Self-stabilizing linker conjugates

Abstract

The present disclosure provides antibody drug conjugate platforms comprising self-stabilizing linker assembly components, and antibody drug conjugates comprising a platform-derived linker payload and an antibody or antigen-binding fragment thereof. In some embodiments, the antibody drug conjugate has the following formula: TIFF2026508145000139.tif48165 or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the variables (e.g., BA, RG, R 1a , R 1b ,r,RS,R 2a , R 2b ,s,R 4 , R 3a , R 3b , t, RE, A, a', W, w', Y, y', PA, x) are as described herein.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2023 / 075152 (filed February 9, 2023), the disclosure of which is incorporated herein by reference in its entirety.

[0002] 2. Field Provided herein are antibody drug conjugate platforms, and antibody drug conjugates (ADCs) comprising the platform and an antibody or antigen-binding fragment thereof, as well as uses of the ADC platforms and ADCs. [Background technology]

[0003] 3.Background The field of antibody-drug conjugates (ADCs) has made great progress, with many ADCs entering the clinic. The linker component of ADCs is a key feature in developing optimized therapeutic agents with high activity at well-tolerated doses. The electrophilic maleimide functional group has proven useful for the preparation of ADCs due to its high specificity for reaction with thiol groups and rapid thiol addition kinetics under mild conditions.

[0004] As several researchers in the field of bioconjugates have pointed out, the thio-substituted product from the reaction of the electrophilic maleimide functionality with free thiols on antibodies is subject to slow elimination, causing the reaction to go backward. When this reversible reaction occurs in purified ADC preparations, the maleimide and thiol regenerated through the elimination process simply react again to reform the intact conjugate, resulting in little or no detectable reaction. However, if other thiols are present, the net effect may be the transfer of the maleimide from the antibody of the ADC to other available thiols. This process has been documented to occur in plasma, with the maleimide of the ADC transferring to cysteine ​​34 of serum albumin (Alley et al., Bioconjugate Chem. 2008, 19, 759-765). This process has also been reported when ADCs are incubated in the presence of excess cysteine ​​or glutathione (Jununtula et al., Nature Biotech, 2012). The present disclosure is directed, inter alia, to bioconjugates that do not undergo this transfer reaction. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Alley et al.,Bioconjugate Chem.2008,19,759-765 [Non-patent document 2] Jununtula et al.,Nature Biotech,2012 Summary of the Invention

[0006] 4. Overview Provided herein are antibody drug conjugate platforms and antibody drug conjugates (ADCs), as well as uses of the ADC platforms for preparing ADCs.

[0007] In some embodiments, provided herein are ADC compounds of formula (I): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the variables (e.g., B A, B R ... 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4 , A, W, Y, r, s, t, a', w', y', x) are as described herein.

[0008] In some embodiments, the platform comprises a linker-payload compound of formula (II): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the variables (e.g., RG, RS, RE, PA, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4 , A, W, Y, r, s, t, a', w', y') are as described herein.

[0009] In some embodiments, the platform comprises a linker compound of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the variables (e.g., RG, RS, RE, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4 , A, r, s, t, a') are as described herein.

[0010] Additional objects and advantages will be set forth in part in the detailed description that follows, and in part will be understood by or may be learned by the practice of the detailed description. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0011] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims.

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments and, together with the detailed description, serve to explain the principles described herein. [Brief explanation of the drawings]

[0013] [Figure 1A] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1B] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1C] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1D] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1E] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1F] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1G] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1H] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1I] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1J] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1K] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 1L] 1 shows maleimide hydrolysis (ring-opening) kinetic curves of conjugator-antibody conjugates. [Figure 2A] Stability data for conjugator-antibody conjugate 3-3 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 2B] Stability data for conjugator-antibody conjugate 3-3 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 2C] Stability data for conjugator-antibody conjugate 3-3 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 2D] Stability data for conjugator-antibody conjugate 3-3 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 3A] Stability data for conjugator-antibody conjugate 3-4 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 3B] Stability data for conjugator-antibody conjugate 3-4 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 3C] Stability data for conjugator-antibody conjugate 3-4 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 3D] Stability data for conjugator-antibody conjugate 3-4 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 4A] Stability data for conjugator-antibody conjugates 3-6 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 4B] Stability data for conjugator-antibody conjugates 3-6 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 4C] Stability data for conjugator-antibody conjugates 3-6 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 4D] Stability data for conjugator-antibody conjugates 3-6 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 5A] Stability data for conjugator-antibody conjugates 3-7 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 5B] Stability data for conjugator-antibody conjugates 3-7 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 5C] Stability data for conjugator-antibody conjugates 3-7 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 5D] Stability data for conjugator-antibody conjugates 3-7 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 6A] Stability data for conjugator-antibody conjugates 3-8 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 6B] Stability data for conjugator-antibody conjugates 3-8 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 6C] Stability data for conjugator-antibody conjugates 3-8 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 6D]Stability data for conjugator-antibody conjugates 3-8 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 7A] Stability data for conjugator-antibody conjugates 3-9 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 7B] Stability data for conjugator-antibody conjugates 3-9 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 7C] Stability data for conjugator-antibody conjugates 3-9 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 7D] Stability data for conjugator-antibody conjugates 3-9 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 8A] Stability data for conjugator-antibody conjugates 3-10 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 8B] Stability data for conjugator-antibody conjugates 3-10 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 8C] Stability data for conjugator-antibody conjugates 3-10 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 8D] Stability data for conjugator-antibody conjugates 3-10 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 9A] Stability data for conjugator-antibody conjugates 3-11 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 9B] Stability data for conjugator-antibody conjugates 3-11 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 9C]Stability data for conjugator-antibody conjugates 3-11 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 9D] Stability data for conjugator-antibody conjugates 3-11 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 10A] Stability data for conjugator-antibody conjugate 3-12 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 10B] Stability data for conjugator-antibody conjugate 3-12 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 10C] Stability data for conjugator-antibody conjugate 3-12 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 10D] Stability data for conjugator-antibody conjugate 3-12 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 11A] Stability data for conjugator-antibody conjugate 3-13 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 11B] Stability data for conjugator-antibody conjugate 3-13 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 11C] Stability data for conjugator-antibody conjugate 3-13 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 11D] Stability data for conjugator-antibody conjugate 3-13 are shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 12A] Stability data for conjugator-antibody conjugate 3-14 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 12B]Stability data for conjugator-antibody conjugate 3-14 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 12C] Stability data for conjugator-antibody conjugate 3-14 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 12D] Stability data for conjugator-antibody conjugate 3-14 is shown in pH 7.4 and 8.0 buffers with and without GSH. [Figure 13A] 1 shows stability data for conjugator-antibody conjugate 3-3 after 168 hours in pH 5.5 histidine buffer. [Figure 13B] 1 shows stability data for conjugator-antibody conjugate 3-3 after 168 hours in pH 5.5 histidine buffer. [Figure 14A] 1 shows stability data for conjugator-antibody conjugate 3-4 after 168 hours in pH 5.5 histidine buffer. [Figure 14B] 1 shows stability data for conjugator-antibody conjugate 3-4 after 168 hours in pH 5.5 histidine buffer. [Figure 15A] 1 shows stability data for conjugator-antibody conjugates 3-6 after 168 hours in pH 5.5 histidine buffer. [Figure 15B] 1 shows stability data for conjugator-antibody conjugates 3-6 after 168 hours in pH 5.5 histidine buffer. [Figure 16A] 1 shows stability data for conjugator-antibody conjugates 3-7 after 168 hours in pH 5.5 histidine buffer. [Figure 16B] 1 shows stability data for conjugator-antibody conjugates 3-7 after 168 hours in pH 5.5 histidine buffer. [Figure 17A]1 shows stability data for conjugator-antibody conjugates 3-8 after 168 hours in pH 5.5 histidine buffer. [Figure 17B] 1 shows stability data for conjugator-antibody conjugates 3-8 after 168 hours in pH 5.5 histidine buffer. [Figure 17C] 1 shows stability data for conjugator-antibody conjugates 3-8 after 168 hours in pH 5.5 histidine buffer. [Figure 17D] 1 shows stability data for conjugator-antibody conjugates 3-8 after 168 hours in pH 5.5 histidine buffer. [Figure 18A] 1 shows stability data for conjugator-antibody conjugate 3-9 after 168 hours in pH 5.5 histidine buffer. [Figure 18B] 1 shows stability data for conjugator-antibody conjugate 3-9 after 168 hours in pH 5.5 histidine buffer. [Figure 19A] 1 shows stability data for conjugator-antibody conjugate 3-10 after 168 hours in pH 5.5 histidine buffer. [Figure 19B] 1 shows stability data for conjugator-antibody conjugate 3-10 after 168 hours in pH 5.5 histidine buffer. [Figure 20A] 1 shows stability data for conjugator-antibody conjugate 3-11 after 168 hours in pH 5.5 histidine buffer. [Figure 20B] 1 shows stability data for conjugator-antibody conjugate 3-11 after 168 hours in pH 5.5 histidine buffer. [Figure 21A] 1 shows stability data for conjugator-antibody conjugate 3-12 after 168 hours in pH 5.5 histidine buffer. [Figure 21B] 1 shows stability data for conjugator-antibody conjugate 3-12 after 168 hours in pH 5.5 histidine buffer. [Figure 22A] 1 shows stability data for conjugator-antibody conjugate 3-13 after 168 hours in pH 5.5 histidine buffer. [Figure 22B] 1 shows stability data for conjugator-antibody conjugate 3-13 after 168 hours in pH 5.5 histidine buffer. [Figure 23A] 1 shows stability data for conjugator-antibody conjugate 3-14 after 168 hours in pH 5.5 histidine buffer. [Figure 23B] 1 shows stability data for conjugator-antibody conjugate 3-14 after 168 hours in pH 5.5 histidine buffer. [Figure 24A] 1 shows stability data for ADCs 4-6 in pH 7.4 or 8.0 GSH buffer. [Figure 24B] 1 shows stability data for ADCs 4-6 in pH 7.4 or 8.0 GSH buffer. [Figure 24C] 1 shows stability data for ADCs 4-6 in pH 7.4 or 8.0 GSH buffer. [Figure 24D] 1 shows stability data for ADCs 4-6 in pH 7.4 or 8.0 GSH buffer. [Figure 25A] 1 shows stability data for ADC 4-10 in GSH buffer at pH 7.4 or 8.0. [Figure 25B] 1 shows stability data for ADC 4-10 in GSH buffer at pH 7.4 or 8.0. [Figure 25C] 1 shows stability data for ADC 4-10 in GSH buffer at pH 7.4 or 8.0. [Figure 25D] 1 shows stability data for ADC 4-10 in GSH buffer at pH 7.4 or 8.0. [Figure 26A] 1 shows stability data for ADC 4-11 in GSH buffer at pH 7.4 or 8.0. [Figure 26B]1 shows stability data for ADC 4-11 in GSH buffer at pH 7.4 or 8.0. [Figure 26C] 1 shows stability data for ADC 4-11 in GSH buffer at pH 7.4 or 8.0. [Figure 26D] 1 shows stability data for ADC 4-11 in GSH buffer at pH 7.4 or 8.0. [Figure 27A] 1 shows stability data for ADC 4-12 in pH 7.4 or 8.0 GSH buffer. [Figure 27B] 1 shows stability data for ADC 4-12 in pH 7.4 or 8.0 GSH buffer. [Figure 27C] 1 shows stability data for ADC 4-12 in pH 7.4 or 8.0 GSH buffer. [Figure 27D] 1 shows stability data for ADC 4-12 in pH 7.4 or 8.0 GSH buffer. [Figure 28A] 1 shows stability data for ADC 4-13 in GSH buffer at pH 7.4 or 8.0. [Figure 28B] 1 shows stability data for ADC 4-13 in GSH buffer at pH 7.4 or 8.0. [Figure 28C] 1 shows stability data for ADC 4-13 in GSH buffer at pH 7.4 or 8.0. [Figure 28D] 1 shows stability data for ADC 4-13 in GSH buffer at pH 7.4 or 8.0. [Figure 29A] 1 shows stability data for ADC 4-14 in GSH buffer at pH 7.4 or 8.0. [Figure 29B] 1 shows stability data for ADC 4-14 in GSH buffer at pH 7.4 or 8.0. [Figure 29C] 1 shows stability data for ADC 4-14 in GSH buffer at pH 7.4 or 8.0. [Figure 29D]1 shows stability data for ADC 4-14 in GSH buffer at pH 7.4 or 8.0. [Figure 30A] 1 shows stability data for ADC 4-16 in GSH buffer at pH 7.4 or 8.0. [Figure 30B] 1 shows stability data for ADC 4-16 in GSH buffer at pH 7.4 or 8.0. [Figure 30C] 1 shows stability data for ADC 4-16 in GSH buffer at pH 7.4 or 8.0. [Figure 30D] 1 shows stability data for ADC 4-16 in GSH buffer at pH 7.4 or 8.0. [Figure 31A] 1 shows stability data for ADC 4-6 in formulation buffer. [Figure 31B] Showing stability data for ADC 4-6 in formulation buffer [Figure 31C] Showing stability data for ADC 4-6 in formulation buffer [Figure 31D] Showing stability data for ADC 4-6 in formulation buffer [Figure 32A] 1 shows stability data for ADC 4-10 in formulation buffer. [Figure 32B] Showing stability data for ADC 4-10 in formulation buffer [Figure 32C] Showing stability data for ADC 4-10 in formulation buffer [Figure 32D] Showing stability data for ADC 4-10 in formulation buffer [Figure 33A] 1 shows stability data for ADC 4-11 in formulation buffer. [Figure 33B] 1 shows stability data for ADC 4-11 in formulation buffer. [Figure 34A] 1 shows stability data for ADC 4-12 in formulation buffer. [Figure 34B]1 shows stability data for ADC 4-12 in formulation buffer. [Figure 35A] 1 shows stability data for ADC 4-13 in formulation buffer. [Figure 35B] 1 shows stability data for ADC 4-13 in formulation buffer. [Figure 36A] 1 shows stability data for ADC 4-14 in formulation buffer. [Figure 36B] 1 shows stability data for ADC 4-14 in formulation buffer. [Figure 37A] 1 shows stability data for ADC 4-16 in formulation buffer. [Figure 37B] 1 shows stability data for ADC 4-16 in formulation buffer. [Figure 38] The figure shows the direct killing activity of the ADC against a cell line (H1650) that highly expresses B7H3. [Figure 39] The figure shows the direct killing activity of the ADC against a cell line (Capan-1) that has low B7H3 expression. [Figure 40] This shows the direct killing activity of the ADC against the B7H3(-) cell line (MB-453). [Figure 41] The figure shows the direct killing activity of the ADC against a cell line (H1650) that highly expresses B7H3. [Figure 42] The figure shows the direct killing activity of the ADC against a cell line (Capan-1) that has low B7H3 expression. [Figure 43] This shows the direct killing activity of the ADC against the B7H3(-) cell line (MB-453). [Figure 44] 1 is a line graph showing the antitumor activity of ADCs in the H1650 model of lung cancer. [Figure 45] 1 is a line graph showing the dose-dependent antitumor activity of ADCs in the H1650 model of lung cancer. [Figure 46] 1 is a line graph showing the rate of payload release from ADCs in mouse plasma. [Figure 47]1 is a line graph showing the rate of payload release from ADCs in human plasma. [Figure 48] 1 is a line graph showing the PK profile of the ADC. DETAILED DESCRIPTION OF THE INVENTION

[0014] 6. Detailed Description Provided herein are antibody-drug conjugates (ADCs), as well as covalent linkers and linker-payload platforms for producing the ADCs. The ADCs can be used to treat diseases or disorders such as cancer, for example, by providing a composition comprising the ADC. The ADCs disclosed herein are more stable than known ADCs.

[0015] Some ADCs (e.g., those using interchain cysteine ​​conjugation with maleimide-based linkers and payloads) are known to undergo deconjugation under physiological conditions due to retromaleimide reactions. ADCs using autohydrolyzing maleimides have been shown to have improved stability and pharmacological properties. In one example, conjugator-antibody conjugate 3-3 (see Table 3 below) underwent maleimide hydrolysis at pH 9.0 for 1–3 days. However, under these harsh conditions, post-translational modifications (e.g., oxidation) and deamination may occur in some antibodies, resulting in changes in physical properties (e.g., hydrophobicity, charge, secondary and / or tertiary structure) and potentially lowering the thermodynamic or kinetic barrier to unfolding. These changes may predispose ADCs to aggregation and other chemical modifications, which may alter binding affinity, half-life, and efficacy.

[0016] The conjugator-antibody conjugates disclosed herein can readily undergo maleimide hydrolysis under mild conditions (e.g., pH 7.0). The conjugators disclosed herein not only conjugate with antibodies under conventional conditions (e.g., pH 6.5-7.0), but also undergo maleimide hydrolysis under conjugation conditions. Buffer exchange with a basic buffer is not required for hydrolysis. Addition of a quenching reagent is sufficient to terminate the conjugation reaction. The conjugates disclosed herein can be buffer exchanged with a formulation buffer after completion of maleimide hydrolysis, as monitored by reduced LCMS.

[0017] 6.1.Definition In this disclosure, the following terms have the following meanings unless otherwise indicated: Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there are a plurality of definitions for terms provided herein, those in this section prevail unless stated otherwise.

[0018] The term "antibody" herein is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting the desired biological activity. Intact antibodies have two main regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region contains complementarity-determining regions (CDRs) that recognize and bind to specific binding sites on a specific antigen. The constant region can be recognized by and interact with the immune system (see, e.g., Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody can be, for example, a human, humanized, or chimeric antibody.

[0019] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population (i.e., the individual antibodies comprising the population are identical except for minor amounts of naturally occurring mutations that may be present). Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0020] An "intact antibody" is one that comprises, in addition to an antigen-binding variable region, a light chain constant domain (CL) and a heavy chain constant domain (CH1, CH2, CH3, and CH4) (depending on the antibody class). The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. "Antibody fragment" includes a portion of an intact antibody comprising the antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, antibody fragment(s), multispecific antibody fragments formed from fragment(s) produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0021] An "antigen" is an entity to which an antibody specifically binds.

[0022] The terms "specific binding" and "specifically bind" mean that an antibody or antibody derivative binds to its corresponding target antigen in a highly selective manner and does not bind to many other antigens. Typically, an antibody or antibody derivative binds to at least about 1 x 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M, which binds to a given antigen with an affinity that is at least two-fold greater than the affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0023] The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent completely.

[0024] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, shrink tumor size, inhibit (i.e., slow or stop to some extent) cancer cell invasion into peripheral organs, inhibit (i.e., slow or stop to some extent) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent a drug can inhibit growth and / or kill existing cancer cells, the drug is cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing the time to progression (TTP) and / or quantifying the response rate (RR).

[0025] The term "substantial" or "substantially" refers to a majority of a mixture or sample, i.e., greater than 50% of the population (e.g., greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population).

[0026] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction inside a cell of a Ligand Drug Conjugate (e.g., an Antibody Drug Conjugate (ADC)) whereby the covalent bond (e.g., linker) between the Drug moiety (D) and the Ligand unit (e.g., an antibody (BA or Ab)) is cleaved, resulting in the release of the free drug from the antibody or another metabolic product of the conjugate inside the cell. Thus, the cleaved portion of the Drug-Linker-Ligand conjugate is an intracellular metabolite.

[0027] The term "cytotoxic activity" refers to the cell-killing, cytostatic, or antiproliferative effect of a Drug-Linker-Ligand conjugate compound or an intracellular metabolic product of the Drug-Linker-Ligand conjugate. Cytotoxic activity can be expressed as an IC value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.

[0028] As used herein, the term "cytotoxic agent" refers to a substance that inhibits the function of cells and / or causes destruction of cells. This term is intended to include radioactive isotopes (e.g., At, I, I, Y, Re, Re, Sm, Bi, P, C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins (e.g., small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin), including synthetic analogs and derivatives thereof.

[0029] The terms "cancer" and "cancerous" refer to or describe a physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal tract cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0030] As used herein, an "autoimmune disease" is a disease or disorder caused by and directed against an individual's own tissues or proteins.

[0031] Examples of a "patient" include, but are not limited to, mammals (e.g., humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, or cats), and birds or poultry. In one embodiment, the patient is a human.

[0032] The terms "treat" or "treatment," unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent recurrence, the purpose of which is to arrest or slow (alleviate) an undesirable physiological change or disorder (e.g., the development or spread of cancer). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder.

[0033] In the context of cancer, the term "treating" includes any or all of inhibiting the growth of tumor cells, cancer cells, or tumors, inhibiting tumor cell or cancer cell replication, reducing the overall tumor burden or reducing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

[0034] In the context of autoimmune disease, the term "treating" includes any or all of inhibiting the replication of cells associated with the autoimmune disease (including, but not limited to, cells that produce autoimmune disease antibodies), reducing the autoimmune antibody load, and ameliorating one or more symptoms of the autoimmune disease.

[0035] As used in this specification and the appended claims, the indefinite articles "a" and "an," and the definite article "the," include plural referents as well as singular, unless the context clearly dictates otherwise.

[0036] As used herein, and unless otherwise specified, the terms "about" and "approximately," when used in reference to an amount or weight percent of a component of a composition, refer to an amount or weight percent that would be recognized by one of ordinary skill in the art as producing an equivalent pharmacological effect to that obtained from the specified amount or weight percent. In certain embodiments, the terms "about" and "approximately," when used in this context, contemplate an amount or weight percent that is within 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percent.

[0037] As used herein, unless otherwise specified, the terms "about" and "approximately," when used in connection with a numerical value or range of values ​​provided to characterize a particular solid form, e.g., a particular temperature or temperature range, e.g., one describing melting, dehydration, desolvation, or glass transition temperature, mass change, e.g., mass change as a function of temperature or humidity, solvent or water content (e.g., in terms of mass or percentage), or peak position, e.g., in analysis by IR or Raman spectroscopy or XRPD, indicate that the value or range of values ​​may deviate to an extent that would be considered reasonable by one of ordinary skill in the art and still describe the solid form. Techniques for characterizing crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), single crystal X-ray diffraction, vibrational spectroscopy such as infrared (IR) and Raman spectroscopy, solid and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In certain embodiments, the terms "about" and "approximately" used in this context indicate that a numerical value or range of values ​​may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the recited numerical value or range of values. For example, in some embodiments, XRPD peak position values ​​may vary by up to ±0.2 degrees 2θ while describing a particular XRPD peak.

