Antibody drug conjugates

Abstract

Antibody-drug conjugate compounds comprising linkers and methods of using such compounds are provided.

Description

1. Technical Field

[0001] Provided herein are novel protein (e.g., antibody) drug conjugates comprising hydrophilic solubilizing groups and / or linkers comprising hydrophilic solubilizing groups, as well as methods of treating diseases, disorders, and conditions comprising administering a protein drug conjugate comprising a hydrophilic solubilizing group and / or a linker thereof. 2. Background technology

[0002] Antibody-drug conjugates (ADCs) are antibodies operably connected to bioactive small molecules (also referred to as toxins or payloads). ADCs selectively deliver potent payloads to target-expressing cells, thereby potentially reducing off-target side effects and / or toxicity and improving therapeutic index. The lipophilicity of many payloads (i.e., drugs) can have an adverse effect on the characteristics of ADCs, so that the payloads cannot be effectively delivered to target cells. The low bioavailability of lipophilic payloads can narrow the therapeutic window for ADC treatment. In addition, the hydrophobicity of the payload may challenge its combination with antibodies, and the combination is a reaction performed under aqueous conditions. Therefore, it is necessary to continuously develop hydrophilic joints for protein conjugates (e.g., ADCs), which will allow the combination of lipophilic payloads, improve the regulation of biological targets, improve bioavailability, and improve therapeutic windows.

[0003] Monoclonal antibody (mAb) therapy is gaining popularity as an adjuvant and first-line treatment for cancer. mAb therapies such as AVASTIN for colon cancer TM (anti-VEGF), RITUXAN for non-Hodgkin lymphoma TM (Rituximab; anti-CD20) and HERCEPTIN for breast cancer TM The success of the first trial (anti-Her2) demonstrated that non-binding antibodies can improve patient survival without significantly increasing the incidence of toxicity.

[0004] Monoclonal antibodies can be combined with therapeutic agents to form antibody-drug conjugates. For example, HERCEPTIN mentioned above TM Antibodies conjugated to maytansine payload to form ADC KADCYLA TM ADCs can exhibit higher efficacy than unbound antibodies. The connection of the antibody to the drug can be direct, or indirect through a linker. The linker can be cleavable or non-cleavable. One component considered important for developing an effective and well-tolerated ADC is the composition and stability of the linker. For some types of ADCs, the linker ideally provides serum stability but selectively releases the drug within the target cell.

[0005] The connection of the linker to the mAb can be achieved in a variety of ways, such as through surface lysines, reductive coupling with oxidized carbohydrates, and through cysteine ​​residues released by reduced interchain disulfide bonds. A variety of ADC connection systems are described in the literature, including hydrazone-, disulfide-, and peptide-based connections. Some hydrazone- and disulfide-based linkers may be unstable in the circulation, resulting in unnecessary release of the drug outside the target tissue. It is believed that such premature release of the drug can lead to systemic or organ-specific toxicity and / or unsatisfactory therapeutic efficacy. Peptide-based linker strategies can provide linkers with higher stability; however, the increase in the associated hydrophobicity of some linkers may lead to aggregation, especially for strongly hydrophobic drugs. This aggregation can lead to many adverse effects, such as ADC precipitation, difficulty in administration, and nonspecific absorption of ADC to non-target tissues, which may affect non-target toxicity and reduce efficacy.

[0006] Exatecan is a drug that is a structural analog of camptothecin with antitumor activity. See Abou-Alfa et al., “Randomized Phase III Study of Exatecan and Gemcitabine Compared with Gemcitabine Alone in Untreated Advanced Pancreatic Cancer,” Journal of Clinical Oncology, 24(27):4441–7, September 20, 2006. Monomethyl auristatin E (MMAE) is a synthetic antitumor agent. It cannot be used as a drug by itself due to its toxicity. MMAE is actually demethyl auristatin E; that is, the N-terminal amino group has only one methyl substituent, rather than two like auristatin E itself. See Dosio et al., “Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components,” Toxins. 3(12):848–883, 2011.

[0007] In the field of small molecule therapeutics, strategies have been developed to provide prodrugs of active chemical entities. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized into the active compound in vivo. Such prodrug strategies can increase the selectivity of the drug for its intended target and reduce side effects.

[0008] Therefore, there remains a need for targeted delivery of toxins to eliminate target cells while reducing toxicity to non-target cells.

[0009] In addition, some antibodies, such as Patritumab, have visually observable aggregation during rapid buffer exchange. Its aggregation temperature (Tagg) detected by dynamic light scattering (DLS) and self-association tendency detected by AC-SINS analysis are both worse than those of a group of well-performing mAbs. The aggregation tendency of Patritumab leads to aggregation of the corresponding ADC.

[0010] Therefore, there is an unmet medical need to generate ADCs with linker systems that provide high levels of linker serum stability and increased solubility, thereby allowing for efficient binding of hydrophobic drugs and effective intracellular delivery of the drugs. 3. Summary of the invention

[0011] Provided herein are ADCs with linker systems that provide high levels of linker serum stability and increased solubility, thereby allowing for efficient binding of hydrophobic drugs and effective intracellular delivery of drugs.

[0012] In one embodiment, the ADC is a compound of formula (I):

[0013]

[0014] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopologue or prodrug thereof, wherein the variables are as defined herein. In one embodiment, the ADC comprises one or more hydrophilic residues.

[0015] In another embodiment, the ADC is a compound of formula (Ia):

[0016]

[0017] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein the variables are as defined herein.

[0018] In another embodiment, the ADC is a compound of formula (Ib):

[0019]

[0020] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein the variables are as defined herein.

[0021] In another embodiment, described herein is a method of treating a disease, illness or condition in a patient in need thereof, comprising administering to the patient a compound described herein. Also provided is the use of the compound described herein for treating the disease, illness or condition described herein. Further provided is the use of the compound described herein for the manufacture of a medicament for treating the disease, illness or condition described herein. In some embodiments, the compound is an antibody-drug conjugate.

[0022] In another embodiment, described herein is a method of preparing an antibody-drug conjugate, comprising the step of contacting a binding agent with a linker-payload compound described herein under conditions suitable for forming a bond between the binding agent and the linker-payload compound. 4. Description of the drawings

[0023] Figure 1 The serial release mode of ADC is shown.

[0024] Figure 2 , Figure 3 , Figure 4 and Figure 5 It is shown that using the tandem release linker design, the cell killing of H358, H441, H1048 and MDA-MB-453 cell lines by ADC-C10, ADC-C11, ADC-C13 and ADC-C14 appears to be similar to that of ADC-C1, indicating that the payload is effectively released inside the cells.

[0025] Figure 6 , Figure 7 , Figure 8 and Fig. 9 It is shown that using the tandem release linker design, the cell killing of H358, H441, H1048 and MDA-MB-453 cell lines by ADC-C3, ADC-C4, ADC-C5 and ADC-C6 appears to be similar to that of ADC-C1, indicating that the payload is effectively released inside the cells.

[0026] Fig.10 , Fig.11 , Fig.12 and Fig.13 It is shown that using the tandem release linker design, the cell killing of H358, H441, H1048 and MDA-MB-453 cell lines by ADC-C7, ADC-C8, ADC-C9 and ADC-C12 appears to be similar to that of ADC-C1, indicating that the payload is effectively released inside the cells.

[0027] Fig.14 Payload release profiles of ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under lysosomal processing are shown.

[0028] Fig.15 Payload release curves of ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under cathepsin B treatment are shown.

[0029] Fig.16 Payload release profiles of ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under lysosomal processing are shown.

[0030] Fig.17 Payload release curves of ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under cathepsin B treatment are shown.

[0031] Fig.18 Payload release profiles of ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under lysosomal processing are shown.

[0032] Fig.19 Payload release curves of ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under cathepsin B treatment are shown.

[0033] Fig. 20 Payload release profiles of ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under human plasma treatment are shown, showing comparable and acceptable payload release rates.

[0034] Fig.21 Payload release profiles of ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under human plasma treatment are shown, showing comparable and acceptable payload release rates.

[0035] Fig. 22 Payload release profiles of ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under human plasma treatment are shown, showing comparable and acceptable payload release rates. 5. Specific implementation methods

[0036] Provided herein are compounds, compositions, ADCs and methods for treating a variety of human cancers, including but not limited to colorectal cancer, gastric cancer, breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, head and neck cancer, pancreatic cancer and cervical cancer. In one embodiment, provided herein are compounds, compositions, ADCs and methods for treating a variety of human cancers.

[0037] In the present disclosure, it is to be understood that the present disclosure is not limited to the specific methods and / or experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0038] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0039] 5.1. Definitions

[0040] When referring to the compounds provided herein, unless otherwise indicated, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. If a term provided herein has multiple definitions, these definitions prevail unless otherwise indicated.

[0041] As used herein and in the specification and appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents unless the context clearly dictates otherwise.

[0042] As used herein and unless otherwise indicated, the terms "about" and "approximately" when used in conjunction with the amount or weight percentage of a component of a composition, mean an amount or weight percentage recognized by those of ordinary skill in the art as providing a pharmacological effect equivalent to that obtained by the specified amount or weight percentage. In certain embodiments, the terms "about" and "approximately" when used in this context, encompass amounts or weight percentages within 30%, within 20%, within 15%, within 10%, or within 5% of the specified amount or weight percentage.

