPi2b antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed., Humana Press, Totowa, NJ, 2003.

[0255] In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast may be suitable expression host cells, particularly fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22: 1409-1414, and Li et al., 2006, Nat. Biotech. 24: 210-215).

[0256] Suitable host cells for expressing glycosylated anti-NaPi2b antibody constructs are generally eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES for producing antigen-binding constructs in transgenic plants. TMMammalian cell lines adapted to growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals NY Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR - CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216) and myeloma cell lines (such as Y0, NS0 and Sp2 / 0). Exemplary mammalian host cell lines suitable for producing antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo ed., Humana Press, Totowa, NJ, 2003).

[0257] In certain embodiments, the host cell can be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell can be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any of these cell lines. In some embodiments, the host cell can be a stable cell line that allows mature glycosylation of the antibody construct.

[0258] Conventional methods can be used to culture host cells containing expression vectors encoding anti-NaPi2b antibody constructs to produce anti-NaPi2b antibody constructs. Alternatively, in some embodiments, host cells containing expression vectors encoding anti-NaPi2b antibody constructs can be used therapeutically or prophylactically to deliver anti-NaPi2b antibody constructs to subjects, or polynucleotides or expression vectors can be administered ex vivo to cells from subjects, and then the cells are returned to the subject.

[0259] Typically, anti-NaPi2b antibody constructs are purified after expression. Proteins can be separated or purified in a variety of ways 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, fractionated chromatography or gel filtration and reverse phase chromatography, performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoresis, immunization, precipitation, dialysis and chromatofocusing techniques. The combination of ultrafiltration and diafiltration techniques with protein concentration is also useful. As is well known in the art, a variety of natural proteins bind to Fc and antibodies, and these proteins can be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be performed by a specific fusion partner. For example, if a GST fusion is used, glutathione resin can be used to purify the antibody, and if a His tag is used, Ni can be used. +2 Affinity chromatography purifies the antibody, or if a flag tag is used, an immobilized anti-flag antibody can be used to purify the antibody. The required degree of purification will vary depending on the use of the anti-NaPi2b antibody construct. In some cases, purification may not be necessary.

[0260] In certain embodiments, the anti-NaPi2b antibody construct is substantially pure. When used with respect to the anti-NaPi2b antibody constructs described herein, the term "substantially pure" (or "substantially purified") means that the antibody construct is substantially or essentially free of components that are usually associated with proteins or interact with proteins as found in its naturally occurring environment (such as natural cells, or in the case of recombinantly produced constructs, host cells). In certain embodiments, substantially pure anti-NaPi2b antibody constructs are protein preparations with 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.

[0261] Certain embodiments of the present disclosure relate to a method for preparing an anti-NaPi2b antibody construct, the method comprising culturing a host cell into which one or more polynucleotides encoding the anti-NaPi2b antibody construct or one or more expression vectors encoding the anti-NaPi2b antibody construct have been introduced under conditions suitable for expressing the anti-NaPi2b antibody construct, and optionally recovering the anti-NaPi2b antibody construct from the host cell (or from the host cell culture medium).

[0262] Post-translational modification

[0263] In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or in vitro after isolating the anti-NaPi2b antibody constructs from host cells.

[0264] Post-translational modifications include various modifications as known in the art (see, e.g., Proteins-Structure and Molecular Properties, 2nd Edition, TECreighton, WH Freeman and Company, New York, 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, Annals of Medicine, 663: 48-62). In those embodiments in which the anti-NaPi2b antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0265] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or proteolytic degradation by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH 4 Specific chemical cleavage.

[0266] Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, treatment of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, and addition or deletion of N-terminal methionine residues produced by prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to allow detection and separation of proteins. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0267] Additional examples of post-translational modifications include acetylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or a lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0268] Camptothecin analogs

[0269] The camptothecin analogs included in the ADC of the present disclosure are compounds having formula (I):

[0270]

[0271] in:

[0272] R 1 Selected from: -H, -CH 3 , -CHF 2 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 、-OCF 3 and -NH 2 ,and

[0273] R2 Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ,

[0274] And among them:

[0275] When R 1 For -NH 2 When R is R 3 or R 4 , and when R 1 No -NH 2 When R is R 4 ;

[0276] R 3 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0277] R 4 Selected from:

[0278] R 5 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl, -aryl and –(C 1 -C 6 alkyl)-aryl;

[0279] R 6 and R 7 Each independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R17 ;

[0280] R 8 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0281] Each R 9 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0282] Each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0283] R 10’ Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0284] R 11 Selected from: -H and -C 1 -C 6 alkyl;

[0285] R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16 and

[0286] R 13Selected from: -H and -C 1 -C 6 alkyl;

[0287] R 14 and R 14’ Each independently selected from: -H, C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0288] R 16 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0289] R 17 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, –(C 1 -C 6 Alkyl)-C 3 -C 8 Heterocycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0290] R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 ;

[0291] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0292] X a and Xb are each independently selected from: NH, O and S, and

[0293] X c Selected from: O, S and S(O) 2 ,

[0294] Provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0295] In some embodiments, the camptothecin analog is a compound of formula (I), provided that when R 1 For NH 2 When R 2 Not H.

[0296] In some embodiments, in the compound of formula (I), R 1 Selected from: -CH 3 , -CF 3 、-OCH 3 、-OCF 3 and NH 2 .

[0297] In some embodiments, in the compound of formula (I), R 1 Yes NH 2 .

[0298] In some embodiments, in the compound of formula (I), R 1 Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 .

[0299] In some embodiments, in the compound of formula (I), R 1 Selected from: -CH 3 , -CF 3 、-OCH 3 and-OCF 3 .

[0300] In some embodiments, in the compound of formula (I), R 2 Selected from: -H, -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0301] In some embodiments, in the compound of formula (I), R 2 Selected from: -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0302] In some embodiments, in the compound of formula (I), R 2 Selected from: -H, -F, -Br and -Cl.

[0303] In some embodiments, in the compound of formula (I), R 2 Selected from: -F, -Br and -Cl.

[0304] In some embodiments, in the compound of formula (I), R 3 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0305] In some embodiments, in the compound of formula (I), R 4 Selected from:

[0306] In some embodiments, in the compound of formula (I), R 5 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C6 alkyl)-aminoaryl.

[0307] In some embodiments, in the compound of formula (I), R 6 and R 7 Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 .

[0308] In some embodiments, in the compound of formula (I), R 8 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0309] In some embodiments, in the compound of formula (I), each R 9 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0310] In some embodiments, in the compound of formula (I), each R 9 Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0311] In some embodiments, in the compound of formula (I), each R9 Independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0312] In some embodiments, in the compound of formula (I), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0313] In some embodiments, in the compound of formula (I), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0314] In some embodiments, in the compound of formula (I), each R 10 Independently selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0315] In some embodiments, in the compound of formula (I), R 10’ Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0316] In some embodiments, in the compound of formula (I), R 11 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0317] In some embodiments, in the compound of formula (I), R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16 .

[0318] In some embodiments, in the compound of formula (I), R 12 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 -S(O) 2 R16 and

[0319] In some embodiments, in the compound of formula (I), R 13 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0320] In some embodiments, in the compound of formula (I), R 14 and R 14’ Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0321] In some embodiments, in the compound of formula (I), R 16 Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0322] In some embodiments, in the compound of formula (I), R 16 Selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0323] In some embodiments, in the compound of formula (I), R 17Selected from: unsubstituted C 1 -C 6 Alkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, -(C 1 -C 6 Alkyl)-C 3 -C 8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0324] In some embodiments, in the compound of formula (I), R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 .

[0325] In some embodiments, in the compound of formula (I), X a and X b are each independently selected from: NH and O.

[0326] Combinations of any of the foregoing embodiments of compounds of formula (I) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0327] In certain embodiments, the compound of formula (I) has formula (II):

[0328]

[0329] in:

[0330] R 2 Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH3 and-OCF 3 ;

[0331] R 20 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl,

[0332] R 5 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0333] R 6 and R 7 Each independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 ;

[0334] R 8 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0335] Each R 9 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1-C 6 alkyl)-aryl;

[0336] Each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0337] R 10’ Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0338] R 11 Selected from: -H and -C 1 -C 6 alkyl;

[0339] R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16 and

[0340] R 13 Selected from: -H and -C 1 -C 6 alkyl;

[0341] R 14 and R 14’ Each independently selected from: -H, C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0342] R 16 Selected from: -C 1 -C 6 Alkyl, -C 3 -C8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0343] R 17 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, –(C 1 -C 6 Alkyl)-C 3 -C 8 Heterocycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0344] R 18 and R 19 Together with the N atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 ;

[0345] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0346] X a and X b are each independently selected from: NH, O and S, and

[0347] X c Selected from: O, S and S(O) 2 ,

[0348] Provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

[0349] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH 3 , -CF 3, -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 .

[0350] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0351] In some embodiments, in the compound of formula (II), R 2 Selected from F and Cl.

[0352] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 , –(C 1 -C 6 alkyl)-aryl,

[0353] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 , –(C 1 -C 6 alkyl)-aryl,

[0354] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 ,

[0355] In some embodiments, in the compound of formula (II), R 20 Selected from: -H, unsubstituted -C 1 -C 6Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , unsubstituted aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 alkyl)-aminoaryl,

[0356] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 , and R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 , –(C 1 -C 6 alkyl)-aryl,

[0357] In some embodiments, in the compound of formula (II), R 2 Selected from: -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 , and R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 , –(C 1 -C 6 alkyl)-aryl,

[0358] In some embodiments, in the compound of formula (II), R2 Selected from: -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 , and R 20 Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 ,

[0359]

[0360] In some embodiments, in the compound of formula (II), R 5 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0361] In some embodiments, in the compound of formula (II), R 6 and R 7 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C(O)R 17 .

[0362] In some embodiments, in the compound of formula (II), R 6 is H, and R 7 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 .

[0363] In some embodiments, in the compound of formula (II), R 6 is H, and R 7 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C(O)R 17 .

[0364] In some embodiments, in the compound of formula (II), R 6 and R 7 Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 .

[0365] In some embodiments, in the compound of formula (II), R 8 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0366] In some embodiments, in the compound of formula (II), each R 9 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0367] In some embodiments, in the compound of formula (II), each R9 Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0368] In some embodiments, in the compound of formula (II), each R 9 Independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0369] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0370] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0371] In some embodiments, in the compound of formula (II), each R 10 Independently selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3-C 8 Cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0372] In some embodiments, in the compound of formula (II), R 10’ Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0373] In some embodiments, in the compound of formula (II), R 11 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0374] In some embodiments, in the compound of formula (II), R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16 .

[0375] In some embodiments, in the compound of formula (II), R 12 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C1 -C 6 Aminoalkyl, -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 -S(O) 2 R 16 and

[0376] In some embodiments, in the compound of formula (II), R 13 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0377] In some embodiments, in the compound of formula (II), R 14 and R 14’ Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0378] In some embodiments, in the compound of formula (II), R 16 Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0379] In some embodiments, in the compound of formula (II), R 16 Selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3-C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0380] In some embodiments, in the compound of formula (II), R 17 For -C 1 -C 6 alkyl.

[0381] In some embodiments, in the compound of formula (II), R 17 Selected from: unsubstituted C 1 -C 6 Alkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, -(C 1 -C 6 Alkyl)-C 3 -C 8 heterocycloalkyl, unsubstituted aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0382] In some embodiments, in the compound of formula (II), R 18 and R 19 Together with the N atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 .

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

[0384] Combinations of any of the foregoing embodiments of compounds of formula (II) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0385] In certain embodiments, the compound of formula (I) has formula (III):

[0386]

[0387] in:

[0388] R 2 Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ;

[0389] R 15 Selected from: -H, -CH 3 , -CHF 2 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ;

[0390] R 4 Selected from:

[0391] R 5 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0392] R 8 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0393] Each R 9 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0394] Each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0395] R 10’ Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0396] R 11 Selected from: -H and -C 1 -C 6 alkyl;

[0397] R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16 and

[0398] R 13 Selected from: -H and -C 1 -C 6 alkyl;

[0399] R 14 and R 14’ Each independently selected from: -H, C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0400] R 16 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C1 -C 6 alkyl)-aryl;

[0401] R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 ;

[0402] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0403] X a and X b are each independently selected from: NH, O and S, and

[0404] X c Selected from: O, S and S(O) 2 .

[0405] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0406] In some embodiments, in the compound of formula (III), R 2 Selected from: -H, -F and -Cl.

[0407] In some embodiments, in the compound of formula (III), R 15 Selected from: -CH 3 , -CF 3 、-OCH 3 and-OCF 3 .

[0408] In some embodiments, in the compound of formula (III), R 15 Selected from: -CH 3 and -OCH 3 .

[0409] In some embodiments, in the compound of formula (III), R2 is selected from: -H, -F and -Cl, and R 15 Selected from: -CH 3 , -CF 3 、-OCH 3 and-OCF 3 .

[0410] In some embodiments, in the compound of formula (III), R 2 is selected from: -H, -F and -Cl, and R 15 Selected from: -CH 3 and -OCH 3 .

[0411] In some embodiments, in the compound of formula (III), R 4 Selected from:

[0412] In some embodiments, in the compound of formula (III), R 5 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0413] In some embodiments, in the compound of formula (III), R 8 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0414] In some embodiments, in the compound of formula (III), each R 9 Independently selected from: -C 1 -C6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0415] In some embodiments, in the compound of formula (III), each R 9 Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0416] In some embodiments, in the compound of formula (III), each R 9 Independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0417] In some embodiments, in the compound of formula (III), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0418] In some embodiments, in the compound of formula (III), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0419] In some embodiments, in the compound of formula (III), each R 10 Independently selected from: unsubstituted -C1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0420] In some embodiments, in the compound of formula (III), R 10' Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0421] In some embodiments, in the compound of formula (III), R 11 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0422] In some embodiments, in the compound of formula (III), R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16 .

[0423] In some embodiments, in the compound of formula (III), R 12 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 -S(O) 2 R 16 and

[0424] In some embodiments, in the compound of formula (III), R 13 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0425] In some embodiments, in the compound of formula (III), R 14 and R 14’ Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0426] In some embodiments, in the compound of formula (III), R 16 Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0427] In some embodiments, in the compound of formula (III), R 16Selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0428] In some embodiments, in the compound of formula (III), R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 .

[0429] In some embodiments, in the compound of formula (III), X a and X b are each independently selected from: NH and O.

[0430] Combinations of any of the foregoing embodiments of compounds of formula (III) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0431] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (I), (II) or (III) is optionally substituted by one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (I), (II) or (III) is optionally substituted by one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl and sulfonamido.

[0432] In certain embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is a compound having formula (I) and is selected from the compounds shown in Table 6 and Table 7.

[0433] In certain embodiments, the camptothecin analog is a compound having the formula (II). In certain embodiments, the camptothecin analog is a compound having the formula (II), wherein R 2 is F, and R 20 H, -(C 1 -C 6 )-OR 5 or In some embodiments, the camptothecin analog is a compound having formula (II), wherein R 2 F; R 20 H, -(C 1 -C 6 )-OR 5 or R 5 is H, and R 18 and R 19 Together with the N atom to which they are bound, they form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, the camptothecin analog is a compound of formula (II), wherein R 2 F; R 20 For-(C 1 -C 6 )-OR 5 , and R 5 is H. In certain embodiments, the camptothecin analog is a compound having formula (II) and is selected from the compounds shown in Table 6.

[0434] In certain embodiments, the camptothecin analog is a compound having the formula (III). In certain embodiments, the camptothecin analog is a compound having the formula (III), wherein R 2 F; R 15 -CH 3 ; R 4 for R 9 For -C 1 -C 6 Hydroxyalkyl, and X a and X b Each is O. In certain embodiments, the camptothecin analog is a compound having formula (III) and is selected from the compounds shown in Table 7.

[0435] In certain embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is Compound 139, Compound 140, Compound 141, or Compound 148. In some embodiments, the camptothecin analog comprised by the ADC according to the present disclosure is Compound 139 or Compound 141.

[0436] Table 6: Exemplary Camptothecin Analogs of Formula (II)

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443] Table 7: Exemplary Camptothecin Analogs of Formula (III)

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450] It should be understood that references throughout this disclosure to compounds of formula (I) include, in various embodiments, compounds of formula (II) and formula (III) as well as the individual compounds shown in Tables 6 and 7, to the same extent as if embodiments of each of these formulas or compounds were specifically recited individually.

[0451] Antibody-drug conjugates

[0452] The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to a camptothecin analog having formula (I). In certain embodiments, the ADC has formula (X):

[0453] T-[L-(D) m ] n

[0454] (X)

[0455] in:

[0456] T is an anti-NaPi2b antibody construct as described herein;

[0457] L is the connector;

[0458] D is a camptothecin analog having formula (I);

[0459] m is an integer between 1 and 4, and

[0460] n is an integer between 1 and 10.

[0461] In certain embodiments, in the conjugate of formula (X), m is between 1 and 2. In some embodiments, m is 1.

[0462] In some embodiments, in the conjugate of formula (X), n is between 1 and 8, such as between 2 and 8. In some embodiments, n is between 4 and 8.

[0463] In certain embodiments, in the conjugate of formula (X), m is between 1 and 2, and n is between 2 and 8, or between 4 and 8. In some embodiments, in the conjugate of formula (X), m is 1, and n is between 2 and 8, or between 4 and 8.

[0464] As shown above and reflected by the parameters m and n in formula (X), the anti-NaPi2b antibody construct "T" can be conjugated to more than one compound "D" of formula (I). Those skilled in the art will understand that although any particular anti-NaPi2b antibody construct T is conjugated to an integer number of compounds D, analyzing the preparation of the conjugate to determine the ratio of compound D to anti-NaPi2b antibody construct T can yield a non-integer result, reflecting a statistical average. This ratio of compound D to targeting moiety T can generally be referred to as the drug-to-antibody ratio or "DAR". Therefore, conjugate preparations with non-integer DARs are intended to be covered by formula (X).

[0465] In certain embodiments, in the conjugate of formula (X), D is a compound of formula (II) or formula (III). In certain embodiments, in the conjugate of formula (X), D is a compound selected from the compounds shown in Table 6 and Table 7. In certain embodiments, in the conjugate of formula (X), D is compound 139, compound 140, compound 141 or compound 148. In some embodiments, in the conjugate of formula (X), D is compound 139 or compound 141.

[0466] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (IV):

[0467]

[0468] in:

[0469] R 1a Selected from: -H, -CH 3 , -CHF 2 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 、-OCF 3 and -NH 2 ;

[0470] R 2a Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ;

[0471] X is -O-, -S- or -NH-, and R 4a Selected from:

[0472] wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or

[0473] X is O, and R 4a -X- is selected from:

[0474] R 5a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0475] R 8a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0476] Each R 9a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl; or R 9a does not exist, and X b =X;

[0477] Each R 10a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl and

[0478] Each R 10a’ Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0479] Each R 10b Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0480] R 11a Does not exist or is -C 1 -C 6 alkyl;

[0481] R 12a Selected from: -C 1 -C 6 Alkyl, -CO 2 R 8a , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16a and

[0482] R 13a Selected from: -H and -C 1 -C 6 alkyl;

[0483] R 14a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0484] R 14a’ Selected from: H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0485] R 16a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0486] R 21 Selected from: -C 1 -C 6 Alkyl, –C 3 -C 8 Cycloalkyl and –(C 1 -C 6Alkyl)-OR 5a ;

[0487] R 22 and R 23 Each is independently selected from: -H, -halogen, -C 1 -C 6 Alkyl and -C 3 -C 8 Cycloalkyl;

[0488] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0489] X a and X b Each independently selected from: NH, O and S;

[0490] X c Selected from: O, S and S(O) 2 ,and

[0491] Indicates the connection point with the linker L.