[0038] An "alkyl" group is a saturated, partially saturated, or unsaturated, straight-chain or branched acyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbon atoms, and in some embodiments 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, -CH=CH(CH), -CH=C(CH), -C(CH)=CH, -C(CH)=CH(CH), C(CHCH)=CH, -C≡CH, -C≡C(CH), -C≡C(CHCH), -CHC≡CH, -CHC≡C(CH), and CHC≡C(CHCH), among others. Alkyl groups can be substituted or unsubstituted. In certain embodiments, when an alkyl group described herein is described as "substituted," the alkyl group can be substituted with halogen (chloro, iodo, bromo, or fluoro), hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonato, phosphine, thiocarbonyl, sulfonyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, B(OH), or O(alkyl)aminocarbonyl, in addition to any substituent(s) as set forth in the compounds and embodiments disclosed herein.

[0039] An "alkenyl" group is a straight-chain or branched acyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms, and containing at least one carbon-carbon double bond. Representative straight-chain and branched (C2-C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, 2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, 3-octenyl, and the like. The double bond of an alkenyl group may be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0040] A "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group of 3 to 10 carbon atoms having a single cyclic ring or multiple fused or bridged rings, optionally substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, by way of example, single ring structures (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.) or multiple ring or bridged ring structures (e.g., adamantyl, etc.). Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, among others. Cycloalkyl groups can be substituted or unsubstituted. Such substituted cycloalkyl groups include, by way of example, cyclohexanone and the like.

[0041] An "aryl" group is an aromatic carbocyclic group of 6 to 14 carbon atoms, having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). In some embodiments, aryl groups contain 6 to 14 carbons, and in others 6 to 12, and sometimes 6 to 10 carbon atoms in the ring portion of the group. Specific aryl groups include phenyl, biphenyl, naphthyl, and the like. Aryl groups can be substituted or unsubstituted. The phrase "aryl group" also includes groups containing condensed rings, for example, fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0042] A "heteroaryl" group is an aryl ring system having 1 to 4 heteroatoms as ring atoms in the heteroaromatic ring system, with the remaining atoms being carbon atoms. In some embodiments, heteroaryl groups contain 5 to 6 ring atoms, and in others 6 to 9 or 6 to 10 atoms, in the ring portion of the group. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[2,3-b]pyridyl), [d]imidazolyl), imidazopyridyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.

[0043] A "heterocyclyl" is an aromatic (also referred to as heteroaryl) or non-aromatic cycloalkyl in which 1 to 4 of the ring carbon atoms are independently replaced with heteroatoms from the group consisting of O, S, and N. In some embodiments, heterocyclyl groups contain 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. Additionally, heterocyclyls may be attached to other groups at any ring atom (i.e., any carbon atom or heteroatom of the heterocyclic ring). Heteroaryl groups may be substituted or unsubstituted. Heterocyclyl groups include unsaturated, partially saturated, and saturated ring systems (e.g., imidazolyl, imidazolinyl, and imidazolidinyl groups). The term "heterocyclyl" includes fused ring species (including ring species containing fused aromatic and non-aromatic groups), such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The term also includes bridged polycyclic ring systems containing heteroatoms (for example, but not limited to, quinuclidyl).Representative examples of heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, and thiomorpholinyl. aryl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithionyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiazolyl benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl, e.g., 1H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, ... Examples of such groups include cinnolidinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups may be mono- or multiply substituted, such as, but not limited to, pyridyl or morpholinyl groups that are 2-, 3-, 4-, 5-, or 6-substituted, or di-substituted with various substituents such as those listed below.

[0044] A "cycloalkylalkyl" group is a radical of the formula: -alkyl-cycloalkyl, where alkyl and cycloalkyl are defined above. Substituted cycloalkylalkyl groups can be substituted on the alkyl, the cycloalkyl, or both the alkyl and the cycloalkyl portions of the group. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups can be mono- or multiply substituted.

[0045] An "aralkyl" group is a radical of the formula: -alkyl-aryl, where alkyl and aryl are defined above. Substituted aralkyl groups can be substituted on the alkyl, the aryl, or both the alkyl and the aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups (e.g., 4-ethyl-indanyl).

[0046] A "heterocyclylalkyl" group is a radical of the formula: -alkyl-heterocyclyl, where alkyl and aryl are defined above. Substituted heterocyclylalkyl groups can be substituted at the alkyl, the heterocyclyl, or both the alkyl and the heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, 4-ethylmorpholinyl, 4-propylmorpholinyl, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyran-4-yl)methyl, (tetrahydro-2H-pyran-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylethyl, and indol-2-ylpropyl.

[0047] "Halogen" is chloro, iodo, bromo, or fluoro.

[0048] A "hydroxyalkyl" group is an alkyl group as defined above substituted with one or more hydroxy groups.

[0049] An "alkoxy" group is O(alkyl), where alkyl is defined as .

[0050] An "alkoxyalkyl" group is an -(alkyl)-O-(alkyl), where alkyl is defined above.

[0051] As used herein, "alkynyl" refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be straight-chained, branched, or cyclic. Alkynyl includes, but is not limited to, radicals having 2 to 20 carbon atoms, i.e., C 2~20 Alkynyl, a radical having 2 to 12 carbon atoms, i.e., C 2~12 Alkynyl, a radical having 2 to 8 carbon atoms, i.e., C 2~8 Alkynyl, a radical having 2 to 6 carbon atoms, i.e., C 2~6Alkynyl and radicals having 2 to 4 carbon atoms, i.e., C 2~4 Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl, and butynyl.

[0052] As used herein, "haloalkyl" refers to an alkyl, as defined above, containing at least one substituent selected from a halogen, e.g., fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, but are not limited to, -CF, -CHCF, -CClF, and -CCl.

[0053] As used herein, "haloalkoxy" refers to an alkoxy as defined above, wherein the alkoxy contains at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.

[0054] As used herein, "arylalkyl" refers to a monovalent moiety that is a radical of an alkyl compound, where the alkyl compound is substituted with an aromatic substituent. That is, the aromatic compound contains a single bond to the alkyl group, and the radical is localized on the alkyl group. The arylalkyl group is attached to the depicted chemical structure via the alkyl group. Arylalkyl can be represented by structures such as B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety, e.g., phenyl. Arylalkyl is optionally substituted. That is, the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include, but are not limited to, benzyl.

[0055] As used herein, "alkylaryl" refers to a monovalent moiety that is a radical of an aryl compound, where the aryl compound is substituted with an alkyl substituent. That is, the aryl compound contains a single bond to the alkyl group, and the radical is localized on the aryl group. The alkylaryl group is attached to the depicted chemical structure via the aryl group. The alkylaryl can be represented by structures such as -B-CH, -B-CH-CH, -B-CH-CH-CH, -B-CH-CH-CH, -B-CH(CH)-CH-CH, -B-CH-CH(CH)-CH, where B is an aromatic moiety, e.g., phenyl. The alkylaryl is optionally substituted. That is, the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl include, but are not limited to, toluyl.

[0056] As used herein, "aryloxy" refers to a monovalent moiety that is a radical of an aromatic compound, where the ring atoms are carbon atoms and the ring is substituted with an oxygen radical, i.e., the aromatic compound contains a single bond to the oxygen atom and the radical is localized at the oxygen atom (e.g., in the case of phenoxy, C6H5-O-). The aryloxy substituent is attached to the compound it substitutes through this oxygen atom. The aryloxy is optionally substituted. Aryloxy includes, but is not limited to, radicals having 6 to 20 ring carbon atoms, i.e., C 6~20 Aryloxy radicals, radicals with 6 to 15 ring carbon atoms, i.e., C 6~15 Aryloxy radicals and radicals with 6 to 10 ring carbon atoms, i.e., C 6~10 Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthroxy.

[0057] An "amino" group is a radical of the formula: NH2.

[0058] A “hydroxylamine” group has the formula: N(R #)OH or NHOH radicals (wherein R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein).

[0059] An "alkoxyamine" group is a group of the formula: -N(R # )O-alkyl or -NHO-alkyl radicals, where R # is as defined above).

[0060] An “aralkoxyamine” group has the formula: N(R # ) O-aryl or NHOaryl radicals (wherein R # is as defined above).

[0061] An "alkylamine" group is a radical of the formula: NH alkyl or N(alkyl) 2 , where each alkyl is independently as defined above.

[0062] An "aminocarbonyl" group is a group of the formula: -C(=O)N(R # )2, -C(=O)NH(R # ), or a radical of C(=O)NH2, where each R # is as defined above).

[0063] An "acylamino" group is a group of the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ) wherein each alkyl and R # are independently as defined above).

[0064] An "O(alkyl)aminocarbonyl" group is a group of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ), or a radical of —O(alkyl)C(═O)NH2, where each R #are independently as defined above).

[0065] An "N-oxide" group is a group of the formula: -N + -O - is a radical of

[0066] A "carboxy" group is a radical of the formula: --C(O)OH.

[0067] A "ketone" group is a group of the formula: C(=O)(R # ) where R # is as defined above).

[0068] An "aldehyde" group is a radical of the formula: --CH(.dbd.O).

[0069] An "ester" group is an ester of the formula: C(=O)O(R # ) or OC(=O)(R # ) where R # is as defined above).

[0070] A "urea" group is a group of the formula: -N(alkyl)C(=O)N(R # )2, -N(alkyl)C(=O)NH(R # ), -N(alkyl)C(=O)NH2, -NHC(=O)N(R # )2, -NHC(=O)NH(R # ), or NHC(=O)NH2 # where each alkyl and R # are independently as defined above).

[0071] An "imine" group is a group of the formula: -N=C(R # )2 or -C(R # )=N(R # ) wherein each R # are independently as defined above).

[0072] An "imido" group is a group of the formula: -C(=O)N(R#)C(=O)(R# ) or N((C=O)(R # ))2, where each R # are independently as defined above).

[0073] A "urethane" group has the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # ), -N(R # )C(=O)O(R # ), or -NHC(=O)O(R # ) wherein each R # are independently as defined above).

[0074] An "amidine" group is a group of the formula: -C(=N(R # ))N(R # )2, -C(=N(R # ))NH(R # ), -C(=N(R # ))NH2, -C(=NH)N(R # )2, -C(=NH)NH(R # ), -C(=NH)NH2, -N=C(R # )N(R # )2, -N=C(R # )NH(R # ), -N=C(R # )NH2, -N(R # )C(R # )=N(R # ), -NHC(R # )=N(R # ), -N(R # )C(R # )=NH, or -NHC(R # )=NH radical (where R # are independently as defined above).

[0075] A "guanidine" group has the formula: -N(R # )C(=N(R # ))N(R # )2, -NHC(=N(R # ))N(R # )2, -N(R# )C(=NH)N(R # )2, -N(R # )C(=N(R # ))NH(R # ), -N(R # )C(=N(R # ))NH2, -NHC(=NH)N(R # )2, -NHC(=N(R # ))NH(R # ), -NHC(=N(R # ))NH2, -NHC(=NH)NH(R # ), -NHC(=NH)NH2, -N=C(N(R # )2)2, -N=C(NH(R # ))2, or -N=C(NH2)2 radical, where R # are independently as defined above).

[0076] An "enamine" group is a group of the formula: -N(R # )C(R # )=C(R # )2, -NHC(R # )=C(R # )2, -C(N(R # )2)=C(R # )2, -C(NH(R # ))=C(R # )2, -C(NH2)=C(R # )2, -C(R # )=C(R # )(N(R # )2), C(R # )=C(R # )(NH(R # )), or -C(R # )=C(R # )(NH2) radical, where R # are independently as defined above).

[0077] An "oxime" group is a group of the formula: -C(=NO(R # ))(R # ), -C(=NOH)(R # ), -CH(=NO(R #)), or a radical of —CH(═NOH), where each R # are independently as defined above).

[0078] A "hydrazide" group has the formula: -C(=O)N(R # )N(R # )2, -C(=O)NHN(R # )2, -C(=O)N(R # )NH(R # ) 、 -C(=O)N(R # )NH2, -C(=O)NHNH(R # )2, or a radical of —C(═O)NHNH2, where R # are independently as defined above).

[0079] A “hydrazine” group has the formula: —N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ) 、 -N(R # )NH2, -NHNH(R # )2, or -NHNH2 radicals (wherein R # are independently as defined above).

[0080] A "hydrazone" group has the formula: -C(=NN(R # )2)(R # )2, -C(=NNH(R # ))(R # )2, -C(=N-NH2)(R # )2, -N(R # )(N=C(R # )2), or -NH(N=C(R # )2) is a radical of the formula (wherein R # are independently as defined above).

[0081] An "azido" group is a radical of the formula -N3.

[0082] An "isocyanate" group is a radical of the formula N=C=O.

[0083] An "isothiocyanate" group is a radical of the formula N=C=S.

[0084] A “cyanate” group is a radical of the formula OCN.

[0085] A “thiocyanate” group is a radical of the formula SCN.

[0086] A "thioether" group has the formula: -S(R # ) where R # are independently as defined above).

[0087] A "thiocarbonyl" group is a group of the formula: -C(=S)(R # ) where R # are independently as defined above).

[0088] A "sulfinyl" group is a group of the formula: -S(=O)(R # ) where R # are independently as defined above).

[0089] A "sulfone" group is a group of the formula: -S(=O)(R # ) where R # are independently as defined above).

[0090] A "sulfonylamino" group is a group of the formula: -NHSO(R # ) or -N(alkyl)SO2(R # ) wherein each alkyl and R # is defined above).

[0091] A "sulfonamide" group is a group of the formula: -S(=O)N(R # )2, -S(=O)2NH(R # ), or —S(═O)NH (wherein each R #are independently as defined above).

[0092] A "phosphonate" group has the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ), or -OP(=O)(OH)(R # ) wherein each R # are independently as defined above).

[0093] A "phosphine" group has the formula: -P(R # )2, where each R # are independently as defined above).

[0094] When a group described herein (with the exception of alkyl groups) is described as being "substituted," that group may be substituted with any suitable substituent(s). Illustrative examples of substituents include those found in the compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro), alkyl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfinyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, oxygen (═O), B(OH), O(alkyl)aminocarbonyl, whether single ring, fused or unfused. Examples include cycloalkyl, which may be multiple non-fused rings (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl); heterocyclyl, which may be single ring or multiple fused or non-fused rings (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl); single ring or multiple fused or non-fused ring aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy.

[0095] As used herein, "pharmaceutically acceptable salt(s)" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases and organic acids or bases.

[0096] As used herein, unless otherwise indicated, the term "solvate" means a compound or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0097] As used herein, unless otherwise indicated, the term "hydrate" means a compound or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0098] As used herein, unless otherwise indicated, the term "prodrug" means a compound derivative that may be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound that contain a biohydrolyzable moiety, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. In certain embodiments, prodrugs of compounds with carboxyl functional groups are lower alkyl esters of the carboxylic acid. Carboxylic acid esters can be formed by esterifying any of the carboxylic acid moieties present on the molecule. Typically, prodrugs are prepared by esterifying the carboxylic acid moieties present on the molecule using well-known methods (e.g., Burger's Medicinal Chemistry and Drug Discovery 6 th (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

[0099] As used herein, and unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" means one stereoisomer that is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound will contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. Compounds may have chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms, including mixtures thereof, are included in the embodiments disclosed herein. The use of stereoisomerically pure forms of such compounds, as well as mixtures of these forms, are encompassed by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or resolved using standard techniques, such as chiral columns or chiral resolving agents.See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN, 1972).

[0100] It should also be noted that the compounds may include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds are isolated as either cis or trans isomers. In other embodiments, the compounds are mixtures of cis and trans isomers.

[0101] "Tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of isomeric forms may vary depending on the environment in which the compound is found, for example, whether the compound is solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other: [ka]

[0102] As will be readily understood by one of ordinary skill in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds are within the scope of the present disclosure.

[0103] It should also be noted that the compounds may contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may contain unnatural proportions of atomic isotopes, such as tritium ( 3 H), iodine-125( 125 I), sulfur-35( 35 S), or carbon-14 ( 14 It may be radiolabeled with a radioisotope such as deuterium ( 2 H), carbon-13( 13 C), or nitrogen-15( 15 The compound may be isotopically enriched, such as with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 120, 121, 132, 133, 144, 150, 165, 170, 171, 182, 191, 192, 193, 194, 195, 196, 197, 198,

[0104] It should be noted that where there is a discrepancy between a depicted structure and the name for that structure, the depicted structure is given weight.

[0105] As used herein, the term "residue" refers to the chemical moiety in a compound that remains after a chemical reaction. For example, the term "amino acid residue" or "N-alkylamino acid residue" refers to the product of amide or peptide coupling of an amino acid or an N-alkylamino acid with a suitable coupling partner, e.g., a water molecule is expelled after amide or peptide coupling of the amino acid or N-alkylamino acid, resulting in the incorporation of the amino acid residue or N-alkylamino acid residue into the product.

[0106] As used herein, "sugar" or "sugar group" or "sugar residue" refers to a carbohydrate moiety that may contain a 3-carbon (triose) unit, a 4-carbon (tetrose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, a "sugar" or "sugar group" or "sugar residue" includes a furanose (e.g., ribofuranose, fructofuranose), or a pyranose (e.g., glucopyranose, galactopyranose), or a combination thereof. In some cases, a "sugar" or "sugar group" or "sugar residue" includes an aldose or ketose, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other "sugars" or "sugar groups" or "sugar residues" include polysaccharides and / or oligosaccharides, including, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, the "sugar" or "sugar group" or "sugar residue" is an amino sugar. In some cases, the "sugar" or "sugar group" or "sugar residue" is a glucamine residue (1-amino-1-deoxy-D-glucitol) (i.e., glucamide) that is attached to the rest of the molecule via the amino group to form an amide bond with the rest of the molecule.

[0107] Certain groups, moieties, substituents, and atoms are represented, for example, with a wavy line crossing the bond(s) to indicate the atom to which the group, moiety, substituent, or atom is bonded, for example, a phenyl group can be represented by a propyl group, shown below: [ka] When substituted with, it has the following structure: [ka]

[0108] Diagrams depicting substituents attached to an acyclic group via a bond between two atoms are meant to indicate that the substituent may be attached to either atom of the bond through which the substituent bond passes, unless otherwise indicated, in accordance with techniques described herein or known in the art to which this disclosure pertains. Thus, for example: [ka] teeth [ka] Includes.

[0109] As used herein, a "binding agent" refers to any molecule (eg, an antibody) that can bind with specificity to a given binding partner (eg, an antigen).

[0110] As used herein, the term "amino acid" refers to an organic compound containing an amino group (-NH2) and a carboxyl group (-COOH), along with a side chain (R group) unique to each amino acid. Amino acids can be proteinogenic or non-proteinogenic. "Proteinogenic" means that the amino acid is one of the 20 naturally occurring amino acids found in proteins. Proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteinogenic" means that the amino acid does not naturally occur in proteins or is not produced directly by cellular machinery (e.g., is a product of post-translational modification). Non-limiting examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, beta-alanine, ornithine, and citrulline.

[0111] As used herein, "peptide" is defined in its broadest sense in its various grammatical forms and refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or other bonds (e.g., esters, ethers, etc.). As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids (including glycine and both D and L optical isomers), as well as amino acid analogs and peptidomimetics. When the peptide chain is short (e.g., two, three, or more amino acids), it is generally referred to as an oligopeptide. When the peptide chain is longer, the peptide is usually referred to as a polypeptide or protein. Full-length proteins, analogs, variants, and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, due to the presence of ionizable amino and carboxyl groups in the molecule, certain peptides can be obtained as acid or base salts or in neutral form. Peptides may be obtained directly from a source organism, or may be produced recombinantly or synthetically.

[0112] The amino acid sequence of an antibody can be numbered using any known numbering scheme, including those described in Kabat et al. ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply sequence differences where they do not exist, and one of skill in the art can readily ascertain sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is generally used when referring to residues within antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra).

[0113] As used herein, the term "anti-HER2 antibody" refers to an antibody that selectively binds to the HER2 receptor (e.g., trastuzumab (Herceptin)). In one embodiment, trastuzumab can be made and used as described in US6407213 and US5821337, the entire disclosures of which are incorporated herein by reference.

[0114] As used herein, the term "anti-HER3 antibody" refers to an antibody that selectively binds to the HER3 receptor (e.g., patritumab). In one embodiment, patritumab can be made and used as described in U.S. Patritumab, the entire disclosure of which is incorporated herein by reference.

[0115] As used herein, the term "anti-PTK7 antibody" refers to an antibody that selectively binds to the PTK7 receptor (e.g., cofetuzumab). In one embodiment, cofetuzumab can be made and used as described in US9777070, the entire disclosure of which is incorporated herein by reference.

[0116] As used herein, the term "ifinatamab" refers to an antibody that selectively binds to the B7H3 receptor. In one embodiment, ifinatamab can be made and used as described in US10117952 or WO2022102695, the entire disclosures of which are incorporated herein by reference.

[0117] As used herein, the term "cytocidal activity" refers to the activity of decreasing or reducing the cell viability of the cell line being tested.

[0118] In the following claims and the preceding description, unless the context requires otherwise, either expressly stated or by necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., to specify the presence of stated features, but are not used to exclude the presence or addition of further features in various embodiments.

[0119] 6.2. Conjugates In various embodiments, the conjugate, or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, comprises a protein bound to at least one payload or payload residue (also referred to herein as a drug unit) and to at least one hydrophilic moiety via a covalent linker. The covalent linker is directly or indirectly attached to each of the protein, payload residue, and hydrophilic moiety. In some embodiments, the protein is a binding agent such as an antibody or antigen-binding fragment thereof.

[0120] In some embodiments, the protein is directly attached to a covalent linker (e.g., a linker) described herein. In such cases, the binding agent is one attachment position away from the covalent linker. The covalent linker can also be directly attached to a payload residue such that the covalent linker is one attachment position away from the payload residue. The payload can be any payload described herein. In some embodiments, the covalent linker is further directly attached to a hydrophilic moiety such that the covalent linker is one attachment position away from the hydrophilic moiety. The hydrophilic moiety can be any hydrophilic moiety (HG) described herein.

[0121] In some embodiments, the binding agent is indirectly attached to the covalent linker such that the binding agent is present at multiple attachment points from the covalent linker. In such cases, the binding agent is attached to the covalent linker through another moiety. For example, the binding agent can be attached to a maleimide group that is attached to a polyethylene glycol group that is attached to the covalent linker.

[0122] In some instances, the covalent linker is further indirectly linked to the payload residue, such that the covalent linker is connected to the payload residue via multiple attachment positions. The covalent linker is linked to the payload via another moiety. For example, the covalent linker may be linked to a dipeptide (for example, but not limited to, Val-Ala or Val-Cit), the dipeptide may be linked to a PAB, and the PAB may be linked to a payload residue.

[0123] In some embodiments, the covalent linker is indirectly attached to the hydrophilic moiety such that the covalent linker is attached to the hydrophilic moiety through multiple attachment points, or the covalent linker is attached to the hydrophilic moiety through another moiety.

[0124] 6.2.1. Aspect 1. Provided herein, for example, are ADCs for use in therapy (e.g., cancer therapy).