[0043] As used herein and unless otherwise indicated, the terms "about" and "approximately" when used in connection with a value or range of values ​​provided to characterize a particular solid form, e.g., a particular temperature or temperature range, such as describing melting, dehydration, desolvation, or glass transition temperature; a change in mass, such as a change in mass as a function of temperature or humidity; solvent or water content, e.g., in mass or percentage; or peak position, such as in analysis by, e.g., IR or Raman spectroscopy or XRPD; indicate that the value or range of values ​​may deviate to an extent that is considered reasonable by one of ordinary skill in the art while still describing the solid form. Techniques used to characterize crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), single crystal X-ray diffraction, vibrational spectroscopy (e.g., infrared (IR) and Raman spectroscopy), solid-state 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" when used in this context indicate that a 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 value or range of values. For example, in some embodiments, the value of an XRPD peak position may vary by as much as ±0.2° 2Θ (or ±0.2 degrees 2Θ) while still describing a specific XRPD peak.

[0044] An "alkyl" group is a saturated, partially saturated or unsaturated straight or branched acyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbons, or 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; while saturated branched alkyl groups 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(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), C(CH2CH3)=CH2, C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and CH2C≡C(CH2CH3), etc. The alkyl group may be substituted or unsubstituted. In certain embodiments, when alkyl groups described herein are referred to as "substituted," they may be substituted with any one or more substituents, such as those found in the exemplary compounds and embodiments disclosed herein; as well as halogen (chloro, iodo, bromo, or fluoro); hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0045] "Alkenyl" is a straight or branched non-cyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms and including at least one carbon-carbon double bond. Representative straight and branched (C2C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -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 etc. The double bond of alkenyl can be non-conjugated, or conjugated with another unsaturated group. Alkenyl can be unsubstituted or substituted.

[0046] "Cycloalkyl" group is a saturated or partially saturated cyclic alkyl group with 3 to 10 carbon atoms, which has a single ring or multiple condensed rings or bridged rings that can be optionally substituted by 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, and in other embodiments, the number of ring carbon atoms is in the range of 3 to 5, 3 to 6 or 3 to 7. Such cycloalkyl groups include, for example, monocyclic structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or polycyclic or bridged ring structures, such as adamantyl, etc. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, etc. Cycloalkyl groups may be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone, etc.

[0047] An "aryl" group is an aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl). In some embodiments, the aryl group contains 6-14 carbons, and in other embodiments, the ring portion of the aryl group contains 6 to 12 or even 6 to 10 carbon atoms. Specific aryl groups include phenyl, biphenyl, naphthyl, etc. The aryl group may be substituted or unsubstituted. The phrase "aryl" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0048] A "heteroaryl" group is an aryl ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl group contains 5 to 6 ring atoms, and in other embodiments, the ring portion of the group contains 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur and nitrogen. In certain embodiments, the heteroaryl ring system is a monocyclic or bicyclic ring. Non-limiting examples include, but are not limited to, groups such as pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, benzothienyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimide), indolyl, azaindolyl (e.g., pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridinyl (e.g., azabenzimidazolyl, 3Himidazo[4,5-b]pyridinyl or 1H-imidazo[4,5-b]pyridinyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridinyl, thionaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolyl, isoquinolyl, tetrahydroquinolyl, quinoxalinyl and quinazolinyl.

[0049] "Heterocyclyl" is an aromatic (also referred to as heteroaryl) or non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently substituted by heteroatoms from the group consisting of O, S and N. In some embodiments, the heterocyclyl group includes 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6 or 3 to 8 ring members. The heterocyclyl group can also be bonded to other groups at any ring atom (i.e., at any carbon atom or heteroatom of the heterocycle). The heterocyclyl group may be substituted or unsubstituted. The heterocyclyl group includes unsaturated, partially saturated and saturated ring systems, such as imidazolyl, imidazolinyl and imidazolidinyl. The phrase heterocyclyl includes fused ring species, including those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[l,4]dioxinyl and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms, such as, but not limited to, quinuclidine. Representative examples of heterocyclyl include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thienyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, ... linyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiolane, dioxy, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiazinyl, dihydrodisulfinyl, homopiperazinyl, quinuclidinyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridinyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiazinyl, benzoxathiazinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[l,3]dioxolyl, pyrazolopyridinyl, imidazopyridinyl (azabenzimidazolyl; for example, 1H-imidazo[4,5-b]pyridinyl or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridinyl, isoxazolopyridinyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolyl, isoquinolyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thionaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridinyl, tetrahydropyrazolopyridinyl, tetrahydroimidazopyridinyl, tetrahydrotriazolopyridinyl and tetrahydroquinolinyl.Representative substituted heterocyclyl groups can be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed below.

[0050] A "cycloalkylalkyl" group is a group of the formula: -alkyl-cycloalkyl, wherein alkyl and cycloalkyl are as defined above. Substituted cycloalkylalkyl may be substituted at the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the group. Representative cycloalkylalkyls include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyls may be monosubstituted or substituted more than once.

[0051] An "aralkyl" group is a group of the formula: -alkyl-aryl, wherein the alkyl and aryl groups are as defined above. Substituted aralkyl groups may be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl and fused (cycloalkylaryl) alkyl groups, such as 4-ethyl-indanyl.

[0052] "Heterocyclylalkyl" group is a group of the formula: -alkyl-heterocyclyl, wherein alkyl and heterocyclyl are as defined above. The substituted heterocyclylalkyl can be substituted at the alkyl, heterocyclyl or alkyl and heterocyclyl parts of the group. Representative heterocyclylalkyl includes but is 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.

[0053] "Halogen" is chlorine, iodine, bromine or fluorine.

[0054] A "hydroxyalkyl" group is an alkyl group as described above substituted with one or more hydroxy groups.

[0055] An "alkoxy" group is O(alkyl) where alkyl is as defined above.

[0056] An "alkoxyalkyl" group is (alkyl)O(alkyl) where alkyl is as defined above.

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

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

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

[0060] An "aralkoxyamine" group is a group of the formula: N(R # )O-aryl or NHO aryl, wherein R # As defined above.

[0061] An "alkylamine" group is a group of the formula: NHalkyl or N(alkyl)2, wherein each alkyl group 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 C(=O)NH2, where each R # 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 group and R # 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 -O(alkyl)C(=O)NH2, wherein each R # Independently as defined above.

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

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

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

[0068] An "aldehyde" group is a group of the formula: -CH(=0).

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

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

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

[0073] A "carbamate" group is a group of the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # )、-N(R # )C(=O)O(R # ) or -NHC(=O)O(R # ), where each R # 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, where each R # Independently as defined above.

[0075] A "guanidine" group is a group of 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, wherein each R # 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), where each R # 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 -CH(=NOH), wherein each R # Independently as defined above.

[0078] A "hydrazide" group is a group of 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 -C(=O)NHNH2, wherein each R # Independently as defined above.

[0079] A "hydrazine" group is a group of the formula: -N(R # )N(R # )2. -NHN(R # )2、-N(R # )NH(R # ) 、 -N(R # )NH2、-NHNH(R # )2 or -NHNH2, where each R # Independently as defined above.

[0080] A "hydrazone" group is a group of 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), where each R # Independently as defined above.

[0081] An "azide" group is a group of the formula: -N3.

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

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

[0084] A "cyanate" group is a group of the formula: OCN.

[0085] A "thiocyanate" group is a group of the formula: SCN.

[0086] A "thioether" group is a group of the formula: -S(R # ), where R # As defined above.

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

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

[0089] A "sulfone" group is a group of the formula: -S(=O)2(R # ), where R # As defined above.

[0090] A "sulfonylamino" group is a group of the formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl group and R # As defined above.

[0091] A "sulfonamide" group is a group of the formula: -S(=O)2N(R # )2 or -S(=O)2NH(R # ) or -S(=O)2NH2, where each R # Independently as defined above.

[0092] A "phosphonate" group is a group of the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ), where each R # Independently as defined above.

[0093] A "phosphine" group is a group of the formula: -P(R # )2, where each R # Independently as defined above.

[0094] When a group described herein (other than an alkyl group) is referred to as "substituted", it may be substituted with any suitable substituent. Illustrative examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chlorine, iodine, bromine or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amine; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (═O); B(OH)2, O(alkyl)aminocarbonyl; cycloalkyl , which may be a monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), or a heterocyclic group, which may be a monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl); a monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolyl, isoquinolyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl or benzofuranyl); aryloxy; aralkyloxy; heterocyclyloxy; and heterocyclylalkoxy.

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

[0096] As used herein, unless otherwise indicated, the term "solvate" means a compound or salt thereof, which further includes a stoichiometric or non-stoichiometric amount of a 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, which 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 can be hydrolyzed, oxidized or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds, including bio-hydrolyzable parts, such as bio-hydrolyzable amides, bio-hydrolyzable esters, bio-hydrolyzable carbamates, bio-hydrolyzable carbonates, bio-hydrolyzable ureides and bio-hydrolyzable phosphate analogs. In certain embodiments, the prodrug of a compound with a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylic acid esters are conveniently formed by any carboxylic acid moiety present on the esterification molecule. Prodrugs can typically be prepared using well-known methods, such as by Burger's Medicinal Chemistry and Drug Discovery 6th edition (Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard, 1985, Harwood Academic Publishers Gmfh) described methods.