[0492] In some embodiments, in the compound of formula (IV), R 1a Selected from: -CH 3 , -CF 3 、-OCH 3 、-OCF 3 and -NH 2 .

[0493] In some embodiments, in the compound of formula (IV), R 1a Selected from: -CH 3 , -CF 3 、-OCH 3 and-OCF 3 .

[0494] In some embodiments, in the compound of formula (IV), R 1a Selected from: -CH 3 、-OCH 3 and NH 2 .

[0495] In some embodiments, in the compound of formula (IV), R 1a Selected from: -CH 3 and -OCH 3 .

[0496] In some embodiments, in the compound of formula (IV), R2a Selected from: -H, -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0497] In some embodiments, in the compound of formula (IV), R 2a Selected from: -H, -F and -Cl.

[0498] In some embodiments, in the compound of formula (IV), R 2a Yes -F.

[0499] In some embodiments, in the compound of formula (IV), X is -O-, -S-, or -NH-, and R 4a Selected from:

[0500] In some embodiments, in the compound of Formula (IV), X is -O- or -NH-.

[0501] In some embodiments, in the compound of formula (IV), each R 9a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0502] In some embodiments, in the compound of formula (IV), each R 9a Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0503] In some embodiments, in the compound of formula (IV), each R 10a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and

[0504] In some embodiments, in the compound of formula (IV), each R 10a Independently selected from: -C 1 -C6 Alkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and

[0505] In some embodiments, in the compound of formula (IV), R 12a Selected from: -C 1 -C 6 Alkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16 .

[0506] In some embodiments, in the compound of formula (IV), R 13a Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0507] In some embodiments, in the compound of formula (IV), R 14a’ Selected from: H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0508] In some embodiments, in the compound of formula (IV), R 16a Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0509] In some embodiments, in the compound of formula (IV), R 22 and R 23 Each is independently selected from: -H, -halogen, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C1 -C 6 Aminoalkyl, -C 1 -C 6 Hydroxyalkyl and -C 3 -C 8 Cycloalkyl.

[0510] In some embodiments, in the compound of formula (IV), X a and X b are each independently selected from: NH and O.

[0511] In some embodiments, in the compound of formula (IV), X a and X b Each is O.

[0512] 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 For -C 1 -C 6 alkyl.

[0513] In some embodiments, in the compound of formula (IV), R 1a -CH 3 or -OCH 3 ;X is O;R 4a for X a and X b Each is O; and R 9a For -C 1 -C 6 alkyl.

[0514] In some embodiments, in the compound of formula (IV), R 1a -CH 3 or -OCH 3 ; R 2a is H or F; X is O; R 4a for X a and X b Each is O; and R 9a For -C 1 -C 6 alkyl.

[0515] Other combinations of any of the foregoing embodiments of compounds of formula (IV) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0516] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (V):

[0517]

[0518] in:

[0519] R 2a Selected from: -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ;

[0520] R 20a Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl,

[0521] R 5 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0522] R 6 and R 7 Each independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 ;

[0523] R 8 Selected from: -H, -C 1 -C 6 Alkyl, -C3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0524] Each R 9 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0525] Each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl and -NR 14 R 14’ ;

[0526] Each R 10’ Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0527] R 11 Selected from: -H and -C 1 -C 6 alkyl;

[0528] R 12 Selected from: -H, -C 1 -C 6 Alkyl, -CO 2 R 8 , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16 and

[0529] R 13 Selected from: -H and -C 1 -C 6 alkyl;

[0530] R 14 and R14’ Each independently selected from: -H, C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0531] R 16 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0532] R 17 Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, –(C 1 -C 6 Alkyl)-C 3 -C 8 Heterocycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0533] R 18 and R 19 Together with the nitrogen atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 ;

[0534] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0535] X a and X b Each independently selected from: NH, O and S;

[0536] X c Selected from: O, S and S(O) 2,and

[0537] Indicates the connection point with the linker L.

[0538] In some embodiments, in the compound of formula (V), R 2a Selected from: -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0539] In some embodiments, in the compound of formula (V), R 2a Selected from: -CF 3 , -F, -Cl and -OCH 3 .

[0540] In some embodiments, in the compound of formula (V), R 2a It's F.

[0541] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , -aryl, -heteroaryl, -(C 1 -C 6 alkyl)-aryl,

[0542] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 , -(C 1 -C 6 alkyl)-aryl,

[0543] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, -C 1 -C 6 Alkyl, -(C1 -C 6 Alkyl)-OR 5 , -(C 1 -C 6 alkyl)-aryl,

[0544] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5 ,

[0545] In some embodiments, in the compound of formula (V), R 20a Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 alkyl)-aminoaryl,

[0546] In some embodiments, in the compound of formula (V), R 6 and R 7 Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C(O)R 17 .

[0547] In some embodiments, in the compound of formula (V), R 6 is H, and R 7 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 .

[0548] In some embodiments, in the compound of formula (V), R 6 is H, and R 7 Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C(O)R 17 .

[0549] In some embodiments, in the compound of formula (V), R 6 and R 7 Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl, -(C 1 -C 6 Alkyl)-OR 5 , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17 .

[0550] In some embodiments, in the compound of formula (V), R 8 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0551] In some embodiments, in the compound of formula (V), each R 9 Independently selected from: -C 1 -C 6Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0552] In some embodiments, in the compound of formula (V), each R 9 Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0553] In some embodiments, in the compound of formula (V), each R 9 Independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0554] In some embodiments, in the compound of formula (V), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0555] In some embodiments, in the compound of formula (V), each R 10 Independently selected from: -C 1 -C 6 Alkyl, -NR 14 R 14’ , -aryl and -(C 1 -C 6 alkyl)-aryl.

[0556] In some embodiments, in the compound of formula (V), R 11 Selected from: -H, unsubstituted -C 1 -C6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0557] In some embodiments, in the compound of formula (V), R 12 Selected from: -H, -C 1 -C 6 Alkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16 .

[0558] In some embodiments, in the compound of formula (V), R 12 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -CO 2 R 8 , unsubstituted -aryl, -aminoaryl, -heteroaryl, -(C 1 -C 6 -S(O) 2 R 16 and

[0559] In some embodiments, in the compound of formula (V), R 13 Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0560] In some embodiments, in the compound of formula (V), R 14 and R 14’ Each independently selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0561] In some embodiments, in the compound of formula (V), R 16 Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0562] In some embodiments, in the compound of formula (V), R 16 Selected from: unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl, unsubstituted -aryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0563] In some embodiments, in the compound of formula (V), R 17 Selected from: unsubstituted -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, -(C 1 -C 6 Alkyl)-C 3 -C 8 heterocycloalkyl, unsubstituted -aryl, -hydroxyaryl, -aminoaryl, -heteroaryl and -(C 1 -C 6 alkyl)-aminoaryl.

[0564] In some embodiments, in the compound of formula (V), R 18 and R 19 Together with the N atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, unsubstituted -C 1 -C6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Aminoalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl and -(C 1 -C 6 Alkyl)-OR 5 .

[0565] In some embodiments, in the compound of formula (V), R 17 Yes-C 1 -C 6 alkyl.

[0566] In some embodiments, in the compound of formula (V), X a and X b are each independently selected from: NH and O.

[0567] In some embodiments, in the compound of formula (V), X a and X b Each is O.

[0568] In some embodiments, in the compound of formula (V), R 20a For –(C 1 -C 6 Alkyl)-OR 5 .

[0569] In some embodiments, in the compound of formula (V), R 20a For –(C 1 -C 6 Alkyl)-OR 5 , and R 5 For H.

[0570] In some embodiments, in the compound of formula (V), R 2a F; R 20a For –(C 1 -C 6 Alkyl)-OR 5 , and R 5 For H.

[0571] Other combinations of any of the foregoing embodiments of compounds of formula (V) are also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0572] Certain embodiments of the present disclosure relate to ADCs having formula (X), wherein D is a compound of formula (VI):

[0573]

[0574] in:

[0575] R 2a Selected from: -H, -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 ;

[0576] X is -O-, -S- or -NH-, and R 25 Selected from: -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5a , -CO 2 R 8a , -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl,

[0577] wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or

[0578] X is O, and R 25 -X- is selected from:

[0579] R 5a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0580] R 6a Selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0581] R 7a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -(C1 -C 6 Alkyl)-OR 5a , -C 3 -C 8 Heterocycloalkyl and -C(O)R 17a ;

[0582] R 8a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0583] Each R 9a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl; or R 9a does not exist, and X b =X;

[0584] Each R 10a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl, –(C 1 -C 6 alkyl)-aryl and

[0585] Each R 10a’ Independently selected from: -H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0586] Each R 10b Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0587] R 11a Does not exist or is -C1 -C 6 alkyl;

[0588] R 12a Selected from: -C 1 -C 6 Alkyl, -CO 2 R 8a , -aryl, -heteroaryl, –(C 1 -C 6 Alkyl)-aryl, -S(O) 2 R 16a and

[0589] R 13a Selected from: -H and -C 1 -C 6 alkyl;

[0590] R 14a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0591] R 14a’ Selected from: H, -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl;

[0592] R 16a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -heteroaryl and –(C 1 -C 6 alkyl)-aryl;

[0593] R 17a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -C 3 -C 8 Heterocycloalkyl, –(C 1 -C 6 Alkyl)-C 3 -C 8 Heterocycloalkyl, -aryl, -heteroaryl and –(C 1 -C6 alkyl)-aryl;

[0594] R 21 Selected from: -C 1 -C 6 Alkyl, –C 3 -C 8 Cycloalkyl and –(C 1 -C 6 Alkyl)-OR 5a ;

[0595] R 22 and R 23 Each is independently selected from: -H, -halogen, -C 1 -C 6 Alkyl and -C 3 -C 8 Cycloalkyl;

[0596] R 24 , R 25 and R 26 Each is -C 1 -C 6 alkyl;

[0597] X a and X b Each independently selected from: NH, O and S;

[0598] X c Selected from: O, S and S(O) 2 ,and

[0599] Indicates the connection point with the linker L.

[0600] In some embodiments, in the compound of formula (VI), R 2a Selected from: -CH 3 , -CF 3 , -F, -Br, -Cl, -OH, -OCH 3 and-OCF 3 .

[0601] In some embodiments, in the compound of formula (VI), R 2a Selected from: -CH 3 , -CF 3 、-F、-Cl、-OCH 3 and-OCF 3 .

[0602] In some embodiments, in the compound of formula (VI), R 2a Selected from: F and Cl.

[0603] In some embodiments, in the compound of formula (VI), R 2a It's F.

[0604] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5a 、-(C 1 -C 6 alkyl)-aryl,

[0605] Or X is O, and R 25 -X- is selected from:

[0606] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5a 、-(C 1 -C 6 alkyl)-aryl,

[0607]

[0608] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from: -C 1 -C 6 Alkyl, -(C 1 -C 6 Alkyl)-OR 5a ,

[0609] In some embodiments, in the compound of formula (VI), X is -O-, -S-, or -NH-, and R 25 Selected from:

[0610] In some embodiments, in the compound of Formula (VI), X is -O- or -NH-.

[0611] In some embodiments, in the compound of formula (VI), R 6a It's H.

[0612] In some embodiments, in the compound of formula (VI), R 6a Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0613] In some embodiments, in the compound of formula (VI), R 7a Selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl and -C(O)R 17a .

[0614] In some embodiments, in the compound of formula (VI), each R 9a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl and -(C 1 -C 6 alkyl)-aryl.

[0615] In some embodiments, in the compound of formula (VI), each R 9a Independently selected from: -C 1 -C 6 Alkyl and -(C 1 -C 6 alkyl)-aryl.

[0616] In some embodiments, in the compound of formula (VI), each R 10a Independently selected from: -C 1 -C 6 Alkyl, -C 3 -C 8 Cycloalkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and

[0617] In some embodiments, in the compound of formula (VI), each R10a Independently selected from: -C 1 -C 6 Alkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and

[0618] In some embodiments, in the compound of formula (VI), R 12a Selected from: -C 1 -C 6 Alkyl, -aryl, -(C 1 -C 6 alkyl)-aryl and -S(O) 2 R 16a .

[0619] In some embodiments, in the compound of formula (VI), R 13a Selected from: -H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl and -C 1 -C 6 Aminoalkyl.

[0620] In some embodiments, in the compound of formula (VI), R 14a’ Selected from: H, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl, -C 3 -C 8 Cycloalkyl and -C 3 -C 8 Heterocycloalkyl.

[0621] In some embodiments, in the compound of formula (VI), R 16a Selected from: -aryl, -heteroaryl and -(C 1 -C 6 alkyl)-aryl.

[0622] In some embodiments, in the compound of formula (VI), R 17a For -C 1 -C 6 alkyl.

[0623] In some embodiments, in the compound of formula (VI), R 22 and R 23 Each is independently selected from: -H, -halogen, unsubstituted -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Hydroxyalkyl, -C 1 -C 6 Aminoalkyl and -C 3 -C 8 Cycloalkyl.

[0624] In some embodiments, in the compound of formula (VI), X a and X b are each independently selected from: NH and O.

[0625] In some embodiments, in the compound of formula (VI), X a and X b Each is O.

[0626] In some embodiments, in the compound of formula (VI), X is O, and R 25 For -C 1 -C 6 alkyl.

[0627] In some embodiments, in the compound of formula (VI), R 2a is F; X is O, and R 25 For -C 1 -C 6 alkyl.

[0628] Other combinations of any of the foregoing embodiments of compounds of formula (VI) are also contemplated, and each combination forms a separate embodiment for purposes of this disclosure.

[0629] In certain embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (IV), (V) or (VI) is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl. In some embodiments, each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl as defined in any one of formula (IV), (V) or (VI) is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxyl, amino, amido, nitro, cyano, azido, alkylthio, sulfo, sulfonyl and sulfonamido.

[0630] In certain embodiments, in an ADC having formula (X), D is a compound of formula (IV), wherein R 1a -CH 3 , and R 2a is F. In some embodiments, in the ADC having formula (X), D is a compound of formula (IV), wherein R 1a -CH 3 ; R 2a is F; X is -O-; R 4a for R 9a For -C 1 -C 6 Alkyl, and X a and X b Each is O.

[0631] In certain embodiments, in an ADC having formula (X), D is a compound of formula (V), wherein R 2a is F, and R 20a H, -(C 1 -C 6 )-OR 5 or In some embodiments, in an ADC having formula (X), D is a compound of formula (V), wherein R 2a F; R 20a H, -(C 1 -C 6 )-OR 5 or R 5 is H, and R 18 and R 19 Together with the N atom to which they are bound, they form an unsubstituted 4-, 5-, 6-, or 7-membered ring. In some embodiments, in the ADC having formula (X), D is a compound of formula (V), wherein R2a F; R 20a For-(C 1 -C 6 )-OR 5 , and R 5 For H.

[0632] In certain embodiments, in an ADC having formula (X), D is a compound of formula (VI), wherein R 2a is F; X is -O-, and R 25 For -C 1 -C 6 alkyl.

[0633] Connector L

[0634] The conjugate of formula (X) includes a linker L, which is a bifunctional or multifunctional moiety capable of connecting one or more camptothecin analogs D to an anti-NaPi2b antibody construct T. A bifunctional (or monovalent) linker L connects a single compound D to a single site on an anti-NaPi2b antibody construct T, while a multifunctional (or multivalent) linker L connects more than one compound D to a single site on an anti-NaPi2b antibody construct T. A linker that connects one compound D to more than one site on an anti-NaPi2b antibody construct T can also be considered multifunctional.

[0635] 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, those described in Bioconjugate Techniques (GTHermanson, 2013, Academic Press). Groups on the anti-NaPi2b antibody construct T and the camptothecin analog D that can be used as target groups for linker connection include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0636] Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. In this case, "self-stabilizing" maleimides as described in Lyon et al., 2014, Nat. Biotechnol., 32: 1059-1062 can also be used.

[0637] Non-limiting examples of functional groups capable of reacting with amines include activated esters such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters, imidoesters such as Traut's reagent, isothiocyanates, aldehydes, and anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include converting carboxyl groups to activated esters using succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), which can then react with amines.

[0638] Non-limiting examples of functional groups capable of reacting with an electrophilic group such as an aldehyde or ketone carbonyl include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0639] In certain embodiments, the linker L may comprise a functional group that allows for bridging of two interchain cysteines on the anti-NaPi2b antibody construct, such as ThioBridge TM linker (Badescu et al., 2014, Bioconjug. Chem. 25: 1124-1136), disulfide maleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12: 3986-3998), disulfide aryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7: 799-802) or dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6: 6645).

[0640] Alternatively, anti-NaPi2b antibody construct T can be modified to include non-natural reactive groups, such as azide, which allows conjugation to the joint via the complementary reactive groups on the joint. For example, the conjugation of the joint and the anti-NaPi2b antibody construct can utilize click chemistry reaction (see, for example, Chio & Bane, 2020, Methods Mol.Biol., 2078: 83-97), such as azide-alkyne cycloaddition (AAC) reaction, which has been successfully used to develop antibody-drug conjugates. AAC reaction can be copper-catalyzed AAC (CuAAC) reaction, which involves the conjugation of azide and straight-chain alkynes; or strain-promoted AAC (SPAAC) reaction, which involves the conjugation of azide and cyclooctyne.

[0641] The linker L can be a cleavable or non-cleavable linker. A cleavable linker is a linker that is easily cleaved under specific conditions, for example, under intracellular conditions (such as in endosomes or lysosomes) or near target cells (such as in tumor microenvironments). Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on the degradation of the antibody in the cell, which generally results in the release of the amino acid-linker-drug moiety.

[0642] The example of cleavable linker includes, for example, a linker comprising an amino acid sequence as a cleavage recognition sequence of a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, for example, an amino acid sequence recognized and cleaved by a protease present in the extracellular matrix near target cells such as cancer cells can be used. The example of extracellular tumor-associated protease includes, for example, plasmin, matrix metalloproteinase (MMP), elastase and kallikrein-related peptidase.

[0643] For conjugates intended to be internalized by cells, the linker L may comprise 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, and legumin.

[0644] The cleavage recognition sequence can be, for example, a dipeptide, a tripeptide, or a tetrapeptide. Non-limiting examples of dipeptide recognition sequences that can 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, Me 3 Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys. Examples of tripeptide and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0645] Additional examples of cleavable linkers include disulfide-containing linkers, such as N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB). Linkers containing disulfide bonds may optionally include additional groups to provide steric hindrance near the disulfide bond to improve the extracellular stability of the linker, for example, including a geminal dimethyl group. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Linkers containing combinations of these functionalities may also be useful, for example, linkers containing both hydrazone and disulfide bonds are known in the art.

[0646] Another example of a cleavable linker is a linker comprising β-glucuronide, which can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des. 8:1391–1403, and International Patent Publication No. WO 2007 / 011968). β-glucuronide can also be used to improve the hydrophilicity of the linker L.

[0647] Another example of a linker that is cleaved internally within the cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, eg, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0648] In certain embodiments, the linker L comprised by the conjugate of formula (X) is a cleavable linker. In some embodiments, the linker L comprises a cleavage recognition sequence. In some embodiments, the linker L may comprise an amino acid sequence recognized and cleaved by a lysosomal protease.