[0125] One embodiment is an ADC compound of formula (I): [ka] or a pharmaceutically acceptable salt, tautomer, isotopologue, or stereoisomer thereof; BA is a binding agent selected from a humanized antibody, a chimeric antibody, a human antibody, or an antigen-binding fragment thereof; RG is the residue of a reactive group, RS is a ring-opening stabilizing group, RE is a ring-opening enhancer, R 1a and R 1b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 1a and R 1b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 2a and R2b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 2a and R 2b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 3a and R 3b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 3a and R 3b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 4 is H, substituted or unsubstituted C 1~4 Alkyl, or substituted or unsubstituted C 3~5 is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is the residue of a Stretcher unit, The subscript a' is 0 or 1, W is the cleavable unit, The subscript w' is 0 or 1, Y is a spacer unit, the subscript y' is 0 or 1, PA is the payload residue, The subscript x is 1 to 15.

[0126] In one embodiment, the subscript x is 1 to 12. In one embodiment, the subscript x is 1 to 10. In one embodiment, the subscript x is 2 to 10. In one embodiment, the subscript x is 3 to 10. In one embodiment, the subscript x is 4 to 10. In one embodiment, the subscript x is 4 to 9. In one embodiment, the subscript x is 4 to 8.

[0127] In some embodiments, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , and R 4 each is independently H.

[0128] In some embodiments, RS is -NR 5a R 5b where R 5a and R 5b each independently represents H or a substituted or unsubstituted C 1~4 It is alkyl.

[0129] In some embodiments, RS is an amino (-NH2) group or -N(CH3)2.

[0130] In some embodiments, RS is an amino group.

[0131] In some embodiments, RE is a bond, —O—, —OC(═O)—, or —OC(═O)NR 6 -, -NHC(=O)NR 6 , -OS(=O)2NR 6 -, -NHS(=O)2NR 6 - or -OC(=O)NHS(=O)NR 6 - and R 6 is H or substituted or unsubstituted C 1~4 It is alkyl.

[0132] In some embodiments, R 6 is H, methyl, ethyl, or isopropyl.

[0133] In some embodiments, RE is —OC(═O)NR 6 -It is.

[0134] In some embodiments, RE is -OC(=O)NH-.

[0135] In some embodiments, RG is [ka] is.

[0136] In some embodiments, RG is [ka] is.

[0137] In some embodiments, r is 0.

[0138] In some embodiments, s is 1.

[0139] In some embodiments, t is 1 or 2.

[0140] In some embodiments, r is 0, s is 1, and t is 1 or 2.

[0141] In some embodiments, A is a bond, —(CH) n -C(=O)-, -CH2-C(=O)-NH-(CH2) n -C(=O)-, -(CH2CH2O) n -CH2CH2-C(=O)-, -CH[-(CH2) n —COOH]—C(═O)—, -CH2-C(=O)-NH-(CH2) n -C(=O)-NH-(CH2) n -C(=O)-, or -C(=O)-(CH2) n -C(=O)-, where each n independently represents an integer of 1, 2, 3, 4, or 5.

[0142] In some embodiments, W w’ is the following formula: [ka] where HG is a hydrophilic moiety or hydrogen.

[0143] In some embodiments, HG is a saccharide, phosphate ester, sulfate ester, phosphodiester, or phosphonate.

[0144] In some embodiments, HG is a saccharide, and the saccharide is β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-αD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, αL-iduronic acid, αD-galactose, αD-glucose, β-D-glucose, αD-mannose, β-D-mannose, αL-fucose, β-D-xylose, neuraminic acid, sulfate, phosphate, carboxyl, amino, or O-acetyl modifications thereof. In some embodiments, HG is selected from β-D-galactose and β-D-glucuronic acid.

[0145] In some embodiments, HG is [ka] is.

[0146] In some embodiments, Y y’ is in PAB units.

[0147] In some embodiments, the Stretcher unit (-A-) is present and extends the framework of the covalent linker, increasing the distance between the self-stabilizing linker assembly and the drug unit (payload or payload residue). The self-stabilizing linker assembly can include components of Formula (I) other than the binder (BA), Stretcher unit (A), cleavable unit (W), Spacer unit (Y), and payload residue (PA). In various embodiments, the self-stabilizing linker includes a reactive group residue (RG), a ring-opening stabilizing group (RS), a ring-opening enhancer (RE), and carbon atoms and R groups connecting RG, RS, and / or RE. The Stretcher unit can connect the self-stabilizing linker assembly to the cleavable unit when the cleavable unit is present, connect the self-stabilizing linker assembly to the spacer unit when the cleavable unit is absent but the spacer unit is present, or connect the self-stabilizing linker assembly to the drug unit when both the cleavable unit and the spacer unit are absent. A Stretcher unit can be attached to multiple cleavable units, spacer units, and / or drug units, where the self-stabilizing linker assembly, cleavable unit, spacer unit, and drug unit can be any self-stabilizing linker assembly, cleavable unit, spacer unit, and drug unit described herein, respectively.

[0148] The Stretcher unit can modify the physicochemical properties of the Drug-Linker depending on the components of the Stretcher unit. In some embodiments, the Stretcher unit can enhance the solubility of the Drug-Linker and can include one or more solubility-enhancing groups, such as ionic groups or water-soluble polymers. The water-soluble polymer is soluble in water at room temperature and can include other polymers, such as polyethyleneimine, in addition to poly(ethylene) glycol groups.

[0149] A Stretcher unit can include one or more Stretcher groups. Examples of Stretcher groups include, for example, -NH-C 1~10 Alkylene-, -NH-C 1~10 Alkylene-NH-C(O)-C1~10 Alkylene-, -NH-C 1~10 Alkylene-C(O)-NH-C 1~10 Alkylene-, -NH-(CH2CH2O) u -, -NH-(CH2CH2O) u -CH2-, -NH-(CH2CH2NH) u -(CH2) u -NH-(CH2CH2NH) u -(CH2) u -NH-C(O)-(CH2) u Examples include -NH-(C3-C8 carbocyclo)-, -NH-(arylene)-, and -NH-(C3-C8 heterocyclo)- (wherein each u is independently 1 to 10).

[0150] In some embodiments, the cleavable unit (-W w’ -) is present and can connect the self-stabilizing linker assembly to the Spacer unit if a Spacer unit is present, or to the Drug unit if a Spacer unit is not present. The self-stabilizing linker assembly can be attached to the Spacer unit or the Drug unit directly from the self-stabilizing linker assembly if a Stretcher unit is not present, or via the Stretcher unit if a Stretcher unit is present.

[0151] In some embodiments, the cleavable unit is directly conjugated to the self-stabilizing linker assembly on one end and to the Drug unit on the other end. In some embodiments, the cleavable unit is directly conjugated to the Stretcher unit on one end and to the Drug unit on the other end. In further embodiments, the cleavable unit is directly conjugated to the Stretcher unit on one end and to the Spacer unit on the other end. In further embodiments, the cleavable unit is directly conjugated to the self-stabilizing linker assembly on one end and to the Spacer unit on the other end. In embodiments, the Stretcher unit and / or the Spacer unit may be absent.

[0152] The cleavable unit can form a cleavable bond with a drug unit (PA) or a spacer unit. Reactive groups for forming a cleavable bond can include, for example, sulfhydryl groups for forming disulfide bonds, aldehyde, ketone, or hydrazine groups for forming hydrazone bonds, carboxyl or amino groups for forming peptide bonds, and carboxyl or hydroxy groups for forming ester bonds.

[0153] Cleavable units can include disulfide-containing linkers that are cleavable through disulfide exchange, acid-labile linkers at acidic pH, or linkers that are cleavable by enzymes such as hydrolases, peptidases, esterases, glucoronidases, etc. Cleavable units can contain one or more cleavage sites.

[0154] In some embodiments, the cleavable unit comprises one or more (e.g., 1-12) amino acids. Cleavable units can include, for example, monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units.

[0155] Each amino acid may be a natural or unnatural amino acid and / or its D- or L-isomer, so long as a cleavable bond is available. In some embodiments, the cleavable unit comprises only natural amino acids. Each amino acid may be a proteinogenic or non-proteinogenic amino acid.

[0156] In some embodiments, each amino acid is independently selected from the group consisting of alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, β-alanine, aminoalkanoic acids, aminoalkynic acids, aminoalkanedioic acids, aminobenzoic acids, aminoheterocycloalkanoic acids, heterocyclocarboxylic acids, citrulline, statins, diaminoalkanoic acids, and derivatives thereof. In some embodiments, each amino acid is independently selected from the group consisting of alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, and selenocysteine. In some embodiments, each amino acid is independently selected from the group consisting of alanine, arginine, aspartic acid, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, and valine.

[0157] In some embodiments, each amino acid is independently selected from the L-isomers of the following naturally occurring amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine. In some embodiments, each amino acid is a D-isomer of the following naturally occurring amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine.

[0158] In embodiments, the cleavable unit is the dipeptide -Val-Cit-, -Phe-Lys-, or -Val-Ala.

[0159] In some embodiments, the cleavable unit comprises one or two terminal amino acids and is attached to the drug unit and / or spacer unit via a functional group present on the terminal amino acid, e.g., its carboxylic acid or amino terminus.

[0160] In some embodiments, the bond between the cleavable unit and the Drug unit can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to release the Drug unit (-PA), which upon release can be protonated in vivo to yield the Drug (PA).

[0161] Useful cleavable units can be designed to optimize selectivity for enzymatic cleavage by particular enzymes (e.g., tumor-associated proteases). In one embodiment, the linkage between the cleavable unit and the drug or spacer unit is a linkage whose cleavage is catalyzed by cathepsin B, C, and / or D, or plasmin proteases.

[0162] In some embodiments, the spacer unit (-Y y’The -) is present and extends the covalent linker framework. The spacer unit can link a cleavable unit and a drug unit, or a stretcher unit and a drug unit, or a self-stabilizing linker assembly and a drug unit. The spacer unit can include one or more self-immolative or non-self-immolative groups. In some embodiments, the spacer unit includes one or more self-immolative groups. In this context, the term "self-immolative group" refers to a bifunctional chemical moiety capable of covalently linking two separate chemical moieties together into a generally stable tripartite molecule. The self-immolative group spontaneously separates from the second chemical moiety upon cleavage of the bond to the first moiety. In other embodiments, the spacer unit is not self-immolative. In such embodiments, some or all of the spacer unit remains attached to the drug unit.

[0163] In some embodiments, -Y y’ - is a self-immolative group that is attached to the cleavable unit through a methylene carbon atom of the self-immolative group and directly to the Drug unit through a carbonate, carbamate, or ether group.

[0164] In some embodiments, -Y y’ - is a p-aminobenzyl alcohol (PAB) unit (e.g., -NH-(C6H4)-CH2-OC(=O)-). The phenylene portion of the PAB unit is optionally substituted with -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano.

[0165] In another embodiment, -Y y’ - is a carbonate group.

[0166] Other examples of self-immolative groups include, but are not limited to, aromatic compounds electronically similar to PAB units, such as 2-aminoimidazole-5-methanol derivatives (see, e.g., Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Suitable spacer units include those that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (see, for example, Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (see, for example, Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (see, for example, Amsberry et al., 1990, J. Org. Chem. 55:5867). Also suitable self-immolative groups include those that undergo cyclization upon amide bond hydrolysis, such as substituted amine-containing drugs (see, for example, Kingsbury et al., 1984, J. Med. Chem. 27:1447).

[0167] Other suitable spacer units are disclosed in U.S. Publication No. 2005-0238649, the disclosure of which is incorporated herein by reference.

[0168] Stretcher, cleavable, and spacer units suitable for use with the linkers, platforms, and ADCs of the present disclosure are described in WO2004 / 010957, WO2007 / 038658, WO2005 / 112919, U.S. Patent Nos. 6,214,345, 7,659,241, 7,498,298, 7,968,687, and 8,163,888, and U.S. Publication Nos. 2009-0111756, 2009-0018086, and 2009-0274713, each of which is incorporated by reference in its entirety for all purposes. Binder

[0169] Provided herein are binding agents (BAs), e.g., for use in the ADCs described herein.

[0170] The compound of formula (I) can include any BA described herein.

[0171] In some embodiments, the BA is an antibody or antigen-binding fragment thereof, eg, a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human B7H3. In some embodiments, the antibody or antigen-binding fragment thereof is ifinatamab.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to PTK7. In some embodiments, the antibody or antigen-binding fragment thereof is cofetuzumab.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to HER3. In some embodiments, the antibody or antigen-binding fragment thereof is patritumab.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to HER2. In some embodiments, the antibody or antigen-binding fragment thereof is trastuzumab.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0178] In some embodiments, the Fc domain is an IgG1 with reduced effector function.

[0179] payload Provided herein are payloads (PAs), e.g., for use in the platforms and / or ADCs described herein.

[0180] The compound of formula (I) can include any PA described herein.

[0181] In some embodiments, each PA is independently a cytotoxic agent.

[0182] In some embodiments, each PA is independently selected from the group consisting of DXd, 7-ethyl-10-hydroxy-camptothecin (SN-38), and monomethylauristatin E (MMAE).

[0183] In some embodiments, each PA independently represents a compound of formula (VI): [ka] and and R 9 and R 10 each independently represents hydrogen, halogen, or substituted or unsubstituted C 1~4 It is alkyl.

[0184] In some embodiments, each PA independently is: [ka] is.

[0185] In some embodiments, the ADC has the following formula: [ka] [ka] and wherein the values ​​of the variables (e.g., BA, PA, A, W, Y, a', w', y', x) are as described above.

[0186] In some embodiments, the ADC has the following formula: [ka] [ka] and wherein the values ​​of the variables (e.g., BA, PA, A, W, Y, a', w', y', x) are as described above.

[0187] 6.2.2. Aspect 2. In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [Table 13-1] [Table 13-2] [Table 13-3] wherein BA is a humanized, chimeric, or human antibody, or antigen-binding fragment thereof, subscript x is 1 to 15, and PA is as described above in embodiment 1. Alternative values ​​for BA are as described herein (e.g., with respect to embodiment 1). Alternative values ​​for the variable subscript x are as described herein (e.g., with respect to compounds of formula (I)).

[0188] In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] wherein BA is a humanized, chimeric, or human antibody, or antigen-binding fragment thereof, subscript x is 1 to 15, and PA is as described above in embodiment 1. Alternative values ​​for BA are as described herein (e.g., with respect to embodiment 1). Alternative values ​​for the variable subscript x are as described herein (e.g., with respect to compounds of formula (I)).

[0189] 6.2.3. Aspect 3. In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [ka] [ka] where Ab is a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof.

[0190] In some embodiments, the Ab is ifinatamab.

[0191] In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [Table 15-1] [Table 15-2] where Ab is a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof.

[0192] In some embodiments, the Ab is ifinatamab.

[0193] In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [ka] [ka] where Ab is a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof, and x is 1 to 15.

[0194] In some embodiments, the Ab is ifinatamab.

[0195] In some embodiments, the ADC compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [Table 16-1] [Table 16-2] where Ab is a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof, and x is 1 to 15.

[0196] In some embodiments, the Ab is ifinatamab.

[0197] 6.2.4. Aspect 4. Also provided herein are platforms, eg, for use in preparing ADCs (eg, ADCs described herein).

[0198] In some embodiments, the platform comprises a linker-payload compound of formula (II): [ka] or a pharmaceutically acceptable salt, tautomer, isotopologue, or stereoisomer thereof; RG is a reactive group, RS is a ring-opening stabilizing group, RE is a ring-opening enhancer, R 1a and R 1b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 1a and R 1b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 2a and R 2b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 2a and R 2b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 3a and R 3b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 3a and R 3btogether with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 4 is H, substituted or unsubstituted C 1~4 Alkyl, or substituted or unsubstituted C 3~5 is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is the residue of a Stretcher unit, The subscript a' is 0 or 1, W is the cleavable unit, The subscript w' is 0 or 1, Y is a spacer unit, the subscript y' is 0 or 1, PA is the payload residue.

[0199] In some embodiments, RG is [ka] is.

[0200] In some embodiments, RG is [ka] is.

[0201] In some embodiments, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , and R 4 each is independently H.

[0202] In some embodiments, RS is an amino group or -NR 5a R 5b where R 5a and R 5b each independently represents H or a substituted or unsubstituted C 1~4 It is alkyl.

[0203] In some embodiments, RS is an amino group or -N(CH3)2.

[0204] In some embodiments, RS is an amino group.

[0205] In some embodiments, RE is a bond, —O—, —OC(═O)—, or —OC(═O)NR 6 -, -NHC(=O)NR 6 -, -OS(=O)2NR 6 -, -NHS(=O)2NR 6 - or -OC(=O)NHS(=O)NR 6 - and R 6 is H or substituted or unsubstituted C 1~4 It is alkyl.

[0206] In some embodiments, R 6 is H, methyl, ethyl, or isopropyl.

[0207] In some embodiments, RE is —OC(═O)NR 6 -It is.

[0208] In some embodiments, RE is -OC(=O)NH-.

[0209] In some embodiments, r is 0.

[0210] In some embodiments, s is 1.

[0211] In some embodiments, t is 1 or 2.

[0212] In some embodiments, A is a bond, —(CH) n -C(=O)-, -CH2-C(=O)-NH-(CH2) n -C(=O)-, -(CH2CH2O)n-CH2CH2-C(=O)-, -CH[-(CH2) n-COOH]-C(=O)-, -CH2-C(=O)-NH-(CH2) n -C(=O)-NH-(CH2) n -C(=O)-, or -C(=O)-(CH2) n -C(=O)-, where each n independently represents an integer of 1, 2, 3, 4, or 5.

[0213] In some embodiments, W w’ is the following formula: [ka] where HG is a hydrophilic moiety or hydrogen.

[0214] In some embodiments, HG is a saccharide, phosphate ester, sulfate ester, phosphodiester, or phosphonate.

[0215] In some embodiments, the saccharide is β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-αD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, αL-iduronic acid, αD-galactose, αD-glucose, β-D-glucose, αD-mannose, β-D-mannose, αL-fucose, β-D-xylose, neuraminic acid, sulfate, phosphate, carboxyl, amino, or O-acetyl modifications thereof. In some embodiments, HG is selected from β-D-galactose and β-D-glucuronic acid.

[0216] In some embodiments, HG is [ka] is.

[0217] In some embodiments, Y y’ is in PAB units.

[0218] In some embodiments, the compound has the following formula: [ka] and wherein the values ​​of the variables (e.g., BA, PA, A, W, Y, a', w', y', x) are as described above.

[0219] 6.2.5. Aspect 5. In some embodiments, the linker payload compound is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof: [Table 17-1] [Table 17-2] wherein PA is as defined above for embodiment 1.

[0220] In some embodiments, the linker payload compound is any one of the following, or a pharmaceutically acceptable salt and / or solvate thereof: [ka] [ka] [ka]

[0221] In some embodiments, the linker payload compound is any one of the following, or a pharmaceutically acceptable salt and / or solvate thereof: [Table 18-1] [Table 18-2] [Table 18-3]

[0222] 6.2.6. Aspect 6. Also provided herein are covalent linkers, e.g., for use in the platforms and / or ADCs described herein.

[0223] In some embodiments (e.g., embodiments of linkers for use in platforms), the linker is a compound of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, isotopologue, or stereoisomer thereof; RG is a reactive group, RS is a ring-opening stabilizing group, RE is a ring-opening enhancer, R 1a and R 1b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 1a and R 1b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 2a and R 2b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 2a and R 2b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 3a and R 3b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 3a and R 3btogether with the atoms to which they are attached, form substituted or unsubstituted C 3~5 forming a cycloalkyl, R 4 is H, substituted or unsubstituted C 1~4 Alkyl, or substituted or unsubstituted C 3~5 is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is a stretcher unit, The subscript a' is either 0 or 1.

[0224] In some embodiments, RG is [ka] is.

[0225] In some embodiments, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , and R 4 each is independently H.

[0226] In some embodiments, RS is an amino group or -NR 5a R 5b where R 5a and R 5b each independently represents H or a substituted or unsubstituted C 1~4 It is alkyl.

[0227] In some embodiments, RS is an amino group or -N(CH3)2.

[0228] In some embodiments, RS is an amino group.

[0229] In some embodiments, RE is a bond, —O—, —OC(═O)—, or —OC(═O)NR 6 -, -NHC(=O)NR 6 -, -OS(=O)2NR6 -, -NHS(=O)2NR 6 - or -OC(=O)NHS(=O)NR 6 - and R 6 is H or substituted or unsubstituted C 1~4 It is alkyl.

[0230] In some embodiments, R 6 is H, methyl, ethyl, or isopropyl.

[0231] In some embodiments, RE is —OC(═O)NR 6 -It is.

[0232] In some embodiments, RE is -OC(=O)NH-.

[0233] In some embodiments, r is 0.

[0234] In some embodiments, s is 1.

[0235] In some embodiments, t is 1 or 2.

[0236] In some embodiments, A is a bond, —(CH) n -C(=O)R 7 , -CH2-C(=O)-NH-(CH2) n -C(=O)R 7 , -(CH2CH2O)n-CH2CH2-C(=O)R 7 , -CH[-(CH2) n -COOH]-C(=O)R 7 , -CH2-C(=O)-NH-(CH2) n -C(=O)-NH-(CH2) n -C(=O)R 7 , or -C(=O)-(CH2) n -C(=O)R 7 wherein each n independently represents an integer of 1, 2, 3, 4, or 5; R 7 is OH or NR 8a R8b and R 8a and R 8b each independently represents H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 3~5 is cycloalkyl, or R 8a and R 8b together with the atoms to which they are attached, form substituted or unsubstituted C 3~5 Forms a cycloalkyl.

[0237] In some embodiments, R 7 is OH, NH2, NHCH3, or N(CH3)2. 6.2.7. Aspect 7.

[0238] In some embodiments, the linker compound is any one of the following, or a pharmaceutically acceptable salt and / or solvate thereof: [Table 19-1] [Table 19-2]

[0239] 6.3. Methods or Processes for Making Conjugates Provided herein are methods for preparing conjugates by contacting a binding agent (BA) with a linker-payload compound under conditions suitable for the formation of a bond between the binding agent and the linker-payload compound. The reaction conditions can be any suitable reaction conditions known in the art. The binding agent can be an antibody, and the bond can form an antibody-drug conjugate.

[0240] Examples of such reactions are given in the Examples below.

[0241] In some embodiments, the method for producing the conjugate comprises treating or contacting a compound with a binding agent under coupling conditions. The compound can comprise a reactive linker attached to at least one payload. The compound can be any linker compound or platform compound disclosed herein.

[0242] Pharmaceutical Compositions Also provided herein are compositions (including pharmaceutical compositions) comprising the ADCs described herein. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise a pharmaceutically acceptable excipient.

[0243] Pharmaceutical compositions according to the present disclosure can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an antibody-drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Generally, pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents, such as EDTA; sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, a sHASEGP is combined with one or more glycosaminoglycanases (eg, chondroitinases).