[0099] Unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" as used herein means that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound with one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound with two chiral centers is substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound contains 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. The compound may have a chiral center and may exist as a racemate, a single enantiomer or diastereomer, and mixtures thereof. All such isomeric forms are included in the embodiments disclosed herein, including mixtures thereof. The embodiments disclosed herein encompass the use of stereoisomerically pure forms of such compounds, as well as the use of mixtures of these forms. For example, a mixture of enantiomers of a particular compound containing equal or unequal amounts can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or split using standard techniques such as chiral columns or chiral splitting 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, EL, Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0100] It should also be noted that the compound may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compound is separated into cis or trans isomers. In other embodiments, the compound is a mixture of cis and trans isomers.

[0101] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is located, and may vary depending on, for example, whether the compound is a 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:

[0102]

[0103] It is readily understood by those skilled in the art that various functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds are within the scope of the present invention.

[0104] It should also be noted that a compound may contain unnatural proportions of atomic isotopes at one or more of its atoms. For example, a compound may contain an isotope such as tritium ( 3 H), iodine-125 ( 125 I), sulfur 35( 35 S) or carbon-14 ( 14 C), or can be radiolabeled with a radioactive isotope such as deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15 N) isotopically enriched. As used herein, "isotopologues" are isotopically enriched compounds. The term "isotope enrichment" refers to an atom having an isotopic composition other than the natural isotopic composition of the atom. "Isotope enrichment" may also refer to a compound in which at least one atom contained therein has an isotopic composition different from the natural isotopic composition of the atom. The term "isotope composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer and inflammatory therapeutic agents), research reagents (e.g., combined with assay reagents) and diagnostic agents (e.g., in vivo imaging agents). All isotopic changes of compounds as described herein (whether or not radioactive) are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotopologues of compounds are provided, for example, isotopologues are deuterium, carbon-13, or nitrogen-15 enriched compounds.

[0105] It should be noted that if there is an inconsistency between the depicted structure and the name of the structure, the depicted structure should be given higher weight.

[0106] The term "effective amount" in relation to a compound means an amount that can completely or partially relieve symptoms, or slow down or stop further development or deterioration of these symptoms. It will be clear to those skilled in the art that the effective amount of the compounds disclosed herein is expected to vary depending on the severity of the indication being treated.

[0107] As used herein, "alkynyl" refers to a monovalent hydrocarbon moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl groups may be optionally substituted and may be straight chain, branched or cyclic. Alkynyl groups include, but are not limited to, groups having 2-20 carbon atoms, i.e., C 2-20 Alkynyl; a group of 2-12 carbon atoms, namely C 2-12 Alkynyl; a group of 2-8 carbon atoms, namely C 2-8 Alkynyl; a group of 2-6 carbon atoms, namely C 2-6 Alkynyl; and groups of 2-4 carbon atoms, i.e., C 2-4 Alkynyl. Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl, and butynyl.

[0108] As used herein, "haloalkyl" refers to an alkyl group as defined above, wherein the alkyl group includes at least one substituent selected from halogen, such as fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Examples of haloalkyl groups include, but are not limited to, -CF3, -CH2CF3, -CCl2F and -CCl3.

[0109] As used herein, "haloalkoxy" refers to an alkoxy group as defined above, wherein the alkoxy group includes at least one substituent selected from halogen, such as F, Cl, Br or I.

[0110] As used herein, "arylalkyl" refers to a monovalent portion of a group that is an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound includes a single bond to the alkyl group, and wherein the group is located on the alkyl group. The arylalkyl group is bonded to the chemical structure shown through the alkyl group. The arylalkyl group can be represented by structures such as B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, wherein B is an aromatic moiety, such as a phenyl group. The arylalkyl group is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl groups include, but are not limited to, benzyl.

[0111] As used herein, "alkylaryl" refers to a monovalent moiety that is a group of an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound includes a single bond to an alkyl group, and wherein the group is located on the aryl group. The alkylaryl group is bonded to the chemical structure shown through the aryl group. The alkylaryl group can be represented by structures such as: -B-CH3, -B-CH2-CH3, -B-CH2-CH2-CH3, -B-CH2-CH2-CH2-CH3, -B-CH(CH3)-CH2-CH3, -B-CH2-CH(CH3)-CH3, wherein B is an aromatic moiety, such as phenyl. The alkylaryl group is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl groups include, but are not limited to, toluoyl.

[0112] As used herein, "aryloxy" refers to a monovalent moiety that is a radical of an aromatic compound, wherein the ring atoms are carbon atoms and wherein the ring is substituted with an oxygen group, i.e., the aromatic compound contains a single bond to an oxygen atom and wherein the radical is located on the oxygen atom, such as C6H5-O- of phenoxy. The aryloxy substituent is bonded to the compound it replaces through this oxygen atom. Aryloxy groups may be optionally substituted. Aryloxy groups include, but are not limited to, those groups having 6 to 20 ring carbon atoms, i.e., C 6-20 Aryloxy; those groups having 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy; and those groups having 6 to 10 ring carbon atoms, namely C 6-10 Aryloxy. Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthracenoxy.

[0113] As used herein, the term "residue" refers to a chemical moiety that remains after a chemical reaction within a compound. For example, the term "amino acid residue" or "N-alkyl amino acid residue" refers to the product of amide coupling or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling partner; wherein, for example, after amide or peptide coupling of an amino acid or N-alkyl amino acid, a water molecule is expelled, thereby producing a product in which an amino acid residue or N-alkyl amino acid residue is incorporated.

[0114] As used herein, "sugar" or "glycosyl" or "sugar residue" refers to a carbohydrate moiety that may include a 3-carbon (triose) unit, a 4-carbon (tetraose) 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, a disaccharide, a trisaccharide, a tetrasaccharide, a pentose, an oligosaccharide, or any other polysaccharide. In some cases, "sugar" or "glycosyl" or "sugar residue" includes a furanose (e.g., ribofuranose, fructofuranose) or a pyranose (e.g., glucopyranose, galactopyranose) or a combination thereof. In some cases, "sugar" or "glycosyl" or "sugar residue" includes an aldose or a 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 "glycosyls" 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 "glycosyl" or "sugar residue" is an amino sugar. In some cases, the "sugar" or "glycosyl" or "sugar residue" is a glucosamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the rest of the molecule via its amino group, thereby forming an amide bond (i.e., glucamide) with the rest of the molecule.

[0115] As used herein, "binding agent" refers to any molecule, such as an antibody, that is capable of specifically binding to a given binding partner (eg, an antigen).

[0116] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group and a side chain (R group), which is specific for each amino acid. Amino acids can be proteinogenic or non-proteinogenic. "Proteogen" means that the amino acid is one of the twenty 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 is not naturally found in proteins, or is not directly produced by cellular mechanisms (e.g., 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-glutamyl acetamide), hydroxyproline, β-alanine, ornithine and citrulline.

[0117] 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 peptide mimetics. The subunits may be connected by peptide bonds, or by other bonds, such as ester bonds, ether bonds, etc. As used herein, the term "amino acid" refers to natural and / or non-natural, proteinogenic or non-proteinogenic, or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. If the peptide chain is shorter, such as two, three or more amino acids, it is usually referred to as an oligopeptide. If the peptide chain is longer, the peptide is typically referred to as a polypeptide or protein. The definition covers full-length proteins, their analogs, mutants and fragments. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. In addition, due to the presence of ionizable amino and carboxyl groups in the molecule, specific peptides can be obtained in acidic or alkaline salts or neutral forms. Peptides can be obtained directly from source organisms, or can be produced by recombinant or synthetic methods.

[0118] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including the scheme described by 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 indicated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme does not mean that there are no differences in the sequence, and a person skilled in the art can easily confirm the sequence position by examining the amino acid sequence of one or more antibodies. Unless otherwise indicated, the "EU numbering scheme" (eg, as reported in Kabat et al., supra) is generally used when referring to residues in the antibody heavy chain constant region.

[0119] As used herein, the term "anti-HER2 antibody" refers to an antibody that selectively binds to a HER2 receptor, such as trastuzumab. In one embodiment, trastuzumab can be manufactured and used as described in US6407213 and US5821337, the entire disclosure of which is incorporated herein by reference.

[0120] As used herein, the term "anti-HER3 antibody" refers to an antibody that selectively binds to the HER3 receptor, such as Pertrastuzumab. In one embodiment, Pertrastuzumab can be made and used as described in US7705130, the entire disclosure of which is incorporated herein by reference.

[0121] As used herein, the term "anti-PTK7 antibody" refers to an antibody that selectively binds to a PTK7 receptor, such as cofiluzumab. In one embodiment, cofiluzumab can be made and used as described in US9777070, the entire disclosure of which is incorporated herein by reference.

[0122] As used herein, the term "ifinatamab" refers to an antibody that selectively binds to a B7H3 receptor. In one embodiment, ifinatamab can be manufactured and used as described in US10117952 or WO2022102695, the entire disclosure of which is incorporated herein by reference.

[0123] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific 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 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.

[0124] As used herein, the term "cell killing activity" refers to an activity that reduces or decreases the cell viability of a test cell line.

[0125] In the following claims and in the foregoing description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" and "comprising" are used in an inclusive sense, i.e. specifying the presence of the stated features in various embodiments of the invention rather than excluding the presence of additional features or adding additional features.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

[0127] 5.2. Conjugate

[0128] In some other examples, described herein is an ADC or a pharmaceutically acceptable salt thereof, comprising: a protein linked to at least one payload moiety and to at least one hydrophilic moiety via a covalent linker, wherein the covalent linker is directly or indirectly bonded to each of the protein, payload moiety, and hydrophilic moiety. In some embodiments, the protein is an antibody or an antigen-binding fragment thereof.