[0649] The cleavable linker may optionally further comprise one or more additional functional groups, such as self-immolative and self-immolative groups, extending groups or hydrophilic moieties.

[0650] Self-decomposition and self-eliminating groups that can be used for joints include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED) and hemiacetal groups. Other examples of self-decomposition groups include, but are not limited to, aromatic compounds similar to PAB or PABC group electronics, such as heterocyclic derivatives, such as 2-aminoimidazole-5-methanol derivatives described in U.S. Patent No. 7,375,078. Other examples include groups that are cyclized when amide bonds are hydrolyzed, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2: 223-227) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J.Org.Chem.55: 5867-5877). Self-decomposition / self-eliminating groups are generally connected to amino or hydroxyl groups on compound D. Self-decomposition / self-eliminating groups are generally included in peptide-based joints alone or in combination, but may also be included in other types of joints.

[0651] Extending groups that can be used in the linker of the drug conjugate include, for example, alkylene groups and extending groups based on aliphatic acids, diacids, amines or diamines, such as diglycolates, malonates, caproates and caproamides. Other extending groups include, for example, extending groups based on glycine and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) extending groups.

[0652] PEG and mPEG extension groups can also be used as hydrophilic parts in joints. For example, PEG or mPEG can be "directly inserted" or included in the joint as a side group to increase the hydrophilicity of the joint (see, for example, U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing joints can be commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that can be optionally incorporated into joint L include, for example, β-glucuronide, sulfonic acid group, carboxylic acid group and pyrophosphate diester.

[0653] In certain embodiments, the ADC of formula (X) may comprise a cleavable linker. In some embodiments, the ADC of formula (X) may comprise a peptide-containing linker. In some embodiments, the ADC of formula (X) may comprise a protease-cleavable linker.

[0654] In some embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XI):

[0655]

[0656] in:

[0657] Z is a functional group capable of reacting with a target group on the anti-NaPi2b antibody construct T;

[0658] Str is an extension group;

[0659] AA 1 and AA 2 Each is independently an amino acid, wherein AA 1 -[AA 2 ] r Formation of protease cleavage sites;

[0660] X is a self-decomposing group;

[0661] q is 0 or 1;

[0662] r is 1, 2, or 3;

[0663] s is 0, 1, or 2;

[0664] # is the connection point with the anti-NaPi2b antibody construct T, and

[0665] % is the connection point with the camptothecin analog D.

[0666] In some embodiments, in the linker of formula (XI), q is 1.

[0667] In some embodiments, in the linker of formula (XI), s is 1. In some embodiments, in the ADC of formula (XI), s is 0.

[0668] In some embodiments, in the linker of formula (XI), r is 1. In some embodiments, in the ADC of formula (XI), r is 3.

[0669] In some embodiments, in the linker of formula (XI):

[0670] Z is Where # is the point of connection to T and * is the point of connection to the rest of the linker.

[0671] In some embodiments, in the linker of formula (XI), Str is selected from:

[0672]

[0673]

[0674] in:

[0675] R is H or C 1 -C 6 alkyl;

[0676] t is an integer between 2 and 10, and

[0677] u is an integer between 1 and 10.

[0678] In some embodiments, in the linker of formula (XI), Str is selected from:

[0679]

[0680] in:

[0681] t is an integer between 2 and 10, and

[0682] u is an integer between 1 and 10.

[0683] In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r is a dipeptide (ie, r=1). In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r having a sequence selected from the group consisting of Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me 3 Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0684] In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r is a tripeptide (ie, r=2). In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r Having a sequence selected from the group consisting of 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.

[0685] In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r is a tetrapeptide (ie, r=3). In some embodiments, in the linker of formula (XI), AA 1 -[AA 2 ] r Having a sequence selected from the group consisting of Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0686] In certain embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XII):

[0687]

[0688] in:

[0689] Z is a functional group capable of reacting with a target group on the anti-NaPi2b antibody construct T;

[0690] Str is an extension group;

[0691] AA 1 and AA 2 Each is independently an amino acid, wherein AA 1 -[AA 2 ] r Formation of protease cleavage sites;

[0692] Y is -NH-CH 2 -;

[0693] q is 0 or 1;

[0694] r is 1, 2, or 3;

[0695] v is 0 or 1;

[0696] # is the connection point with the anti-NaPi2b antibody construct T, and

[0697] % is the connection point with the camptothecin analog D.

[0698] In some embodiments, in the linker of formula (XII), q is 1.

[0699] In some embodiments, in the linker of formula (XII), v is 0. In some embodiments, in the ADC of formula (XII), s is 1.

[0700] In some embodiments, in the linker of formula (XII), r is 1. In some embodiments, in the ADC of formula (XII), r is 3.

[0701] In some embodiments, in the linker of formula (XII):

[0702] Z is Where # is the point of connection to T and * is the point of connection to the rest of the linker.

[0703] In some embodiments, in the linker of formula (XII), Str is selected from:

[0704]

[0705] in:

[0706] R is H or C 1 -C 6 alkyl;

[0707] t is an integer between 2 and 10, and

[0708] u is an integer between 1 and 10.

[0709] In some embodiments, in the linker of formula (XII), Str is selected from:

[0710]

[0711] in:

[0712] t is an integer between 2 and 10, and

[0713] u is an integer between 1 and 10.

[0714] In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r is a dipeptide (ie, r=1). In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r having a sequence selected from the group consisting of Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me 3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0715] In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r is a tripeptide (ie, r=2). In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r Having a sequence 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.

[0716] In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r is a tetrapeptide (ie, r=3). In some embodiments, in the linker of formula (XII), AA 1 -[AA 2 ] r Having a sequence selected from the group consisting of Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0717] In some embodiments, in the linker of formula (XII), Y is -NH-CH 2 In some embodiments, in the linker of formula (XII), v is 1 and Y is -NH-CH 2 .

[0718] In some embodiments, the ADC of formula (X) may comprise a disulfide bond-containing linker. In some embodiments, in the ADC of formula (X), m is 1, and the linker L is a cleavable linker having formula (XIII):

[0719]

[0720] in:

[0721] Z is a functional group capable of reacting with a target group on the anti-NaPi2b antibody construct T;

[0722] Q is -(CH 2 ) p -or-(CH 2 CH 2 O) q -, wherein p and q are each independently an integer between 1 and 10;

[0723] Each R is independently H or C 1 -C 6 alkyl;

[0724] n is 1, 2 or 3;

[0725] # is the connection point with the anti-NaPi2b antibody construct T, and

[0726] % is the connection point with the camptothecin analog D.

[0727] In some embodiments, the ADC of formula (X) may comprise a β-glucuronide-containing linker.

[0728] Various non-cleavable joints are known in the art for connecting drugs to targeting moieties, and can be used in ADCs of the present disclosure in certain embodiments. Examples of non-cleavable joints include having N-succinimidyl esters or N-sulfosuccinimidyl ester moieties for reacting with anti-NaPi2b antibody constructs and joints based on maleimido or haloacetyl moieties for reacting with camptothecin analogs, or vice versa. Examples of such non-cleavable joints are based on sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylates (sulfo SMCC). Sulfo-SMCC conjugation usually occurs via maleimide groups, which react with the sulfhydryl (thiol, -SH) on camptothecin analogs, and sulfo-NHS esters are reactive to primary amines (such as found in lysine and at the N-terminal of proteins or peptides) on anti-NaPi2b antibody constructs. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminohexanoate) ("long chain" SMCC or LC-SMCC), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), epsilon-maleimidobutyric acid N-succinimidyl ester (EMB), and succinimidyl-maleimidobutyrate (SMB). Examples include iminohexanoic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl) isocyanate (PMPI). Other examples include those containing haloacetyl-based functional groups such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido) propionate (SBAP).

[0729] Non-limiting examples of drug-linkers comprising camptothecin analogs of formula (I) are shown in Table 8, Table 9, and Table 10. Non-limiting examples of conjugates comprising these drug-linkers are shown in Table 11, Table 12, and Table 13. In certain embodiments, the ADC of formula (X) comprises a drug-linker selected from the drug-linkers shown in Table 8, Table 9, and Table 10. In certain embodiments, the ADC of formula (X) is selected from the conjugates shown in Table 11, Table 12, and Table 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 the conjugates shown in Table 11, Table 12, and Table 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 the conjugates shown in Table 11, Table 12, and Table 13, wherein T is an anti-FRα antibody construct, and n is between 4 and 8.

[0730] In certain embodiments, the ADC of formula (X) comprises a drug-linker (L-(D)) selected from the group consisting of m ): MT-GGFG-AM-Compound 139, MC-GGFG-AM-Compound 139, MT-GGFG-Compound 140, MC-GGFG-Compound 140, MT-GGFG-AM-Compound 141, MC-GGFG-AM-Compound 141, MT-GGFG-Compound 141, MC-GGFG-Compound 141, MT-GGFG-Compound 148 and MC-GGFG-Compound 148, and n is 4 or 8. In some embodiments, the ADC of formula (X) comprises a drug-linker (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.

[0731] Preparation of ADC

[0732] 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 commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal Pharma Solutions (Grangemouth, UK).

[0733] Typically, the preparation of ADCs involves first preparing a drug-linker DL comprising one or more camptothecin analogs of formula (I) and a linker L, and then conjugating the drug-linker DL to an appropriate group on an anti-NaPi2b antibody construct T. However, the connection of the linker L to the anti-NaPi2b antibody construct T and the subsequent connection of the anti-NaPi2b antibody construct-linker TL to one or more camptothecin analogs D of formula (I) are still alternative methods that can be used in some embodiments.

[0734] In any of the above methods, suitable groups on the compound D of formula (I) for connecting the linker L include, but are not limited to, thiol groups, amine groups, carboxylic acid groups, and hydroxyl groups. In some embodiments of the present disclosure, the linker L is connected to the compound D of formula (I) via a hydroxyl or amine group on the compound.

[0735] In any of the above methods, suitable groups on the anti-NaPi2b antibody construct T for attaching the linker L include sulfhydryl 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.

[0736] For example, the anti-NaPi2b antibody construct T may include one or more naturally occurring thiol groups, thereby allowing the anti-NaPi2b antibody construct T to bond to the linker L through the sulfur atom of the thiol group. Alternatively, the anti-NaPi2b antibody construct T may include one or more lysine residues, which may be chemically modified to introduce one or more thiol groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio) propionate ("SPDP") and 2-iminothiolane hydrochloride (Traut reagent). Alternatively, the anti-NaPi2b antibody construct T may include one or more carbohydrate groups, which may be chemically modified to include one or more thiol groups.

[0737] Carbohydrate groups on anti-NaPi2b antibody construct T can also be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which can then react with linker L, e.g., via a hydrazine or hydroxylamine group on linker L.

[0738] Anti-NaPi2b antibody construct T can also be modified to include additional cysteine ​​residues (see, e.g., U.S. Pat. Nos. 7,521,541, 8,455,622, and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow site-specific conjugation. Alternatively, the anti-NaPi2b antibody construct T can be modified to include a non-natural reactive group, such as an azide, which allows conjugation to the linker via a complementary reactive group on the linker, for example by click chemistry (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078: 83-97). Another option is to use GlycoConnectTM Technology (Synaffix BV, Nijmegen, Netherlands) involves enzymatic remodeling of antibody glycans to allow attachment of linkers via metal-free click chemistry (see, eg, European Patent No. EP 2 911 699).

[0739] Other protocols for modifying proteins to attach or associate a linker L are known in the art and include those described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002).

[0740] Alternatively, ADCs can be prepared using transglutaminases, especially bacterial transglutaminases (BTG) from Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49: 9995-9997). BTG forms an amide bond between the side chain carboxamide of glutamine (amine acceptor, usually on antibodies) and an alkylene amino group (amine donor, usually on drug-linker), which can be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies can also be modified to include a peptide or "tag" containing glutamine, which allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US 2013 / 0230543 and International (PCT) Publication No. WO 2016 / 144608).

[0741] A similar conjugation method utilizes the enzyme transpeptidase A. In this approach, the antibody is typically modified to contain a transpeptidase A recognition motif (LPXTG, where X is any natural amino acid), and the drug-linker is designed to contain an oligoglycine motif (usually GGG) to allow transpeptidase A-mediated transpeptidation (see, e.g., Beerli et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0742] Once conjugation is complete, the average number of compounds of formula (I) conjugated to the anti-NaPi2b antibody construct (i.e., "drug-antibody ratio" or DAR) can be determined by standard techniques such as UV / VIS spectroscopy, ELISA-based techniques, chromatographic techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS), and MALDI-TOF MS. In addition, the distribution of drug-linked forms (e.g., containing zero, one, two, three, etc., of the anti-NaPi2b antibody construct T of compound D of formula (I)) can also be optionally analyzed. Various techniques for measuring DAR distribution are known in the art, including MS (with or without accompanying chromatographic separation steps), hydrophobic interaction chromatography, reversed-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Wakankar et al., 2011, mAbs, 3: 161-172).

[0743] Pharmaceutical composition

[0744] For therapeutic use, the ADC of the present disclosure is generally formulated as a pharmaceutical composition. Therefore, certain embodiments of the present disclosure relate to a pharmaceutical composition comprising an ADC as described herein and a pharmaceutically acceptable carrier, diluent or excipient. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

[0745] The pharmaceutical composition can be formulated for administration to a subject, for example, by oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spraying. The term "parenteral" as used herein includes subcutaneous injection, as well as intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will generally be formulated into a form suitable for administration to a subject, such as a syrup, elixir, tablet, lozenge, pastille, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granules, emulsion, injection or solution. The pharmaceutical composition can be provided as a unit dose formulation.

[0746] In certain embodiments, the pharmaceutical composition comprising the ADC is formulated for parenteral administration, for example, in the form of a lyophilized formulation or an aqueous solution. Such pharmaceutical compositions can be provided, for example, in a unit dose injectable form.

[0747] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl paraben or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0748] In certain embodiments, the composition comprising ADC can be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be prepared using suitable dispersants or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions can contain ADC in non-toxic parenteral acceptable diluents or carriers. Acceptable diluents and carriers that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile fixed oils can be used as carriers. To this end, various mild fixed oils can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections. Adjuvants, such as local anesthetics, preservatives, and / or buffers can also be included in injectable solutions or suspensions.

[0749] In certain embodiments, the composition comprising ADC can be formulated for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent 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 form, for example, as a dried lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a sachet) indicating the amount of the active agent. When the composition needs to be administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0750] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0751] How to use

[0752] Certain embodiments of the present disclosure relate to therapeutic uses of the ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents.

[0753] Certain embodiments of the present disclosure relate to inhibiting abnormal cancer cell or tumor cell growth; inhibiting cancer cell or tumor cell proliferation in a subject, or treating cancer, comprising administering an ADC described herein. In certain embodiments, the ADC described herein can be used to treat cancer. Therefore, some embodiments of the present disclosure relate to the use of ADC as an anticancer agent.

[0754] Certain embodiments of the present disclosure relate to methods of inhibiting proliferation of cancer or tumor cells, comprising contacting the cells with an ADC as described herein, e.g., an ADC of formula (X). Some embodiments relate to methods of killing cancer or tumor cells, comprising contacting the cells with an ADC as described herein, e.g., an ADC of formula (X).

[0755] Some embodiments relate to methods of treating a subject with cancer by administering an ADC as described herein, e.g., an ADC of Formula (X), to the subject. In this case, treating the subject may result in one or more of the following: a reduction in tumor size, a slowing or prevention of an increase in tumor size, a prolonged disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of subsequent occurrences of a tumor (e.g., metastasis), a prolonged time to progression, a reduction in one or more adverse symptoms associated with a tumor, and / or a prolonged overall survival time of a subject with cancer.

[0756] Certain embodiments relate to the use of an ADC as described herein, e.g., an ADC of formula (X), in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of an ADC as described herein, e.g., an ADC of formula (X), in a method of inhibiting cancer cell proliferation and / or killing cancer cells in vitro. Some embodiments relate to the use of an ADC as described herein, e.g., an ADC of formula (X), in a method of inhibiting cancer cell proliferation and / or killing cancer cells in a subject with cancer.

[0757] Examples of cancers that can be treated in certain embodiments are carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also commonly referred to as "solid tumors". Examples of common solid tumors that can be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC) and colorectal cancer. Various forms of lymphomas can also lead to the formation of solid tumors and therefore can also be considered solid tumors in some cases. Typically, the cancer to be treated is a cancer that expresses NaPi2b.

[0758] Certain embodiments relate to a method of inhibiting the growth of NaPi2b-positive tumor cells, the method comprising contacting the cells with an ADC as described herein (e.g., an ADC of formula (X)). The cells may be in vitro or in vivo. In certain embodiments, the ADC may be used in a method of treating a subject's NaPi2b-positive cancer or tumor.

[0759] 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, an ADC as described herein can be used in a method for treating ovarian cancer or lung cancer. In one embodiment, an ADC as described herein can be used in a method for treating NSCLC.

[0760] Drug kit

[0761] Certain embodiments relate to a pharmaceutical kit comprising an ADC as described herein, eg, an ADC of Formula (X).

[0762] The kit will typically include a container for containing the ADC and a label and / or package insert on or accompanying the container. The label or package insert contains instructions typically included in the commercial packaging of the therapeutic product, providing information about the indications, usage, dosage, administration, contraindications and / or warnings for using such therapeutic products. The label or package insert may also include a notice in the form of a governmental agency regulation for the manufacture, use or sale of regulated drugs or biological products, which reflects the approval of the use or sale of the manufacturing agency for human or animal administration. 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 pierceable by a hypodermic needle.

[0763] In addition to the container holding the ADC, the kit may optionally include one or more additional containers containing other components of the kit, for example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution), other buffers or diluents.

[0764] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The container may be made of various materials such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dry form (such as a powder or granules), and the kit may additionally contain a suitable solvent for reconstituting the lyophilized or dried component.

[0765] The kit may also include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.

[0766] Tables 8 to 13

[0767] Table 8: Exemplary Drug-Linker (DL) Structures Comprising Camptothecin Analogs of Formula (I) Having a C7 Bond

[0768]

[0769]

[0770]

[0771] Table 9: Exemplary Drug-Linker (DL) Structures Comprising Camptothecin Analogs of Formula (I) Having a C10 Bond

[0772]

[0773]

[0774]

[0775]

[0776] Table 10: Exemplary Drug-Linker (DL) Structures Comprising Camptothecin Analogs of Formula (I) Having a C7 or C10 Bond

[0777]

[0778]

[0779]

[0780] Table 11: Exemplary conjugate (DC) structures comprising camptothecin analogs of formula (I) having a C7 bond

[0781]

[0782]

[0783]

[0784] Table 12: Exemplary conjugate (DC) structures comprising camptothecin analogs of formula (I) having a C10 bond

[0785]

[0786]

[0787]

[0788] Table 13: Exemplary conjugate (DC) structures comprising camptothecin analogs of formula (I) having a C7 or C10 bond

[0789]

[0790]

[0791]

[0792] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way.

[0793] Example

[0794] The following examples 1 to 3 illustrate various methods for preparing camptothecin analogs of formula (I). It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known in the art. It should also be understood that those skilled in the art will be able to use the method described below or similar methods, by using appropriate starting components and modifying synthesis parameters as needed to prepare other compounds of formula (I) not specifically described below. Typically, starting components can be obtained from commercial sources, such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (ThermoFisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., or synthesized according to sources known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013) or prepared as described herein.