[0244] Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0245] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody drug conjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0246] Formulations to be used for in vivo administration are generally sterile. Sterilization is readily accomplished, for example, by filtration through sterile filtration membranes.

[0247] 6.5.How to use In some embodiments, provided herein are methods of treating a disease or disorder (e.g., cancer) in a subject (e.g., patient) in need thereof, comprising administering to the patient an effective amount of an ADC disclosed herein.

[0248] The antibody drug conjugates disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if necessary for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection (e.g., intravenous or subcutaneous injection), depending in part on whether administration is brief or chronic. Various administration schedules are contemplated, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0249] The antibody drug conjugates of the present disclosure may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to medical practitioners. [Example]

[0250] 7. Working Example The following examples are intended to be illustrative and should not be construed as limiting in any way. Unless otherwise specified, experimental methods in the following examples are conventional. Unless otherwise specified, reagents and materials were commercially available. All solvents and chemicals used were of analytical grade or chemical purity. Solvents were redistilled before use. Anhydrous solvents were prepared according to standard or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin-layer chromatography (TLC) were commercially available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd., China. Both were eluted with petroleum ether (60-90°C) / ethyl acetate (v / v) and visualized with iodine or molybdophosphate solutions in ethanol unless otherwise specified. All extraction solvents were dried over anhydrous Na2SO4 unless otherwise specified. 1H NMR spectra were recorded on a Bruck-400, Varian 400MR nuclear magnetic resonance spectrometer (TMS (tetramethylsilane) was used as an internal standard). Coupling constants are given in Hertz. Peaks are reported as singlets (s), doublets (d), triplets (t), quartets (q), quintets (p), sextets (h), septets (hept), multiplets (m), or combinations thereof. br means broad. LC / MS data were recorded using an Agilent 1100, 1200 high-performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and an ion trap (ESI source) detecting at 214 nm and 254 nm. All compound names, except for reagents, were generated by ChemDraw® 18.0.

[0251] For the sake of brevity, certain abbreviations are used herein, such as the one-letter abbreviations for amino acids. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 20]

[0252] In the examples below, the following abbreviations are used: [Table 21-1] [Table 21-2]

[0253] UPLC analysis method Method A: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold 0.2 min, 10% to 95% B 5.8 min, 95% B hold 0.5 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm.

[0254] Method B: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.5 min, 10% to 90% B for 2.5 min, 90% B hold for 0.2 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm Method C: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.2 min, 10% to 90% B for 1.3 min, 90% B hold for 0.3 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0255] Commercially available compounds 1-1 and 1-3 were purchased from MedChemExpress.

[0256] Example 1-2 [ka] Step 1: (S,Z)-4-((2-((tert-butoxycarbonyl)amino)-1-carboxyethyl)amino)-4-oxobut-2-enoic acid (1-2c)

[0257] A mixture of 1-2a (445.0 mg, 2.18 mmol) and 1-2b (213.7 mg, 2.18 mmol) in glacial acetic acid (10 mL) was prepared. The mixture was stirred at room temperature for 2.5 h. DCM / hexane (1:1, 30 mL) was added to the reaction mixture, followed by precipitation of a white solid. The solid was then filtered and coevaporated twice with toluene to remove AcOH, affording 1-2c (530 mg, approximately 80.4% yield) as a white solid.

[0258] MS (ESI) m / z: 301.2 [M−H].

[0259] Step 2: (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (1-2d)

[0260] To a suspension of 1-2c (530 mg, 1.75 mmol) in toluene (18 mL) and DMA (1 mL) was added TEA (532.3 mg, 5.26 mmol) and 4 Å molecular sieves, and the mixture was then heated to 120 °C and stirred at this temperature for 4 h. The mixture was concentrated in vacuo to remove toluene and then purified by preparative HPLC (FA). The fraction was lyophilized to give 1-2d (201 mg, 40.3% yield) as a white powder.

[0261] MS (ESI) m / z: 304.3 [M+Na] + .

[0262] 1H NMR (400 MHz, DMSO) δ 7.09 (s, 2H), 6.99 (t, J = 6.4 Hz, 1H), 4.60 (dd, J = 10.8, 4.0 Hz, 1H), 3.59-3.53 (m, 1H), 3.46-3.38 (m, 1H), 1.32 (s, 9H).

[0263] Step 3: (S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethyl-4,8-dioxo-3,12,15,18,21-pentaoxa-5,9-diazatetracosan-24-oic acid (1-2f)

[0264] To a solution of 1-2d (25 mg, 0.09 mmol) in DMF (1 mL) were added TSTU (26.5 mg, 0.09 mmol) and DIPEA (45.5 mg, 0.35 mmol). The mixture was stirred at room temperature for 10 min. 1-2d was converted to the activated ester. 1-2e was added. The mixture was stirred at room temperature for 1 h. The reaction mixture was purified by preparative HPLC (FA 0.1%), and the fractions were lyophilized to give 1-2f (22 mg, 46.5% yield) as a white powder.

[0265] MS (ESI) m / z: 554.5 [M+Na] + .

[0266] Step 4: (S)-1-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecane-19-oic acid TFA salt (1-2)

[0267] A mixture of 1-2f (22.0 mg, 0.04 mmol) in DCM / TFA (1:1, 2 mL) was stirred at room temperature for 1 h. The mixture was concentrated in vacuo to give the crude product, which was then dissolved in water and lyophilized. 1-2 (17.5 mg, TFA salt) was obtained as a yellow foamy solid.

[0268] MS (ESI) m / z: 432.4 [M+H] + .

[0269] Examples 1-4 [ka] Step 1: tert-Butyl (S)-(2-amino-3-hydroxypropyl)carbamate (1-4b)

[0270] To a mixture of 1-4a (2.0 g, 6.17 mmol) (synthesis reference: Journal of Medicinal Chemistry, 1998, vol. 41, #15, pp. 2786-2805) in MeOH (10 mL) was added wet Pd / C (106 mg). The black suspension was purged with a H balloon three times and then stirred under a H balloon at room temperature for 1 h. The mixture was filtered through a syringe head and washed with MeOH / H O (5:1, 10 mL). The combined organic layers were concentrated in vacuo to give 1-4b (1.3 g, crude) as a colorless oil.

[0271] MS (ESI) m / z: 191.2 [M+H] + .

[0272] Step 2: (S,Z)-4-((1-((tert-butoxycarbonyl)amino)-3-hydroxypropan-2-yl)amino)-4-oxobut-2-enoic acid (1-4d)

[0273] To a solution of 1-4b (934 mg, 4.91 mmol) in DCM (15 mL) was added 1-4c (481.43 mg, 4.91 mmol) in DCM (20 mL) dropwise at room temperature. The mixture was concentrated in vacuo to give a residue, which was dissolved in ACN and purified by preparative HPLC (FA 0.1%). The fractions were concentrated in vacuo to give 1-4d (1010 mg, 71.4% yield) as a colorless oil.

[0274] MS (ESI) m / z: 311.3 [M+Na] + .

[0275] 1 H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 7.4 Hz, 1H), 6.36 (s, 2H), 5.22 (t, J = 6.3 Hz, 1H), 3.99 (m, 1H), 3.81 (dd, J = 12.2, 2.8 Hz, 1H), 3.64 (dd, J = 12.2, 3.2 Hz, 1H), 3.45 - 3.24 (m, 3H), 1.45 (s, 9H).

[0276] Step 3: tert-Butyl (S)-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-hydroxypropyl)carbamate (1-4e)

[0277] To a solution of 1-4d (1010.0 mg, 3.50 mmol) in toluene (40 mL) was added 4 Å molecular sieves (300 mg) and TEA (1063.5 mg, 10.51 mmol). The suspension was purged with a N balloon three times and heated to 120 °C overnight. The mixture was filtered, washed with EtOAc (50 mL), washed with 1 N KHSO (30 mL × 2), dried over NaSO, filtered, and purified by FCC (petroleum ether / EtOAc = 0% to 65%) as the mobile phase. The fractions were concentrated in vacuo to give 1-4e (470 mg, 49% yield) as an off-white solid.

[0278] MS (ESI) m / z: 293.2 [M+Na] + .

[0279] 1 H NMR (400 MHz, cdcl3) δ 6.71 (s, 2H), 4.93 (m, 1H), 4.28 (td, J = 9.6, 4.8 Hz, 1H), 3.90 (d, J = 5.2 Hz, 2H), 3.66 (dt, J = 14.8, 7.6 Hz, 1H), 3.46 - 3.39 (m, 1H), 1.40 (s, 9H).

[0280] Step 4: (S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethyl-4,10-dioxo-3,9,14,17,20,23-hexaoxa-5,11-diazahexacosane-26-oic acid (1-4 g)

[0281] To a solution of 1-4e (110 mg, 0.41 mmol) in DMF (3 mL) were added DSC (114.7 mg, 0.45 mmol) and DIPEA (57.9 mg, 0.45 mmol). The mixture was stirred at room temperature for 2 hours, and the mixture became brown. 1-4f (140.4 mg, 0.53 mmol) was added and stirred at room temperature for 30 minutes. The mixture was purified by preparative HPLC (FA), and the fractions were lyophilized to give 1-4g (64 mg, 28% yield) as a white solid.

[0282] MS (ESI) m / z: 584.5 [M+Na] + .

[0283] Step 5: (S)-1-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-oxo-4,9,12,15,18-pentaoxa-6-azahenicosan-21-oic (TFA salt) (1-4)

[0284] A solution of 1-4g (32 mg, 0.057 mmol) in TFA / DCM (1:1, 1 mL) was stirred at room temperature for 10 min. The mixture was concentrated in vacuo, dissolved in water / ACN (2 mL), and lyophilized to give 1-4 (25.2 mg) as a pale yellow oil.

[0285] MS (ESI) m / z: 462.4 [M+H] + .

[0286] Examples 1-5 [ka] Step 1: tert-Butyl (S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethyl-4,10-dioxo-3,9-dioxa-5,11-diazatetradecane-14-oate (1-5b)

[0287] To a solution of 1-4e (107 mg, 0.40 mmol) in DMF (3 mL) was added DSC (121.7 mg, 0.48 mmol) and DIPEA (76.8 mg, 0.59 mmol). The mixture was stirred at room temperature for 2 h, resulting in a brown mixture. 1-5a (93.5 mg, 0.52 mmol) was added and stirred at room temperature for 30 min. This was purified by preparative HPLC (FA 0.1%), and the fractions were lyophilized to give 1-5b (69 mg, 40% yield) as a white solid.

[0288] MS (ESI) m / z: 464.4 [M+Na] + .

[0289] Step 2: (S)-3-(((3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)carbonyl)amino)propanoic acid HCl salt (1-5c)

[0290] A solution of 1-5b (69 mg, 0.16 mmol) in 4 M HCl / EtOAc (3 mL) was stirred in an ice bath for 1 h, and the mixture was concentrated in vacuo to give 1-5c (55 mg, crude) as an off-white solid.

[0291] MS (ESI) m / z: 286.2 [M+H] + .

[0292] Step 3: (S)-3-(((3-(dimethylamino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)carbonyl)amino)propanoic acid (1-5)

[0293] To a suspension of 1-5c (55 mg, 0.19 mmol) in DCM was added 30% formaldehyde in water (5.8 g, 200 equiv.). The mixture was stirred at room temperature for 10 min, then NaBH(OAc) (102.1 mg, 0.48 mmol) was added, followed by stirring at room temperature for 1 h. The DCM was removed using N bubbling, and the residue was purified by preparative HPLC (FA) to give 1-5 (13.4 mg, 22% yield) as a pale yellow oil.

[0294] MS (ESI) m / z: 314.3 [M+H] + .

[0295] Examples 1-6 [ka] Step 1: Ethyl (S)-3-(2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propoxy)propanoate (1-6b)

[0296] To a mixture of 1-4a (9.3 g, 28.7 g), TBAB (924.2 mg, 2.87 mmol), and 1-6a (26.0 g, 143.4 mmol) in DCM (100 mL) was added 50 wt% aqueous NaOH (40 mL) dropwise in an ice bath. The mixture was warmed to room temperature and stirred at room temperature overnight. The mixture was acidified to pH = 3 with 1N KHSO in an ice bath and extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over NaSO, filtered, and concentrated in vacuo to give 1-6b (12 g, crude) as a yellow oil.

[0297] MS (ESI) m / z: 447.4 [M+Na] + .

[0298] Step 2: (S)-3-(2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propoxy)propanoic acid (1-6c)

[0299] To a solution of 1-6b (12 g, crude) in MeOH (50 mL) was added 2N LiOH (10 mL). The mixture was stirred at room temperature for 4 h. The mixture was acidified to pH = 3 with 1N KHSO4 in an ice bath and extracted with EA (100 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC (EA / PE = 40% to 100%) to give 1-6c (2.7 g, crude) as a colorless, clear oil.

[0300] MS (ESI) m / z: 419.3 [M+Na] + .

[0301] Step 3: (S)-3-(3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanoic acid (1-6d)

[0302] To a solution of 1-6c (2 g, crude) in MeOH (30 mL) was added wet Pd / C (200 mg), and the mixture was stirred at room temperature for 30 min. The black suspension was filtered through a syringe head, washed with HO, and concentrated in vacuo to give a residue, which was then dissolved in water (30 mL) and extracted with EA (50 mL × 3). The aqueous phase was concentrated in vacuo to give 1-6d (930 mg) as a clear, colorless oil.

[0303] MS (ESI) m / z: 263.4 [M+H] + .

[0304] Step 4: (S)-3-(3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanoic acid (1-6f)

[0305] To a mixture of 1-6d (930 mg, crude) in saturated NaHCO (10 mL) was added 1-6e (827.3 mg, 5.9 mmol) in an ice bath, stirred for 20 min, and then warmed to room temperature over 1 h. The mixture was acidified to pH = 3 with 1N KHSO in an ice bath and extracted with EA (20 mL × 3). The combined organic layer was dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue, which was purified by preparative HPLC (FA) to give 1-6f (189 mg) as a white solid.

[0306] MS (ESI) m / z: 365.3 [M+H] + .

[0307] Step 5: (S)-3-(3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanoic acid HCl salt (1-6)

[0308] A solution of 1-6f (26 mg, 0.08 mmol) in 4N HCl / EtOAc (1 mL) was stirred at room temperature for 1 h. The suspension was filtered and washed with EtOAc to give 1-6 (12.1 mg) as a white solid.

[0309] MS (ESI) m / z: 243.2 [M+H] + .

[0310] 1H NMR (400 MHz, dmso) δ 12.25 (s, 1H), 8.16 (s, 3H), 7.12 (s, 1H), 4.47 - 4.37 (m, 1H), 3.69 (d, J = 10.8 Hz, 2H), 3.66 - 3.57 (m, 2H), 3.34 (m, 1H), 3.15 (d, J = 11.6 Hz, 1H), 2.45 (t, J = 6.4 Hz, 2H).

[0311] Examples 1-7 [ka] Step 1: Ethyl (S)-3-(3-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)propoxy)propanoate (1-7b)

[0312] To a mixture of 1-7a (5.0 g, 15.41 mmol) (purchased from WUXI), TBAB (496.9 mg, 1.54 mmol), and 1-6a (14.0 g, 77.07 mmol) in DCM (50 mL) was added 50 wt% aqueous NaOH (7.2 g, 77.07 mmol) dropwise. The mixture was stirred overnight at room temperature. The mixture was diluted with DCM (100 mL) and extracted with DCM (50 mL × 2). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. This was purified by silica gel column chromatography (PE / EA = 0% to 30%), and the fractions were concentrated in vacuo to give 1-7b (3.06 g, purity approximately 46.8%) as a colorless, clear oil.

[0313] MS (ESI) m / z: 447.4 [M+Na] + .

[0314] 1H NMR (400 MHz, CDCl3) δ 7.32 (m, 5H), 5.35 (s, 1H), 5.18-5.04 (m, 2H), 4.19 -4.09 (dq, J = 6.8, 1.6 Hz 2H), 3.82 (m, 1H), 3.69 (m, 2H), 3.56 (dd, J = 9.2, 3.6 Hz, 1H), 3.47 (dd, J = 9.6, 5.2 Hz, 1H), 3.45-3.37 (m, 1H), 3.36-3.25 (m, 1H), 2.55 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H).

[0315] Step 2: (S)-3-(3-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)propoxy)propanoic acid (1-7c)

[0316] To a solution of 1-7b (3.06 g, 7.21 mmol) in MeOH (30 mL) was added 4N NaOH (2.7 mL, 10.8 mmol), followed by stirring at room temperature for 12 h. The mixture was diluted with water (30 mL), acidified with 1N KHSO to pH = 3 in an ice bath, and extracted with EtOAc (50 mL × 4). The combined organic layers were washed with brine (50 mL × 3), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give 1-7c (2.8 g, 98% yield) as a clear, colorless oil.

[0317] MS (ESI) m / z: 419.3 [M+Na] + .

[0318] Step 3: (S)-3-(3-amino-2-((tert-butoxycarbonyl)amino)propoxy)propanoic acid (1-7d)

[0319] To a solution of 1-7c (2.8 g, 7.1 mmol) in MeOH (30 mL) was added wet Pd / C (250 mg), which was then stirred at room temperature for 30 min. The black suspension was filtered through a syringe head, washed with HO, and then concentrated in vacuo to give 1-7d (1.85 g) as a white solid.

[0320] MS (ESI) m / z: 263.2 [M+H] + .

[0321] 1 H NMR (400 MHz, d2o) δ 4.1~3.9 (m, 1H), 3.73 (t, J = 6.0 Hz, 2H), 3.64 (dd, J = 10.4, 4.4 Hz, 1H), 3.57 (dd, J = 10.4, 5.2 Hz, 1H), 3.22 (dd, J = 13.2, 4.4 Hz, 1H), 3.08 (dd, J = 13.2, 8.0 Hz, 1H), 2.45 (t, J = 6.0 Hz, 2H), 1.44 (s, 9H).

[0322] Step 4: (S)-3-(2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanoic acid (1-7e)

[0323] To a solution of 1-7d (500 mg, 1.91 mmol) in 1 N NaHCO (10 mL) was added 1-6e (397.7 mg, 2.86 mmol). The mixture was stirred at 0 °C for 30 min and then slowly warmed to room temperature over 2 h. The mixture was acidified to pH = 3 with 1 N KHSO in an ice bath and extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give 1-7e (560 g, 85.8% yield) as a white solid.

[0324] MS (ESI) m / z: 365.3 [M+Na] + .

[0325] 1 H NMR (400 MHz, cdcl3) δ 6.69 (s, 2H), 5.06 (d, J = 9.2 Hz, 1H), 4.08~3.97 (m, 1H), 3.82~3.68 (m, 3H), 3.59 (dd, J = 14.0, 3.6 Hz, 2H), 3.51 (dd, J = 9.6, 4.4 Hz, 1H), 2.64 (t, J = 6.2 Hz, 1H), 1.38 (s, 9H).

[0326] Step 5: tert-Butyl (S)-(1-(3-(dimethylamino)-3-oxopropoxy)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propan-2-yl)carbamate (1-7f)

[0327] To a solution of 1-7e (50 mg, 0.15 mmol) in DMF (2 mL) were added HATU (55.5 mg, 0.15 mmol), DIPEA (56.6 mg, 0.44 mmol), and dimethylamine hydrochloride (11.9 mg, 0.15 mmol). The mixture was stirred at room temperature for 30 min. The mixture was diluted with EtOAc (20 mL), washed with brine (15 mL × 3), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0-5%) to give 1-7f (35 mg, 64.9% yield) as a yellow oil.

[0328] MS (ESI) m / z: 392.4 [M+Na] + .

[0329] Step 6: (S)-3-(2-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)-N,N-dimethylpropanamide TFA salt (1-7)

[0330] A solution of 1-7f (35 mg, 0.095 mmol) in DCM / TFA (1:1, 2 mL) was stirred at room temperature for 10 min. The mixture was concentrated in vacuo to give a residue, which was then dissolved in water (20 mL) and extracted with EtOAc (20 mL × 3). The aqueous phase was lyophilized to give 1-7 (38.5 mg) as a brown oil.

[0331] MS (ESI) m / z: 392.4 [M+Na] + .

[0332] 1H NMR (400 MHz, dmso) δ 8.05 (s, 3H), 7.09 (s, 2H), 3.73 - 3.55 (m, 6H), 3.53 - 3.42 (m, 1H), 2.97 (s, 3H), 2.83 (s, 3H), 2.58 (t, J = 6.4 Hz, 2H).

[0333] Examples 1-8 [ka] Step 1: tert-Butyl (S)-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-(((4-nitrophenoxy)carbonyl)oxy)propyl)carbamate (1-8a)

[0334] To a solution of 1-4e (81 mg, 0.3 mmol) and bis(4-nitrophenyl)carbonate (100.2 mg, 0.33 mmol) in DMF (2.3 mL) was added DIPEA (81.6 mL, 0.45 mmol) at room temperature. After the addition, the mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc, and the organic layer was washed with saturated aqueous NH4Cl, HO, and brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. The crude product was triturated with MTBE and filtered to give the desired product 1-8a (91 mg, 70% yield) as a white solid.

[0335] MS (ESI) m / z: 458.4 [M+Na] + .

[0336] Step 2: (9H-Fluoren-9-yl)methyl (2-(dimethylamino)-2-oxoethyl)(methyl)carbamate (1-8c)

[0337] To a solution of 1-8b (1.0 g, 3.2 mmol) in DMF (15 mL) was added HATU (1.34 g, 3.53 mmol), DIPEA (1.24 g, 9.63 mmol), and dimethylamine hydrochloride (314 mg, 3.85 mmol). The reaction was stirred at room temperature for 30 min. The mixture was diluted with EtOAc (100 mL), washed with brine (15 mL × 3), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0-5%) to give 1-8c (1.75 g, >100% yield) as a yellow oil containing 1-hydroxy-7-azabenzotriazole (HOAt).

[0338] MS (ESI) m / z: 339.3 [M+H] + .

[0339] Step 3: N,N-Dimethyl-2-(methylamino)acetamide (1-8d)

[0340] To a solution of 1-8c (100.0 mg, 0.30 mmol) in DMF (3 mL) was added EtNH (252 mg, 3.0 mmol). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and co-evaporated twice with toluene to give 1-8d as a brown solid, which was used directly in the next step.

[0341] Step 4: (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl(2-(dimethylamino)-2-oxoethyl)(methyl)carbamate (1-8f)

[0342] To a solution of 1-8e (50.0 mg, 0.115 mmol) in DMF (1 mL) was added crude solid 1-8d and DIPEA (44.5 mg, 0.345 mmol) at room temperature. The mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with DMF and acidified with 0.1 N HCl. The mixture was purified by preparative HPLC (FA), and the fractions were lyophilized to give 1-8f (17.2 mg, 36.3% yield) as a pale yellow solid.