[0129] As shown herein, in some instances, the binding agent is directly bonded to a covalent linker, such as a linker described herein. This means that the binding agent is one bond away from the covalent linker described herein. In some of these instances, the covalent linker is also directly bonded to the effective load portion. This means that the covalent linker is one bond away from an effective load (such as, but not limited to, Dxd, MMAE or any effective load described herein). In some of these instances, the covalent linker is also directly bonded to a hydrophilic moiety. This means that the covalent linker is one bond away from a hydrophilic residue (such as, for example, a hydrophilic residue described herein). In some of these instances, the covalent linker is a covalent linker described herein.

[0130] In other examples, the binding agent is indirectly bonded to the covalent linker. This means that the binding agent is more than one bond away from the covalent linker.

[0131] This also means that the binding agent is bonded to the covalent linker through another part. For example, the binding agent can be bonded to a maleimide group, which is bonded to a polyethylene glycol group, which is bonded to a covalent linker. In some of these examples, the covalent linker is also indirectly bonded to the payload portion. This means that the covalent linker is more than one bond away from the payload (such as, but not limited to, Dxd, MMAE, or any of its stereoisomers, or any payload described herein). This also means that the covalent linker is bonded to the payload through another part. For example, the covalent linker can be bonded to a dipeptide, such as, but not limited to, Val-Ala or Val-Cit, which can be bonded to PAB, and PAB can be bonded to the payload. In some of these examples, the covalent linker is also indirectly bonded to the hydrophilic part. This means that the covalent linker is more than one bond away from the hydrophilic part (such as, for example, a hydrophilic residue described herein). This also means that the covalent linker is bonded to the hydrophilic part through another part.

[0132] In some cases, the hydrophilic residue comprises a terminal hydrophilic group. In some cases, the hydrophilic residue comprises at least one sugar residue. In some cases, the hydrophilic residue comprises a sugar residue. In some cases, the hydrophilic residue comprises a terminal sugar residue. In further cases, the hydrophilic residue comprises more than one sugar residue. In some cases, the hydrophilic residue comprises more than one terminal sugar residue.

[0133] In another embodiment, the effective load provided herein is a chromophore functional group, for which the compounds provided herein can be used to detect, monitor or study the interaction of cell binding molecules with target cells. A chromophore functional group is a functional group having the ability to absorb certain light, such as ultraviolet light, fluorescence, infrared light, near infrared light, visible light. The pigment cell functional group is a functional group selected from the following: classes or subclasses of yellow pigment cells, red pigment cells, iridophores, white pigment cells, melanophores and blue pigment cells; classes or subclasses of fluorophore molecules, which are fluorescent compounds that re-emit light under illumination; classes or subclasses of visual phototransduction molecules; classes or subclasses of luminescent molecules; classes or subclasses of luminescent molecules; and classes or subclasses of fluorescein compounds.

[0134] 5.2.1. Aspect 1

[0135] Described herein are compounds according to formula (I):

[0136]

[0137] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0138] wherein BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding fragment of an antibody;

[0139] RG is a reactive group residue; SP is a spacer group residue; each of A, B and C is independently an amino acid residue in each case; HG is a hydrophilic residue or hydrogen; the subscripts a, c and p are independently 0 or 1 in each case; PA is a payload residue; the subscript x is from 1 to 15; and PAB represents -NH-CH2-O-.

[0140] In one embodiment, BA is an antibody. In one embodiment, the antibody is a humanized, chimeric or human antibody or an antigen-binding fragment of an antibody. In one embodiment, the antibody is a humanized, chimeric or human anti-HER2 or anti-HER3 antibody or an antigen-binding fragment of an anti-HER2 or anti-HER3 antibody. In one embodiment, the antibody is a monoclonal antibody.

[0141] In one embodiment, BA is an antibody. In one embodiment, the antibody is a humanized, chimeric or human antibody or an antigen-binding fragment of ifenatumab, cofiltuzumab, paltrazumab or trastuzumab. In one embodiment, the antibody is a humanized, chimeric or human antibody or an antigen-binding fragment of ifenatumab. In one embodiment, the antibody is ifenatumab, cofiltuzumab, paltrazumab or trastuzumab or an antigen-binding fragment of ifenatumab, cofiltuzumab, paltrazumab or trastuzumab. In one embodiment, the antibody is ifenatumab or an antigen-binding fragment of ifenatumab.

[0142] In certain embodiments, the antibodies described herein bind to one or more receptors selected from the group consisting of HER2, HER3, CD7, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD46, CD70, CD71, CD74, CD79b, CD123, CD138, CD142, CD166, CD205, CD228, CCR2, CA6, placental cadherin, CEA, CEACAM5, C4.4a, DLL3, EGFR, EGFRVIII, ENPP3, EphA2, EphrinA, FLOR1, FGFR2, GCC, cKIT, LIV1, LY6E, MSLN, MUC16, NaPi2b, Nectin4, gpNMB, PSMA, SLITRK6, STEAP1, TROP2, 5T4, SSEA4, GloboH, Gb5, STn, Tn, B7H3, BCMA, MUC1, cMet, ROR1MSLN, FRa, CLDN18.2, CLDN6, PTK7 and Axl. In certain embodiments, the antibodies as described herein bind to one or more receptors selected from the group consisting of B7H3, MUC1, FGFR2b, CLL1, CCR7, GPC1 and GPC3.

[0143] In certain embodiments, the antibodies as described herein bind to a CEA receptor. In certain embodiments, the antibodies as described herein bind to one or more receptors selected from the group consisting of: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21 receptors.

[0144] In certain embodiments, an antibody as described herein binds to the CEACAM5 receptor.

[0145] In certain embodiments, an antibody as described herein binds to the CEACAM6 receptor.

[0146] In certain embodiments, the antibodies described herein are bispecific antibodies.

[0147] In certain embodiments, RG is -(Succinimidyl-3-yl-N)-,

[0148]

[0149] In certain embodiments, RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 The electron withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0150] In certain embodiments, RG is

[0151] In certain embodiments, RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 The electron withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0152] In certain embodiments, SP is -(CH2) 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)-, wherein each of n independently represents an integer from 1 to 8.

[0153] In certain embodiments, SP is (CH2) n -C(═O)- or -CH2-C(═O)-NH-(CH2) n -C(═O)-; and each of n independently represents an integer of 5 or 2.

[0154] In certain embodiments, RG is SP is -(CH2) n -C(═O)-; and n is an integer of 1, 2, 3, 4 or 5; preferably n is an integer of 1 or 2.

[0155] In certain embodiments, the HG is selected from the group consisting of: a sugar, a phosphate, a sulfate, a phosphodiester, and a phosphonate.

[0156] In certain embodiments, the sugar is selected from the group consisting of β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-aD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, aL-iduronic acid, aD-galactose, aD-glucose, β-D-glucose, aD-mannose, β-D-mannose, aL-fucose, β-D-xylose, neuraminic acid or any analogue or modification thereof, or sulfate, phosphate, carboxyl, amino or O-acetyl modifications thereof; preferably β-D-galactose and b-glucuronic acid.

[0157] In certain embodiments, HG is or a stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein each m is independently 0 or 1.

[0158] In certain embodiments, HG is or stereoisomers, enantiomers, isotopomers or prodrugs thereof.

[0159] In certain embodiments, each PA independently represents a chromophore functional group.

[0160] In certain embodiments, each chromophore functional group is a functional group independently selected from the class or subclass of yellow pigment cells, red pigment cells, iridophores, white pigment cells, melanophores, and blue pigment cells; the class or subclass of fluorophore molecules, which are fluorescent compounds that re-emit light upon illumination; the class or subclass of visual phototransduction molecules; the class or subclass of luminophore molecules; the class or subclass of luminescent molecules; and the class or subclass of luciferin compounds.