[0795] abbreviation

[0796] The following abbreviations are used throughout the Examples section: BCA: bicinchoninic acid; Boc: di-tert-butyl dicarbonate; CE-SDS: capillary electrophoresis sodium dodecyl sulfate; DCM: dichloromethane; DTPA: diethylenetriaminepentaacetic acid; DIPEA: N,N-diisopropylethylamine; DMF: dimethylformamide; DMMTM: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc: fluorenylmethoxycarbonyl; HATU: Tetramethyluronium azabenzotriazole hexafluorophosphate; HIC: hydrophobic interaction chromatography; HOAt: 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; LC / MS: liquid chromatography-mass spectrometry; MC: maleimidocaproyl; MT: maleimidotriglycolate; NMM: N-methylmorpholine; PNP: p-nitrophenol; RP-UPLC-MS: reversed-phase ultra-performance chromatography-mass spectrometry; SEC: size exclusion chromatography; TCEP: tris(2-carboxyethyl)phosphine; Tfp: tetrafluorophenyl; TLC: thin layer chromatography; TFA: trifluoroacetic acid.

[0797] General Chemistry Procedures

[0798] General Procedure 1: Conversion of Chlorides to Amines

[0799] To a stirred solution of the chloride compound in dimethylformamide (0.05M to 0.1M) is added the appropriate secondary amine (3 equiv.) Upon completion (typically 1 to 3 hours as determined by LC / MS), the reaction mixture is purified by reverse phase HPLC to afford the desired product after lyophilization.

[0800] General Procedure 2: Conversion of amines to amides

[0801] To a stirred solution of the amine compound in dimethylformamide (0.05-0.1 M) was added triethylamine (1.2 eq), the appropriate carboxylic acid (1.1 eq), followed by a solution of DMMTM (2 eq) in water (1 M). Upon completion (determined by LC / MS, typically 16 h), the reaction mixture was purified by reverse phase HPLC to provide the desired product after lyophilization.

[0802] General Procedure 3: Conversion of amines to sulfonamides

[0803] To a stirred solution of the amine compound in dimethylformamide (0.05M to 0.1M) was added DIPEA (3 equiv.) followed by the appropriate sulfonyl chloride. Upon completion (as determined by LC / MS, typically 16 hours), the reaction mixture was purified by reverse phase HPLC to provide the desired product after lyophilization.

[0804] General Procedure 4: Two-step conversion of amines to ureas (Synthetic Scheme IV)

[0805] Step 1: Add p-nitrophenyl carbonate (1 equivalent) to a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05M to 0.1M), 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 reverse phase HPLC to provide the desired PNP-carbamate intermediate after lyophilization. This intermediate can be used to generate a single analog or divided into multiple batches to generate multiple analogs in the second step. Step 2: Add appropriate primary amine (3 equivalents) to the PNP-carbamate intermediate (0.1M to 0.2M) in dimethylformamide. After completion (determined by LC / MS, typically 1 hour), the reaction mixture is purified by reverse phase HPLC to provide the desired product after lyophilization.

[0806] General Procedure 5: Conversion of Amines to Carbamates

[0807] To a stirred solution of the amine compound in dichloromethane or dimethylformamide (0.05M to 0.1M) is added p-nitrophenyl carbonate (1 equivalent) followed by triethylamine (2 equivalents). After completion (determined by LC / MS, typically 1 to 4 hours), the appropriate alcohol is added to the resulting PNP-carbamate intermediate. After completion (determined by LC / MS, typically 1 to 16 hours), the reaction mixture is purified by reverse phase HPLC to provide the desired product after lyophilization.

[0808] General Procedure 6: Removal of Boc Protecting Group

[0809] To a stirred solution of the Boc-protected amine compound in dichloromethane (0.1 M) was added TFA (20 vol%). After completion (typically 1 hour as determined by LC / MS), the reaction mixture was concentrated in vacuo to afford a crude solid or purified as described in General Procedure 9.

[0810] General Procedure 7: Copper-mediated amide coupling

[0811] To a rapidly stirred solution (0.02 M) of Boc-GGFG-OH (3 eq) and HOAt (3 eq) in a 10% v / v mixture of dimethylformamide in dichloromethane was added EDC (HCl salt, 3 eq). After 5 min, a solution (0.02 M) of the amine-containing payload (1 eq) in a 10% v / v mixture of dimethylformamide in dichloromethane was added, followed immediately by the addition of CuCl 2 (4 equiv.) After completion (typically 1 to 16 h as determined by LC / MS), the reaction mixture was concentrated in vacuo to afford a crude solid or purified by preparative HPLC to afford the desired product after lyophilization.

[0812] General Procedure 8: MT Placement

[0813] To a stirred solution of the amine compound (1 eq.) in dimethylformamide (about 0.02 M) was added a solution of MT-OTfp (1.2 eq. to 1.5 eq.) in acetonitrile (about 0.02 M), followed by DIPEA (10 μL, 4 eq.). Upon completion (determined by LC / MS, typically 1 to 16 hours), the reaction mixture was concentrated in vacuo to provide a crude solid, which was purified by preparative HPLC to provide the desired product after lyophilization.

[0814] General Procedure 9: Compound Purification

[0815] Flash chromatography : The crude reaction product was used Snap Ultra columns (10 g, 25 g, 50 g or 100 g) (Biotage, Charlotte, NC) were used for purification. Isolera TM The elution was performed on an automated rapid system (Biotage, Charlotte, NC) using a linear gradient of ethyl acetate / hexane or methanol / dichloromethane. Alternatively, the elution was performed using Reverse phase flash purification was performed on a SnapUltra C18 column (12 g, 30 g, 60 g or 120 g) eluting with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was isolated by removing the organic solvent by rotary evaporation or lyophilizing the acetonitrile / water mixture.

[0816] Preparative HPLC: Reverse phase HPLC of crude compound was performed using 5-μm C18 The HPLC-MS / MS ratio was 40.1:1 (150×30 mm) column (Phenomenex, Torrance, CA) was run on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) and eluted with a linear gradient of 0.1% TFA in acetonitrile / 0.1% TFA in water. The purified compound was isolated by lyophilization of the acetonitrile / water mixture.

[0817] General Procedure 10: Compound Analysis

[0818] LC / MS: Monitor the reaction for completion and use 2.6-μm C18 The purified compound was analyzed on an Agilent 1290 HPLC / 6120 single quadrupole LC / MS system (Agilent Technologies, Inc., Santa Clara, CA) (30×3 mm) column (Phenomenex, Torrance, CA) eluted with a 10% to 100% linear gradient of 0.1% formic acid / acetonitrile / 0.1% formic acid / water.

[0819] NMR: 1 H 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).

[0820] Example 1: Preparation of Camptothecin Analogs Having a Methyl Group at the C10 Position

[0821] 1.1: (S)-11-(Chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 1.1)

[0822]

[0823] The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

[0824] 1.2: (S)-11-(Aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 1.2)

[0825]

[0826] The title compound was prepared according to the procedure provided in Li et al., 2019, ACS Med. Chem. Lett., 10(10): 1386-1392.

[0827] 1.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 100)

[0828]

[0829] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 3.6 mg, 26% yield).

[0830] LC / MS: C 26 H 26 FN 3 O 5 Calculated value m / z = 479.2, found value [M+H] + =480.4.

[0831] 1 H NMR (300 MHz, CDCl 3)δ8.20(d,J=8.0Hz,1H),7.82(d,J=10.4Hz,1H),7.67(s,1H),5.77(d,J=16.4Hz,1H),5.42(s,2H),5.33(d,J=16.4Hz,1H ), 4.26 (s, 2H), 3.81 (t, J = 4.7Hz, 4H), 2.82–2.76 (m, 4H), 2.57 (d, J = 1.7Hz, 3H), 1.99–1.82 (m, 2H), 1.06 (t, J = 7.4Hz, 3H).

[0832] 1.4: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 102)

[0833]

[0834] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 3.6 mg, 21% yield).

[0835] LC / MS: C 32 H 31 FN 4 O 6 Calculated value m / z = 618.2, found value [M+H] + =619.4.

[0836] 1 H NMR (300 MHz, CDCl 3 )δ8.07(d,J=7.9Hz,1H),7.88–7.44(m,7H),5.73(d,J=16.4Hz,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=11.6,7.0Hz,2H),1.04(t,J=7.3Hz,3H).

[0837] 1.5: (S)-11-((4-((4-aminophenyl)sulfonyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 104)

[0838]

[0839] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 4.7 mg, 27% yield).

[0840] LC / MS: C 32 H 32 FN 5 O 6 Calculated value m / z = 633.2, found value [M+H] + =634.4.

[0841] 1 H NMR (300MHz, MeOD) δ8.32(d,J=8.0Hz,1H),7.85(d,J=10.5Hz,1H),7.65(s,1H),7.46(d,J=8.7Hz,2H),6.74(d,J=8.7Hz,2H),5.61(d,J= 16.5Hz,1H),5.44(s,2H),5.41(d,J=16.5Hz,1H),4.51(s,2H),3.22–3.07(m,8H),2.58(s,3H),2.03–1.93(m,2H),1.02(t,J=7.3Hz,3H).

[0842] 1.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 106)

[0843]

[0844] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and N-methylpiperazine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 3.6 mg, 25% yield).

[0845] LC / MS: C 27 H 29 FN 4 O 4 Calculated value m / z = 492.2, found value [M+H] + =493.4.

[0846] 1.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 108)

[0847]

[0848] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 3.7 mg, 23% yield).

[0849] LC / MS: C 32 H 32 FN 5 O 4 Calculated value m / z = 569.2, found value [M+H] + =570.4.

[0850] 1H NMR (300MHz, MeOD) δ8.39(d,J=8.1Hz,1H),7.79(d,J=10.6Hz,1H),7.21(d,J=9.0Hz,2H),7.14(d,J=9.0Hz,2H),5.62(d,J=16.4Hz,1H),5.49(s, 2H),5.41(d,J=16.4Hz,1H),4.45(s,2H),3.44–3.38(m,4H),3.06–3.00 (m,4H),2.58(d,J=1.8Hz,3H),2.00–1.89(m,2H),1.03(t,J=7.3Hz,3H).

[0851] 1.8: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 110)

[0852]

[0853] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and piperidine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 1.5 mg, 11% yield).

[0854] LC / MS: C 27 H 28 FN 3 O 4 Calculated value m / z = 477.2, found value [M+H] + =478.2.

[0855] 1 H NMR (300MHz, MeOD) δ8.34(d,J=7.6Hz,1H),7.94(d,J=10.3Hz,1H),7.70(s,1H),5.63(d,J=16.4Hz,1H),5.52(s,2H),5.44( d,J=16.5Hz,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=7.4Hz,3H).

[0856] 1.9: (S)-tert-butyl 4-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazine-1-carboxylate (Compound 111)

[0857]

[0858] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and tert-butyl piperazine-1-carboxylate. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 6.6 mg, 40% yield).

[0859] LC / MS: C 31 H 35 FN 4 O 6 Calculated value m / z = 578.2, found value [M+H] + =579.4.

[0860] 1.10: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-11-(piperazin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 112)

[0861]

[0862] The title compound was prepared according to General Procedure 6 starting from compound 111 (5.0 mg) to afford the title compound as an off-white solid (TFA salt, 4.4 mg).

[0863] LC / MS: C 26 H 27 FN 4 O 4 Calculated value m / z = 478.2, found value [M+H] + =479.2.

[0864] 1.11: (S)-4-ethyl-8-fluoro-4-hydroxy-11-(((R)-2-(hydroxymethyl)morpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 113)

[0865]

[0866] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and (R)-morpholin-2-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 4.6 mg, 32% yield).

[0867] LC / MS: C 27 H 28 FN 3 O 6 Calculated value m / z = 509.2, found value [M+H] + =510.4.

[0868] 1.12: (4S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)thiomorpholino)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 114)

[0869]

[0870] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and thiomorpholin-3-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 1.5 mg, 12% yield).

[0871] LC / MS: calculated value m / z = 525.6 (C 27 H 28 FN 3 O 5 S), measured value [M+H] + =526.5.

[0872] 1 H NMR (300 MHz, 10% D 2 O / CD 3CN)8.36(d,J=8.1Hz,1H),7.83(d,J=10.7Hz,1H),7.50(s,1H),5.57(d,J=16 .4Hz,1H),5.52–5.29(m,3H),5.02(d,J=14.6Hz,1H),4.71–4.54(m,1H),4.27 (dd,J=12.4,5.0Hz,1H),3.98(dd,J=12.3,3.4Hz,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=7.4Hz,3H).

[0873] 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 115)

[0874]

[0875] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 2-oxa-5-azabicyclo[2.2.1]heptan-4-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 3.5 mg, 29% yield).

[0876] LC / MS: C 28 H 28 FN 3 O 6 Calculated value m / z = 521.5, found value [M+H] + =522.5.

[0877] 1 H NMR (300 MHz, 10% D 2 O / CD 3CN)δ8.36(d,J=7.9Hz,1H),7.86(dd,J=10.6,5.0Hz,1H),7.50(d,J=1.8Hz,1H ),5.63–5.49(m,2H),5.37(dd,J=17.8,14.1Hz,2H),5.05(s,2H),4.63(d,J=2. 5Hz,1H),4.55(d,J=10.7Hz,1H),4.33(s,2H),3.92(d,J=10.7Hz,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=7.4Hz,3H).

[0878] 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 116)

[0879]

[0880] The compound 1.1 (10 mg) and 3-(hydroxymethyl)-1λ were prepared according to the general procedure 1. 6 The title compound was prepared starting with -thiomorpholine-1,1-dione. Purification was accomplished as described in General Procedure 9, using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 0.2 mg, 2% yield).

[0881] LC / MS: calculated value m / z = 557.6 (C 27 H 28 FN 3 O 7 S), measured value [M+H] + =558.4.

[0882] 1 H NMR (300 MHz, 10% D 2 O / CD 3CN)δ8.44(d,J=8.2Hz,1H),7.80(d,J=11.0Hz,1H),7.50(s,1H),5.58(d, J=16.5Hz,1H),5.45–5.26(m,3H),4.60(d,J=14.9Hz,1H),4.33(d,J=14. 7Hz,1H),3.88(d,J=4.8Hz,2H),3.41-2.85(m,4H),2.53(s,2H),2.19(p, J=2.5Hz,2H),1.74(p,J=2.5Hz,2H),1.27(s,2H),0.95(t,J=7.4Hz,3H).

[0883] 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 117)

[0884]

[0885] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 3-azabicyclo[3.1.1]heptan-6-ol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 1.3 mg, 11% yield).

[0886] LC / MS: C 28 H 28 FN 3 O 5 Calculated value m / z = 505.5, found value [M+H] + =506.6.

[0887] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ8.25(d,J=7.9Hz,1H),7.87(d,J=10.6Hz,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=7.3Hz,3H).

[0888] 1.16: (S)-4-Ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 118)

[0889]

[0890] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 3-fluoroazetidin-3-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 1.4 mg, 12% yield).

[0891] LC / MS: calculated value m / z = 497.5 (C 26 H 25 F 2 N 3 O 5 ), measured value [M+H] + =498.4.

[0892] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ8.24(d,J=7.9Hz,1H),7.85(d,J=10.7Hz,1H),7.50(s,1H),5.57(d,J=16.5Hz,1H),5.48–5.28(m, 3H), 4.98 (s, 2H), 4.44–4.14 (m, 4H), 3.78 (d, J = 14.9Hz, 2H), 2.01-1.86 (m, 2H), 0.95 (t, J = 7.4Hz, 3H).

[0893] 1.17: (S)-4-ethyl-8-fluoro-4-hydroxy-11-((3-(hydroxymethyl)azetidin-1-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 119)

[0894]

[0895] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and azetidin-3-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 0.5 mg, 4.5% yield).

[0896] LC / MS: C 26 H 26 FN 3 O 5 Calculated value m / z = 479.5, found value [M+H] + =480.4.

[0897] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ8.23(d,J=7.8Hz,1H),7.90(d,J=10.6Hz,1H),7.53(s,1H),5.58(d,J=16.5Hz,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=3.9Hz,2H),2.58(s,3H),2.01-1.86(m,2H),0.96(t,J=7.4Hz,3H).

[0898] 1.18: (4S)-11-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 120)

[0899]

[0900] The title compound was prepared starting from compound 1.1 (10 mg) and 4,4-difluoropiperidin-3-ylmethanol according to General Procedure 1. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the title compound as an off-white solid (TFA salt, 4 mg, 32% yield).

[0901] LC / MS: calculated value m / z = 543.5 (C 28 H28 F 3 N 3 O 5 ), measured value [M+H] + =544.4.

[0902] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ8.25(d,J=8.0Hz,1H),7.77(dd,J=10.7,1.4Hz,1H),7.47(s,1H),5.55(d,J=16.5Hz,1H),5.42–5.25(m,3H),4.66(d, J=3.2Hz,2H),3.90–3.77(m,1H),3.71–3.45(m,4H),2.24(q,J=11.8,9.2Hz,2H),2.01-1.86(m,2H),0.94(t,J=7.4Hz,3H).

[0903] 1.19: (S)-4-ethyl-8-fluoro-4-hydroxy-11-((1-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-7-yl)methyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 121)

[0904]

[0905] The title compound was prepared according to General Procedure 1 starting from compound 1.1 (10 mg) and 7-azabicyclo[2.2.1]hept-1-ylmethanol. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 0.8 mg, 6.6% yield).

[0906] LC / MS: C 29 H 30 FN 3 O 5 Calculated value m / z = 519.6, found value [M+H] + =520.4.

[0907] 1 H NMR (300 MHz, 10% D 2 O / CD 3CN)δ8.22(s,1H),7.92(d,J=10.7Hz,1H),7.54(s,1H),5.59(dd,J=17.6,7.6Hz,2H),5.33(t,J=17.4Hz,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=14.0Hz,5H),0.96(t,J=7.4Hz,3H).

[0908] 1.20: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 122)

[0909]

[0910] The title compound was prepared according to General Procedure 3 starting from compound 1.2 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound as an off-white solid (0.8 mg, 7% yield).

[0911] LC / MS: C 23 H 22 FN 3 O 6 Calculated value for S m / z = 487.1, found value [M+H] + =488.2.

[0912] 1 H NMR (300MHz, MeOD) δ8.33(d,J=8.1Hz,1H),7.83(d,J=10.8Hz,1H),7.68(s,1H),5.62(d,J=16.3Hz,1H),5.52(s ,2H),5.42(d,J=16.4Hz,1H),4.87(s,2H),3.06(s,3H),2.59(s,3H),2.06-1.93(m,2H),1.03(t,J=7.4Hz,3H).

[0913] 1.21: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-(4-nitrophenyl)methanesulfonamide (Compound 124)

[0914]

[0915] The title compound was prepared according to General Procedure 3 starting from compound 1.2 (20 mg) and (4-nitrophenyl)methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound (5.0 mg, 17% yield) as an off-white solid.

[0916] LC / MS: C 29 H 25 FN 4 O 8 Calculated value for S m / z = 608.1, found value [M+H] + =609.2.