[0343] MS (ESI) m / z: 435.4 [M+Na] + .

[0344] Step 5: (S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl(2-(dimethylamino)-2-oxoethyl)(methyl)carbamate (1-8)

[0345] A solution of 1-8f (7.7 mg, 0.021 mmol) in TFA / DCM (1:1, 0.6 mL) was stirred at room temperature for 60 min. The mixture was concentrated in vacuo, dissolved in water / ACN (1:1, 2 mL), and lyophilized to give 1-8 (7.8 mg, approximately 100% yield) as a white solid.

[0346] MS (ESI) m / z: 313.3 [M+H] + .

[0347] Examples 1-9 [ka] Step 1: Benzyl (3-(dimethylamino)-3-oxopropyl)carbamate (1-9b)

[0348] To a solution of 1-9a (1.116 g, 5 mmol) in DMF (25 mL) was added HATU (2.09 g, 5.5 mmol), DIPEA (1.94 g, 15 mmol), and dimethylamine hydrochloride (489 mg, 6 mmol). The reaction was stirred at room temperature for 30 min. The mixture was diluted with EtOAc (100 mL), washed with brine (15 mL × 3), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0-5%) to give 1-9b (0.987 g, 79% yield) as a yellow oil.

[0349] MS (ESI) m / z: 251.2 [M+H] + .

[0350] Step 2: 3-Amino-N,N-dimethylpropanamide (1-9c)

[0351] To a suspension of 1-9b (500 mg, 2.0 mmol) in MeOH (5 mL) was added Pd / C (50 mg, 10% wt / wt) at room temperature. The atmosphere was replaced with H2, and the mixture was stirred at room temperature for 2 h. The mixture was filtered through a Celite pad, and the filtrate was concentrated in vacuo to give crude product 1-9c (233 mg, approximately 100% yield) as a colorless oil, which was used directly in the next step.

[0352] MS (ESI) m / z: 117.0 [M+H] + .

[0353] Step 3: 3-(Isopropylamino)-N,N-dimethylpropanamide (1-9d)

[0354] To a solution of 1-9c (233 mg, 2.0 mmol) and acetone (232.3 mg, 4.0 mmol) in MeOH (9 mL) was added NaBHCN (510.8 mg, 8.0 mmol) at room temperature. The reaction was stirred at room temperature for 18 h. The mixture was concentrated in vacuo, and the residue was redissolved in DCM (20 mL), washed with saturated NaHCO (10 mL), HO (10 mL), brine (10 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give the crude desired product 1-9d (164 mg, 52% yield) as a pale green oil.

[0355] MS (ESI) m / z: 159.1 [M+H] + .

[0356] Step 4: (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl(3-(dimethylamino)-3-oxopropyl)(isopropyl)carbamate (1-9e)

[0357] To a solution of 1-8e (30.0 mg, 0.115 mmol) in DMF (1 mL) was added crude oil 1-9d (33 mg, 0.21 mmol) and DIPEA (27 mg, 0.21 mmol) at room temperature. The reaction was stirred at room temperature for 30 min. The reaction mixture was diluted with DMF and acidified with 0.1 N HCl. The mixture was purified by preparative HPLC (FA), and the fractions were lyophilized to afford 1-9e (22.5 mg, 58.8% yield) as a white solid.

[0358] MS (ESI) m / z: 477.4 [M+Na] + .

[0359] Step 5: (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl (3-(dimethylamino)-3-oxopropyl)(isopropyl)carbamate trifluoroacetate (1-9)

[0360] A solution of 1-9e (22.5 mg, 0.0495 mmol) in TFA / DCM (1:1, 1.2 mL) was stirred at room temperature for 60 min. The mixture was concentrated in vacuo, dissolved in water / ACN (1:1, 2 mL), and lyophilized to give 1-9 (23.2 mg, approximately 100% yield) as a white solid.

[0361] MS (ESI) m / z: 355.3 [M+H] + .

[0362] Examples 1-10 [ka] Step 1: Benzyl tert-butyl(3-oxopropane-1,2-diyl)(S)-dicarbamate (1-10a)

[0363] A solution of (COCl)2 (3.9 g, 30.83 mmol) in DCM (50 mL) was cooled to -70 °C, and DMSO (3.6 g, 46.24 mmol) was added dropwise, followed by stirring at -70 °C for 30 min. 1-4a (5.0 g, 15.41 mmol) was dissolved in DMSO (10 mL), added via syringe, and then stirred at -70 °C for 30 min. TEA (7.8 g, 77.1 mmol) was added dropwise, followed by slowly warming to room temperature. The mixture was poured into water (100 mL) and extracted with EtOAc (50 mL × 2). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC using EA / PE (1%-50%) as the mobile phase, and the fractions were concentrated under vacuum to give 1-10a (4.86 g, 97.8% yield) as a yellow oil.

[0364] 1H NMR (400 MHz, cdcl3) δ 9.64 (s, 1H), 7.39 - 7.30 (m, 5H), 5.93 (s, 1H), 4.86 (s, 1H), 4.35-4.22(m, 1H), 3.77 - 3.64 (m, 1H), 3.56 (dt, J = 14.8, 4.8 Hz, 1H), 1.40 (s, 9H).

[0365] Step 2: Ethyl (R)-6-(((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)hept-4-enoate (1-10c)

[0366] To a suspension of 1-10b (6.5 g, 14.3 mmol) in toluene (30 mL), t-BuOK (1 M solution in THF, 14.5 mL, 14.5 mmol) was added dropwise at 0 °C. The mixture turned dark over 30 min. 1-10a (2.0 g, 6.2 mmol) in toluene (20 mL) was added dropwise at -70 °C and slowly warmed to room temperature. The mixture was quenched by adding saturated NH4Cl (50 mL). After separation, the toluene phase was washed with brine (30 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give a residue. Purification by FCC (EA / PE = 0% to 30% to 50%) afforded 1-10c (808 mg, 30.8% yield) as a white solid.

[0367] MS (ESI) m / z: 443.4 [M+Na]+.

[0368] 1 H NMR (400 MHz, cdcl3) δ 7.40 - 7.27 (m, 5H), 5.72~5.60 (m, 0.2 H), 5.60 - 5.46 (m, 0.8H), 5.40 (dd, J = 16.0 Hz, 6.4 Hz, 0.2H), 5.34~5.20 (m, 1.8 H), 4.95~4.70 (m, 1 H), 5.16~5.01 (m, 2H), 4.60~4.40 (m, 1 H),4.12 (q, J = 7.2 Hz, 2H), 3.39 - 3.08 (m, 2H), 2.6~2.2 (m, 4H), 1.42 (s, 9H), 1.24 (t, J = 7.2 Hz, 1H).

[0369] Step 3: (R)-6-(((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)hept-4-enoic acid (1-10d)

[0370] To a solution of 1-10c (808.0 mg, 1.92 mmol) in MeOH (10 mL) was added 4N NaOH (1.92 mL, 7.69 mmol). The mixture was stirred at room temperature for 3.5 hours. The mixture was diluted with water (20 mL), acidified with saturated KHSO to pH = 3, and extracted with EtOAc (30 mL × 3). The combined organic layer was washed with brine (50 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give 1-10d (781 mg) as a white solid.

[0371] MS (ESI) m / z: 415.4 [M+Na] + .

[0372] Step 4: (R)-6-Amino-7-((tert-butoxycarbonyl)amino)heptanoic acid (1-10e)

[0373] To a solution of 1-10d (781.0 mg, 1.99 mmol) in MeOH (8 mL) was added wet Pd / C (67 mg) and stirred at room temperature for 24 h. The black suspension was filtered with a syringe head, washed with MeOH / HO (5:1, 30 mL), and then concentrated in vacuo to give 1-10e (510 mg, 98.5% yield) as a white solid.

[0374] MS (ESI) m / z: 261.3 [M+H] + .

[0375] Step 5: (R)-7-((tert-butoxycarbonyl)amino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoic acid (1-10f)

[0376] To a mixture of 1-10e (510 mg, 1.96 mmol) in 1 N NaHCO3 (10 mL) was added 1-6e (455.8 mg, 2.94 mmol) at 0 °C, stirred at 0 °C for 30 min, and then warmed to room temperature over 3 h. The reaction was acidified with saturated KHSO4 to pH = 3 and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated to give a residue, which was purified by FCC (MeOH / DCM = 0% to 5%). The fractions were concentrated in vacuo to give 1-10f (590 mg, 85% yield) as a pale yellow oil.

[0377] MS (ESI) m / z: 363.3 [M+Na] + .

[0378] Step 6: tert-Butyl (R)-(7-(dimethylamino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7-oxoheptyl)carbamate (1-10g)

[0379] To a solution of 1-10f (53 mg, 0.16 mmol) in DMF (2 mL) were added HATU (71.6 mg, 0.19 mmol), DIPEA (44.3 mg, 0.34 mmol), and dimethylamine hydrochloride (14.0 mg, 0.17 mmol). The mixture was stirred at room temperature for 30 min. The mixture was diluted with EtOAc (20 mL), washed with brine (15 mL × 3), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0-5%) to give 1-10g (53 mg, 92.1% yield) as a yellow oil.

[0380] MS (ESI) m / z: 390.4 [M+Na] + .

[0381] Step 7: (R)-7-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N,N-dimethylheptanamide TFA salt (1-10)

[0382] A solution of 1-10g (30 mg, 0.08 mmol) in DCM / TFA (1:1, 2 mL) was stirred at room temperature for 10 minutes. The mixture was concentrated in vacuo to give a residue, which was then dissolved in water (20 mL) and extracted with EtOAc (20 mL × 3). The aqueous phase was lyophilized to give 1-10 (33 mg) as a brown oil.

[0383] MS (ESI) m / z: 268.3 [M+H] + .

[0384] Examples 1-11 [ka] Step 1: Methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate (1-11b)

[0385] 1-11a (4.00 g, 9.08 mmol) and K2CO3 (1.38 g, 9.99 mmol) were added to DMF (10 mL), followed by dropwise addition of CHI (2.58 g, 18.16 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 20 min, warmed to 25 °C, and further stirred at 25 °C for 30 min. After the reaction was complete, the reaction mixture was diluted with EA (50 mL) and washed with brine (25 mL × 3) and HO (25 mL × 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give 1-11b (4.08 g, quantitative) as a pale yellow solid.

[0386] MS (ESI) m / z: 477.4 [M+Na] + .

[0387] Step 2: (9H-Fluoren-9-yl)methyl tert-butyl(4-hydroxybutane-1,3-diyl)(S)-dicarbamate (1-11c)

[0388] 1-11b (1.00 g, 2.20 mmol) was dissolved in THF (55 mL) and EtOH (55 mL), followed by the successive addition of NaBH (499 mg, 13.20 mmol) and LiCl (616 mg, 14.52 mmol) at 0 °C. The resulting mixture was stirred at 25 °C for 1.5 h. After the reaction was complete, saturated aqueous NH Cl (10 mL) was added to quench the reaction. The reaction mixture was diluted with H O (50 mL) and extracted with EA (35 mL × 3). The combined organic layer was washed with brine (30 mL × 2) and water (30 mL × 2), dried over anhydrous Na SO , filtered, and concentrated under reduced pressure to give 1-11c as a white solid (938 mg, quantitative). The product was used directly in the next step without purification.

[0389] MS (ESI) m / z: 449.4 [M+Na] + .

[0390] 1 H NMR (400 MHz, d6-DMSO) δ 7.86 (d, J=7.2 Hz, 2H), 7.70-7.67 (m, 2H), 7.39 (t, J=7.2 Hz, 2H), 7.32-7.29 (m, 2H), 7.07-7.05 (m, 1H), 6.67 (t, J=5.2 Hz, 1H), 6.51 (s, 1H), 4.62 (br s, 1 H), 4.29-4.17 (m, 3H), 3.41-3.38 (m, 1H), 3.25-3.24 (m, 1H), 2.98-2.95 (m, 1H), 2.86-2.83 (m, 1H), 1.33 (s, 9H).

[0391] Step 3: (9H-Fluoren-9-yl)methyl tert-butyl (4-(((4-nitrophenoxy)carbonyl)oxy)butane-1,3-diyl) (S)-dicarbamate (1-11d)

[0392] 1-11c (850 mg, 1.87 mmol) and bis(4-nitrophenyl)carbonate (1.14 g, 3.74 mmol) were dissolved in DMF (5 mL), followed by the addition of DIPEA (363 mg, 2.81 mmol). The resulting mixture was stirred at 25 °C for 1.5 h. After the reaction was complete, the reaction mixture was diluted with EA (100 mL) and washed with brine (35 mL × 2) and water (35 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 1-11d as a white solid (890 mg, 80.4% yield).

[0393] MS (ESI) m / z: 614.4 [M+Na] + .

[0394] Step 4: (9H-Fluoren-9-yl)methyl tert-butyl (4-(((3-(dimethylamino)-3-oxopropyl)carbamoyl)oxy)butane-1,3-diyl) (S)-dicarbamate (1-11f)

[0395] 1-11d (360 mg, 0.61 mmol) and 1-11e (141 mg, 1.22 mmol) were dissolved in DMF (5 mL), followed by the addition of DIPEA (157 mg, 1.22 mmol). The resulting mixture was stirred at 25 °C for 2.5 h. After the reaction was complete, the reaction mixture was concentrated and purified by flash column chromatography (DCM / MeOH) to give 1-11f (260 mg, 75.1% yield) as a pale yellow solid.

[0396] MS (ESI) m / z: 592.5 [M+Na] + .

[0397] Step 5: tert-Butyl (S)-(3-amino-4-(((3-(dimethylamino)-3-oxopropyl)carbamoyl)oxy)butyl)carbamate (1-11g)

[0398] 1-11f (260 mg, 0.46 mmol) was dissolved in DMF (3 mL), followed by the addition of EtNH (334 mg, 4.57 mmol). The resulting mixture was stirred at 25 °C for 15 min. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to give 1-11g as a light brown syrup (158 mg, quantitative), which was used directly in the next step.

[0399] MS (ESI) m / z: 347.4 [M+H] + .

[0400] Step 6: (S)-4-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl(3-(dimethylamino)-3-oxopropyl)carbamate (1-11i)

[0401] 1-11g (64 mg, 0.18 mmol) and 1-11h (43 mg, 0.28 mmol) were dissolved in a mixture of ACN (2 mL) and aqueous NaHCO (1 M, 4 mL). The mixture was first stirred at 0 °C for 30 min and then at 25 °C for 40 min. After the reaction was complete, the reaction mixture was adjusted to pH 4 with aqueous KHSO (2 M), diluted with EA (60 mL), and washed with brine (30 mL) and HO (30 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated to give a yellow oil as the crude product, which was purified by flash column chromatography (DCM / MeOH) to give 1-11i (60 mg, 76.2% yield) as a white solid.

[0402] MS (ESI) m / z: 449.5 [M+Na] + .

[0403] 1H NMR (400 MHz, CD3OD) δ 6.79 (s, 2H), 6.56-6.54 (m, 1H), 4.34-4.31 (m, 2H), 4.23-4.16 (m, 1H), 3.31-3.30 (m, 2H), 3.01-2.99 (m, 5H), 2.92 (s, 3H), 2.55-2.52 (m, 2H), 2.17-2.08 (m, 1H), 1.86-1.77 (m, 1H), 1.41 (s, 9H).

[0404] Step 7: (S)-4-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl(3-(dimethylamino)-3-oxopropyl)carbamate (1-11)

[0405] 1-11i (55 mg, 0.13 mmol) was dissolved in DCM (3 mL), followed by the addition of TFA (1 mL). The resulting mixture was stirred at 25 °C for 20 min. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to give 1-11 as a clear oil (TFA salt, 54 mg, quantitative).

[0406] MS (ESI) m / z: 327.3 [M+H] + .

[0407] 1 H NMR (400 MHz, d6-DMSO) δ 7.78 (s, 3H), 7.05 (s, 2H), 4.28-4.13 (m, 3H), 3.12 (dd, J1=12.8 Hz, J1=6.8 Hz, 2H), 2.91 (s, 3H), 2.79-2.73 (m, 5H), 2.39 (t, J=7.2 Hz, 2H), 2.12-2.07 (m, 1H), 1.96-1.90 (m, 1H).

[0408] Examples 1-12 [ka] Step 1: Methyl 3-aminopropanoate (1-12b)

[0409] SOCl2 (13.4 g, 112.2 mmol) was added dropwise to a suspension of compound 1-12a (5 g, 56.1 mmol) in MeOH (100 mL). The mixture was stirred at 70 °C for 16 h. The mixture was concentrated to give the crude compound. The crude compound was further purified by flash column chromatography (eluted with DCM / MeOH = 0 to 20%). Compound 1-12b (3.2 g, 55.3%) was obtained as an off-white solid.

[0410] MS (ESI) m / z: 130.1 [M+Na] + .

[0411] Step 2: Methyl 3-isocyanatopropanoate (1-12c)

[0412] A solution of compound 1-12b (500 mg, 4.85 mmol) in CHCl (5 mL) and saturated NaHCO (aq) (5 mL) was degassed, and the flask was cooled in an ice-water bath. Triphosgene (1.44 g, 4.85 mmol) was added in one portion at 0 °C under an inert atmosphere. The reaction was stirred from 0 °C to room temperature for 2.5 h. The reaction was diluted with water (100 mL) and poured into a separatory funnel. The layers were separated, and the aqueous layer was extracted with CHCl. ​​The combined organic extracts were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give crude compound 1-12c (426 mg, 68.1% yield) as a yellow liquid, which was used directly in the next reaction without purification.

[0413] Step 3: Benzyl tert-butyl (3-(1,3-dioxoindolin-2-yl)propane-1,2-diyl) (S)-dicarbamate (1-12e)

[0414] Triphenylphosphine (2.91 g, 11.1 mmol) and phthalimide (1.63 g, 11.1 mmol) were added to a flask containing dry THF (30 mL). Compound 1-12d (3 g, 9.25 mmol) was added, and the flask was cooled to 0 °C. Diisopropyl azodicarboxylate (DIAD) (2.24 mg, 11.1 mmol) was added dropwise, and the mixture was stirred at 0 °C for 30 min and then at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (eluted with PE / EA = 0 to 70%) to give compound 1-12e (3.6 g, 85.9%) as a white solid.

[0415] MS (ESI) m / z: 476.4 [M+Na] + .

[0416] Step 4: Benzyl tert-butyl(3-aminopropane-1,2-diyl) (R)-dicarbamate (1-12f)

[0417] Compound 1-12e (1.3 g, 2.87 mmol) was dissolved in methanol (30 mL) and hydrazine monohydrate (359 mg, 5.73 mmol) was added. The reaction mixture was refluxed for 2 h and then cooled to room temperature. The precipitate formed was filtered, and the filtrate was washed with methanol. The filtrate was concentrated under reduced pressure, and the remaining solid was purified by flash column chromatography (eluted with DCM / MeOH = 0-20%) to give compound 1-12f (640 mg, 69.0% yield) as a colorless oil.

[0418] MS (ESI) m / z: 324.3 [M+H] + .

[0419] Step 5: Methyl (R)-7-(((benzyloxy)carbonyl)amino)-2,2-dimethyl-4,10-dioxo-3-oxa-5,9,11-triazatetradecane-14-oate (1-12 g)

[0420] To a solution of compound 1-12f (500 mg, 1.55 mmol) in THF (10 mL) were added 1-12c (399 mg, 3.09 mmol) and EtN (312.9 mg, 3.09 mmol) at 0 °C, and the mixture was allowed to warm to room temperature for 4 h. The mixture was diluted with EA (100 mL) and washed with brine (50 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by flash column chromatography (eluted with PE / EA = 0% to 40%) to give compound 1-12g (335 mg, 47.9% yield) as a white solid.

[0421] MS (ESI) m / z: 453.5 [M+H] + .

[0422] Step 6: (R)-7-(((benzyloxy)carbonyl)amino)-2,2-dimethyl-4,10-dioxo-3-oxa-5,9,11-triazatetradecan-14-oic acid (1-12h)

[0423] To a solution of compound 1-12g (335 mg, 0.74 mmol) in MeOH-HO (6 mL, v:v=3:1) was added LiOH (35.5 mg, 1.48 mmol). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated to remove MeOH and diluted with HO (5 mL). The pH of the mixture was adjusted to 5 with HOAc and extracted with EA (50 mL × 3). The organic layers were combined, dried, and concentrated to give compound 1-12h (289 mg, 88.9% yield) as a white solid.

[0424] MS (ESI) m / z: 439.4 [M+H] + .

[0425] Step 7: Benzyl tert-butyl (3-(3-(3-(dimethylamino)-3-oxopropyl)ureido)propane-1,2-diyl) (R)-dicarbamate (1-12i)

[0426] To a solution of compound 1-12h (180 mg, 0.41 mmol) in DCM (6 mL) was added MeNH.HCl (67.0 mg, 0.82 mmol), 1-ethyl-3-(3-dimethylaminopropyl)urea (EDCI) (102.3 mg, 0.534 mmol), HOBt (72.1 mg, 0.534 mmol), and EtN (83.08 mg, 0.82 mmol). The mixture was stirred at room temperature for 16 h. The mixture was diluted with EA (100 mL) and washed with 1 N HCl (50 mL × 3), saturated NaHCO (50 mL × 3), and brine (50 mL × 3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated to give crude product 1-12i (131 mg, 68.6% yield) as a white solid.

[0427] MS (ESI) m / z: 466.5 [M+H] + .

[0428] Step 8: tert-Butyl (R)-(2-amino-3-(3-(3-(dimethylamino)-3-oxopropyl)ureido)propyl)carbamate (1-12j)

[0429] To a solution of compound 1-12i (131 mg, 0.28 mmol) in MeOH (4 mL) was added Pd / C (10%, 26 mg). The mixture was stirred under H at room temperature for 4 hours. The mixture was filtered and concentrated to give crude product 1-12j (93 mg, crude) as a colorless oil.

[0430] MS (ESI) m / z: 332.4 [M+H] + .

[0431] Step 9: tert-Butyl (R)-(3-(3-(3-(dimethylamino)-3-oxopropyl)ureido)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)carbamate (1-12l)

[0432] To a solution of compound 1-12j (93 mg, 0.281 mmol) in NaHCO3 (1N, 5 mL) and THF (5 mL) was added 1-12k (132 mg, 0.842 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 minutes, then warmed to room temperature and stirred for 1 hour. The mixture was purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5um 19*250 mm; Mobile phase: A-water (0.1% TFA): B-acetonitrile; Flow rate: 20 mL / min). Compound 1-12l (32 mg, yield 27.7%) was obtained as a white solid.

[0433] MS (ESI) m / z: 412.4 [M+H] + .