[0161] In certain embodiments, each PA is independently a cytotoxic agent, wherein the cytotoxic agent contains at least one hydroxyl group. Examples of cytotoxic agents include, but are not limited to, 10-deacetyl-7-xylosylpaclitaxel, 17-AAG, 17-AEP-GA, 17-AH-Geldanamycin, 17-DMAP-GA, 17-GMB-APA-GA, 4-methyl-Ahtea-Puwainaphycin F, 9-hydroxyellipticine, hydrochloride, Actinomycin X2, Aerolysinin 1, Aeruginosin 865, soil Agrochelin A, Agrochelin B, α-Amanitin, α-Amanitin-from fungal fermentation, Ansamitocin P-3, Ansatrienin B, Aphidicolin, Apoptolidin, Auristatin E, Auristatin F, AZD4547, free base, AZD8055, free base, Bafilomycin A1, β-Amanitin, Boc-Nme-Val-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Do e, Boc-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Phe-Ome, Calicheamicin, Campathecin, Chaetocin B, Chaetoglobosin, Chaetocin C13, Chlamydocin, Cinerubin B, Colchicine, Combretastatin-A4, Compound CL0485, Cordycepin, Nostoc yptophycin), Cucurbitacin B, Cucurbitacin E, Curvulin, Cyclopamine, Free Base, D8-MMAE, Daun02, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride, DGN462, DL-dithiothreitol / DTT, DL-dithiothreitol / DTT-Biotech Grade, DL-dithiothreitol / DTT-Pure Grade, DM1, DM4, Dolastatin 10, Dolastatin 15, DOXO-EMCH, Doxorubicin, Doxorubicin Hydrochloride,Duocarmycin DM, Duocarmycin MA, Duocarmycin TM, Englerin A, Epothilone A, Epothilone B, ε-amanitin, Ferulenol, Fumagillin, γ-amanitin, Geldanamycin, Glucopiericidin A, Gramicidin A, Herboxidiene, HL-100-AL1-R01 (H-3137), Hygrolidin, Hypothemycin, Ilimaquinone, Isatropolone A, Isofistularin-3, Ixabepilone, Free Base, JW 55, Lactacystin, Luisol A, Maytansine DM3, Maytansinoid AP-3, Maytansinol, Mechercharmycin A, Mensacarcin, Methotrexate, Microcolin B, Microcystin LR, MMAD, MMAD hydrochloride, MMAF, MMAF hydrochloride, Monomethylauristatin E, Monomethylauristatin F methyl ester, Mycotoxin A, Myoseverin, Mytoxin B, N-acetylcalicheamicin g1, Nemorubicin, Nocuolin A, Okilactomycin, Oligomycin B, Paclitaxel, PF-06380101, Carboxydihydroxyphalloidin, Phalloidin, Phytosphingosine, Piericidin A, Pironetin, PNU-159682, Podophyllotoxin, Polyketomycin, Pseudolaric acid B, Pseurotin A, Pyrrolobenzodiazepine Dimer, Quinaldopeptin, Rachelmycin,Rebeccamycin, Ro 5-3335, Safracin B, Sandramycin, Sanguinarine, Saporin, Seco-duocarmycin SA, Sinefungin, Taltobulin, Taltobulin hydrochloride, Taltobulin trifluoroacetate, Telomestatin, Thiocolchicine, Tolytoxin, Tripolin A, Triptolide, Tubastatin A, Tubulysin A, Tubulysin IM-1, Tubulysin IM-2, Tubulysin IM-3, and Tubulysin M. In certain embodiments, each PA is a cytotoxic agent designed to induce target cell death after being internalized and released by tumor cells. In certain embodiments, each PA is a cytotoxic agent designed to induce target cell death after being internalized by tumor cells. In certain embodiments, each PA is a small molecule drug with high systemic toxicity.

[0162] In certain embodiments, each PA is independently selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), Mertansine (Maytansine DM1 / DM4), Paclitaxel, Docetaxel, Epothilone B, Epothilone A, CYT997, Auristatin tyramine phosphate, Auristatin aminoquinoline (Auristatin aminoquinoline), Halocombstatins, Calicheoamicin theta, 7-ethyl-10-hydroxy-camptothecin (SN-38), Pyrrolobenzodiazepine (PBD), Pancratistatin, Cyclophosphonates, Cribrostatin-6, Kitastatin, Turbostatin 1-4, Halocombstatins, Eribulin, Hemiasterlin, PNU and Silstatins.

[0163] In certain embodiments, each PA independently represents formula (VI):

[0164]

[0165] Where R 2 and R 3 Each of which is independently hydrogen, halogen, or substituted or unsubstituted C 1-4 alkyl.

[0166] In one embodiment, R 2 and R 3 It's hydrogen.

[0167] In one embodiment, R 2 and R 3 It's methyl.

[0168] In one embodiment, R 2 is a methyl group, and R 3 It's F.

[0169] In one embodiment, R 2 and R 3 The attached carbon is in the S configuration.

[0170] In one embodiment, R 2 and R 3 The attached carbon is in the R configuration.

[0171] In certain embodiments, each PA independently represents

[0172] In certain embodiments, each PA independently represents

[0173]

[0174] In one embodiment, subscript x is from 2 to 12. In one embodiment, subscript x is from 4 to 12. In one embodiment, subscript x is from 6 to 12. In one embodiment, subscript x is from 8 to 12. In one embodiment, subscript x is from 9 to 11.

[0175] 5.2.2. Aspect 2

[0176] Described herein are compounds according to formula (Ia):

[0177]

[0178] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

[0179] In certain embodiments, BA, RG, SP, HG, PA, and x are as provided herein.

[0180] 5.2.3. Aspect 3

[0181] Described herein are compounds according to formula (Ib):

[0182]

[0183] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

[0184] In certain embodiments, BA, RG, SP, HG, PA, and x are as provided herein.

[0185] 5.2.4. Aspect 4

[0186] Described herein are compounds according to formula (II):

[0187]

[0188] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

[0189] In certain embodiments, RG is Succinimide-N-,

[0190]

[0191] In certain embodiments, RG is Wherein EWG is an electron withdrawing group selected from the group consisting of: -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 , and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0192] In certain embodiments, RG is

[0193] In certain embodiments, RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 electron-withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0194] In certain embodiments, SP, A, B, C, HG, PAB, PA, a, c, and p are as provided herein.

[0195] 5.2.5. Aspect 5

[0196] Described herein are compounds according to formula (IIa):

[0197]

[0198]

[0199] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

[0200] In certain embodiments, RG is SP is -(CH2) n -C(═O)-; and n is an integer of 1, 2, 3, 4 or 5; preferably n is an integer of 1 or 2.

[0201] In certain embodiments, RG, SP, HG, and PA are as provided herein.

[0202] 5.2.6. Aspect 6

[0203] Described herein are compounds according to formula (IIb):

[0204]

[0205] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

[0206] In certain embodiments, RG is SP is -(CH2) n -C(═O)-; and n is an integer of 1, 2, 3, 4 or 5; preferably n is an integer of 1 or 2.

[0207] In certain embodiments, RG, SP, HG, and PA are as provided herein.

[0208] 5.2.7. Aspect 17

[0209] Described herein is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure of formula (III):

[0210]

[0211] in

[0212] R2 and R 3 Each of is independently hydrogen, halogen or alkyl.

[0213] In some embodiments, the ligand-drug conjugate comprises a structure of formula (E2):

[0214]

[0215] in

[0216] BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding fragment of an antibody;

[0217] L is a covalent linker as described herein; and

[0218] x is as provided herein.

[0219] In some embodiments, the antibody is ifenatumab, cofiluzumab, or trastuzumab. In one embodiment, the antibody is ifenatumab.

[0220] In one embodiment, R 2 and R 3 It's hydrogen.

[0221] In one embodiment, R 2 and R 3 It's methyl.

[0222] In one embodiment, R 2 is a methyl group, and R 3 It's F.

[0223] In one embodiment, R 2 and R 3 The attached carbon is in the S configuration.

[0224] In one embodiment, R 2 and R 3 The attached carbon is in the R configuration.

[0225] In one embodiment, the covalent linker L has the structure of formula (IL):

[0226]

[0227] In one embodiment, the covalent linker L has the structure of formula (Ia-L):

[0228]

[0229] In one embodiment, the covalent linker L has the structure of formula (Ib-L):

[0230]

[0231] In certain embodiments, RG, SP, HG, PAB, A, B, C, a, c, and p are as provided herein.

[0232] 5.2.8. Aspect 18

[0233] Described herein are compounds having the formula (IV):

[0234]

[0235] or a pharmaceutically acceptable solvate, stereoisomer or derivative thereof, wherein

[0236] L is a covalent linker as described herein;

[0237] R 2 and R 3 Each of is independently hydrogen, halogen or alkyl.

[0238] In one embodiment, R 2 and R 3 It's hydrogen.

[0239] In one embodiment, R 2 and R 3 It's methyl.

[0240] In one embodiment, R 2 is a methyl group, and R 3 It's F.

[0241] In one embodiment, R 2 and R 3 The attached carbon is in the S configuration.

[0242] In one embodiment, R 2 and R 3 The attached carbon is in the R configuration.

[0243] In one embodiment, the covalent linker L has the structure of formula (II-L):

[0244]

[0245] In one embodiment, the covalent linker L has the structure of formula (IIa-L):

[0246]

[0247] In one embodiment, the covalent linker L has the structure of formula (IIb-L):

[0248]

[0249] In certain embodiments, RG, SP, HG, PAB, A, B, C, a, c, and p are as provided herein.

[0250] 5.3. Payload

[0251] Provided herein is a compound or a pharmaceutically acceptable solvate, stereoisomer or derivative thereof, wherein the compound is a compound of formula (V):

[0252]

[0253] or a pharmaceutically acceptable solvate, stereoisomer or derivative thereof, wherein

[0254] R 2 and R 3 Each of is independently hydrogen, halogen or alkyl.

[0255] In one embodiment, R 2 and R 3 It's hydrogen.

[0256] In one embodiment, R 2 and R 3 It's methyl.

[0257] In one embodiment, R 2 is a methyl group, and R 3 It's F.

[0258] In one embodiment, R 2 and R 3 The attached carbon is in the S configuration.

[0259] In one embodiment, R 2 and R 3 The attached carbon is in the R configuration.

[0260] 5.4. Method or process for making conjugate

[0261] In some embodiments, a method for preparing an antibody-drug conjugate is described herein, comprising the step of contacting a binding agent with a linker-payload compound under conditions suitable for forming a bond between the binding agent and the linker-payload compound. Also provided is a method for preparing a compound of Formula (I), Formula (Ia) or Formula (Ib) under conditions suitable for forming a bond between the binding agent and the linker-payload compound.

[0262] In certain embodiments, the antibody is reacted with a reactive linker-payload or treated with a reactive linker-payload to form an antibody-payload conjugate. The reaction may be carried out under conditions deemed suitable by those skilled in the art. In certain embodiments, the antibody is contacted with a reactive linker-payload compound under conditions suitable for forming a bond between the antibody and the linker-payload compound. Suitable reaction conditions are well known to those skilled in the art.

[0263] Examples of such reactions are provided in the Examples below.