[0917] 1 H NMR (300 MHz, CDCl 3 )δ8.02–7.92(m,3H),7.74(d,J=10.5Hz,1H),7.65(s,1H),7.33(d,J=8.6Hz,2H),5.66(d,J=16.8Hz,1H),5.28(d,J=16.5 Hz,1H),5.14(d,J=5.4Hz,2H),4.67(s,2H),4.28(d,J=6.3Hz,2H),3.39(s,3H),2.03–1.83(m,2H),1.04(t,J=7.4Hz,3H).

[0918] 1.22: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 125)

[0919]

[0920] The title compound was prepared according to General Procedure 3 starting from compound 1.2 (10 mg) and benzenesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 50% CH3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (9.8 mg, 73% yield) as an off-white solid.

[0921] LC / MS: C 28 H 24 FN 3 O 6 Calculated value for S m / z = 549.6, found value [M+H] + =550.6.

[0922] 1 H NMR (300MHz, DMSO-d6) δ8.60(t,J=6.2Hz,1H),8.17(d,J=8.1Hz,1H),7.83(d,J =10.8Hz,1H),7.71(dd,J=7.1,1.7Hz,2H),7.66–7.48(m,2H),7.46(dd,J=8.3,6 .8Hz,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 = 6.2Hz, 2H), 2.48 (s, 3H), 1.98–1.76 (m, 2H), 0.89 (t, J = 7.3Hz, 3H).

[0923] 1.23: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (Compound 1.23)

[0924]

[0925] The title compound was prepared according to General Procedure 3 starting from compound 1.2 (75 mg) and 4-nitrobenzenesulfonyl chloride. A 12 g C18 column was used as described in General Procedure 9 with 5% to 75% CH 3 CN / H 2 Purification of the title compound was accomplished by gradient elution with 0 + 0.1% TFA to afford the title compound as an off-white solid (37.8 mg, 47% yield).

[0926] LC / MS: C 28 H 23 FN 4 O 8 Calculated value for S m / z = 594.6, found value [M+H] + =595.2.

[0927] 1.24: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 127)

[0928]

[0929] To a solution of compound 1.23 (37.8 mg, 0.064 mmol) in methanol (6.4 mL) was added platinum 1% vanadium 2% on carbon (75 mg). The flask was heated with H 2 Purge, then incubate at room temperature in H 2 Stir under atmosphere for 45 min.The mixture was filtered through a pad of celite, washed with DMF and the filtrate was evaporated to give the title compound (30 mg, 84% yield) as a pale yellow solid.

[0930] LC / MS: C 28 H 24 FN 4 O 6 Calculated value for S m / z = 564.6, found value [M+H] + =565.2.

[0931] 1 H NMR (300MHz, DMSO-d6) δ8.13(d,J=8.2Hz,1H),8.02(t,J=6.2Hz,1H),7.88(d,J=10.8Hz,1H),7.48–7.35(m,2H),7.31(d,J =8.4Hz,1H),6.63–6.45(m,2H),5.45(s,2H),5.36(s,2H),4.50(d,J=6.3Hz,2H),1.98–1.75(m,2H),0.89(t,J=7.3Hz,3H).

[0932] 1.25: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 129)

[0933]

[0934] The title compound was prepared according to General Procedure 3 starting from compound 1.2 (20 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 25% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (1.3 mg, 13% yield).

[0935] LC / MS: C 24 H 24 FN 3 O 7 Calculated value for S m / z = 517.1, found value [M+H] + =518.2.

[0936] 1 H NMR (300MHz, DMSO-d6) δ8.30(d,J=8.4Hz,1H),7.91(d,J=10.9Hz,1H),7.84(t,J=6.3Hz,1H),7.33(s,1H),5.50-5.33(m,4H),5 .07(t,J=5.4Hz,1H),4.78(d,J=6.0Hz,2H),4.07(s,3H),3.80(dt,J=6.3Hz,J=5.8Hz,2H),1.86(m,2H),0.87(d,J=7.3Hz,3H).

[0937] 1.26: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 131)

[0938]

[0939] To a solution of chlorosulfonyl isocyanate (3 μL) in dichloromethane (1 mL) was added tert-butyl alcohol (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 was stirred for 1 hour, then concentrated to dryness. Preparative HPLC purification of the intermediate Boc compound was completed as described in General Procedure 9, with 10% to 50% CH 3 CN / H 20 + 0.1% TFA gradient elution. To the purified solid in dichloromethane (1 mL) was added trifluoroacetic acid (200 μL). The reaction was stirred for 16 hours and then concentrated to dryness to give the title compound (7.5 mg, 48% yield) as an off-white solid.

[0940] LC / MS: C 22 H 21 FN 4 O 6 Calculated value for S: m / z = 488.1, found [M+H] + =489.0.

[0941] 1 H NMR(300MHz,MeOD)δ8.25(d,J=8.1Hz,1H),7.73(d,J=10.7Hz,1H),7.62(s,1H),5.59(d,J=16.4Hz,1H),5.45 (s,2H),5.39(d,J=16.4Hz,1H),4.81(s,2H),2.55(d,J=1.7Hz,3H),2.07–1.89(m,2H),1.03(t,J=7.4Hz,3H).

[0942] 1.27: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamic acid 4-nitrophenyl ester (Compound 1.27)

[0943]

[0944] The title PNP-carbamate intermediate compound was prepared starting from compound 1.2 (24 mg) according to the first step of General Procedure 4. A 12 g column C18 column was used as described in General Procedure 9 with 10% to 50% CH 3 CN / H 2 Purification was accomplished by gradient elution with 0.05% TFA to afford the title compound (14 mg, 53% yield) as an off-white solid.

[0945] LC / MS: C 29 H 23 FN 4 O 8 Calculated value for S m / z = 574.2, found value [M+H] + =575.2

[0946] 1.28: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (Compound 132)

[0947]

[0948] The title compound was prepared according to General Procedure 4 starting from compound 1.2 (25 mg) and aqueous methylamine as the primary amine (500 μL, 40 wt% in water). In this case, the crude intermediate PNP-carbamate was used. Preparative HPLC purification was accomplished as described in General Procedure 9 with 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound (8.9 mg, 31% yield) as an off-white solid.

[0949] LC / MS: C 24 H 23 FN 4 O 5 Calculated value m / z = 466.2, found value [M+H] + =467.2.

[0950] 1 H NMR (300MHz, MeOD) δ8.26(d,J=8.2Hz,1H),7.79(d,J=10.7Hz,1H),7.66(s,1H),5.61(d,J=16.3Hz,1H),5.48(s ,2H),5.41(d,J=16.4Hz,1H),4.97(s,2H),2.73(s,3H),2.57(s,3H),2.08–1.93(m,2H),1.03(t,J=7.4Hz,3H).

[0951] 1.29: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (Compound 134)

[0952]

[0953] The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 12% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (0.6 mg, 20% yield).

[0954] LC / MS: C 30 H 28 FN 5 O 5 Calculated value m / z = 557.2, found value [M+H] + =558.4.

[0955] 1 H NMR (300MHz, MeOD) δ8.25(d,J=8.1Hz,1H),7.80(d,J=10.8Hz,1H),7.67(s,1H),7.43(d,J=8.2Hz,2H),7.24(d,J=8.3Hz,2H),5.63(d,J=16 .4Hz,1H),5.48(s,2H),5.43(d,J=16.4Hz,1H),5.01(s,2H),4.37(s,2H),2.56(d,J=1.7Hz,3H),2.05–1.94(m,2H),1.03(t,J=7.3Hz,3H).

[0956] 1.30: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (Compound 136)

[0957]

[0958] The title compound was prepared according to the second step of General Procedure 4 using compound 1.27 (4 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (2.4 mg, 66% yield).

[0959] LC / MS: C 25 H 25 FN4 O 6 Calculated value m / z = 496.2, found value [M+H] + =497.2.

[0960] 1 H NMR (300MHz, MeOD) δ8.08(d,J=8.0Hz,1H),7.74(d,J=10.5Hz,1H),7.68(s,1H),5.64(d,J=16.4Hz,1H),5.41(s,2H),5.31(d,J =16.4Hz,1H),4.96(s,2H),3.63(t,J=5.2Hz,2H),3.29(t,J=5.3Hz,2H),2.54(s,3H),1.98–1.87(m,2H),1.01(t,J=7.4Hz,3H).

[0961] 1.31: (S)-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 138)

[0962]

[0963] The title compound was prepared according to General Procedure 5 starting from compound 1.2 (50 mg) and reacting methanol with the intermediate PNP-carbamate. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (3.5 mg, 6% yield).

[0964] LC / MS: C 24 H 22 FN 3 O 6 Calculated value m / z = 467.2, found value [M+H] + =468.2.

[0965] 1H NMR (300MHz, MeOD) δ8.17(d,J=8.2Hz,1H),7.77(d,J=10.5Hz,1H),7.69(s,1H),5.65(d,J=16.5Hz,1H),5.48(s,2H) ,5.33(d,J=16.4Hz,1H),4.86(d,J=5.6Hz,2H),3.65(s,3H),2.56(s,3H),2.02–1.89(m,2H),1.02(t,J=7.4Hz,3H).

[0966] 1.32: (S)-2-Hydroxyethyl ((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 139)

[0967]

[0968] The title compound was prepared according to General Procedure 5 starting from compound 1.2 (18 mg) and reacting 1,2-ethanediol with the intermediate PNP-carbamate. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound (4.2 mg, 19% yield) as an off-white solid.

[0969] LC / MS: C 25 H 24 FN 3 O 7 Calculated value m / z = 497.2, found value [M+H] + =498.2.

[0970] 1 H NMR (300MHz, DMSO) δ8.23(d,J=8.2Hz,1H),7.78(d,J=10.7Hz,1H),7.40(s,1H),5.47(d,J=16.5Hz,1H),5.42(s,2H),5.34(d, J=16.4Hz,1H),4.77(s,2H),3.99(t,J=4.9Hz,2H),3.64–3.38(m,2H),2.48(s,3H),2.02–1.67(m,2H),0.89(t,J=7.3Hz,3H).

[0971] Example 2: Preparation of Camptothecin Analogs Having a Methoxy Group at the C10 Position

[0972] 2.1: 1-(2-amino-4-fluoro-5-methoxyphenyl)-2-chloroethane-1-one (Compound 2.1)

[0973]

[0974] A solution of 3-fluoro-4-methoxyaniline (10 g, 71 mmol) in DCM (100 mL) was cooled to 0°C. First, 1 M BCl 3 1M chloro(diethyl)aluminate in DCM (71 mL, 71 mmol) was then added, followed by 1M chloro(diethyl)aluminate in DCM (71 mL, 71 mmol), and finally 2-chloroacetonitrile (6.4 g, 85 mmol). The solution was heated at reflux for 3 hours, cooled to room temperature and quenched by adding 2M HCl aqueous solution. The resulting heterogeneous mixture was heated at reflux for 1 hour, cooled to room temperature, and then washed with Na 2 CO 3 The pH was adjusted to about 12. The layers were separated and the aqueous layer was extracted with DCM (3 x 100 mL). The combined organic layers were washed with Na 2 SO 4 Dry, concentrate and flash purify as described in General Procedure 9 eluting with 0% to 20% EtOAc / hexanes to afford the title compound (6 g, 28 mmol, 39% yield).

[0975] LC / MS: C 9 H 9 ClFNO 2 Calculated value m / z = 217.1, found value [M+H] + =218.1.

[0976] 1 H NMR (400 MHz, CDCl 3 )δ7.19(d,J=9.2Hz,1H),6.44(d,J=12.8Hz,1H),4.59(s,2H),3.86(s,3H)

[0977] 2.2: (S)-11-(Chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 2.2)

[0978]

[0979] 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-pyrans [3,4-f] indolizine-3,6,10 (4H)-trione (2 g, 7.6 mmol) in toluene. The solution was heated at 140 ° C for 3 hours and then cooled to room temperature. The product as a yellow precipitate was collected by filtration to obtain the title compound (1.27 g, 2.85 mmol, 37.5% yield).

[0980] LC / MS: C 22 H 18 CIF 2 O 5 Calculated value m / z = 445.2, found value [M+H] + =445.1.

[0981] 1 H NMR(400MHz, DMSO-d6)δ7.99(d,J=12.0Hz,1H)7.80(d,J=9.2Hz,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=7.2Hz,3H)

[0982] 2.3: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 101)

[0983]

[0984] The title compound was prepared according to General Procedure 1 starting from compound 2.2 (10 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 41% to 60% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (5.6 mg, 20% yield) as an off-white solid.

[0985] LC / MS: C 26 H 26 FN 3 O 6 Calculated value m / z = 495.2, found value [M+H] + =496.4.

[0986] 1 H NMR(300MHz,MeOD)δ7.84–7.70(m,2H),7.59(s,1H),5.62(d,J=16.3Hz,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=7.4Hz,3H).

[0987] 2.4: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 103)

[0988]

[0989] The title compound was prepared according to General Procedure 1 starting from compound 2.2 (10 mg) and 1-(phenylsulfonyl)piperazine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (2.5 mg, 14% yield).

[0990] LC / MS: C 32 H 31 FN 4 O 7 Calculated value for S m / z = 634.2, found [M+H] + =635.4.

[0991] 2.5: (S)-11-((4-((4-aminophenyl)sulfonyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 105)

[0992]

[0993] The title compound was prepared according to General Procedure 1 starting from compound 2.2 (10 mg) and 4-(piperazin-1-ylsulfonyl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H2 Gradient elution with 0.05% TFA afforded the title compound (4.0 mg, 23% yield) as an off-white solid.

[0994] LC / MS: C 32 H 32 FN 5 O 7 Calculated value for S: m / z = 649.2, found [M+H] + =650.4.

[0995] 1 H NMR (300MHz, DMSO) δ8.08(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=7.3Hz,3H).

[0996] 2.6: (S)-4-Ethyl-8-fluoro-4-hydroxy-9-methoxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 107)

[0997]

[0998] The title compound was prepared according to General Procedure 1 starting from compound 2.2 (10 mg) and N-methylpiperazine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (2.1 mg, 19% yield).

[0999] LC / MS: C 27 H 29 FN 4 O 5 Calculated value m / z = 508.2, found value [M+H] + =509.4.

[1000] 2.7: (S)-11-((4-(4-aminophenyl)piperazin-1-yl)methyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 109)

[1001]

[1002] The title compound was prepared according to General Procedure 1 starting from compound 2.2 (10 mg) and 4-(piperazin-1-yl)aniline. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (3.2 mg, 20% yield).

[1003] LC / MS: C 32 H 32 FN 5 O 5 Calculated value m / z = 585.2, found value [M+H] + =586.4.

[1004] 1 H NMR(300MHz,MeOD)δ7.83–7.74(m,2H),7.62(s,1H),7.06(d,J=8.9Hz,2H),6.98(d,J=8.9Hz,2H) ,5.65(d,J=16.4Hz,1H),5.36(s,2H),5.27(d,J=16.4Hz,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=7.4Hz,3H).

[1005] 2.8: (S)-11-(Aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 2.8)

[1006]

[1007] To a solution of compound 2.2 (250 mg, 0.56 mmol) in ethanol (7 mL) was added hexamethylenetetramine (236 mg, 1.7 mmol) followed by iPr 2NEt (100 μL, 0.56 mmol). The solution was heated to reflux for 5 h, cooled to room temperature and quenched with 12 M aqueous HCl (60 μL). The solution was concentrated to about 1 / 2 volume, and 1 M aqueous HCl (1.5 mL) was added, stirred for 5 min, and then concentrated to give a brown residue. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column with 5% to 40% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (179 mg, 75% yield) as a light yellow solid.

[1008] LC / MS: C 22 H 20 FN 3 O 5 Calculated value m / z = 425.4, found value [M+H] + =426.2

[1009] 2.9: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 123)

[1010]

[1011] The title compound was prepared according to General Procedure 3 starting from compound 2.8 (10 mg) and methanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 5% to 65% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (8.5 mg, 91% yield) as an off-white solid.

[1012] LC / MS: C 23 H 22 FN 3 O 7 Calculated value for S m / z = 503.1, found value [M+H] + =504.2.

[1013] 1H NMR (300MHz, DMSO-d6) δ7.98(d,J=12.1Hz,1H),7.89(t,J=6.4Hz,1H),7.80(d,J=9.1Hz,1H),7.28(s,1H),5.42 (s,2H),5.39(s,2H),4.77(d,J=6.4Hz,2H),4.06(s,3H),3.06(s,3H),1.95-1.73(m,2H),0.88(d,J=7.3Hz,3H).

[1014] 2.10: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 126)

[1015]

[1016] The title compound was prepared according to General Procedure 3 starting from compound 2.8 (7.5 mg) and benzenesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 5% to 70% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound (4.6 mg, 46% yield) as an off-white solid.

[1017] LC / MS: C 28 H 24 FN 3 O 7 Calculated value for S m / z = 565.6, found value [M+H] + =566.2.

[1018] 1 H NMR (300MHz, DMSO-d6) δ8.59(t,J=6.3Hz,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=8.3Hz,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=7.3Hz,2H).

[1019] 2.11: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4-nitrobenzenesulfonamide (Compound 2.11)

[1020]

[1021] The title compound was prepared according to General Procedure 3 starting from compound 2.8 (12 mg) and 4-nitrobenzenesulfonyl chloride. A 12 g C18 flash column was used as described in General Procedure 9 and the column was heated from 5% to 75% CH 3 CN / H 2 Purification was accomplished by gradient elution with 0.05% TFA to afford the title compound (9.7 mg, 71% yield) as a light yellow solid.

[1022] LC / MS: C 28 H 23 FN 4 O 9 Calculated value for S m / z = 610.6, found value [M+H] + =611.5.

[1023] 2.12: (S)-4-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)benzenesulfonamide (Compound 128)

[1024]

[1025] To a solution of compound 2.11 (9.7 mg, 0.016 mmol) in methanol (1.6 mL) was added platinum 1% vanadium 2% on carbon (15 mg). The flask was heated with H 2 Purge, then incubate at room temperature in H 2 Stir under atmosphere for 45 min.The mixture was filtered through a pad of celite, washed with DMF and the filtrate was evaporated to give the title compound (1.5 mg, 16% yield) as a light yellow solid.

[1026] LC / MS: C 28 H 25 FN 4 O 7 Calculated value for S m / z = 580.6, found value [M+H] + =581.4.

[1027] 1H NMR (300MHz, MeOD) δ7.77(d,J=11.0Hz,1H),7.58(s,1H),7.48(d,J=8.6Hz,1H),6.61(d,J=8.6Hz,1H),5.59(d,J=16.3Hz,1H),5.39(d ,J=16.4Hz,1H),5.30(s,1H),4.56(s,1H),4.10(d,J=3.7Hz,3H),2.04–1.91(m,2H),1.31(s,1H),1.02(t,J=7.3Hz,3H),0.90(s,1H).

[1028] 2.13: (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 130)

[1029]

[1030] The title compound was prepared according to General Procedure 3 starting from compound 2.8 (8 mg) and 2-hydroxyethanesulfonyl chloride. Preparative HPLC purification was accomplished as described in General Procedure 9 using 15% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (2.2 mg, 22% yield).

[1031] LC / MS: C 24 H 24 FN 3 O 8 Calculated value for S m / z = 533.1, found value [M+H] + =534.2.

[1032] 1 H NMR(300MHz, DMSO-d6)δ7.99(d,J=12.2Hz,1H),7.89-7.79(m,2H),7.29(s,1H),5.43(s,2H),5.40(s,2H),4.76(d, J=6.4Hz,2H),4.06(s,3H),3.81(t,J=6.3Hz,2H),3.34(t,J=6.3Hz,2H),1.94-1.75(m,2H),0.87(d,J=7.4Hz,3H).