[0434] Step 10: (S)-3-(3-(3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)ureido)-N,N-dimethylpropanamide acetate (1-12)

[0435] To a solution of compound 1-12l (30 mg, 0.073 mmol) in DCM (10 mL) was added TFA (1 mL). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated to give crude compound 1-12 (19 mg, 83.7% yield) as a white solid.

[0436] MS (ESI) m / z: 312.4 [M+H] + .

[0437] 1 H NMR (400 MHz, DMSO): δ 7.90 (br s, 3H), 7.04 (s, 2H), 6.32-6.29 (m, 1H), 5.89 (br s, 1H), 4.20-4.17 (m, 1H), 3.37-3.23 (m, 4 H), 3.15-3.12 (m, 3H), 3.09 (s, 3H), 2.73 (s, 3H), 2.36-2.33 (m, 2H).

[0438] Examples 1-13 [ka] 4-Amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl(3-(dimethylamino)-3-oxopropyl)carbamate (1-13) 1-13 (TFA salt, 178 mg, quantitative) was synthesized according to the synthetic procedure of Example 1-11.

[0439] MS (ESI) m / z: 327.4 [M+H] + .

[0440] 1 H NMR (400 MHz, d6-DMSO): δ 8.01 (br s, 3H), 7.04 (s, 2H), 6.83 (t, J=6.0 Hz, 1H), 4.28-4.23 (m, 1H), 3.87 (t, J=6.0 Hz, 2H), 3.34-3.29 (m, 1H), 3.17-3.12 (m, 3H), 2.94 (s, 3H), 2.81 (s, 3H), 2.43 (t, J=7.2 Hz, 2H), 2.13-2.06 (m, 1H), 1.98-1.93 (m, 1H).

[0441] Examples 1-14 [ka] (R)-3-(4-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butoxy)propanoic acid (1-14e)

[0442] Following the synthetic procedure of Example 1-7, 1-13e (110 mg) was synthesized as a white solid.

[0443] MS (ESI) m / z: 379.3 [M+Na] + .

[0444] 1H NMR (400 MHz, cdcl3) δ 6.68 (s, 2H), 4.87 (s, 1H), 4.42-4.26 (m, 1H), 3.70-3.56 (m, 3H), 3.50 3.52-3.47 (m, 1H), 3.43-3.36 (m, 1H), 3.31 (dt, J=14.0, 4.4 Hz, 1H), 2.56 (t, J=6.0 Hz, 1H), 3.32-2.12 (m, 1H), 1.97-1.92 (m, 1H), 1.40 (s, 9H).

[0445] (R)-3-(4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butoxy)-N,N-dimethylpropanamide (TFA salt) (1-14)

[0446] Following the synthetic procedure of Example 1-7, 1-14 (9.6 mg) was synthesized as a brown oil.

[0447] MS (ESI) m / z: 384.4 [M+H] + .

[0448] Reference compounds 2-1 and 2-3 were synthesized according to the methods reported in Patent No. US10,973,924B2 and Patent Application Publication No. US2020 / 345863A1.

[0449] Example 2-2 [ka] Step 1: (9H-Fluoren-9-yl)methyl((6S,15S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-15-benzyl-24-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7,10,13,16,19,24-heptaoxo-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (2-2c)

[0450] Compound 2-2c (20 mg, yield 36.8%) was synthesized according to Step 3 of Example 1-2.

[0451] MS (ESI) m / z: 1386.9 [M+Na] + .

[0452] Step 2: (S)-2-amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)pentanediamide (2-2d)

[0453] Compound 2-2c (20 mg, 36.8% yield) was synthesized according to Step 5 of Example 1-11. (17 mg, quantitative)

[0454] MS (ESI) m / z: 1142.8 [M+H] + .

[0455] Step 3: (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)pentanediamide(2-2)

[0456] To a mixture of 1-3 (5 mg, 0.021 mmol) and HATU (8 mg, 0.021 mmol) in DMF (1 mL) was added DIPEA (5 mg, 0.035 mmol). The mixture was allowed to react at room temperature for 10 minutes. 2-2d (20 mg, 0.018 mmol) was added, and the mixture was stirred at this temperature for an additional 15 minutes. The mixture was filtered, and the filtrate was purified using preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 um 19*250 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) to give 2-2 (4 mg, 16.9% yield).

[0457] MS (ESI) m / z: 1372.8 [M+Na] + .

[0458] Examples 2-4 [ka] Step 1: (2R,3R,4S,5S,6S)--2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(((2-(benzyloxy)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-4c)

[0459] A mixture of 2-4a (150 mg, 0.25 mmol, purchased from MedChemExpress), 2-4b (148 mg, 0.37 mmol, commercially available), and 4Å MS (800 mg) in dry DCE (5 mL) was stirred at room temperature for 30 min. AgOTf (83 mg, 0.32 mmol) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 14 h. The reaction solution was diluted with EtOAc (10 mL), filtered through Celite, and washed with EtOAc (5 mL × 3). The organic phase was washed with saturated NaHCO3 (20 mL), concentrated, and purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100, then DCM / MeOH = 100 / 0 to 95 / 5) to give 2-4c (28 mg, 12.2% yield) as a pale yellow solid.

[0460] MS (ESI) m / z: 942.5 [M+Na] + .

[0461] Step 2: (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxa-4,7,10-triazatetradecan-14-oic acid (2-4d)

[0462] To a mixture of 2-4c (114 mg, 0.12 mmol) and 10% Pd / C (26 mg, 0.012 mmol) was added MeOH (3 mL) under a nitrogen atmosphere. The reaction solution was stirred at room temperature under a H atmosphere for 1 h. The solution was filtered and concentrated in vacuo to give 2-4d (103 mg, 12.4% yield) as a white solid.

[0463] MS (ESI) m / z: 852.5 [M+Na] + .

[0464] Step 3: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-4e)

[0465] To a solution of 2-4d (103 mg, 0.12 mmol), exatecan mesylate (64 mg, 0.12 mmol), and HATU (47 mg, 0.12 mmol) in dry DMF (2 mL) was added DIPEA (61 μL, 0.36 mmol). The solution was stirred at room temperature for 1 h, added to HO (20 mL), and extracted with DCM / MeOH (10:1, 11 mL × 3). The organic phase was concentrated and purified by flash column chromatography (eluent: DCM / MeOH = 100 / 0 to 95 / 5) to give 2-4e (97 mg, 64.0% yield) as a light brown solid.

[0466] MS (ESI) m / z: 1269.5 [M+Na] + .

[0467] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-4f)

[0468] To a solution of 2-4e (30 mg, 0.024 mmol) in MeOH / HO (0.5 / 0.5 mL) was added TEA (167 μL, 1.2 mmol). The solution was stirred at room temperature for 9 hours. After the reaction was complete, the mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 um 19*250 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to give 2-4f (15 mg, 70.6% yield) as a pale yellow solid.

[0469] MS (ESI) m / z: 885.5 [M+H] + .

[0470] Step 5: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-3-methylbutanamido)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-4)

[0471] To a solution of 1-3 (8.3 mg, 0.038 mmol) and HATU (11 mg, 0.029 mmol) in dry DMF (0.3 mL) was added DIPEA (5 μL, 0.029 mmol). The resulting mixture was stirred at room temperature for 15 min and then added dropwise to a solution of 2-4f (17 mg, 0.019 mmol) in dry DMF (0.7 mL). The resulting mixture was further stirred at room temperature for 15 min. HOAc was added to adjust the pH to 5. The solution was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 um 19*250 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to give 2-4 (13 mg, 63.5% yield) as a beige solid.

[0472] MS (ESI) m / z: 1093.5 [M+H] + .

[0473] Examples 2-5 [ka] Step 1: (2R,3R,4S,5S,6S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-5b)

[0474] A mixture of 2-5a (834 mg, 2 mmol), 2-4b (950 mg, 2.4 mmol), and 4Å MS (3000 mg) was added to dry DCM (30 mL) and stirred at room temperature for 30 min. AgOTf (617 mg, 2.4 mmol) was added under a nitrogen atmosphere and stirred at room temperature for 16 h. The reaction solution was diluted with EtOAc (30 mL), filtered through Celite, and washed with EtOAc (20 mL × 3). The organic phase was washed with saturated NaHCO3 (20 mL), concentrated, and purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100) to give 2-5b (470 mg, 32.1% yield) as a pale yellow solid.

[0475] MS (ESI) m / z: 756.3 [M+Na] + .

[0476] Step 2: (2R,3R,4S,5S,6S)-2-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-5c)

[0477] To a solution of 2-5b (470 mg, 0.64 mmol) in DMF (5 mL) was added EtN (1420 μL, 1011 mg, 12.8 mmol). The mixture was stirred at room temperature for 0.5 h. Upon completion of the reaction, the mixture was concentrated in vacuo to give 2-5c (327 mg, crude) as a yellow solid.

[0478] MS (ESI) m / z: 512.4 [M+H] + .

[0479] Step 3: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-5e)

[0480] To a solution of 2-5d (327 mg) in DMF (5 mL) were added HATU (291 mg, 0.76 mmol) and DIPEA (223 μL, 165 mg, 1.27 mmol). The resulting yellow solution was stirred at room temperature for 5 min, and then 2-5c (239 mg, 0.70 mmol) was added. The mixture was stirred at room temperature for an additional 60 min. Upon completion of the reaction, the solvent was evaporated, and the residue was purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100) and preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*250 mm; Mobile phase: A - water (0.1% formic acid): B - acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to give 2-5e (300 mg, 56.2% yield) as a white solid.

[0481] MS (ESI) m / z: 833.4 [M+H] + .

[0482] Step 4: N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (2-5f)

[0483] To a solution of 2-5e (300 mg, 0.36 mmol) in MeOH (10 mL) and THF (8 mL), wet Pd / C (30 mg) was added, purged with a H balloon three times, and stirred at room temperature for 2 h. Upon reaction completion, the mixture was filtered through a syringe filter head, and the filtrate was concentrated in vacuo to give 2-5f (265 mg, crude) as a white solid.

[0484] MS (ESI) m / z: 765.3 [M+Na] + .

[0485] Step 5: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-((2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate(2-5h)

[0486] To a solution of 2-5f (265 mg) in DMF (5 mL) was added HATU (120 mg, 0.42 mmol) and DIPEA (124 μL, 92 mg, 0.71 mmol). The resulting yellow solution was stirred at room temperature for 5 min, and then 2-5g (227 mg, 0.39 mmol) was added. The mixture was stirred at room temperature for 60 min. Upon completion of the reaction, the solvent was evaporated, and the residue was purified by flash column chromatography (eluent: DCM / MeOH = 95 / 5 to 90 / 10) to give 2-5h (280 mg, 59.6% yield) as a brown solid.

[0487] MS (ESI) m / z: 1326.4 [M+Na] + .

[0488] Step 6: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-amino-3-methylbutanamide)-3-((2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-5i)

[0489] To a solution of 2-5h (280 mg, 0.21 mmol) in DMF (5 mL) was added EtN (478 μL, 340 mg, 4.29 mmol). The mixture was stirred at room temperature for 0.5 h. Upon completion of the reaction, the mixture was concentrated in vacuo to give 2-5i (220 mg, crude) as a white solid.

[0490] MS (ESI) m / z: 1082.4 [M+H] + .

[0491] Step 7: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-((2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-5j)

[0492] To a solution of 2-5i (220 mg) in MeOH / HO (5 / 5 mL) was added NaCO (151 mg, 1.42 mmol), and the mixture was stirred at room temperature for 9 hours. The solution was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 um 19*250 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to give 2-5j (45 mg, 23.8% yield) as a white solid.

[0493] MS (ESI) m / z: 942.3 [M+H] + .

[0494] Step 8: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamide)-3-methylbutanamide)-3-((2-(((2-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo So-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-5)

[0495] To a solution of 1-3 (5.76 mg, 0.026 mmol) and HATU (9.576 mg, 0.025 mmol) in dry DMF (1 mL) was added DIPEA (7.3 μL, 5.4 mg, 0.042 mmol), and the mixture was stirred at room temperature for 15 min. The solution was added dropwise to a solution of dry 2-5j (20 mg, 0.021 mmol) in DMF (1 mL) and stirred at room temperature for 15 min. The solution was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 um 19*250 mm; Mobile phase: A - water (0.1% formic acid): B - acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to give 2-5 (7.4 mg, 31.0% yield) as a white solid.

[0496] MS (ESI) m / z: 1172.4 [M+Na] + .

[0497] Examples 2-6 [ka] Step 1: tert-Butyl ((10S,21S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,19-dioxa-5,8,11,14,17-pentaazadocosan-22-yl)carbamate (2-6b)

[0498] To a mixture of 1-4e (205.7 mg, 0.76 mmol) in DMF (3 mL) were added DSC (195.0 mg, 0.76 mmol) and DIPEA (118.1 mg, 0.91 mmol). The mixture was stirred at room temperature for 2 h. 2-6a (500.7 mg, 0.57 mmol, purchased from MedChemExpress) was added, followed by stirring at room temperature for 10 min. The mixture was filtered, and the filtrate was purified by preparative HPLC (FA) to give 2-6 (230 mg) as a dark yellow solid.

[0499] MS (ESI) m / z: 1160.8 [M+Na] + .

[0500] Step 2: (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)carbamate (2-6)

[0501] To a mixture of 2-6b (30.0 mg, 0.026 mmol) in DCM (2 mL) was added ZnBr (237.7 mg, 1.06 mmol), and the mixture was heated to 45 °C for 16 h. The mixture was concentrated in vacuo to remove DCM, then dissolved in DMSO / HO and purified by preparative HPLC (FA). The fraction was lyophilized to give 2-6 (12.6 mg, 45% yield) as a white solid.

[0502] MS (ESI) m / z: 1037.8 [M+H] + .

[0503] Examples 2-7 [ka] Step 1: N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (2-7a)

[0504] To a mixture of 2-5b (4.1 g, 4.92 mmol) in MeOH (50 mL), THF (100 mL), and DCM (20 mL) was added wet Pd / C (400 mg, 10% purity). The black suspension was purged with a H balloon three times and then stirred at room temperature for 1 h. The black suspension was filtered through a pad of Celite and washed with MeOH (200 mL). The combined organic layers were concentrated in vacuo to give 2-7a (3650 mg, 99.8% yield) as an off-white solid.

[0505] MS (ESI) m / z: 743.6 [M+H] + .

[0506] Step 2: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-((2-(benzyloxy)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-7c)

[0507] To a solution of 2-7a (3.65 g, 4.92 mmol) and 2-7c (1.66 g, 4.92 mmol) in DMF (50 mL) was added HATU (1.87 g, 4.92 mmol) and DIPEA (1.59 g, 12.29 mmol). The mixture was stirred at room temperature for 30 min. After the reaction was complete, the mixture was purified by FCC (MeOH / DCM = 0-10%), and the fractions were concentrated in vacuo to give 2-7c (3.8 g, 86.9% yield) as an off-white foamy solid.

[0508] MS (ESI) m / z: 890.7 [M+H] + .

[0509] Step 3: N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serylglycine (2-7d)

[0510] To a mixture of 2-7c (3.8 g, 4.27 mmol) in MeOH (150 mL) and DCM (50 mL) was added wet Pd / C (400 mg, 10% purity). The black suspension was purged with a H balloon three times and then stirred at room temperature for 40 min. The black suspension was filtered through a pad of Celite and washed with MeOH (150 mL). The combined organic layers were concentrated in vacuo to give 2-7d (3.3 g, 96.6% yield) as an off-white solid.

[0511] MS (ESI) m / z: 800.7 [M+H] + .

[0512] Step 4: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-((acetoxymethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-7e)

[0513] To a solution of 2-7d (3.3 g, 4.13 mmol) in DMF (30 mL) was added Pb(OAc) (2.74 g, 6.19 mmol), Cu(OAc) (74.9 mg, 0.41 mmol), and HOAc (247.8 mg, 4.13 mmol). The resulting dark mixture was purged with a N balloon three times and stirred at 65 °C for 40 min. The mixture was diluted with EtOAc (300 mL), washed with brine (100 mL × 3), dried over NaSO, filtered, and concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0-10%), and the fractions were concentrated in vacuo to give 2-7e (2.8 g, 83.4% yield) as a pale yellow solid.

[0514] MS (ESI) m / z: 836.6 [M+Na] + .

[0515] Step 5: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutanamide)-3-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7g)

[0516] A white suspension of 2-7e (301 mg, 0.37 mmol), 2-7f (177 mg, 0.37 mmol), and 4 Å molecular sieves (500 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf) (218 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension was filtered through a pad of Celite and washed with EA. The combined organic layers were washed with saturated NaHCO (30 mL) and brine (30 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue, which was purified using a silica gel column (MeOH / DCM = 0% to 5%). The fractions were concentrated in vacuo to give 2-7g (353 mg, 77.5% yield) as a white foam.

[0517] MS (ESI) m / z: 1254.8 [M+Na] + .

[0518] Step 6: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutanamide)-3-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7h)

[0519] Following Step 6 of Example 2-5, 2-7h (65 mg, 54.3% yield) was synthesized as a white solid.

[0520] MS (ESI) m / z: 870.7 [M+H] + .

[0521] Step 7: (2S,3S,4S,5R,6R)-6-(((7S,12S,15S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyra No[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)carbamoyl)-12-isopropyl-2,2-dimethyl-4,10,13-trioxo-3,9-dioxa-5,11,14-triazahexadecan-16-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7i)

[0522] Following Step 1 of Example 2-6, 2-7i (18 mg, 40.1% yield) was synthesized as a white solid.

[0523] MS (ESI) m / z: 1166.9 [M+H] + .

[0524] Step 8: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-((((S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)carbonyl)amino)-3-methylbutanamide)-3-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7)

[0525] Following Step 2 of Example 2-6, 2-7 (4.7 mg, 50.1% yield) was synthesized as a clear syrup.

[0526] MS (ESI) m / z: 1066.8 [M+H] + .

[0527] Examples 2-8 [ka] Step 1: (5S,8S)-1-(9H-Fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecan-11-yl acetate (2-8b)

[0528] To a mixture of 2-8a (5.0 g, 10.70 mmol) in DMF (100 mL) was added Cu(OAc) (738.2 mg, 4.06 mmol), Pb(OAc) (5.41 g, 12.2 mmol), and glacial acetic acid (1.4 mL, 1.5 g, 24.3 mmol). The resulting dark blue mixture was purged with a N balloon three times and then heated at 70 °C for 1 h, resulting in a green mixture. The mixture was diluted with EtOAc (1000 mL), washed with brine (300 mL × 3), dried over NaSO, filtered, and the filtrate was concentrated under vacuum to give the crude product. This was triturated with MTBE / PE (10 / 1, 500 mL), filtered, and the filter cake was then dried under high vacuum to give 2-8b (4.4 g, 85.4% yield) as a pale yellow solid.

[0529] MS (ESI) m / z: 504.5 [M+Na] + .

[0530] 1 H NMR (400 MHz, dmso) δ 8.89 (t, J = 6.8 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.75 (t, J = 6.4 Hz, 2H), 7.43 (dd, J = 14.4, 7.6 Hz, 3H), 7.35-1.30(m, 2H), 5.13-5.05 (m, 2H), 4.35 - 4.19 (m, 4H), 3.88 (dd, J = 8.8, 7.2 Hz, 1H), 1.98 (s, 2H), 1.26 - 1.17 (m, 4H), 0.86 (dd, J = 9.8, 6.8 Hz, 6H).

[0531] Step 2: Benzyl (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8,14,14-trimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecan-15-oate (2-8d)

[0532] A white suspension of 2-8b (300 mg, 0.623 mmol), 2-8c (259.6 mg, 1.246 mmol), and 4 Å molecular sieves in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf) (368.0 mg, 0.748 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension was filtered through a pad of Celite and washed with EtOAc (30 mL). The combined organic layers were washed with saturated NaHCO (30 mL) and brine (30 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue, which was purified by silica gel column chromatography (MeOH / DCM = 0% to 5%). The fractions were concentrated in vacuo to give 2-8d (274 mg, 69.8% yield) as a white solid.

[0533] MS (ESI) m / z: 652.6 [M+Na] + .

[0534] Step 3: Benzyl 3-(((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)methoxy)-2,2-dimethylpropanoate (2-8e)

[0535] To a solution of 2-8d (274.0 mg, 0.44 mmol) in DMF (5 mL) was added EtN (477.3 mg, 5.53 mmol). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and co-evaporated twice with toluene to give 2-8e (275.3 mg, crude) as a brown oil.

[0536] MS (ESI) m / z: 430.4 [M+Na] + .

[0537] Step 4: Benzyl (5S,8S,11S)-5-(3-((((2S,3R,4S,5R)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11,17,17-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-oate (2-8f)

[0538] To a solution of 2-8e (275.3 mg, crude) and 2-2b (282.3 mg, 0.52 mmol) in DMF (5 mL) was added HATU (198.2 mg, 0.52 mmol) and DIPEA (168.4 mg, 1.30 mmol). The mixture was stirred at room temperature for 10 min. The mixture was purified by reserve phase (C18, 60 g, 30%-70%), and the fractions were freeze-dried to give 2-8f (370 mg, 91.5% yield) as a brown solid.

[0539] MS (ESI) m / z: 953.8 [M+Na] + .

[0540] Step 5: (5S,8S,11S)-5-(3-((((2S,3R,4S,5R)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11,17,17-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-oic acid (2-8 g)

[0541] To a mixture of 2-8f (370 mg, 0.388 mmol) in DMF / MeOH (1:1, 15 mL) was added Pd / C (35 mg). The black suspension was purged with a H balloon three times and then stirred under a H balloon at room temperature for 6 h. The reaction mixture was filtered through a Celite pad and washed with DMF (15 mL) and MeOH (50 mL). The combined organic layers were concentrated in vacuo and coevaporated three times with toluene to give 2-8g (350 mg, crude) as a light brown solid.

[0542] MS (ESI) m / z: 863.7 [M+Na] + .

[0543] Step 6: (9H-Fluoren-9-yl)methyl((6S,9S,12S)-1-((2S,3R,4S,5R)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-9-isopropyl-12,18,18-trimethyl-3,7,10,13,19-pentaoxo-16-oxa-2,8,11,14-tetraazanonadecane-6-yl)carbamate (2-8h)

[0544] Following Step 3 of Example 2-4, 2-8h (276.1 mg, 65% yield) was synthesized as a pale yellow solid.

[0545] MS (ESI) m / z: 1282.1 [M+Na] + .

[0546] Step 7: (S)-2-amino-N5-(((2S,3R,4S,5R)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 ,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-8i)

[0547] To a solution of 2-8h (276.1 mg, 0.22 mmol) in DMF (5 mL) was added EtN (240.6 mg, 3.29 mmol). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and coevaporated twice with toluene to give 2-8i (278.3 mg, crude) as a brown solid.

[0548] MS (ESI) m / z: 1036.9 [M+H] + .