[0264] In some embodiments, described herein is a method of making a conjugate, comprising treating a compound with a binding agent or contacting a compound with a binding agent under coupling conditions, wherein the compound comprises a reactive linker bonded to at least one payload portion, and wherein the compound reacted with the binding agent is a compound of Formula (II), Formula (IIa) or Formula (IIb) or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof.

[0265] 5.5. Pharmaceutical Compositions

[0266] Provided herein is a pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0267] 5.6. Usage

[0268] In some embodiments, described herein is a method of treating a disease or condition in a patient in need thereof, the method comprising administering to the patient a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0269] In some embodiments, described herein is a method of treating or preventing a disease, disorder or condition selected from the group consisting of: a proliferative disorder, a neurodegenerative disorder, an immunological disorder, an autoimmune disease, an inflammatory disorder, a skin disease, a metabolic disease, a cardiovascular disease, and a gastrointestinal disease, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0270] In some embodiments, a method for treating a proliferative disease, metabolic disease, inflammation or neurodegenerative disease in a subject is described herein, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, a method for treating a proliferative disease in a subject is described herein, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0271] In some embodiments, described herein is a method of treating a metabolic disease in a subject, the method comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0272] In some embodiments, described herein is a method of treating inflammation in a subject, the method comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0273] In some embodiments, described herein is a method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0274] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0275] 6. Examples

[0276] The following examples are intended to be exemplary only and should not be considered to be limited in any way. Unless otherwise stated, the experimental methods in the embodiments described below are conventional methods. Unless otherwise stated, reagents and materials are all commercially available. All solvents and chemicals used are of analytical grade or chemical purity. Solvents are all redistilled before use. Anhydrous solvents are all prepared according to standard methods or reference methods. Silica gel (100 mesh-200 mesh) for column chromatography and silica gel (GF254) for thin layer chromatography (TLC) are commercially available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd. in China; unless otherwise stated, all are eluted with petroleum ether (60°C-90°C) / ethyl acetate (v / v) and developed by ethanol solutions of iodine or molybdophosphoric acid. Unless otherwise stated, all extraction solvents are dried over anhydrous Na2SO4. 1 H NMR spectra were recorded on a Bruck-400, Varian 400 MR NMR spectrometer with TMS (tetramethylsilane) as internal standard. Coupling constants are given in Hertz. Peaks are reported as singlet (s), doublet (d), triplet (t), quadruple (q), pentapole (p), hexapole (h), heptapole (hept), multiplet (m) or a combination thereof; br represents broad peak. LC / MS data were recorded using an Agilent 1100, 1200 high performance liquid chromatograph-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) detecting at 214 nm and 254 nm and an ion trap (ESI source). All compound names except reagents are by 18.0 build.

[0277] For the sake of brevity, certain abbreviations are used herein. One example is the use of single-letter abbreviations to represent amino acid residues. The amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0278] List of amino acid abbreviations

[0279]

[0280]

[0281] In the following examples, the following abbreviations are used:

[0282] List of abbreviations:

[0283]

[0284]

[0285] Example 1

[0286]

[0287] Step 1

[0288] Benzyl (((3aR,4R,6S,6aS)-6-(2-amino-2-oxoethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)carbamate (1b)

[0289] PyBOP (3.42 g, 6.57 mmol), HOBt (887 mg, 6.57 mmol), DIPEA (2.12 g, 16.42 mmol) and NH4Cl (2.93 g, 54.7 mmol) were added to a suspension of 1a (2.01 g, 5.47 mmol) in DMF (20 mL). The resulting brown suspension was stirred overnight at 50 ° C. After the reaction was completed, the mixture was quenched with water (20 mL) and extracted with EtOAc (30 mL * 3). The combined organic layer was washed with brine (30 mL * 3), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue, which was purified by silica gel column chromatography (DCM / MeOH = 100 / 0 to 90 / 10) to give 20a (1.81 g, 90.6% yield) as a light yellow solid.

[0290] MS (ESI) m / z: 365.4 [M+H] +

[0291] Step 2

[0292] 2-((3aS,4S,6R,6aR)-6-(Aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)acetamide (1c)

[0293] 1c (1.80 g, 4.94 mmol) was dissolved in a mixed solvent of THF (15 mL) and MeOH (15 mL), and then Pd / C (wet basis, 10%, 450 mg) was added. The resulting mixture was stirred at 25 ° C for 3 h. After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give 1c (1.14 g, crude) in the form of a gray syrup. The crude product was used directly in the next step without purification.

[0294] MS (ESI) m / z: 231.4 [M+H] +

[0295] Step 3

[0296] N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-(((3aR,4R,6S,6aS)-6-(2-amino-2-oxoethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-L-glutamic acid benzyl ester (1e)

[0297] To a mixture of 1c (900 mg, crude) and 1d (2.18 g, 3.91 mmol) (commercially available in THF (20 mL) was added saturated aqueous NaHCO3 solution (5 mL). The mixture was stirred at rt for 1 h. After completion of the reaction, the mixture was diluted with water (20 mL), extracted with EtOAc (20 mL*2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to give a brown oil, which was purified by flash column (DCM / MeOH=100 / 0 to 85 / 15) to give 1e (2.15 g, 82.1% yield) as a white solid.

[0298] MS (ESI) m / z: 672.6 [M+H] +

[0299] Step 4

[0300] N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-(((3aR,4R,6S,6aS)-6-(2-amino-2-oxoethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-L-glutamine (1f)

[0301] 1e (2.10 g, 3.13 mmol) was dissolved in a mixed solvent of THF (20 mL) and MeOH (20 mL), and then Pd / C (wet basis, 10%, 525 mg) was added. The resulting mixture was stirred at 25 ° C for 3 h. After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give 1f (1.87 g, crude product) as a white solid, which was used directly in the next step without purification.

[0302] MS (ESI) m / z: 582.5 [M+H] +

[0303] Step 5

[0304] N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-L-glutamine (1)

[0305] 1f (1.80 g, 3.09 mmol) was dissolved in DCM (20 mL), and the solution was cooled to 0 ° C, and then TFA / H2O (20 mL, v / v=9:1) mixed solvent was added dropwise. The resulting mixture was stirred for another 1 h at 0 ° C. After the reaction was completed, the reaction mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.05% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min)) to obtain 1 (630 mg, 37.6%) as a white solid.

[0306] MS (ESI) m / z: 542.5 [M+H] +

[0307] Example 2

[0308]

[0309] 2 was synthesized according to standard protocols for solid phase peptide synthesis (Muriel et al., Methods and protocols of modern solid phase peptide synthesis, Mol. Biotechnol., 2006, 33, 239-254) using readily commercially available starting materials.

[0310] Example 3

[0311]

[0312] 3 was purchased from ShangHai HaoYuan MedChemExpress CO.LTD.

[0313] Example 4

[0314]

[0315] 4 was synthesized according to a reported procedure (Wei et al., Synthesis and Evaluation of Camptothecin Antibody Drug Conjugates, ACS Med. Chem. Lett. 2019, 10, 1386-1392; US 9808537 B2) using readily commercially available starting materials.

[0316] Example 5

[0317]

[0318] 5 was purchased from ShangHai HaoYuan MedChemExpress CO.LTD.

[0319] Other reactants or reagents that are readily commercially available are not specifically listed or numbered.

[0320] Example 6

[0321]

[0322] Step 1

[0323] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanylglycylglycine (6b)

[0324] Pb(OAc)4(1.62g, 3.66mmol) and HOAc(348μL, 6.10mmol) were added to a mixture of 2(1.60g, 3.05mmol), Cu(OAc)2(221mg, 1.22mmol) in DMF(20mL). The resulting black mixture was purged with N2 balloon 3 times and then stirred at 70°C for 1h, and the black mixture became a dark blue mixture. After the reaction was completed. The mixture was diluted with EtOAc(100mL), then washed with brine, dried over Na2SO4, filtered and concentrated under vacuum to give 6b(1.40g, 85.2% yield) as a white solid.

[0325] MS (ESI) m / z: 561.5 [M+Na] +

[0326] Step 2

[0327] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid benzyl ester (6d)

[0328] To 6b (0.60 g, 1.12 mmol) and Molecular sieves were added to a solution of THF (6 mL) with 6c (0.56 g, 3.34 mmol) and Sc (OTf) (0.55 g, 1.12 mmol). The resulting yellow suspension was stirred overnight at rt. After the reaction was complete, the reaction mixture was filtered and the filter cake was washed with THF. The combined THF phases were then quenched with saturated NaHCO (50 mL) aqueous solution and extracted with EtOAc (100 mL). After separation, the combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (eluent: DCM / MeOH=100 / 0 to 85 / 15) to obtain 6d (600 mg, 83.5% yield) as a yellow solid.

[0329] MS (ESI) m / z: 667.5 [M+Na] +

[0330] Step 3

[0331] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (6e)

[0332] To a solution of 6d (215 mg, 0.33 mmol) in MeOH, wet Pd / C (20 mg) was added. The mixture was then purged with H2 balloon 3 times and stirred at rt for 2 h. After the reaction was completed. The mixture was filtered through a syringe filter and the filtrate was concentrated under vacuum to obtain 6e (185 mg, crude product) as a white solid.

[0333] MS (ESI) m / z: 578.4 [M+Na] +

[0334] Step 4

[0335] ((10S,13S)-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)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (6 g)

[0336] HBTU (190 mg, 0.5 mmol) and DIPEA (170 μL, 129 mg, 1 mmol) were added to a solution of 6e (185 mg, 0.33 mmol) in DMF (2 mL). The resulting yellow solution was stirred at rt for 5 min, and then 3 (195 mg, 0.37 mmol) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 6g (150 mg, 46.3% yield) as a white powder.