[1033] 2.14: (S)-4-nitrophenyl ((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 2.14)

[1034]

[1035] The title PNP-carbamate intermediate compound was prepared according to the first step of General Procedure 4 starting from compound 2.8 (65 mg) and using a 1:1 mixture of dimethylformamide and dichloromethane as solvent. Flash purification was accomplished as described in General Procedure 9 using a 12 g C12 column with 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (61 mg, 86% yield). This intermediate was isolated and used to generate the following compounds.

[1036] LC / MS: C 29 H 23 FN 4 O 9 Calculated value m / z = 590.1, found value [M+H] + =591.2.

[1037] 2.15: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (Compound 133)

[1038]

[1039] The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and aqueous methylamine (500 uL, 40 wt% in water) as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 47% to 60% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (5.8 mg, 20% yield) as an off-white solid.

[1040] LC / MS: C 24 H 23 FN 4 O 6 Calculated value m / z = 482.2, found value [M+H]+ =483.2.

[1041] 1 H NMR (300MHz, DMSO-d6) δ8.00–7.87(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).

[1042] 2.16: (S)-1-(4-aminobenzyl)-3-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)urea (Compound 135)

[1043]

[1044] The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and 4-(aminomethyl)aniline as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 2.1 mg, 12% yield).

[1045] LC / MS: C 30 H 28 FN 5 O 6 Calculated value m / z = 573.2, found value [M+H] + =574.2.

[1046] 1 H NMR (300MHz, MeOD) δ7.79(d,J=11.9Hz,1H),7.74(d,J=9.0Hz,1H),7.59(s,1H),7.43(d,J=8.2Hz,2H),7.25(d,J=8.2Hz,2H) ,5.61(d,J=16.3Hz,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=7.4Hz,3H).

[1047] 2.17: (S)-1-((4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (Compound 137)

[1048]

[1049] The title compound was prepared according to the second step of General Procedure 4 using compound 2.14 (15 mg) as the PNP-carbamate and hydroxyethylamine as the primary amine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 12% to 60% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (1.5 mg, 20% yield).

[1050] LC / MS: C 25 H 25 FN 4 O 7 Calculated value m / z = 512.2, found value [M+H] + =513.2.

[1051] 1 H NMR (300MHz, MeOD) δ7.93(d,J=12.1Hz,1H),7.88(d,J=9.2Hz,1H),7.56(s,1H),5.62(d,J=16.2Hz,1H),5.52(s,2H),5.45(d,J =16.3Hz,1H),4.98(s,2H),4.17(s,3H),3.59(t,J=5.6Hz,2H),3.28(t,J=5.6Hz,2H),2.10–1.91(m,2H),1.05(t,J=7.3Hz,3H).

[1052] Example 3: Preparation of Camptothecin Analogs Having an Amino Group at the C10 Position

[1053] 3.1: 5-Bromo-4-fluoro-2-nitrobenzaldehyde (Compound 3.1)

[1054]

[1055] to HNO at 0°C 3 (121.2 mL, 67% purity, 2.0 equiv) in H 2 SO 43-Bromo-4-fluorobenzaldehyde (180 g, 1.0 equiv.) was added to the stirred solution (500 mL) in 4% paraformaldehyde (M+H) (5% paraformaldehyde). After the addition was complete, the ice bath was removed and the reaction 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.

[1056] 1 H NMR (400 MHz, CDCl 3 )δ10.39(s,1H),8.23(d,J=6.8Hz,1H),7.91(d,J=7.6Hz,1H).

[1057] 3.2: tert-Butyl (2-fluoro-5-formyl-4-nitrophenyl)carbamate (Compound 3.2)

[1058]

[1059] Compound 3.1 (219 g, 1.0 equivalent), tert-butyl carbamate (124 g, 1.2 equivalent), Cs 2 CO 3 (575 g, 2.0 eq.), Pd 2 (dba) 3 A mixture of 2-nitro-1-pyrrolidone (40 g, 0.05 eq.) and XPhos (84 g, 0.2 eq.) in toluene (2000 mL) was degassed and heated with N 2 The mixture was then purged for three cycles at 90 °C and N 2 The reaction mixture was stirred under a 4% CO atmosphere for 15 hours. 2 O (800 mL) and extracted with EtOAc (300 mL×2). The combined organic layers were washed with brine (200 mL×2), then dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=100:1 to 20:1) to give the title compound (140 g, 56% yield) as a yellow solid.

[1060] 1 H NMR (400MHz, DMSO-d6) δ10.24(s,1H),9.94(s,1H),8.42(d,J=7.6Hz,1H),8.16(d,J=10.8Hz,1H),1.50(s,9H)

[1061] 3.3: tert-Butyl (4-amino-2-fluoro-5-formylphenyl)carbamate (Compound 3.3)

[1062]

[1063] Compound 3.2 (100 g, 1.0 eq.) was added in H 2 To the solution of O (300 mL) and EtOH (1200 mL) was added NH 4 Cl (30.5 g, 1.62 eq.) was added in portions at 80 °C. Iron (78.6 g, 4.0 eq.) was added 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 (SiO 2 , petroleum ether:ethyl acetate=1:0 to 0:1), TLC (petroleum ether) to give the title compound (19.0 g, 21% yield) as a yellow solid.

[1064] LC / MS: C 12 H 15 FN 2 O 3 Calculated value m / z = 254.1, found value [M+H] + =255.0.

[1065] 1 H NMR (400MHz, DMSO-d6) δ9.73 (s, 1H), 8.57 (s, 1H), 7.58 (d, J = 4.8Hz, 1H), 7.21 (s, 2H), 6.53 (d, J = 12.8Hz, 1H), 1.43 (s, 9H).

[1066] 3.4: (S)-tert-butyl (4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.4)

[1067]

[1068] A mixture of compound 3.3 (4.20 g, 1.2 eq.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.5 g, 1 eq.) and TsOH (monohydrate, 253 mg, 0.1 eq.) in toluene (350 mL) was stirred for 2 hours at 110 ° C. The reaction solution was cooled to 25 ° C, filtered, and the solid was washed with methyl tert-butyl ether (30 mL) and then dried in vacuo. The title compound (4.5 g, 62% yield) was obtained as a yellow solid.

[1069] LC / MS: C 25 H 24FN 3 O 6 Calculated value m / z = 481.2, found value [M+H] + =482.1.

[1070] 1 H NMR (400MHz, DMSO-d6) δ9.49(s,1H),8.65(s,1H),8.43(d,J=8.4Hz,1H),7.95(d,J=12.0Hz,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=7.2Hz,3H)

[1071] 3.5: (S)-tert-butyl (4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.5)

[1072]

[1073] To a mixture of compound 3.4 (4.00 g) in MeOH (360 mL) was added FeSO 4 (heptahydrate, 1.2 g), H 2 SO 4 (280mL) in H 2 The reaction mixture was heated at 65 °C while H 2 O 2 (24 mL, 30% purity) and then stirred for 0.5 h. The reaction solution was cooled to 25 °C and then filtered to obtain the title compound as a yellow solid (1.53 g, 33.2% yield). H 2 O (400 mL), and then saturated with Na 2 S 2 O 3 The aqueous solution was quenched. 2 CO 3 The aqueous solution adjusted the pH to 7-8, then the solution was concentrated and filtered. The solid was triturated with MeOH (30 mL) at 55°C for 1 h, then filtered to give a second crop of the title compound (1.09 g, 26% yield) as a brown solid.

[1074] LC / MS: C 26 H 26 FN 3 O7 Calculated value m / z = 511.2, found value [M+H] + =512.2.

[1075] 1 H NMR(300MHz,d6-DMSO)δ9.47(s,1H),8.47(d,J=7.6Hz,1H),7.94(d,J=12.0Hz,1H),7.29(d,J=1.6Hz,1H),6.49(s,1H),5 .86-5.76(m,1H),5.42(s,2H),5.38(s,2H),5.16(d,J=4.4Hz,2H),1.90-1.83(m,2H),1.52(s,9H),0.88(t,J=6.4Hz,3H).

[1076] 3.6: (S)-tert-butyl (4-ethyl-8-fluoro-11-formyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.6)

[1077]

[1078] In a 50 mL round-bottom flask containing compound 3.5 (150 mg, 0.293 mmol), DCM (2.9 mL) was added, followed by Dess-Martin periodinane (0.56 g, 1.32 mmol) and water (15.8 μL, 0.88 mmol). The solution was stirred at room temperature for 18 hours, then diluted with DCM and washed with saturated NaHCO 3 Aqueous and brine washes. The layers were separated and the combined organic layers were evaporated onto celite. Flash purification was accomplished as described in General Procedure 9 using a 10 g silica column eluting with 0% to 10% DCM / MeOH to give the title product as an orange powder (42.5 mg, 28%).

[1079] LC / MS: C 26 H 24 FN 3 O 7 Calculated value m / z = 509.2, found value [M+H] + =510.4.

[1080] 1H NMR (300 MHz, acetone-d6) δ11.10 (s, 1H), 9.68 (d, J = 8.6 Hz, 1H), 8.81 (s, 1H), 8.04 (d, J = 11.9 Hz, 1H), 7.63 (s, 1H), 5.73 (s, 2H), 5.69 (d, J = 16.2 Hz, 1H), 5.42 (d, J = 16.2 Hz, 1H), 2.02-1.95 (m, 2H), 8.47 (d, J=7.6Hz,1H),7.94(d,J=12.0Hz,1H),7.29(d,J=1.6Hz,1H),6.49(s,1H),5.86-5.76(m,1H),5.42 (s,2H),5.38(s,2H),5.16(d,J=4.4Hz,2H),1.90-1.83(m,2H),1.52(s,9H),0.88(t,J=6.4Hz,3H).

[1081] 3.7: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 140)

[1082]

[1083] The title compound was prepared according to General Procedure 6 starting from compound 3.4 (40 mg) to afford the title compound as a red solid (TFA salt, 36 mg, 87% yield).

[1084] LC / MS: C 20 H 16 FN 3 O 4 Calculated value m / z = 381.1, found value [M+H] + =382.2.

[1085] 1 H NMR (300MHz, DMSO) δ8.28(s,1H),7.72(d,J=12.5Hz,1H),7.21(d,J=7.3Hz,1H),5.43(d,J= 16.2Hz, 1H), 5.34 (d, J = 16.2Hz, 1H), 5.17 (s, 2H), 1.92–1.74 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[1086] 3.8: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(hydroxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 141)

[1087]

[1088] The title compound was prepared according to General Procedure 6 starting from compound 3.5 (5 mg) to afford the title compound as a red solid (TFA salt, 4.1 mg, 78% yield).

[1089] LC / MS: C 21 H 18 FN 3 O 5 Calculated value m / z = 411.2, found value [M+H] + =412.2.

[1090] 1 H NMR(300MHz,MeOD)δ7.71(d,J=12.2Hz,1H),7.60(s,1H),7.29(d,J=9.5Hz,1H),5.61(d,J=16.3Hz ,1H),5.47(s,2H),5.40(d,J=16.3Hz,1H),5.25(s,2H),2.03–1.94(m,2H),1.03(t,J=7.4Hz,3H).

[1091] 3.9: (S)-tert-butyl (11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.9)

[1092]

[1093] To a stirred solution of compound 3.5 (100 mg) in dichloromethane (5 mL) was added a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL). After 1 hour, a solution of thionyl chloride (14 μL) in dichloromethane (0.1 mL) was added. After another hour, the reaction was diluted with dichloromethane (10 mL) and toluene (1 mL) and then concentrated in vacuo to give the title compound as a red solid, which was used in subsequent reactions without further purification.

[1094] LC / MS: C 26 H 25 CIF 3 O6 Calculated value m / z = 529.1, found value [M+H] + =530.2.

[1095] 3.10: (S)-tert-butyl (11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.10)

[1096]

[1097] To a solution of compound 3.9 (100 mg) in ethanol (500 μL) was added hexamethylenetetramine (79 mg) followed by DIPEA (99 μL). The solution was heated at 60°C for 16 hours and then concentrated to dryness in vacuo. Flash purification was accomplished as described in General Procedure 9 using a 12 g C18 column with 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as an off-white solid (TFA salt, 29 mg, 24% yield).

[1098] LC / MS: C 26 H 27 FN 4 O 6 Calculated value m / z = 510.2, found value [M+H] + =511.4.

[1099] 1 H NMR(300MHz,MeOD)δ8.88(d,J=8.2Hz,1H),7.96(d,J=11.9Hz,1H),7.62(s,1H),5.60(d,J=16.4Hz,1H), 5.48(s,2H),5.41(d,J=16.4Hz,1H),4.80(s,2H),2.07–1.89(m,2H),1.64(s,9H),1.02(t,J=7.3Hz,3H).

[1100] 3.11: (S)-9-amino-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 145)

[1101]

[1102] The title compound was prepared according to General Procedure 6 starting from compound 3.10 (2.1 mg) to afford the title compound as a red solid (TFA salt, 1.8 mg, 100% yield).

[1103] LC / MS: C 21 H 19 FN 4 O 4 Calculated value m / z = 410.1, found value [M+H] + =411.2.

[1104] 1 H NMR(300MHz,MeOD)δ7.82(d,J=12.1Hz,1H),7.60(s,1H),7.37(d,J=9.1Hz,1H),5.61(d,J=16.3Hz ,1H),5.42(s,2H),5.41(d,J=16.3Hz,1H),4.69(s,2H),2.08–1.94(m,2H),1.03(t,J=7.4Hz,3H).

[1105] Example 3.12: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 3.12)

[1106]

[1107] The title compound was prepared according to General Procedure 1 starting from compound 3.9 (150 mg) and morpholine. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound as a red solid (TFA salt, 103 mg, 52% yield).

[1108] LC / MS: C 30 H 33 FN 4 O 7 Calculated value m / z = 580.2, found value [M+H] + =581.4.

[1109] 1H NMR (300MHz, MeOD) δ9.06(d,J=8.3Hz,1H),7.93(d,J=12.0Hz,1H),7.66(s,1H),5.63(d,J=16.3Hz,1H),5.51(s,2H),5. 43(d,J=16.4Hz,1H),4.92(s,2H),3.84(s,4H),3.10(s,4H),1.99(d,J=5.5Hz,2H),1.63(s,9H),1.03(t,J=7.4Hz,3H).

[1110] 3.13: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(morpholinomethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 142)

[1111]

[1112] The title compound was prepared according to General Procedure 6 starting from compound 3.12 (45 mg) to afford the title compound as a red solid (TFA salt, 37 mg, 99% yield).

[1113] LC / MS: C 25 H 25 FN 4 O 5 Calculated value m / z = 480.2, found value [M+H] + =481.4.

[1114] 1 H NMR(300MHz,MeOD)δ7.73(d,J=12.0Hz,1H),7.54(s,1H),7.48(d,J=9.2Hz,1H),5.60(d,J=16.3Hz,1H),5.4 7–5.34(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=7.3Hz,3H).

[1115] 3.14: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(piperidin-1-ylmethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 148)

[1116]

[1117] To a 5 mL flask containing compound 3.6 (37 mg, 0.067 mmol) was added dichloromethane (1.45 mL), followed by acetic acid (18.69 μL, 0.327 mmol), piperidine (21.52 μL, 0.218 mmol) and sodium triacetoxyborohydride (23.0 mg, 0.109 mmol). The solution was then stirred at room temperature for 2 hours, quenched by adding water + 0.1% TFA and DMF (1: 1, 1.0 mL), and partially evaporated. Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and 5% to 40% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the Boc-protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to give the title compound as a yellow solid (TFA salt, 32.5 mg, 98% yield).

[1118] LC / MS: C 26 H 27 FN 4 O 4 Calculated value m / z = 478.2, found value [M+H] + =479.4.

[1119] 1 H NMR(300MHz,MeOD)δ7.78(d,J=12.1Hz,1H),7.56(s,1H),7.41(d,J=9.1Hz,1H),5.60(d,J=16.4Hz,1H),5.4 7–5.35(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=7.4Hz,3H).

[1120] 3.15: (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-methylpiperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 149)

[1121]

[1122] To a 2 mL vial containing compound 3.6 (15 mg, 0.029 mmol) was added dichloromethane (0.59 mL), acetic acid (7.58 μL, 0.132 mmol) and N-methylpiperazine (4.90 μL, 0.044 mmol). The solution was stirred at room temperature for 4 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% aqueous TFA (0.5 mL). Purification was accomplished as described in General Procedure 9 using a 12 g C18 flash column and 5% to 40% CH 3 CN / H 2 Gradient elution with 0.05% TFA gave the Boc protected intermediate as a yellow powder. This intermediate was deprotected according to General Procedure 6 to give the title product as a yellow solid (TFA salt, 1.5 mg, 7.1% yield).

[1123] LC / MS: C 26 H 28 FN 5 O 4 Calculated value m / z = 493.2, found value [M+H] + =494.4.

[1124] 1 H NMR(300MHz,MeOD)δ7.68(d,J=12.2Hz,1H),7.56(s,1H),7.53(d,J=9.5Hz,1H),5.60(d,J=16.3Hz,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=7.4Hz,3H).

[1125] 3.16: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-((4-(phenylsulfonyl)piperazin-1-yl)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 153)

[1126]

[1127] The Boc-protected precursor of the title compound was prepared starting from compound 3.9 (10 mg) and 1-(phenylsulfonyl)piperazine according to General Procedure 1. Preparative HPLC was performed as described in General Procedure 9 with 35% to 44% CH3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the Boc protected intermediate as a yellow powder. This intermediate was then deprotected according to General Procedure 6 to afford the title compound (TFA salt, 2.4 mg, 17% yield over 2 steps).

[1128] LC / MS: C 31 H 30 FN 5 O 6 Calculated value for S: m / z = 619.2, found [M+H] + =520.4.

[1129] 1H NMR (300MHz, MeOD) δ7.81-7.60(m,7H),7.34(s,1H),5.51(d,J=16.4Hz,1H),5.35(d,J=16.4Hz,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=7.4Hz,3H).

[1130] 3.17: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (Compound 147)

[1131]

[1132] The title compound was prepared starting from compound 3.10 (8 mg) and acetic acid according to General Procedure 2 followed by General Procedure 6. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 O + 0.1% TFA gradient elution gave the title compound as a red solid (4.0 mg, 56% yield).

[1133] LC / MS: C 23 H 21 FN 4 O 5 Calculated value m / z = 452.2, found value [M+H] + =453.2.

[1134] 1H NMR(300MHz,MeOD)δ7.69(d,J=12.1Hz,1H),7.56(s,1H),7.38(d,J=9.3Hz,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=7.4Hz,3H).

[1135] 3.18: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)methanesulfonamide (Compound 146)

[1136]

[1137] The title compound was prepared starting from compound 3.10 (8 mg) and methanesulfonyl chloride according to General Procedure 3 followed by General Procedure 6. Preparative HPLC purification of the intermediate Boc protected compound was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 O + 0.1% TFA gradient elution gave the title compound as a red solid (4.4 mg, 57% yield).

[1138] LC / MS: C 22 H 21 FN 4 O 6 Calculated value for S: m / z = 488.1, found [M+H] + =489.2.