[0549] Step 8: (9H-Fluoren-9-yl)methyl((8S,11S,14S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4-oxa-6,9,12,15-tetraazaoctadecan-18-yl)carbamate (2-8k)

[0550] To a solution of 2-8i (49.9 mg, 0.048 mmol) and 2-8j (15.0 mg, 0.048 mmol) in DMF (3 mL) was added HATU (18.3 mg, 0.048 mmol) and DIPEA (18.7 mg, 0.145 mmol). The mixture was stirred at room temperature for 10 min. The mixture was purified by preparative HPLC (FA 0.1%), and the fractions were freeze-dried to give 2-8k (42 mg, purity 65.6%) as a white solid.

[0551] MS (ESI) m / z: 1352.0 [M+Na] + .

[0552] Step 9: (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3-aminopropanamide)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di Hexo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-8l)

[0553] To a solution of 2-8k (42 mg, 0.032 mmol) in DMF (5 mL) was added EtN (34.7 mg, 0.474 mmol). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and coevaporated twice with toluene to give 2-8l (43 mg, crude) as a brown solid.

[0554] MS (ESI) m / z: 1107.9 [M+H] + .

[0555] Step 10: tert-Butyl((2S,7S,10S,13S)-7-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-20-(((1S,9S)-9-ethyl-5-fluoro-9-hydro Oxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-isopropyl-13,19,19-trimethyl-5,8,11,14,20-pentaoxo-4,17-dioxa-6,9,12,15-tetraazaicosyl)carbamate (2-8m)

[0556] To a solution of 1-4e (120 mg, 0.444 mmol) in DMF (3 mL) was added bis(4-nitrophenyl)carbonate (148.7 mg, 0.489 mmol) and DIPEA (94.7 mg, 0.733 mmol). The resulting pale yellow mixture was stirred at room temperature for 2 h. This was purified by preparative HPLC (FA), and the fractions were concentrated in vacuo to give the activated carbonate (81.6 mg, 42.2% yield) as a white solid. To the activated carbonate (13.8 mg, 0.032 mmol) and 2-8l (42.5 mg, crude) in DMF (3 mL) was added DIPEA (8.2 mg, 0.063 mmol). The resulting pale yellow mixture was stirred at room temperature for 1 h. The reaction mixture was purified by preparative HPLC (FA 0.1%) and the fractions were lyophilized to give 2-8m (28.1 mg, 0.02 mmol) as a pale yellow solid.

[0557] MS (ESI) m / z: 1427.1 [M+Na] + .

[0558] Step 11: (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl((6S,9S,12S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-9-isopropyl-12,18,18-trimethyl-3,7,10,13,19-pentaoxo-16-oxa-2,8,11,14-tetraazanonadecane-6-yl)carbamate (2-8)

[0559] To a suspension of 2-8m (28.1 mg, 0.02 mmol) in DCM (3 mL) was added ZnBr (179.7 mg, 0.798 mmol) and stirred at 45 °C for 4 h. The mixture was concentrated in vacuo, dissolved in DMSO, and then purified by preparative HPLC (0.1% FA). The fraction was lyophilized to give 2-8 (15.7 mg, 60.4% yield) as a pale yellow solid.

[0560] MS (ESI) m / z: 1304.1 [M+H] + .

[0561] Examples 2-9 [ka] (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl((17S,20S,23S)-17-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-30-(((1S,9S)-9-ethyl-5-fluoro-9-hydro Oxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-20-isopropyl-23,29,29-trimethyl-15,18,21,24,30-pentaoxo-3,6,9,12,27-pentaoxa-16,19,22,25-tetraazatriacontyl)carbamate (2-9)

[0562] Following the synthetic procedure of Example 2-8, 2-9 (14.4 mg, 61.5% yield) was synthesized as a pale yellow solid.

[0563] MS (ESI) m / z: 1480.2 [M+H] + .

[0564] Example 2-10 [ka] Step 1: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(((3-(benzyloxy)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-10a)

[0565] A white suspension of 2-7e (300 mg, 0.37 mmol), 2-8c (154 mg, 0.74 mmol), and 4 Å molecular sieves (500 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf) (217.9 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension was filtered through a pad of Celite and washed with EA. The combined organic layers were washed with saturated NaHCO (30 mL) and brine (30 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue, which was purified by silica gel column chromatography (MeOH / DCM = 0% to 5%). The fractions were concentrated in vacuo to give 2-10a (275 mg, 77.5% yield) as a white foamy solid.

[0566] MS (ESI) m / z: 984.8 [M+Na] + .

[0567] Step 2: (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-14,14-dimethyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxa-4,7,10-triazapentadecan-15-oic acid (2-10b)

[0568] To a solution of 2-10a (275 mg, 0.29 mmol) in MeOH (10 mL) was added wet Pd / C (55 mg, 10% purity). The black suspension was purged with a H balloon three times and then stirred at room temperature for 2 h. The mixture was filtered through a syringe head, washed with MeOH (15 mL), and concentrated in vacuo to give 2-10b (230 mg, crude) as a white foamy solid.

[0569] MS (ESI) m / z: 894.6 [M+Na] + .

[0570] Step 3: (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2-10c)

[0571] To a mixture of 2-10b (230 mg, crude), exatecan mesylate (139.9 mg, 0.26 mmol), and HATU (100.3 mg, 0.26 mol) in DMF (5 mL) was added DIPEA (102.3 mg, 0.79 mmol). The resulting brown mixture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc (20 mL), washed with brine (20 mL × 3), dried over NaSO, filtered, and concentrated in vacuo to give a residue. This was purified by FCC (MeOH / DCM = 0% to 3%) and concentrated in vacuo to give 2-10c (325 mg, 95.6% yield) as an off-white foamy solid.

[0572] MS (ESI) m / z: 1289.9 [M+H] + .

[0573] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10d)

[0574] To a solution of 2-10c (325 mg, 0.25 mmol) in DMF (5 mL) was added EtN (523.2 mg, 5.06 mmol). The mixture was stirred at room temperature for 20 minutes. After LCMS showed the reaction was complete, the mixture was concentrated in vacuo to give the crude product. This was dissolved in MeOH (6 mL), KCO (174.7 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then, HO (2 mL) was added to the mixture, and the mixture was stirred at room temperature for 30 minutes. The mixture was acidified with saturated KHSO to pH = 3 at 0 °C, filtered, and purified by preparative HPLC (0.1% FA). The fraction was lyophilized to give 2-10d (140 mg, 59.7% yield) as a pale yellow solid.

[0575] MS (ESI) m / z: 927.4 [M+H] + .

[0576] 1H NMR (400 MHz, d6-DMSO) δ 9.56 (s, 1H), 8.39 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.54 (dd, J = 13.2, 7.2 Hz, 1H), 5.43 (s, 2H), 5.18 (dd, J = 41.6, 18.8 Hz, 2H), 5.09-5.02 (m, 1H), 4.96 (s, 1H), 4.62 (dd, J = 10.0, 6.8 Hz, 1H), 4.56-4.44 (m, 2H), 4.19 (d, J = 7.6 Hz, 1H), 3.82 (dd, J = 10.8, 6.8 Hz, 1H), 3.61 (dd, J = 11.6, 6.4 Hz, 2H), 3.17-3.05 (m, 4H), 2.94 (t, J = 8.0 Hz, 1H), 2.39 (s, 3H), 2.11 (dt, J = 21.3, 7.6 Hz, 2H), 2.03-1.93 (m, 2H), 1.92-1.78 (m, 3H), 1.12 (d, J = 8.0 Hz, 6H), 0.87 (dd, J = 13.0, 6.6 Hz, 9H).

[0577] Step 5: (2S,3S,4S,5R,6R)-6-(((6S,13S,16S)-6-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-16-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-13-isopropyl-2,2-dimethyl-4,11,14-trioxo-3,8-dioxa-5,12,15-triazaheptadecan-17-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10e)

[0578] To a solution of 1-7e (6.7 mg, 0.02 mmol) in DMF (2 mL) were added HATU (8.6 mg, 0.02 mmol) and DIPEA (5.2 mg, 0.04 mmol). The mixture was stirred at room temperature for 10 min. 2-10d (15 mg, 0.02 mmol) was added and stirred at room temperature for 30 min. The mixture was purified by preparative HPLC (FA) to give 2-10e (9.1 mg, 44.4% yield) as an off-white solid.

[0579] MS (ESI) m / z: 1252.1 [M+H] + .

[0580] Step 6: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-((S)-2-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanamide)-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13 -Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10)

[0581] To a suspension of 2-10e (9.1 mg, 0.007 mmol) in DCM (1.5 mL) was added ZnBr (32.4 mg, 0.14 mmol), followed by stirring at 45 °C for 8 h. The mixture was concentrated in vacuo, dissolved in DMSO, and then purified by preparative HPLC (0.1% FA). The fraction was lyophilized to give 2-10 (7.1 mg, 85.7% yield) as a white solid.

[0582] MS (ESI) m / z: 1151.9 [M+H] + .

[0583] Example 2-11 [ka] Step 1: tert-Butyl 3-(isopropylamino)propanoate (2-11b)

[0584] To a solution of 2-11a (908 mg, 5.0 mmol) and acetone (580 mg, 10.0 mmol) in MeOH-HO (1:1, 20 mL) was added NaBHCN (1.28 g, 20.0 mmol) at room temperature. The reaction was stirred at room temperature for 18 h. The mixture was concentrated in vacuo, and the residue was redissolved in DCM (20 mL), washed with saturated NaHCO (10 mL), HO (10 mL), brine (10 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give the crude target product 1-11b (1.21 g) as a colorless oil, which was used directly in the next step.

[0585] MS (ESI) m / z: 188.2 [M+H] + .

[0586] Step 2: 3-(Isopropylamino)propanoic acid (2-11c)

[0587] A solution of 2-11b (600 mg, 2.5 mmol) in TFA / DCM (5:2, 7 mL) was stirred overnight at room temperature. The mixture was concentrated in vacuo, and the residue was redissolved in water. The aqueous layer was washed with EtOAc and lyophilized to give 2-11c (444.4 mg, 77.9% yield) as a white solid.

[0588] MS (ESI) m / z: 132.1 [M+H] + .

[0589] Step 3: (S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-11-isopropyl-2,2-dimethyl-4,10-dioxo-3,9-dioxa-5,11-diazatetradecan-14-oic acid (2-11d)

[0590] To a solution of 2-11c (12.3 mg, 0.094 mmol) and 1-8e (20 mg, 0.046 mmol) in DMF (1 mL) was added DIPEA (30 mg, 0.23 mmol) and HOBt (2 mg, 0.014 mmol) at room temperature. The reaction was stirred at room temperature for 16 min. The reaction mixture was diluted with DMF and acidified with 0.1 N HCl. The mixture was purified by preparative HPLC (FA), and the fractions were lyophilized to afford 2-11d (6.8 mg, 34.6% yield) as a pale yellow solid.

[0591] MS (ESI) m / z: 450.48 [M+Na] + .

[0592] Step 4: (2S,3S,4S,5R,6R)-6-(((7S,16S,19S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-19-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]i Indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-11,16-diisopropyl-2,2-dimethyl-4,10,14,17-tetraoxo-3,9-dioxa-5,11,15,18-tetraazaicosan-20-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-11e)

[0593] To a solution of 2-11d (6.8 mg, 0.0159 mmol) in DMF (0.5 mL) was added HATU (6.0 mg, 0.0159 mmol), DIPEA (4.7 mg, 0.0363 mmol), and 2-10d (13.4 mg, 0.0145 mmol). The reaction was stirred at room temperature for 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with AcOH. This was purified by preparative HPLC (FA). The fractions were lyophilized to afford 2-11e (5.1 mg, 26.4% yield) as a white solid.

[0594] MS (ESI) m / z: 1358.9 [M+Na] + .

[0595] Step 5: (2S,3S,4S,5R,6R)-6-(((2S,5S,14S)-15-amino-14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d e]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5,10-diisopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazapentadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-11)

[0596] To a suspension of 2-11e (5.1 mg, 0.007 mmol) in DCM (1.5 mL) was added ZnBr (32.4 mg, 0.14 mmol), followed by stirring at 45 °C for 21 h. The mixture was concentrated in vacuo, dissolved in 0.1% / ACN (80:20, 1 mL), and purified by preparative HPLC (0.1% TFA). The fraction was lyophilized to afford 2-11 (2.2 mg, 46.6% yield) as a white solid.

[0597] MS (ESI) m / z: 1236.8 [M+H] + .

[0598] Example 2-12 [ka] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-((R)-7-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanamide)-3-methylbutanamide)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-12)

[0599] Following the synthetic procedure of Example 2-10, 2-12 (5.8 mg, 45% yield) was synthesized as a white solid.

[0600] MS (ESI) m / z: 1149.9 [M+H] + .

[0601] Example 2-13 [ka] Step 1: (2S,3S,4S,5R,6R)-6-(((8S,11S)-11-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-1-(9H-fluoren-9-yl)-8-isopropyl-4-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-13b)

[0602] To a solution of 2-13a (11.1 mg, 0.0356 mmol) in DMF (1 mL) was added HATU (13.5 mg, 0.0356 mmol), DIPEA (10.5 mg, 0.081 mmol), and 2-10d (30 mg, 0.0323 mmol). The reaction was stirred at room temperature for 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with AcOH. This was purified by preparative HPLC (FA). The fractions were lyophilized to afford 2-13b (14.6 mg, 36.9% yield) as a pale yellow solid.

[0603] MS (ESI) m / z: 1221.0 [M+H] + .

[0604] Step 2: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(2-(methylamino)acetamido)butanamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-13c)

[0605] To a solution of 2-13b (14.6 mg, 0.012 mmol) in DMF (0.3 mL) was added EtNH (8.8 mg, 0.12 mmol). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo and coevaporated twice with toluene to give 2-13c as a brown solid, which was used directly in the next step.

[0606] Step 3: (2S,3S,4S,5R,6R)-6-(((7S,15S,18S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-18-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]i Indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-15-isopropyl-2,2,11-trimethyl-4,10,13,16-tetraoxo-3,9-dioxa-5,11,14,17-tetraazanonadecane-19-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-13d)

[0607] To a mixture of 2-4e (3.6 mg, 0.0132 mmol) in DMF (0.2 mL) was added DSC (3.5 mg, 0.0136 mmol) and DIPEA (2.3 mg, 0.018 mmol). The mixture was stirred at room temperature for 3 h. Crude solid 2-13c was then added and stirred at room temperature for an additional 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with AcOH. This was purified by preparative HPLC (FA) to give 2-13d (5.4 mg, 34.8% yield) as a pale yellow solid.

[0608] MS (ESI) m / z: 1294.9 [M+H] + .

[0609] Step 4: (2S,3S,4S,5R,6R)-6-(((2S,5S,13S)-14-amino-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de ]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-9-methyl-4,7,10-trioxo-11-oxa-3,6,9-triazatetradecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-13)

[0610] To a suspension of 2-13d (5.4 mg, 0.0042 mmol) in DCM (0.5 mL) was added ZnBr (24 mg, 0.11 mmol), followed by stirring at 45 °C for 24 h. The mixture was concentrated in vacuo, dissolved in 0.1% / ACN (80:20, 1 mL), and purified by preparative HPLC (0.1% TFA). The fraction was lyophilized to afford 2-13 (1.3 mg, 26% yield) as a white solid.

[0611] MS (ESI) m / z: 1194.9 [M+H] + .

[0612] Example 2-14 [ka] (R)-3-(((4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butoxy)carbonyl)amino)propanoic acid (2-14a)

[0613] 2-14a (280 mg, 89.6% yield) was synthesized according to the synthetic procedure of Example 1-13.

[0614] MS (ESI) m / z: 422.3 [M+Na] + .

[0615] 1 H NMR (400 MHz, d6-DMSO) δ 12.48 (br s, 1H), 7.03-6.99 (m, 2H), 6.94 (s, 2H), 4.08-4.03 (m, 3H), 3.86-3.83 (m, 2H), 3.17-3.11 (m, 2H), 2.35 (t, J=7.2 Hz, 2H), 2.14-2.09 (m, 1H), 1.91-1.84 (m, 1H), 1.32 (s, 9H).

[0616] (2S,3S,4S,5R,6R)-6-(((2S,5S,15R)-16-amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pi Lano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazahexadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid--formic acid (2-14)

[0617] 2-14 (13 mg, 64.0% yield) was synthesized according to the synthetic procedure of Example 2-12.

[0618] MS (ESI) m / z: 1208.9 [M+H] + .

[0619] Example 2-15 [ka] Step 1: tert-Butyl((8S,11S,14S,21S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy -4-Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4,19-dioxa-6,9,12,15-tetraazadocosan-22-yl)carbamate (2-15a)

[0620] To a solution of 1-6f (45 mg, 0.13 mmol) in DMF (3 mL) was added HATU (55.0 mg, 0.15 mmol) and DIPEA (34.1 mg, 0.26 mmol). The mixture was stirred at room temperature for 10 min. 2-8i (147 mg, crude) was added to the mixture and stirred at room temperature for 5 min. The mixture was purified by preparative HPLC (FA 0.1%) to give 2-8r (93 mg, 52% yield) as a pale yellow solid.

[0621] MS (ESI) m / z: 1384.1 [M+Na] + .

[0622] Step 2: (S)-2-(3-((S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)propanamide)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro 2-9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-15)

[0623] To a suspension of 2-8r (52 mg, 0.038 mmol) in DCM (4 mL) was added ZnBr (258.2 mg, 1.147 mmol), followed by stirring at 45 °C for 8 h. The mixture was concentrated in vacuo, dissolved in DMSO, and then purified by preparative HPLC (0.1% FA). The fraction was lyophilized to give 2-15 (29.2 mg) as a pale yellow solid.

[0624] MS (ESI) m / z: 1261.0 [M+H] + .

[0625] Example 2-16 [ka] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-((R)-4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butoxy)propanamido)-3-methylbutanamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-diox So-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-16)

[0626] Following the synthetic procedure of Example 2-10, 2-16 (13.4 mg, 79.1% yield) was synthesized as a white solid.

[0627] MS (ESI) m / z: 1165.7 [M+H] + .

[0628] The compounds of Examples 1-2 and 1-4 to 1-14 and Reference Compounds 1-1 and 1-3 are shown in Table 1 below. The compounds of Examples 2-2 and 2-4 to 2-16 and Reference Compounds 2-1 and 2-3 are shown in Table 2 below.

[0629] [Table 1-1] [Table 1-2]

[0630] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0631] Preparation and Characterization of Conjugator-Antibody Conjugates and Antibody-Drug Conjugates Preparation of DAR8 antibody-drug conjugate / conjugator-antibody conjugate. Antibody in conjugation buffer (concentration 0.5–25 mg / mL, PBS buffer pH 6.0–8.5) was incubated at reduced temperature (0–40°C) for 10 min. 8–15 equivalents of TECP solution (5 mM stock in PBS buffer) were added to the reaction mixture, and the reduction reaction was allowed to proceed at reduced temperature for 1–8 h. After the reduction mixture was cooled to 0–25°C, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0–25% v / v) and conjugator-linker-payload (see Table 2) or conjugator (see Table 1) stocks (10–25 equivalents, 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0–25°C for 1–3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was buffer exchanged (spin desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., histidine acetate buffer, pH 5.5-6.5, optionally supplemented with additives (e.g., sucrose, trehalose, Tween® 20, 60, 80)).

[0632] After the conjugation step, the buffer solution of the ADC (or conjugator-antibody conjugate) was exchanged into a ring-opening buffer (pH 6.5-9.0, PBS, boric acid, or Tris buffer), and the solution was left at 22 or 37 °C for 1 to 48 hours. The maleimide ring-opening process was monitored by reduction LCMS. Upon completion of conjugation maleimide hydrolysis, the resulting ADC was buffer exchanged into a basic Tris buffer (pH 8.0-8.5) or an acidic histidine-acetate buffer (pH 5.0-6.5) by dialysis.

[0633] The conjugator-antibody conjugates and ADCs prepared according to the above methods are shown in Tables 3 and 4, respectively. The ring opening times are shown in Table 3 below.

[0634] Maleimide hydrolysis was monitored and measured by LC-MS. LC-MS analysis was performed under the following conditions: LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac(TM) RP, 2.1*50mm, 4μm, 1,500Å, Thermo Scientific(TM) Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) in water Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient Program 1: 25% B to 25% B (0 min to 2 min), 25% B to 50% B (2 min to 18 min), 50% B to 90% B (18 min to 18.1 min), 90% B to 90% B (18.1 min to 20 min), 90% B to 25% B (20 min to 20.1 min), 25% B to 25% B (20.1 min to 25 min) Gradient Program 2 Injection sample amount: 2 μg MS parameters: Intact and denatured MS data were acquired in HMR mode with a setting of R=15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0635] ADC characterization. The ADCs were characterized using the following analytical methods. The drug-to-antibody ratio (DAR) of the ADCs was quantified by LCMS or HIC. The SEC purity of all the ADCs produced was >95%.

[0636] LCMS method: LC-MS analysis was carried out under the following measurement conditions. LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac(TM) RP, 2.1*50mm, 4μm, 1,500Å, Thermo Scientific(TM) Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) in water Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient program: 25% B to 25% B (0 min to 2 min), 25% B to 50% B (2 min to 18 min), 50% B to 90% B (18 min to 18.1 min), 90% B to 90% B (18.1 min to 20 min), 90% B to 25% B (20 min to 20.1 min), 25% B to 25% B (20.1 min to 25 min) Injection sample amount: 1 μg MS parameters: Intact and denatured MS data were acquired in HMR mode with a setting of R=15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0637] HIC method: HPLC analysis was carried out under the following measurement conditions. Method 1 HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID x 3.5 cm, 2.5 μm butyl non-porous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0 Gradient program: 0% B to 0% B (0 min to 2 min), 0% B to 100% B (2 min to 15 min), 100% B to 100% B (15 min to 16 min), 100% B to 0% B (16 min to 17 min), 0% B to 0% B (17 min to 20 min) Injection sample amount: 20 μg Method 2 HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: MABPac HIC-10, 5 μm, 4.6 × 10 mm (Thermo) Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20% B to 20% B (0 min to 1 min), 0% B to 0% B (1 min to 35 min), 20% B to 20% B (35 min to 40 min) Flow rate: 0.5mL / min Sample preparation: Samples were diluted to 0.5 mg / mL with the initial mobile phase.

[0638] SEC method for quantifying ADC purity: HPLC analysis was performed under the following measurement conditions: HPLC system: Waters H-Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6×150mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropyl alcohol, pH 7.0 Gradient program: 10 min isocratic elution, flow rate 0.3 mL / min Injection sample amount: 20 μg

[0639] ADC Hydrophobicity Assessment: ADCs with higher hydrophobicity would be evident from later retention times from HIC (hydrophobic interaction column) chromatography. The results, using the DAR8 peak as a reference, are shown in Table 4.