[0337] MS (ESI) m / z: 972.5 [M+H] +

[0338] Step 5

[0339] (S)-2-amino-N-((S)-1-((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)-1-oxopropan-2-yl)-3-methylbutanamide (6h)

[0340] To a solution of compound 6g (30 mg, 0.03 mmol) in DMF (2 mL) was added Et2N (30 μL, 22.59 mg, 0.31 mmol). The mixture was stirred at rt for 1 h. After the reaction was complete, the mixture was concentrated under vacuum to give 6h (24 mg, crude) as a yellow oil.

[0341] Step 6

[0342] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((10S,13S)-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)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)hexanamide (6)

[0343] HBTU (23 mg, 0.06 mmol) and DIPEA (16 μL, 12 mg, 0.09 mmol) were added to a solution of 6i (13 mg) in DMF (2 mL). The resulting yellow solution was stirred at rt for 5 min, and then 6h (24 mg) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 6 (18 mg, 61.5% yield) as a white powder.

[0344] MS (ESI) m / z: 943.6 [M+H] +

[0345] Example 7

[0346]

[0347] Step 1

[0348] (S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)-N-((S)-1-((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)-1-oxopropan-2-yl)-3-methylbutanamide (7)

[0349] To a solution of 7a (19 mg) in DMF (2 mL) was added HBTU (31 mg, 0.08 mmol) and DIPEA (22 μL, 16 mg, 0.12 mmol). The resulting yellow solution was stirred at rt for 5 min, and then 6h (30 mg) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 7 (20 mg, 50.7% yield) as a white powder.

[0350] MS (ESI) m / z: 958.5 [M+H] +

[0351] Example 8

[0352]

[0353] Step 1

[0354] (10S,13S)-13-Amino-10,14-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazapentadecanoic acid benzyl ester (8b)

[0355] To a solution of 6d (600 mg, 0.93 mmol) in DMF (5 mL) was added Et2NH (948 μL, 681 mg, 9.31 mmol). The mixture was stirred at rt for 1 h. After the reaction was completed, the mixture was concentrated under vacuum to give 8b (395 mg, crude) as a yellow solid.

[0356] Step 2

[0357] (5S,8S,11S)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oic acid benzyl ester (8d)

[0358] EDCI (214 mg, 1.12 mmol), HOBt (151 mg, 1.12 mmol) and DIPEA (238 μL, 180 mg, 1.4 mmol) were added to a solution of 1 (604 mg, 1.12 mmol) in THF (5 mL). The resulting yellow solution was stirred at rt for 15 min, and then 8b (395 mg, 0.93 mmol) was added. The mixture was stirred at rt for 16 h. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 8d (350 mg, 40.8% yield) as a white powder.

[0359] MS (ESI) m / z: 968.7 [M+Na] +

[0360] Step 3

[0361] (5S,8S,11S)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oic acid (8e)

[0362] To a solution of 8d (190 mg, 0.33 mmol) in MeOH / THF, wet Pd / C (20 mg) was added, then purged with H2 balloon 3 times and stirred at rt for 3 h. After the reaction was complete, the mixture was filtered with Pd / C through a syringe filter and the filtrate was concentrated under vacuum to give 8e (172 mg, crude) as a white solid.

[0363] Step 4

[0364] ((6S,9S,12S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-21-(((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-methyl-3,7,10,13,16,21-hexaoxo-19-oxa-2,8,11,14,17-pentaazahesin-6-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (8 g)

[0365] To a solution of 8e (172 mg, 0.33 mmol) in DMF (2 mL) was added HBTU (109.75 mg, 0.29 mmol) and DIPEA (68.5 uL, 51.95 mg, 0.4 mmol). The resulting yellow solution was stirred at rt for 5 min, and then 3 (128 mg, 0.24 mmol) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 8g (100 mg, 39.1% yield) as a white powder.

[0366] MS (ESI) m / z: 1272.51 [M+Na] +

[0367] Step 5

[0368] (S)-2-amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((10S,13S)-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)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)glutaramide (8h)

[0369] To a solution of 8g (30 mg, 0.02 mmol) in DMF (2 mL) was added Et2N (24 μL, 17 mg, 0.24 mmol). The mixture was stirred at rt for 1 h. After the reaction was complete, the mixture was concentrated under vacuum to give 8h (25 mg, crude) as a yellow oil.

[0370] Step 6

[0371] (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)-N1-((10S,13S)-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)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadeca-13-yl)glutaramide (8)

[0372] HBTU (18 mg, 0.05 mmol) and DIPEA (12 μL, 9.1 mg, 0.07 mmol) were added to a solution of 8i (11 mg) in DMF (2 mL). The resulting yellow solution was stirred at rt for 5 min, and then 8h (25 mg) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 8 (8.8 mg, 29.7% yield) as a white powder.

[0373] MS (ESI) m / z: 1281.8 [M+Na] +

[0374] Example 9

[0375]

[0376] Step 1

[0377] (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-N1-((10S,13S)-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)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadeca-13-yl)glutaramide (9)

[0378] To a solution of 9b (13 mg) in DMF (2 mL) was added HBTU (24 mg, 0.06 mmol) and DIPEA (16 μL, 12 mg, 0.09 mmol). The resulting yellow solution was stirred at rt for 5 min, and then 9a (40 mg) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 9 (18 mg, 45.8% yield) as a white powder.

[0379] MS (ESI) m / z: 1266.8 [M+Na] +

[0380] Example 10

[0381]

[0382] Step 1

[0383] ((10S,13S)-1-hydroxy-10,14-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazapentadeca-13-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (10b)

[0384] To a solution of 3 (160 mg, 0.25 mmol) in anhydrous THF (2 mL) was added LiAlH4 (1 M in THF, 500 μL, 0.5 mmol) at -78 °C. The reaction mixture was stirred at -78 °C for 1 h, then quenched with H2O (100 μL) and passed through celite. The filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography (eluent: DCM / MeOH=100 / 0 to 90 / 10) to give 10b (120 mg, 89.6% yield) as a white solid.

[0385] MS (ESI) m / z: 563.4 [M+Na] +

[0386] Step 2

[0387] ((10S,13S)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadeca-13-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (10c)

[0388] Oxalyl chloride (95 μL, 140 mg, 1.1 mmol) was added to a solution of 10b (120 mg, 0.22 mmol) in anhydrous DMSO (157 μL, 172 mg, 2.2 mmol) at -78 °C. The reaction mixture was stirred at -78 °C for 30 min, then EtN (457 μL, 334 mg, 3.3 mmol) was added. The mixture was stirred at -78 °C for 30 min, then warmed to rt and diluted with DCM (10 mL). The organic solution was subsequently washed with saturated NaHCO (1 mL), H2O (1 mL) and brine (1 mL). The organic phase was collected and dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to obtain 10c as a light yellow oil. The crude product was directly used in the next step without further purification.

[0389] MS (ESI) m / z: 561.4 [M+Na] +

[0390] Step 3

[0391] ((3R,4S,7S,10S,21S,24S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,21,25-tetramethyl-6,9,17,20,23-pentaoxo-2,14-dioxa-5,8,11,16,19,22-hexaazacosin-24-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (10d)

[0392] 5 (26.56 mg, 0.037 mmol) was added to a solution of 10c (20 mg, 0.037 mmol) in anhydrous MeOH (1 mL). The reaction mixture was stirred at rt for 15 min, and then NaBHCN (12 mg, 0.19 mmol) was added. The mixture was stirred at rt for 12 h. After the reaction was completed, the mixture was quenched with H2O and then purified by preparative HPLC (TFA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.05% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 10d (11 mg, 24.5% yield) as a white powder.

[0393] MS (ESI) m / z: 1242.1 [M+H] +

[0394] Step 4

[0395] (S)-2-((2S,13S,16S)-16-amino-2-isopropyl-3,13,17-trimethyl-9,12,15-trioxo-6-oxa-3,8,11,14-tetraazaoctadecanoyl)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutanamide (10e)

[0396] To a solution of 10d (10 mg, 0.008 mmol) in DMF (0.5 mL) was added EtNH (8.4 μL, 5.8 mg, 0.08 mmol). The mixture was stirred at rt for 0.5 h. After the reaction was complete, the mixture was concentrated under vacuum to give 10e (10 mg) as a light yellow solid, which was used directly in the next step without further purification.

[0397] MS (ESI) m / z: 1018.8 [M+H] +

[0398] Step 5

[0399] N-((3R,4S,7S,10S,21S,24S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,21,25-tetramethyl-6,9,17,20,23-pentaoxo-2,14-dioxa-5,8,11,16,19,22-hexaazacosin-24-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (10)

[0400] HBTU (6.07 mg, 0.016 mmol) and DIPEA (4.19 μL, 3.11 mg, 0.024 mmol) were added to a solution of 10e (8.15 mg, 0.008 mmol) in DMF (0.5 mL), followed by 10f (3.38 mg, 0.016 mmol). The mixture was stirred at rt for 30 min. After the reaction was complete, the mixture was purified by preparative HPLC (FA) (method: column: XBridgePrep C18 OBD 5um 19*250 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 10 (5 mg, 51.5% yield) as a white powder.