[1139] 1 H NMR(300MHz,MeOD)δ7.74(d,J=12.2Hz,1H),7.60(s,1H),7.49(d,J=9.3Hz,1H),5.61(d,J=16.2Hz,1H), 5.45(s,2H),5.40(d,J=16.2Hz,1H),4.78(s,2H),3.05(s,3H),2.08–1.94(m,2H),1.03(t,J=7.4Hz,3H).

[1140] 3.19: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyethane-1-sulfonamide (Compound 150)

[1141]

[1142] The title compound was prepared starting 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 intermediate Boc-protected compound was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 O + 0.1% TFA gradient elution gave the title compound as a red solid (1 mg, 16% yield).

[1143] LC / MS: C 23 H 23 FN 4 O 7 Calculated value for S m / z = 518.5, found value [M+H] + =519.5.

[1144] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ7.77–7.61(m,1H),7.48–7.30(m,2H),5.53(d,J=16.3Hz,1H),5.31(d,J=15.4Hz,3H),4.69(s,2H),3 .97(dd,J=6.6,4.9Hz,2H),3.39(t,J=5.8Hz,2H),2.93(s,1H),1.99-1.83(m,2H),0.94(t,J=7.3Hz,3H).

[1145] 3.20: (S)-4-nitrophenyl ((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 3.20)

[1146]

[1147] To a solution of compound 3.10 (10 mg, 0.02 mmol) in DMF (400 μL, 0.05 M) was added 4-nitrophenyl carbonate (12 mg, 0.04 mmol) and diisopropylethylamine (6.8 μL, 0.04 mmol). The solution was stirred at room temperature for about 30 minutes and then used directly in the subsequent reaction.

[1148] 3.21: (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)carbamate (Compound 143)

[1149]

[1150] The title compound was prepared by adding MeOH (100 μL) to a 200 μL solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9 with 10% to 60% CH 3 CN / H 2 Gradient elution of O + 0.1% TFA. The title compound was obtained as a red solid according to General Procedure 6 (2.1 mg, 47% yield).

[1151] LC / MS: C 23 H 21 FN 4 O 6 Calculated value m / z = 468.4, found value [M+H] + =468.3.

[1152] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ7.72(d,J=12.2Hz,1H),7.41(d,J=18.1Hz,1H),6.96(s,1H),5.52(d,J=3.6Hz,1H),5.39–5.23(m, 3H), 4.82 (s, 1H), 4.73 (s, 1H), 3.63 (d, J = 1.2Hz, 3H), 1.56 (s, 3H), 1.27 (s, 2H), 0.94 (t, J = 7.4Hz, 3H).

[1153] 3.22: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylurea (Compound 144)

[1154]

[1155] The reaction mixture was prepared by adding methylamine hydrochloride (10 mg) to a 200 μL solution of compound 3.20, followed by the addition of iPr 2 NEt (5 μL) was used to prepare the title compound. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9 with 10% to 60% CH 3 CN / H 2 O + 0.1% TFA gradient elution. The title compound was obtained as a red solid according to General Procedure 6 (2.9 mg, 64.5% yield).

[1156] LC / MS: C 23 H 21 FN 5 O 5 Calculated value m / z = 467.5, found value [M+H] + =468.5.

[1157] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ8.13(d,J=9.2Hz,1H),7.92(s,1H),7.73(d,J=12.3Hz,1H),7.52–7.35(m,2H),6.94(d,J=9.2Hz,2H),5.55(d,J=16 .5Hz,2H),5.44–5.27(m,4H),4.85(s,2H),4.78(s,1H),1.56(d,J=2.5Hz,3H),1.27(s,2H),0.93(q,J=11.7,9.5Hz,3H).

[1158] 3.23: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-(2-hydroxyethyl)urea (Compound 151)

[1159]

[1160] The title compound was prepared by adding ethanolamine (100 μL) to a 200 μL solution of compound 3.20. The solution was stirred at room temperature for 30 minutes. Preparative HPLC purification of the intermediate Boc-protected compound was accomplished as described in General Procedure 9 with 10% to 60% CH 3 CN / H 2 Gradient elution of O + 0.1% TFA. The title compound was obtained as a red solid according to General Procedure 6 (0.5 mg, 8.5% yield).

[1161] LC / MS: C 24 H 24 FN 5 O 6 Calculated value m / z = 497.5, found value [M+H] + =498.5.

[1162] 1 H NMR (300 MHz, 10% D 2 O / CD 3 CN)δ7.77–7.61(m,1H),7.48–7.30(m,2H),5.53(d,J=16.3Hz,1H),5.31(d,J=15.4Hz,1H),5.19(s,2H),4.69(s ,2H),3.97(dd,J=6.6,4.9Hz,2H),3.39(t,J=5.8Hz,2H),2.93(s,1H),2.01-1.83(m,2H),0.94(t,J=7.3Hz,3H).

[1163] 3.24: (S)-9-Amino-11-(azidomethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 152)

[1164]

[1165] To a stirred solution of compound 3.5 (100 mg) in 2 mL of dichloromethane was added thionyl chloride (35 mL, 2.5 equiv). The solution was stirred at room temperature for 20 minutes before further thionyl chloride (35 mL, 2.5 equiv) was added. After 20 minutes, toluene (1 mL) was added and the reaction mixture was then concentrated in vacuo. The crude solid was suspended in DMSO (1 mL) and sodium azide (19 mg, 1.5 equiv) was added. The solution was stirred at room temperature for 16 hours. Purification was accomplished as described in General Procedure 9 with 5% to 50% CH 3 CN / H2 Gradient elution with 0.05% TFA afforded the title compound as an off-white solid (20 mg, 23% yield).

[1166] LC / MS: C 21 H 17 FN 6 O 4 Calculated value m / z = 436.1, found value [M+H] + =437.2.

[1167] 1 H NMR(300MHz,MeOD)δ7.75(d,J=12.2Hz,1H),7.60(s,1H),7.38(d,J=9.3Hz,1H),5.61(d, J=16.3Hz,1H),5.46–5.35(m,3H),5.07(s,2H),2.03–1.97(m,2H),1.03(t,J=7.3Hz,3H).

[1168] 3.25: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (Compound 164)

[1169]

[1170] The title compound was prepared according to General Procedure 2 starting from compound 145 (10 mg) and glycolic acid. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 45% CH 3 CN / H 2 O + 0.1% TFA gradient elution to obtain the title compound as a yellow solid (6.9 mg, 60% yield).

[1171] LC / MS: C 23 H 21 FN 4 O 6 Calculated value m / z = 468.1, found value [M+H] + =469.2.

[1172] 1H NMR (300MHz, MeOD)7.70(d,J=12.2Hz,1H),7.60(s,1H),7.42(d,J=9.4Hz,1H),5.62(d,J=16.3Hz,1H),5.43( s,2H),5.36(d,J=16.2Hz,1H),4.95(d,J=5.9Hz,2H),4.08(s,2H),2.04–1.90(m,1H),1.03(t,J=7.4Hz,3H).

[1173] 3.26: (S)-1-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3-methylthiourea (Compound 161)

[1174]

[1175] To a solution of compound 145 (9 mg, 1.0 eq.) in DMF (1 mL) was added thiocarbonyldiimidazole (6 mg, 1.5 eq.) followed by DIPEA (8 μL, 2.0 eq.). The resulting solution was stirred at 25 °C for 2 hours after which complete conversion to the isothiocyanate intermediate was observed. Methylammonium chloride (3 mg, 2.0 eq.) was then added and the reaction mixture was heated at 60 °C for 30 minutes. Preparative HPLC purification was accomplished as described in General Procedure 9 with 10% to 45% CH 3 CN / H 2 O + 0.1% TFA gradient elution gave the title compound as a yellow solid (2.3 mg, 22% yield).

[1176] LC / MS: C 23 H 22 FN 5 O 4 Calculated value for S m / z = 483.1, found value [M+H] + =484.2.

[1177] 1 H NMR(300MHz,MeOD)δ7.70(d,J=12.0Hz,1H),7.60(s,1H),7.38(d,J=9.3Hz,1H),5.62(d,J=16.2Hz,1H), 5.36(s,2H),5.31(d,J=16.2Hz,1H),5.30(s,2H),3.04(s,3H),1.99–1.90(m,2H),1.02(t,J=7.4Hz,3H).

[1178] 3.27: (S)-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)thiocarbamic acid S-(2-hydroxyethyl) ester (Compound 160)

[1179]

[1180] The title compound was prepared starting from compound 145 (10 mg) and 2-mercaptoethanol according to General Procedure 5. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 45% CH 3 CN / H 2 O + 0.1% TFA gradient elution gave the title compound as a yellow solid (4.2 mg, 43% yield).

[1181] LC / MS: C 24 H 23 FN 4 O 6 Calculated value for S: m / z = 514.1, found [M+H] + =515.2.

[1182] 1 H NMR (300MHz, MeOD) δ7.71(d,J=12.1Hz,1H),7.60(s,1H),7.36(d,J=9.4Hz,1H),5.62(d,J=16.3Hz,1H),5.42(s,2H),5.35(d, J=16.2Hz,1H),4.88(d,J=4.6Hz,2H),3.68(t,J=6.4Hz,2H),3.03(t,J=6.5Hz,2H),2.04–1.92(m,2H),1.03(t,J=7.4Hz,3H).

[1183] 3.28: (S)-9-Amino-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 154)

[1184]

[1185] To a 5 mL flask containing compound 140 (50 mg) were added water (0.72 mL), FeSO 4(heptahydrate, 11.0 mg) and propionaldehyde (74 μL). The resulting suspension was cooled to -15 °C using an ice-salt bath, and sulfuric acid (0.40 mL) was then added dropwise. Hydrogen peroxide (95 μL) was then added dropwise. The mixture was stirred at -15 °C for 10 minutes, then warmed to room temperature and stirred for 2 hours. The reaction mixture was diluted with water (30 mL) and the resulting suspension was extracted with DCM (3×30 mL). The organic phase was then evaporated to dryness. Preparative HPLC purification was completed as described in General Procedure 9 with 25% to 70% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (2.4 mg, 4.4% yield) as a dark orange solid.

[1186] LC / MS: C 22 H 20 FN 3 O 4 Calculated value m / z = 410.1, found value [M+H] + =410.2.

[1187] 1 H NMR (300MHz, MeOD) δ7.63(d,J=12.3Hz,1H),7.55(s,1H),7.36(d,J=9.4Hz,1H),5.57(d,J=16.4Hz,1H),5.37(d,J =16.4Hz,1H),5.21(s,2H),3.13(q,J=7.7Hz,2H),2.02–1.90(m,2H),1.38(t,J=7.7Hz,3H),1.01(t,J=7.3Hz,3H).

[1188] 3.29: (S)-tert-butyl (11-((carbamoyloxy)methyl)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.29)

[1189]

[1190] Compound 3.5 (15 mg) was added to a 5 mL conical flask containing a solution of chlorosulfonyl isocyanate (7.7 μL) in dimethylformamide (0.29 mL) at -20°C. The resulting suspension was stirred at -20°C for 5 minutes. Water (59 μL) was added and the reaction mixture was allowed to warm to room temperature and stirred for 2 hours, then heated at 70°C for 1 hour. The reaction mixture was cooled to room temperature and partially evaporated. Preparative HPLC purification was performed as described in General Procedure 9 with 40% to 55% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound as a dark orange solid (5.1 mg, 31% yield).

[1191] LC / MS: C 27 H 27 FN 4 O 8 Calculated value m / z = 555.2, found value [M+H] + =555.2.

[1192] 1 H NMR(300MHz, DMSO-d6)δ9.53(s,1H),8.56(d,J=8.5Hz,1H),8.00(d,J=12.0Hz,1H),7.31(s,1H),7.11-6.62(m,2H),6.5 2(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=7.2Hz,3H).

[1193] 3.30: (S)-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)carbamic acid methyl ester (Compound 169)

[1194]

[1195] The title compound was prepared according to General Procedure 6 starting from compound 3.29 (5.1 mg) to afford the title compound as a yellow powder (TFA salt, 3.8 mg, 73% yield).

[1196] LC / MS: C 22 H 19 FN 4 O 6 Calculated value m / z = 455.1, found value [M+H] +=455.2.

[1197] 1 H NMR (300MHz, DMSO-d6) δ7.79(d,J=12.4Hz,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=7.3Hz,3H).

[1198] 3.31: ((S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(methoxymethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 155)

[1199]

[1200] MeOH / dioxane (1:1) (9.8 mL) and sulfuric acid (0.73 mL) were added to a 50 mL flask containing compound 3.5 (30 mg). The reaction mixture was then stirred at reflux 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 purified by MgSO 4 Preparative HPLC purification was performed as described in General Procedure 9 using 25% to 40% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound as a dark orange solid (5.1 mg, 16% yield).

[1201] LC / MS: C 22 H 20 FN 3 O 5 Calculated value m / z = 426.1, found value [M+H] + =426.2.

[1202] 1 H NMR(300MHz,DMSO-d6)δ7.75(d,J=12.3Hz,1H),7.24(d,J=9.9Hz,1H),7.20(s,1H),6.47(s,1H),6.30-5.9 2(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=7.3Hz,3H).

[1203] 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]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 158)

[1204]

[1205] In a 5mL conical flask containing compound 3.6 (15mg), dichloromethane (0.6mL) was added, followed by 3-azabicyclo [3.1.1] heptane-6-ol (10mg) and acetic acid (7.6μL). The reactant was stirred at room temperature and sodium triacetoxyborohydride (9.4mg) 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 completed as described in General Procedure 9, with 20% to 50% CH 3 CN / H 2 Gradient elution with 0.05% 4% TFA gave the Boc-protected title compound as a yellow powder. Deprotection was performed according to General Procedure 6 and the resulting residue was purified by preparative HPLC as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 O + 0.1% TFA gradient elution afforded the title compound as a yellow powder (TFA salt, 7.1 mg, 39% yield).

[1206] LC / MS: C 27 H 27 FN 4 O 5 Calculated value m / z = 507.2, found value [M+H] + =507.4.

[1207] 1 H NMR(300MHz, DMSO-d6)δ7.85(d,J=12.1Hz,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=7.3Hz,3H).

[1208] 3.33: (S)-9-amino-4-ethyl-8-fluoro-11-((3-fluoro-3-(hydroxymethyl)azetidin-1-yl)methyl)-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 159)

[1209]

[1210] In a 5 mL conical flask containing compound 3.6 (15 mg), dichloromethane (0.6 mL) was added, followed by (3-fluoroazetidine-3-yl)methanol (9.3 mg) and acetic acid (7.6 μL). The reactants were 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 completed as described in General Procedure 9, with 20% to 50% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the Boc-protected title compound as a yellow powder. Deprotection was then performed according to General Procedure 6. The resulting residue was purified by preparative HPLC as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound as a yellow powder (TFA salt, 1.8 mg, 10% yield).

[1211] LC / MS: C 25 H 24 F 2 N 4 O 5 Calculated value m / z = 499.2, found value [M+H] + =499.4.

[1212] 1 H NMR(300MHz,DMSO-d6)δ7.82(d,J=12.4Hz,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=7.3Hz,3H).

[1213] 3.34: tert-Butyl-(S)-(4-ethyl-8-fluoro-4-hydroxy-11-((methylamino)methyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Compound 3.34)

[1214]

[1215] To a stirred solution of compound 3.9 (210 mg) in DMF (5 mL) was added sodium iodide (5.9 mg) followed by methylammonium chloride (107 mg). The reaction mixture was then stirred at room temperature overnight. Reverse phase purification was accomplished as described in General Procedure 9 using a 30 g C18 column with 10% to 65% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (15.0 mg, 7.2% yield) as a yellow solid.

[1216] LC / MS: C 27 H 29 FN 4 O 6 Calculated value m / z = 524.2, found value [M+H] + =525.4.

[1217] 3.35: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-N-methylacetamide (Compound 165)

[1218]

[1219] The Boc-protected form of the title compound was prepared starting from compound 3.34 (6.4 mg) and glycolic acid according to General Procedure 2. Preparative HPLC purification was accomplished as described in General Procedure 9 using 20% ​​to 50% CH 3 CN / H 2 Gradient elution of O + 0.1% TFA. Deprotection was then performed according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.0 mg, 28% yield).

[1220] LC / MS: C 24 H 23 FN 4 O 6 Calculated value m / z = 482.2, found value [M+H] +=483.2.

[1221] 1 H NMR (300MHz, DMSO-d6) δ7.79(d,J=12.3Hz,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=7.3Hz,3H).

[1222] 3.36: (S)-N-((9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-N-methylmethanesulfonamide (Compound 166)

[1223]

[1224] The Boc-protected form of the title compound was prepared starting from compound 3.34 (8.0 mg) and methanesulfonyl chloride according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 9 with 10% to 50% CH 3 CN / H 2 Gradient elution with O + 0.1% TFA. Deprotection was then performed according to General Procedure 6 to afford the title compound as a yellow powder (TFA salt, 2.6 mg, 34% yield).

[1225] LC / MS: C 23 H 23 FN 4 O 6 Calculated value for S m / z = 502.1, found value [M+H] + =503.2.

[1226] 1 H NMR(300MHz,DMSO-d6)δ7.81(d,J=12.3Hz,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).

[1227] 3.37: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(2-methoxyethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 170)

[1228]

[1229] To a 10 mL round-bottom flask containing compound 3.4 (62.0 mg) were added water (0.89 mL), FeS O 4 (heptahydrate, 18.0 mg) and 3-methoxypropanal (113.0 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 minutes, then allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was then diluted with water (30 mL) and the resulting suspension was extracted with DCM (3×30 mL). The organic phase was evaporated to dryness. Preparative HPLC purification was performed as described in General Procedure 9 with 25% to 45% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as a dark orange solid (TFA salt, 3.1 mg, 4.4% yield).

[1230] LC / MS: C 23 H 22 FN 3 O 5 Calculated value m / z = 440.2, found value [M+H] + =440.2.

[1231] 1 H NMR (300MHz, DMSO-d6) δ7.75(d,J=12.4Hz,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=6.5Hz,2H),3.24(s,3H),3.23(t,J=6.5Hz,2H),1.96-1.76(m,2H),0.88(t,J=7.3Hz,3H).

[1232] 3.38: (S)-N-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)acetamide (Compound 171)

[1233]

[1234] To a 25 mL round bottom flask containing acetic acid (0.071 mL) in dimethylformamide (0.69 mL) was added N-methylmorpholine (0.343 mL), HOAt (0.142 g) and HATU (0.435 g). After stirring at room temperature for 5 minutes, the solution was added to a 10 mL conical flask containing compound 140 (0.127 g). The solution was stirred at room temperature for 24 hours and then directly purified by preparative HPLC as described in General Procedure 9 with 25% to 45% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (43.0 mg, 38% yield) as a bright yellow powder.

[1235] LC / MS: C 22 H 18 FN 3 O 5 Calculated value m / z = 424.1, found value [M+H] + =424.2.

[1236] 1 H NMR (300MHz, DMSO-d6) δ10.13(s,1H),8.73(d,J=8.5Hz,1H),8.61(s,1H),7.96(d,J=912.1Hz,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=7.3Hz,3H).

[1237] 3.39: tert-Butyl (5-formyl-2-methoxy-4-nitrophenyl)carbamate (Compound 3.39)

[1238]

[1239] To a solution of compound 3.2 (1.3 g, 1.0 eq.) in MeOH (12 mL) was added sodium methoxide (0.74 g, 3.0 eq.) 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 in vacuo to give the title compound (1.2 g, 89% yield) as an orange solid.