[0640] The ring-opening times measured by the LCMS method described above for monitoring ring hydrolysis were compared with the RO T 1 / 2 The time (h) until 50% of the maleimide rings were opened is shown in Table 3.

[0641] [Table 3-1] [Table 3-2]

[0642] [Table 4-1] [Table 4-2]

[0643] Antibody information Ifinatamab (MABX-9001a) (anti-B7H3 antibody)

[0644] Light chain sequence

[0645] EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1)

[0646] Heavy chain sequence

[0647] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0648] Conjugator-antibody conjugate hydrolysis kinetics Maleimide hydrolysis (ring-opening) kinetics of conjugator-antibody conjugates are shown in Figures 1A-1L and Table 3 (reaction conditions at pH 7.0, hydrolysis T 1 / 2 ) and are shown in Table 5. The data in Table 5 was generated by the LCMS method described above and includes the time (h) for greater than 95% of the conjugated maleimide rings to be opened after antibody conjugation ("RO Completion Time (>95%) (h)"). Results for conjugator-antibody conjugate 3-1 show that approximately 0% of the maleimide rings were opened after 24 hours, and results for conjugator-antibody conjugate 3-3 show that approximately 28% of the maleimide rings were opened after 24 hours. [Table 5-1] [Table 5-2] [Table 5-3]

[0649] This data demonstrates that the conjugator-antibody conjugates disclosed herein (e.g., 3-4 through 3-14) readily undergo maleimide hydrolysis under mild conditions (e.g., pH 7.0). The conjugators disclosed herein not only conjugate with antibodies under conventional conditions (e.g., pH 6.5-7.0), but also undergo maleimide hydrolysis under conjugation conditions. This potentially reduces ADC production costs, as buffer exchange with a basic buffer is not required for hydrolysis. The addition of a quenching reagent is sufficient to quench the conjugation reaction. The conjugates disclosed herein can be buffer exchanged with a formulation buffer after maleimide hydrolysis is complete, as monitored by reduced LCMS.

[0650] Stability of autohydrolyzing conjugator-antibody conjugates After the conjugation and hydrolysis steps described above (see "Preparation of DAR8 Antibody Drug Conjugate / Conjugator-Antibody Conjugate"), the conjugator-antibody conjugate was incubated in formulation buffer (pH 5.5, 20 mM histidine buffer) or glutathione (GSH) buffer (pH 7.4 or 8.0), and the solution was left at 22 or 37°C for 1 to 168 hours. The hydrolysis status was monitored by reduced LCMS. The results are shown in Tables 6 and 7 and Figures 2A to 23B. [Table 22]

[0651] [Table 6-1] [Table 6-2]

[0652] The results showed that the DAR of all conjugator-antibody conjugates remained around 8.0 when incubated in buffer with or without GSH for 18 hours.

[0653] [Table 7]

[0654] The results, shown in Table 7, demonstrate that the conjugator-antibody conjugates of the present disclosure retain greater than 96% of their hydrolyzed form after 168 hours in acidic formulation buffer.

[0655] ADC: Post-conjugation maleimide hydrolysis After maleimide hydrolysis, ADCs 4-4 and 4-5 (see Table 4) readily adhered to the Slide-A-Lyzer™ dialysis kit or ultrafiltration Amicon® membrane during the buffer exchange process with the formulation buffer, resulting in low recovery (<40%). Without being limited to any mechanism or mode of action, it was suspected that maleimide hydrolysis may have generated eight additional negative charges, potentially causing changes in the physical properties of the ADCs, such as making them sticky on the purification column and ultrafiltration Amicon® membrane. ADCs 4-7 through 4-16 showed >80% recovery during purification, and no adhesive behavior was observed on either the Slide-A-Lyzer™ dialysis kit or the ultrafiltration Amicon® membrane.

[0656] The results of the stability assessment of the ADC in GSH solution or formulation buffer are shown in Tables 8 and 9, and Figures 24A-37B, respectively.

[0657] [Table 8]

[0658] The results show that for ADCs 4-6, 4-10 to 4-14, and 4-16, no deconjugation events were observed after incubation in GSH solution (pH 7.4 or 8.0, 18 hours).

[0659] [Table 9]

[0660] The results showed that no deconjugation events were observed for ADCs 4-6, 4-10 to 4-14, and 4-16 after 1 week of storage in the formulation buffer.

[0661] cell line NCI-H1650 (ATC, CRL-5883). NCI-H1650 is a cell line exhibiting epithelial morphology isolated from the lung tissue of a 27-year-old male smoker with stage III bronchoalveolar carcinoma in 1987. NCI-H1650 was purchased from ATCC. The basal medium for NCI-H1650 is ATCC-formulated RPMI-1640 medium (ATCC 30-2001). To create a complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0662] Capan-1 (ATCC, HTB-79). Capan-1 is a cell line with epithelial morphology isolated from the pancreas of a 40-year-old Caucasian male with pancreatic adenocarcinoma. Capan-1 was purchased from ATCC. The basal medium for Capan-1 is ATCC-formulated Iscove's Modified Dulbecco's Medium (Cat. No. 30-2005). To create a complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 20%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0663] MDA-MB-453 (SIBS). MDA-MB-453 was derived from the pleural effusion of a 48-year-old female patient with metastatic breast cancer that had invaded the lymph nodes, brain, and both the pleural and pericardial cavities. MDA-MB-453 was purchased from SIBS. The basal medium for MDA-MB-453 was RPMI 1640 medium with HEPES (Gibco; 22400105). To create a complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710). [Table 10]

[0664] Direct killing of ADC in NCI-H1650, Capan-1, and MDA-MB-453 cancer cell lines The direct killing of ADCs was evaluated in NCI-1650, Capan-1, and MDA-MB-453 cancer lines. Cells (NCI-1650 or MDA-MB-453 (2E3 / well) or Capan-1 (4E3 / well)) were seeded into 3D 96-well plates (Corning: 4520) at 80 μl / well and incubated overnight at 37°C, 5% CO2. Fresh growth medium containing various concentrations of ADC was added at 40 μl / well and incubated at 37°C, 5% CO2 for 6 days. Cell viability was detected with 3D reagent (Promega; G9683), 100 μl / well. The 3D plates were incubated at room temperature for 30 minutes to allow the luminescence signal to stabilize. The plates were analyzed using a microplate reader.

[0665] The data are summarized in Tables 11-12 and Figures 38-43. [Table 11] [Table 12]

[0666] In vivo efficacy testing of ADCs

[0667] Female BALB / c nude mice were treated with 3 x 10 cells per 200 μL of PBS / Matrigel on the right flank. 6 H1650 cells were subcutaneously implanted. After inoculation, tumor volume was quantified twice a week in two dimensions using a caliper and calculated using the formula: V = 0.5(a × b 2 ) to mm 3 The tumor size was approximately 200 mm 3 Once the tumor volume reached a mean volume of 14 mm, mice were randomly assigned to six groups of eight mice each and administered vehicle, ADC4-B, ADC4-10, ADC4-12, ADC4-14, or ADC4-16 intravenously at 1 or 3 mg / kg twice weekly. Partial regression (PR) was defined as three consecutive tumor volume measurements <50% of the starting tumor volume on the first day of treatment, and complete regression (CR) was defined as three consecutive tumor volume measurements >14 mm. 3The tumor growth inhibition (TGI) was calculated using the following formula:

number

[0668] The results are shown in Figures 44 and 45. The test ADCs with ring-opening (RO) conjugators (i.e., ADC4-10, ADC4-12, ADC4-14, and ADC4-16) demonstrated potent antitumor activity in a human lung cancer model, and the activity was superior to that of the ADC without the RO conjugator (i.e., ACD4-B) (Figure 44). The RO-conjugated ADCs demonstrated dose-dependent antitumor activity (Figure 45).

[0669] Plasma stability of ADC Incubation of ADC with plasma: ADC was diluted in mouse or human plasma to give a final solution of 100 μg / mL ADC in plasma. Samples were incubated at 37°C. Aliquots (100 μL) were taken at five time points (0, 4, 24, 72, and 168 hours). Samples were frozen at -80°C until analysis.

[0670] The plasma payload concentration was measured under the following conditions. Equipment: LC-MS / MS (Triple Quad 6500 plus) Monitor: MRM Column: Advanced Materials Technology, HALO AQ-C18 2.7μm 90Å, 50*2.1mm Column temperature: 40℃ Mobile phase A: H2O-0.1% FA Mobile phase B: ACN-0.1% FA Gradient programs for DXd and Topol i analog: 2% B to 2% B (0 min to 0.2 min), 2% B to 98% B (0.2 min to 1.2 min), 98% B to 98% B (1.2 min to 2.0 min), 98% B to 2% B (2.0 min to 2.01 min), 2% B to 2% B (2.01 min to 4.0 min). Injection sample volume: 10 μL (DXd or Topol i analog)

[0671] The results are shown in Figures 46 and 47. The ADC with the maleimide hydrolysis RO conjugator exhibited low payload release rates in mouse plasma (Figure 46) and human plasma (Figure 47).

[0672] Mouse PK study After a single intravenous administration of the ADC to H1650 tumor-bearing or non-tumor-bearing mice, blood samples were collected at 0.0833, 2, 24, 72, 120, and 168 hours, followed by centrifugation (4°C, 3000 x g, 7 minutes) to separate the plasma. The ADC concentration was measured using an in-house developed Meso Scale Discovery (MSD) ligand binding assay. Briefly, a His-tagged B7H3 extracellular domain fusion protein was used as the capture reagent, and a biotin-labeled anti-payload Ab was used as the detection reagent for the ADC. The plasma concentration of the payload was measured using the same method as above.

[0673] The results are shown in Figure 48. The ADC with the maleimide hydrolyzable RO conjugator demonstrated good ADC PK and low payload exposure in the H1650 efficacy model.

[0674] Although the foregoing disclosure has been presented in some detail by way of illustration and example for purposes of clarity of understanding, certain minor changes and modifications will be apparent to those skilled in the art. Therefore, the specification and examples should not be construed as limiting.

[0675] It is to be understood that if any publication is mentioned herein, such mention does not constitute an admission that the publication forms part of the general knowledge in the art in any country.

[0676] The disclosures of all non-patent publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

Claims

1. Compounds of formula (I): 【Chemistry 61】 or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein: BA is a binding agent selected from a humanized antibody, a chimeric antibody, a human antibody, or an antigen-binding fragment thereof; RG is the residue of a reactive group; RS is a ring-opening stabilizing group; RE is a ring-opening enhancer; R １ａ and R １ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R １ａ and R １ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ２ａ and R ２ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ２ａ and R ２ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ３ａ and R ３ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ３ａ and R ３ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ４ is H, substituted or unsubstituted C １～４ Alkyl, or substituted or unsubstituted C ３～５ is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is a residue of a Stretcher unit; the subscript a' is 0 or 1; W is the cleavable unit, The subscript w' is 0 or 1; Y is a spacer unit, the subscript y' is 0 or 1; PA is the payload residue; the subscript x is 1 to 15.

2. R １ａ , R １ｂ , R ２ａ , R ２ｂ , R ３ａ , R ３ｂ , and R ４ 2. The compound of claim 1, wherein each of is independently H.

3. RS is an amino group or -NR ５ａ R ５ｂ and In the formula, R ５ａ and R ５ｂ each independently represents H or substituted or unsubstituted C １～４ 3. The compound of claim 1 or claim 2, wherein the compound is alkyl.

4. RS is an amino group or -N(CH ３ ) ２ 4. The compound of claim 3, wherein:

5. The compound of claim 4, wherein RS is an amino group.

6. RE is a bond, —O—, —OC(═O)—, -OC(=O)NR ６ -, -NHC(=O)NR ６ -, -OS (=O) ２ NR ６ -, -NHS (=O) ２ NR ６ -, or -OC(=O)NHS(=O) ２ NR ６ - and R ６ is H, or substituted or unsubstituted C １～４ The compound according to any one of claims 1 to 5, which is alkyl.

7. R ６ The compound of claim 6 , wherein is methyl, ethyl, or isopropyl.

8. RE is -OC(=O)NR ６ The compound according to claim 7, wherein

9. The compound of claim 8, wherein RE is -OC(=O)NH-.

10. RG, 【Transformation 62】 The compound according to any one of claims 1 to 9,

11. RG, 【Transformation 63】 The compound according to any one of claims 1 to 9,

12. The compound according to any one of claims 1 to 11, wherein r is 0.

13. The compound according to any one of claims 1 to 11, wherein s is 1.

14. The compound according to any one of claims 1 to 11, wherein t is 1 or 2.

15. A is -(CH ２ ) ｎ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, -(CH ２ CH ２ O) n-CH ２ CH ２ -C(=O)-, -CH[-(CH ２ ) ｎ -COOH] -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, or -C(=O)-(CH ２ ) ｎ 15. The compound of any one of claims 1 to 14, wherein the compound is -C(=O)-, wherein each n independently represents an integer of 1, 2, 3, 4, or 5.

16. W ｗ’ is the following formula: 【Chemistry 64】 It is one of the 16. The compound of any one of claims 1 to 15, wherein HG is a hydrophilic moiety or hydrogen.

17. 17. The compound of claim 16, wherein HG is a saccharide, phosphate ester, sulfate ester, phosphodiester, or phosphonate.

18. 18. The compound of claim 17, wherein HG is a saccharide, and the saccharide is β-D-galactose, N-acetyl-P-D-galactosamine, N-acetyl-α-D-galactosamine, N-acetyl-P-D-glucosamine, β-D-glucuronic acid, α-L-iduronic acid, α-D-galactose, α-D-glucose, β-D-glucose, a-D-mannose, β-D-mannose, α-L-fucose, β-D-xylose, neuraminic acid, sulfate, phosphate, carboxyl, amino, or O-acetyl modifications thereof.

19. HG, 【Transformation 65】 17. The compound of claim 16, wherein:

20. Y ｙ’ The compound according to any one of claims 1 to 19, wherein is a p-aminobenzyl alcohol (PAB) unit.

21. The compound has the following formula: 【Chemistry 66-1】 【Chemistry 66-2】 2. The compound of claim 1, wherein

22. The compound has the following formula: 【Chemistry 67-1】 【Chemistry 67-2】 【Chemistry 67-3】 【Chemistry 67-4】 【Chemistry 67-5】 2. The compound of claim 1, wherein

23. 23. The compound of any one of claims 1 to 22, wherein BA is ifinatamab, cofetuzumab, patritumab, or trastuzumab, or an antigen-binding fragment of ifinatamab, cofetuzumab, patritumab, or trastuzumab.

24. 23. The compound of any one of claims 1 to 22, wherein the BA is a humanized, chimeric, or human antibody, or an antigen-binding fragment thereof, that binds to one or more receptors selected from HER2, HER3, PTK7, or B7H3.

25. The compound is 【Chemistry 68-1】 【Chemistry 68-2】 and 2. The compound of claim 1, wherein Ab is ifinatamab.

26. The compound is 【Chemistry 69-1】 【Chemistry 69-2】 and 2. The compound of claim 1, wherein Ab is ifinatamab.

27. 27. A pharmaceutical composition comprising a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, and a pharmaceutically acceptable excipient.

28. Compound of formula (II): 【Transformation 70】 or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer, wherein RG is a reactive group, RS is a ring-opening stabilizing group; RE is a ring-opening enhancer; R １ａ and R １ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R １ａ and R １ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ２ａ and R ２ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ２ａ and R ２ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ３ａ and R ３ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ３ａ and R ３ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ４ is H, substituted or unsubstituted C １～４ Alkyl, or substituted or unsubstituted C ３～５ is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is a residue of a Stretcher unit; the subscript a' is 0 or 1; W is the cleavable unit, The subscript w' is 0 or 1; Y is a spacer unit, the subscript y' is 0 or 1; PA is the payload residue.

29. R １ａ , R １ｂ , R ２ａ , R ２ｂ , R ３ａ , R ３ｂ , and R ４ 29. The compound of claim 28, wherein each of is independently H.

30. RS is an amino group or -NR ５ａ R ５ｂ and In the formula, R ５ａ and R ５ｂ each independently represents H, or substituted or unsubstituted C １～４ 30. The compound of claim 28 or claim 29, which is alkyl.

31. RS is an amino group or -N(CH ３ ) ２ 31. The compound of claim 30, wherein:

32. 32. The compound of claim 31, wherein RS is an amino group.

33. RE is a bond, —O—, —OC(═O)—, -OC(=O)NR ６ -, -NHC(=O)NR ６ -, -OS (=O) ２ NR ６ -, -NHS (=O) ２ NR ６ -, or -OC(=O)NHS(=O) ２ NR ６ - and R ６ is H or substituted or unsubstituted C １～４ The compound of any one of claims 28 to 32, which is alkyl.

34. R ６ 34. The compound of claim 33, wherein is H, methyl, ethyl, or isopropyl.

35. RE is -OC(=O)NR ６ The compound of claim 34, wherein

36. 36. The compound of claim 35, wherein RE is -OC(=O)NH-.

37. RG, 【Chemistry 71】 The compound according to any one of claims 28 to 36,

38. The compound of any one of claims 28 to 37, wherein r is 0.

39. The compound of any one of claims 28 to 38, wherein s is 1.

40. The compound according to any one of claims 28 to 39, wherein t is 1 or 2.

41. A is -(CH ２ ) ｎ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, -(CH ２ CH ２ O) ｎ -CH ２ CH ２ -C(=O)-, -CH[-(CH ２ ) ｎ -COOH] -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, or -C(=O)-(CH ２ ) ｎ 41. The compound of any one of claims 28 to 40, wherein each n independently represents an integer of 1, 2, 3, 4, or 5.

42. W ｗ’ is the following formula: 【Chemistry 72】 42. The compound of any one of claims 28 to 41, wherein HG is a hydrophilic moiety or hydrogen.

43. 43. The compound of claim 42, wherein HG is a saccharide, phosphate ester, sulfate ester, phosphodiester, or phosphonate.

44. 44. The compound of claim 43, wherein HG is a saccharide, and the saccharide is β-D-galactose, N-acetyl-P-D-galactosamine, N-acetyl-α-D-galactosamine, N-acetyl-P-D-glucosamine, β-D-glucuronic acid, α-L-iduronic acid, α-D-galactose, α-D-glucose, β-D-glucose, a-D-mannose, β-D-mannose, α-L-fucose, β-D-xylose, neuraminic acid, sulfate, phosphate, carboxyl, amino, or O-acetyl modifications thereof.

45. HG, 【Transformation 73】 43. The compound of claim 42, wherein:

46. Y ｙ’ The compound of any one of claims 28 to 45, wherein is a PAB unit.

47. The compound has the following formula: 【Chemistry 74-1】 【Chemistry 74-2】 29. The compound of claim 28, wherein

48. The compound has the following formula: 【Chemistry 75-1】 【Chemistry 75-2】 The compound according to any one of claims 28 to 47, wherein

49. The compound is 【Chemistry 76-1】 【Chemistry 76-2】 【Chemistry 76-3】 The compound according to any one of claims 28 to 48,

50. The compound of any one of claims 1 to 26 and 28 to 48, wherein each PA is independently a cytotoxic agent.

51. 51. The compound of claim 50, wherein PA is independently selected from the group consisting of DXd, 7-ethyl-10-hydroxy-camptothecin (SN-38), and monomethylauristatin E (MMAE).

52. PA is independently a compound of formula (VI): 【Chemical 77】 represents In the formula, R ９ and R １０ each independently represents hydrogen, halogen, or substituted or unsubstituted C １～４ 51. The compound of claim 50, which is alkyl.

53. PA becomes independent, 【Transformation 78】 51. The compound of claim 50, wherein

54. Compounds of formula (III): 【Chemistry 79】 or a pharmaceutically acceptable salt, tautomer, isotopologue, or stereoisomer thereof. [In the formula, RG is a reactive group, RS is a ring-opening stabilizing group; RE is a ring-opening enhancer; R １ａ and R １ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R １ａ and R １ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ２ａ and R ２ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ２ａ and R ２ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ３ａ and R ３ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ３ａ and R ３ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ forming a cycloalkyl, R ４ is H, substituted or unsubstituted C １～４ Alkyl, or substituted or unsubstituted C ３～５ is cycloalkyl, each of r, s, and t is independently 0, 1, or 2; A is a stretcher unit, the subscript a' is 0 or 1.

55. RG, 【Chemistry 80】 55. The compound of claim 54, wherein:

56. R １ａ , R １ｂ , R ２ａ , R ２ｂ , R ３ａ , R ３ｂ , and R ４ each independently is H. The compound of any of claims 54 or 55.

57. RS is an amino group or -NR ５ａ R ５ｂ wherein R ５ａ and R ５ｂ each independently represents H or substituted or unsubstituted C １～４ 57. The compound of any one of claims 54 to 56, which is alkyl.

58. RS is an amino group or -N(CH ３ ) ２ 58. The compound of claim 57, wherein:

59. 59. The compound of claim 58, wherein RS is an amino group.

60. RE is a bond, —O—, —OC(═O)—, -OC(=O)NR ６ -, -NHC(=O)NR ６ -, -OS (=O) ２ NR ６ -, -NHS (=O) ２ NR ６ -, or -OC(=O)NHS(=O) ２ NR ６ - and R ６ is H or substituted or unsubstituted C １～４ 60. The compound of any one of claims 54 to 59, which is alkyl.

61. R ６ 61. The compound of claim 60, wherein is H, methyl, ethyl, or isopropyl.

62. RE is -OC(=O)NR ６ The compound of claim 61, wherein

63. 63. The compound of claim 62, wherein RE is -OC(=O)NH-.

64. 64. The compound of any one of claims 54 to 63, wherein r is 0.

65. 65. The compound of any one of claims 54 to 64, wherein s is 1.

66. 66. The compound of any one of claims 54 to 65, wherein t is 1 or 2.

67. A is a bond, -(CH ２ ) ｎ -C(=O)R ７ , -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)R ７ , -(CH ２ CH ２ O) ｎ -CH ２ CH ２ -C(=O)R ７ , -CH [-(CH ２ ) ｎ -COOH]-C(=O)R ７ , -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-NH-(CH ２ ) ｎ -C(=O)R ７ , or —C(═O)—(CH ２ ) ｎ -C(=O)R ７ and each n independently represents an integer of 1, 2, 3, 4, or 5; R ７ is OH or NR ８ａ R ８ｂ and R ８ａ and R ８ｂ each independently represents H, substituted or unsubstituted C １～４ Alkyl, substituted or unsubstituted C ３～５ cycloalkyl, or R ８ａ and R ８ｂ together with the atoms to which they are attached, represent a substituted or unsubstituted C ３～５ 67. The compound of any one of claims 54 to 66, which forms a cycloalkyl.

68. R ７ But OH, NH ２ , NHCH ３ , or N(CH ３ ) ２ 68. The compound of claim 67, wherein:

69. The compound is 【Chemistry 81】 55. The compound of claim 54, wherein:

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