[1240] LC / MS: C13 H 16 N 2 O 6 Calculated value m / z = 296.10, found value [M+H] + =297.1.

[1241] 1 H NMR(300MHz,MeOD)δ10.29(s,1H),8.61(s,1H),7.73(s,1H),4.08(s,3H),1.57(s,9H)

[1242] 3.40: tert-Butyl (4-amino-5-formyl-2-methoxyphenyl)carbamate (Compound 3.40)

[1243]

[1244] Compound 3.39 (500 mg, 1 eq.) was dissolved in MeOH (10 mL) and H 2 Add B 2 (OH) 4 The resulting mixture was cooled to 0 ° C and 5M NaOH aqueous solution (2.75mL) was added under stirring within 10 minutes. The reaction mixture was stirred for another 5 minutes and then quenched by pouring the solution into ice (40mL). The resulting mixture was extracted with DCM (3×50mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Rapid purification was completed as described in General Procedure 9, using a 25g silica gel column and eluted with 10% to 50% hexane / EtOAc to give the title compound (386mg, 86%) as an orange solid.

[1245] LC / MS: C 13 H 18 N 2 O 4 Calculated value m / z = 266.1, found value [M+H] + =297.2.

[1246] 3.41: (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 168)

[1247]

[1248] A mixture of compound 3.40 (385 mg, 1.0 equiv) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (362 mg, 0.95 equiv), TsOH (monohydrate, 25 mg, 0.1 equiv) and toluene (30 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred for 2 hours at 110 °C. The reaction mixture was then cooled to 25 °C and concentrated in vacuo. Purification was accomplished as described in General Procedure 9 using a 25 g silica gel column and eluting with a 0% to 50% DCM / MeOH gradient to give a Boc-protected intermediate as a red solid. The material was then deprotected according to General Procedure 6 and subsequently purified by preparative HPLC as described in General Procedure 9 using 20% ​​to 65% CH 3 CN / H 2 Gradient elution with 0.05% TFA + 0.1% TFA afforded the title compound as a red solid (TFA salt, 300 mg, 53% yield).

[1249] LC / MS: C 21 H 19 N 3 O 5 Calculated value m / z = 393.2, found value [M+H] + =393.2.

[1250] 1 H NMR(300MHz,MeOD)δ8.27(s,1H),7.62(s,1H),7.42(s,1H),7.11(s,1H),5.61(d,J=16.2Hz,1H ), 5.38 (d, J = 16.2Hz, 1H), 5.24 (s, 2H), 4.11 (s, 3H), 2.06–1.91 (m, 2H), 1.04 (t, J = 7.4Hz, 3H).

[1251] 3.42: 5-Bromo-2-nitro-4-(trifluoromethyl)benzaldehyde (Compound 3.42)

[1252]

[1253] to HNO at 0°C 3 (2.0 g, 1.4 mL, 67% purity, 2 eq.) in H 2 SO 43-Bromo-4-(trifluoromethyl)benzaldehyde (4 g, 1 eq.) was added to a stirred solution in 4% paraformaldehyde (8 mL). After the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 5 hours. The mixture was poured into ice (100 mL) and the precipitate was extracted with DCM (3×100 mL). The combined organic fractions were then washed with brine (50 mL) and purified by Na 2 SO 4 Drying and concentration in vacuo gave the title compound as a yellow solid (4.4 g, 93% yield).

[1254] LC / MS: C 8 H 3 F 3 NO 3 Calculated value m / z = 296.90, found value [M+H] + =298.0.

[1255] 1 H NMR (300MHz, MeOD) δ10.35(s,1H),8.29(s,1H),8.23(s,1H).

[1256] 3.43: tert-Butyl (5-formyl-4-nitro-2-(trifluoromethyl)phenyl)carbamate (Compound 3.43)

[1257]

[1258] Compound 3.42 (800 mg, 1 equivalent), tert-butyl carbamate (378 mg, 1.2 equivalents), Cs 2 CO 3 (1.7 g, 2 equivalents), Pd 2 (dba) 3 (122 mg, 0.05 eq.) and dicyclohexyl[2',4',6'-tri(propan-2-yl)[1,1'-biphenyl]-2-yl]phosphane ( A mixture of 2-X XPhos (256 mg, 0.2 eq.) in toluene (5 mL) was degassed and heated to 4 °C with N 2 The mixture was then purged for three cycles at 90 °C and N 2 The reaction mixture was stirred under a 4% CO atmosphere for 15 hours. 2 The mixture was stirred for 2 hours at 4 ℃ for 10 minutes. The mixture was diluted with 4% 4-D- 4-piperidin- ate (25 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (2×25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification was accomplished according to General Procedure 9 using a 25 g silica gel column and eluting with 0% to 25% DCM / MeOH to give the title compound (750 mg, 84% yield) as an orange solid.

[1259] LC / MS: C 13 H 13 FN 2 O 5 Calculated value m / z = 334.1, found value [MH] - =333.1.

[1260] 3.44: tert-Butyl (4-amino-5-formyl-2-(trifluoromethyl)phenyl)carbamate (Compound 3.44)

[1261]

[1262] Compound 3.43 (750 mg, 1 eq.) was dissolved in MeOH (16 mL) and H 2 Add B to the solution in O (1.6 mL) 2 (OH) 4 The resulting mixture was cooled to 0 ° C and 5M NaOH aqueous solution (2.75mL) was added under stirring within 10 minutes. The reaction mixture was stirred for another 5 minutes and then quenched by pouring the solution into ice (50mL). The resulting mixture was extracted with DCM (3×75mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Rapid purification was completed as described in General Procedure 9, using a 25g silica gel column and eluted with 10% to 50% hexane / EtOAc to give the title compound (460mg, 67%) as an orange solid.

[1263] LC / MS: C 13 H 15 F 3 N 2 O 3 Calculated value m / z = 304.1, found value [M+H] + =305.2

[1264] 3.45: (S)-9-amino-4-ethyl-4-hydroxy-8-(trifluoromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 167)

[1265]

[1266] A mixture of compound 3.44 (460 mg, 1 eq.) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (378 mg, 0.95 eq.), TsOH (monohydrate, 26 mg, 0.1 eq.) and toluene (35 mL) in a 250 mL round-bottom flask equipped with a Dean-Stark apparatus was stirred at 110 °C for 2 hours. The reaction mixture was then cooled to 25 °C and concentrated in vacuo. Purification was accomplished as described in General Procedure 9 using a 25 g silica gel column and eluting with a 0% to 50% DCM / MeOH gradient to give a Boc-protected intermediate as a red solid. The material was then deprotected according to General Procedure 6 and subsequently purified by preparative HPLC as described in General Procedure 9 using 20% ​​to 65% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (6.2 mg, 48%) as a yellow solid.

[1267] LC / MS: C 21 H 16 F 3 N 3 O 4 Calculated value m / z = 431.1, found value [M+H] + =432.2.

[1268] 1 H NMR (300MHz, MeOD) δ8.29(s,1H),8.27(s,1H),7.59(s,1H),7.24(s,1H),5.59(d,J=16. 3Hz, 1H), 5.39 (d, J = 16.3Hz, 1H), 5.28 (s, 2H), 2.00–1.89 (m, 2H), 1.03 (t, J = 7.4Hz, 3H).

[1269] Example 4: Preparation of drug-linker

[1270] 4.1: (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine 2,5-dioxopyrrolidin-1-yl ester (Compound 4.1)

[1271]

[1272] The title compound was prepared according to the procedure described in Chinese Patent Publication No. CN105218644.

[1273] 4.2: (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (Fmoc-GGF-OH; compound 4.2)

[1274]

[1275] To a solution of L-phenylalanine (965 mg) in acetonitrile (10 mL) and dimethylformamide (0.5 mL) was added DIPEA (1.51 mL) followed by compound 4.1 (1.3 g). After 1 hour, the reaction was concentrated to dryness. Flash purification was accomplished as described in General Procedure 9 with 10% to 50% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (430 mg, 30% yield) as a white solid.

[1276] LC / MS: C 28 H 71 N 3 O 6 Calculated value for S m / z = 501.2, found value [M+H] + =502.4.

[1277] 1 H NMR (300MHz, DMSO) δ8.16(d,J=8.1Hz,1H),8.04(t,J=5.8Hz,1H),7.90(d,J=7.5 Hz,2H),7.72(d,J=7.4Hz,2H),7.59(t,J=6.0Hz,1H),7.54–7.39(m,2H),7.33(t ,J=7.6Hz,2H),7.28–7.13(m,5H),4.44(td,J=8.5,5.1Hz,1H),4.33–4.13(m,3H ),3.83–3.59(m,4H),3.06(dd,J=13.7,5.1Hz,1H),2.88(dd,J=13.8,9.0Hz,1H).

[1278] 4.3: 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (MT-OTfp; compound 4.3)

[1279]

[1280] The title compound was prepared according to the procedure described in International Patent Publication No. WO 2017 / 054080.

[1281] 4.4: (3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propionyl)glycylglycyl-L-phenylalanine (Compound 4.4)

[1282]

[1283] To a solution of compound 4.3 (1.61 g, 3.58 mmol) in DMF (35 mL) was added Gly-Gly-Phe (1 g, 3.58 mmol) in one portion, followed by iPr 2 NEt (1.25 mL, 7.2 mmol). The solution was stirred at room temperature for 1 hour and then evaporated to dryness. Purification was accomplished as described in General Procedure 9 using a 30 g C18 flash column with 10% to 90% CH 3 CN / H 2 Gradient elution with 0.05% TFA afforded the title compound as a white solid (400 mg, 20% yield).

[1284] LC / MS: C 26 H 34 N 4 O 10 Calculated value m / z = 562.6, found value [MH] - =561.5.

[1285] 1 H NMR (300 MHz, CDCl 3 )δ7.60(t,J=5.6Hz,2H),7.41(d,J=7.7Hz,1H),7.32–7.07(m,5H),6.70(s,2H),6.33–6.07(m,3H),4.72(td,J=7.6,5.3Hz,1H),4.12–3.7 8(m,4H),3.72(ddd,J=15.2,6.9,4.8Hz,5H),3.60(dd,J=11.6,6.1Hz,10H),3.12(ddd,J=48.2,14.0,6.5Hz,2H),2.52(d,J=11.7Hz,2H).

[1286] 4.5: (S)-11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (Compound 4.5)

[1287]

[1288] The title compound was prepared according to the procedure described in US Patent Publication No. US2017 / 021031.

[1289] 4.6: (S)-11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (Compound 4.6)

[1290]

[1291] The title compound was prepared according to the procedure described in US Patent Publication No. US2017 / 021031 using Fmoc-GGFGG-OH as the starting peptide.

[1292] 4.7: tert-butyl (2-((2-(((S)-1-((2-((4-((4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (Compound 4.7)

[1293]

[1294] The title compound was prepared starting from compound 104 (20 mg) according to General Procedure 7. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (14 mg, 42% yield) as a white solid.

[1295] LC / MS: C 52 H 58 N 9 O 12 Calculated value for S: m / z = 1051.4, found [M+H] + =1052.6.

[1296] 4.8: (S)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)-N-(2-((4-((4-(((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)piperazin-1-yl)sulfonyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (MT-GGFG-Compound 104)

[1297]

[1298] The title compound was prepared starting from compound 4.7 (14 mg) according to Procedure 6 followed by Procedure 8. Preparative HPLC purification was accomplished as described in General Procedure 9 using 10% to 60% CH 3 CN / H 2 Gradient elution with 0.05% O + 0.1% TFA afforded the title compound (9.1 mg, 56% yield) as a white so...

Claims

1. An antibody-drug conjugate having formula (X): T-[L-(D) m ] n (X) in: m is an integer between 1 and 4; n is an integer between 1 and 10; T is an anti-NaPi2b (sodium-dependent phosphate transporter 2B) antibody construct, which comprises an antigen binding domain that binds to human NaPi2b, wherein the antigen binding domain comprises: a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence as shown in SEQ ID NO:7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence as shown in SEQ ID NO:8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence as shown in SEQ ID NO:9, and b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence as shown in SEQ ID NO: 19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence as shown in SEQ ID NO: 20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence as shown in SEQ ID NO: 18; L is a connector, and D is a compound of formula I: in: R 1 is 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-C6alkyl)-aryl; R 4 Selected from: R 5 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl, -aryl and -(C1-C6 alkyl)-aryl; R 6 and R 7 Each is independently selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5 , -C3-C8 heterocycloalkyl and -C(O)R 17 ; R 8 Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; Each R 9 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-C6alkyl)-aryl; R 10’ Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 11 Selected from: -H and -C1-C6 alkyl; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and R 13 Selected from: -H and -C1-C6 alkyl; R 14 and R 14’ Each 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 atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 ; R 24 , R 25 and R 26 Each is -C1-C6 alkyl; X a and X b are each independently selected from: NH, O and S, and X c Selected from: O, S and S(O)2, Provided that the compound is not (S)-9-amino-11-butyl-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione.

2. The antibody-drug construct of claim 1, wherein the antigen binding domain comprises: a) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:24 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:29; b) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:24 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:30; c) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:26 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:30; d) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:25 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:30; e) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:27 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:30; f) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:27 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:29; g) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:26 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:29; h) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:25 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:29; i) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:27 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; j) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:26 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; k) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:25 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; l) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:24 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; or m) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:31 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:

32.

3. The antibody-drug conjugate according to claim 1 or 2, wherein 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: wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or X is O, and R 4a -X- is selected from: R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; Each R 9a R is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; or 9a does not exist, and X b =X; Each R 10a are 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 Absent or -C1-C6 alkyl; R 12a Selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and R 13a Selected from: -H and -C1-C6 alkyl; R 14a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 16a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 21 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-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 -C1-C6 alkyl; X a and X b Each independently selected from: NH, O and S; X c is selected from: O, S and S(O)2, and Indicates the connection point with the linker L.

4. The antibody-drug conjugate according to claim 3, wherein R 1a Selected from: -CH3, -CF3, -OCH3, -OCF3 and -NH2.

5. The antibody-drug conjugate according to claim 3, wherein R 1a Selected from: -CH3, -OCH3 and NH2.

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

7. The antibody-drug conjugate according to any one of claims 3 to 6, wherein X is -O-, -S- or -NH-, and R 4a Selected from:

8. The antibody-drug conjugate according to claim 1 or 2, wherein 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, 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 heterocycloalkyl 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 are 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; R 12 Selected from: -H, -C1-C6 alkyl, -CO2R 8 , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16 and R 13 Selected from: -H and -C1-C6 alkyl; R 14 and R 14’ Each 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 atom to which they are bound, they form a 4-, 5-, 6- or 7-membered ring having 0 to 3 substituents selected from the group consisting of halogen, -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-OR 5 ; R 24 , R 25 and R 26 Each is -C1-C6 alkyl; X a and X b Each independently selected from: NH, O and S; X c is selected from: O, S and S(O)2, and Indicates the connection point with the linker L.

9. The antibody-drug conjugate according to claim 8, wherein R 2a For F.

10. The antibody-drug conjugate according to claim 8 or 9, wherein R 20a Selected from: -H, -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5 , -(C1-C6 alkyl)-aryl, 11. The antibody-drug conjugate according to claim 1 or 2, wherein 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, wherein * is the point of attachment to X, and wherein p is 1, 2, 3 or 4; or X is O, and R 25 -X- is selected from: R 5a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 6a Selected from: -H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 7a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -(C1-C6 alkyl)-OR 5a , -C3-C8 heterocycloalkyl and -C(O)R 17a ; R 8a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; Each R 9a R is independently selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; or 9a does not exist, and X b =X; Each R 10a are 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 Absent or -C1-C6 alkyl; R 12a Selected from: -C1-C6 alkyl, -CO2R 8a , -aryl, -heteroaryl, -(C1-C6 alkyl)-aryl, -S(O)2R 16a and R 13a Selected from: -H and -C1-C6 alkyl; R 14a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 14a’ Selected from: H, -C1-C6 alkyl, -C3-C8 cycloalkyl and -C3-C8 heterocycloalkyl; R 16a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 17a Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-C3-C8 heterocycloalkyl, -aryl, -heteroaryl and -(C1-C6 alkyl)-aryl; R 21 Selected from: -C1-C6 alkyl, -C3-C8 cycloalkyl and -(C1-C6 alkyl)-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 -C1-C6 alkyl; X a and X b Each independently selected from: NH, O and S; X c is selected from: O, S and S(O)2, and Indicates the connection point with the linker L.

12. The antibody-drug conjugate according to claim 11, wherein R 2a Selected from: -CH3, -CF3, -F, -Br, -Cl, -OH, -OCH3 and -OCF3.

13. The antibody-drug conjugate according to claim 11, wherein R 2a For F.

14. The conjugate according to any one of claims 11 to 13, wherein X is -O-, -S- or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, Or X is O, and R 25 -X- is selected from:

15. The conjugate according to any one of claims 11 to 13, wherein X is -O-, -S- or -NH-, and R 25 Selected from: -C1-C6 alkyl, -(C1-C6 alkyl)-OR 5a , -(C1-C6 alkyl)-aryl, 16. The antibody-drug conjugate of any one of claims 1 to 15, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of halogen, acyl, acyloxy, alkoxy, carboxyl, hydroxy, amino, amido, nitro, cyano, azido, alkylthio, thio, sulfonyl, sulfonamido, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

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

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

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

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

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

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

23. The antibody-drug conjugate according to any one of claims 20 to 22, wherein L has: (a) Formula (XI) in: Z is a functional group capable of reacting with a target group on the anti-NaPi2b antibody construct T; Str is an extension group; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r Forming a protease cleavage site; X is a self-degrading group; q is 0 or 1; r is 1, 2, or 3; s is 0, 1, or 2; # is the connection point with the anti-NaPi2b antibody construct T, and % is the connection point with the camptothecin analog D, or (b) Formula (XII) in: Z is a functional group capable of reacting with a target group on the anti-NaPi2b antibody construct T; Str is an extension group; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r Forming a protease cleavage site; Y is -NH-CH2-; q is 0 or 1; r is 1, 2, or 3; v is 0 or 1; # is the connection point with the anti-NaPi2b antibody construct T, and % is the connection point with the camptothecin analog D.

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

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

26. The antibody-drug conjugate according to claim 1 or 2, wherein L-(D) in formula (X) is: MC-GGFG-AM-Compound 139 MT-GGFG-AM-Compound 139 MT-GGFG-AM-Compound 141 MC-GGFG-AM-Compound 141 MT-GGFG-Compound 141 MC-GGFG-Compound 141 27. The antibody-drug conjugate according to any one of claims 1 to 26, wherein m is between 1 and 2.

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

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

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

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

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

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

51.

34. The antibody-drug conjugate of claim 33, wherein L-(D) in formula (X) is: MC-GGFG-AM-Compound 139 35. An antibody-drug conjugate having the following structure: wherein n is 4; and T is an anti-NaPi2b (sodium-dependent phosphate transporter 2B) antibody construct, which comprises an antigen binding domain that binds to human NaPi2b, wherein the antigen binding domain comprises: a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence as shown in SEQ ID NO:7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence as shown in SEQ ID NO:8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence as shown in SEQ ID NO:9, and b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence as shown in SEQ ID NO:19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence as shown in SEQ ID NO:20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence as shown in SEQ ID NO:

18.

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

29.

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

51.

38. The antibody-drug construct of claim 36, 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.

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

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

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

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

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

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

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

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

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

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

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