[0401] MS (ESI) m / z: 1212.6 [M+H] +

[0402] Embodiment 11

[0403]

[0404] Step 1

[0405] (5R,8S)-1-(9H-fluoren-9-yl)-8-(hydroxymethyl)-5-isopropyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecanoic acid (11f)

[0406] To a solution of 4 (100 mg, 0.17 mmol) in MeOH was added wet Pd / C (10 mg), then purged with H2 balloon 3 times and stirred at rt for 2 h. After the reaction was complete, the mixture was filtered through a syringe filter with Pd / C and the filtrate was concentrated under vacuum to give 11f (90 mg, crude) as a white solid.

[0407] MS (ESI) m / z: 536.4 [M+Na] +

[0408] Step 2

[0409] 2-(((S)-2-((R)-2-amino-3-methylbutyramido)-3-hydroxypropionamido)methoxy)acetic acid (11 g)

[0410] To a solution of 11f (90 mg, 0.175 mmol) in DMF (2 mL) was added Et2N (180 uL, 128 mg, 1.75 mmol). The mixture was stirred at rt for 0.5 h. After the reaction was complete, the mixture was concentrated under vacuum to give 11g (57 mg, crude) as a yellow solid.

[0411] MS (ESI) m / z: 292.4 [M+H] +

[0412] Step 3

[0413] (7S,10R)-17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7-(hydroxymethyl)-10-isopropyl-6,9,12-trioxo-3-oxa-5,8,11-triazaheptadecanoic acid (11i)

[0414] DIPEA (24 uL, 19 mg, 0.15 mmol) was added to a solution of 11g (29 mg, 0.098 mmol) and 11h (45 mg, 0.15 mmol) in DMF (2 mL). The mixture was stirred at rt for 1.5 h. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 11i (18 mg, 25.3% yield) in a white powder.

[0415] MS (ESI) m / z: 507.4 [M+Na] +

[0416] Step 4

[0417] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(((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-hydroxy-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (11)

[0418] HATU (15 mg, 0.039 mmol) and DIPEA (12 μL, 9.6 mg, 0.074 mmol) were added to a solution of 11i (18 mg, 0.037 mmol) in DMF (2 mL). The resulting yellow solution was stirred at rt for 5 min, and then 3 (20 mg, 0.037 mmol) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 11 (29 mg, 78.4% yield) as a white powder.

[0419] MS (ESI) m / z: 902.6 [M+H] +

[0420] Example 12

[0421]

[0422] Step 1

[0423] (7S,10R)-17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7-(hydroxymethyl)-10-isopropyl-6,9,12,16-tetraoxo-3-oxa-5,8,11,15-tetraazaheptadecanoic acid (12b)

[0424] TSTU (41 mg, 0.14 mmol) and DIPEA (24 uL, 19 mg, 0.15 mmol) were added to a solution of 11 g (29 mg, 0.098 mmol) and 12a (33 mg, 0.15 mmol) in DMF. The mixture was stirred at room temperature for 1.5 hours. After the reaction was completed. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 12b (23 mg, 31.4% yield) as a white powder.

[0425] MS (ESI) m / z: 522.4 [M+Na] +

[0426] Step 2

[0427] (S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)-N-((S)-1-(((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-hydroxy-1-oxopropan-2-yl)-3-methylbutanamide (12)

[0428] Compound 12 (13 mg, 32.9% yield) was synthesized according to the synthetic procedure of Step 6 of Example 11.

[0429] MS (ESI) m / z: 917.6 [M+H] +

[0430] Embodiment 13

[0431]

[0432] Step 1

[0433] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-3-(((2-(benzyloxy)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (13b)

[0434] To a solution of compound 4 (420 mg, 0.70 mmol) in DCM (12 mL) were added 13a (486 mg, 1.18 mmol) and To the mixture was added 13b (183 mg, 28.2% yield) of 13a (400 mg, 16.4% yield) and 13b (50 mL, 34.3% yield) in 4% paraformaldehyde (Mercury ether) (20% yield) at 4 ° C. Molecular sieve (800 mg). The mixture was stirred at rt for 30 min. AgOTf (250 mg, 0.97 mmol) was added to the mixture and stirred for 4 h at rt. After the reaction was completed, the mixture was filtered and the filtrate was diluted with EA (200 mL), washed with saturated NaHCO (50 mL*3) and brine (50 mL*3). The organic layer was dried over anhydrous NaSO, filtered and concentrated. The residue was purified by flash column chromatography (eluent: PE / EA=100 / 0 to 70 / 30) to obtain 13b (183 mg, 28.2% yield) as a white solid.

[0435] MS (ESI) m / z: 956.5 [M+Na] +

[0436] Step 2

[0437] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxa-4,7,10-triazatetradec-14-oic acid (13c)

[0438] To a solution of compound 13b (183 mg, 0.20 mmol) in MeOH (6 mL) was added Pd / C (10%, 20 mg). The mixture was stirred under H2 atmosphere (15 psi) for 4 h. The mixture was filtered through a celite pad and concentrated to give compound 13c (165 mg, crude) as a white solid.

[0439] MS (ESI) m / z: 866.4 [M+Na] +

[0440] Step 3

[0441] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carboxy)amino)-3-methylbutyramido)-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-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (13e)

[0442] To a solution of compound 13c (165 mg, crude) in DMF (4 mL) was added 3 (109 mg, 0.21 mol), HATU (112 mg, 0.29 mmol) and DIEA (101 mg, 0.78 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to give 13f (124 mg, 50.2% yield) as a white solid.

[0443] MS (ESI) m / z: 1283.6 [M+Na] +

[0444] Step 4

[0445] (S)-2-amino-N-((S)-1-(((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)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propan-2-yl)-3-methylbutanamide (13f)

[0446] To a solution of compound 13e (124 mg, 0.098 mmol) in MeOH (6 mL) was added KCO (136 mg, 0.98 mmol). The mixture was stirred at rt for 4 h. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridgePrep C18 OBD 5um19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to obtain 13f (42 mg, 49.1% yield) as a white solid.

[0447] MS (ESI) m / z: 893.5 [M+Na] +

[0448] Step 5

[0449] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(((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)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (13)

[0450] To a solution of compound 13g (11 mg, 0.048 mmol) in DMF (1.5 mL) was added HATU (17 mg, 0.043 mmol) and DIEA (6.2 mg, 0.048 mmol). The mixture was stirred at rt for 15 min. Compound 13f (21 mg, 0.024 mmol) was added to the mixture and stirred at rt for 15 min. The mixture was filtered and prepared using HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to purify the filtrate to obtain 13 (6.3 mg, 24.6% yield) as a white solid.

[0451] MS (ESI) m / z: 1086.6 [M+Na] +

[0452] Embodiment 14

[0453]

[0454] Step 1

[0455] Compound 14 (3.6 mg, 13.8% yield) was synthesized according to the synthetic procedure of Step 5 of Example 13.

[0456] MS (ESI) m / z: 1101.6 [M+Na] +

[0457] Embodiment 15

[0458]

[0459] Step 1

[0460] (2R,3R,4S,5S,6R)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15c)

[0461] To a solution of compound 15a (200 mg, 0.48 mmol) in DCM (8 mL) were added 15b (335 mg, 0.81 mmol) and To the mixture was added 4-nitro-dioic acid (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (2-nitro-2-yl)-2-nitropropene (4-nitro-1-yl)-2-nitropropene (

[0462] MS (ESI) m / z: 770.4 [M+Na] +

[0463] Step 2

[0464] (2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15d)

[0465] To a solution of compound 15c (230 mg, 0.31 mmol) in DMF (6 mL) was added EtNH (450 mg, 602 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 mL*3) to give 15d (162 mg, crude) as a brown solid.

[0466] MS (ESI) m / z: 526.4 [M+H] +

[0467] Step 3

[0468] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-3-(benzyloxy)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15f)

[0469] To a solution of compound 15d (162 mg, 0.31 mmol) in DMF (4 mL) was added 15e (126 mg, 0.37 mol), HATU (176 mg, 0.46 mmol) and DIEA (120 mg, 0.93 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated and the residue was purified by flash column chromatography (eluent: PE / EA=100 / 0 to 70 / 30) to give compound 15f (145 mg, 55.6% yield) as a white solid.

[0470] MS (ESI) m / z: 869.5 [M+Na] +

[0471] Step 4

[0472] N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-L-serine (15 g)

[0473] To a solution of compound 15f (100 mg, 1.35 mmol) in MeOH (6 mL) was added Pd / C (10%, 20 mg). The mixture was stirred under H2 atmosphere (15 psi) for 4 h. The mixture was filtered through a celite pad and concentrated to give compound 15g (95 mg, crude) as a white solid.

[0474] MS (ESI) m / z: 779.5 [M+Na] +

[0475] Step 5

[0476] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-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-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15i)

[0477] To a solution of compound 15g (95 mg, crude) in DMF (4 mL) was added 15h (80 mg, 0.14 mol), HATU (72 mg, 0.19 mmol) and DIEA (49 mg, 0.38 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to give 15i (117 mg, 70.4% yield) as a white solid.

[0478] MS (ESI) m / z: 1340.5 [M+Na] +

[0479] Step 6

[0480] (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-((S)-2-((S)-2-amino-3-methylbutyramido)-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)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15j)

[0481] Compound 15j (116 mg, crude) was synthesized according to the synthetic procedure of Step 2 of Example 15.

[0482] Step 7

[0483] (S)-2-amino-N-((S)-1-((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)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propan-2-yl)-3-methylbutanamide (15k)

[0484] To a solution of compound 15j (116 mg, 0.11 mmol) in MeOH (4 mL) was added KCO (44 mg, 0.32 mmol). The mixture was stirred at rt for 3 h. The mixture was filtered and prepared using HPLC (method: column: XBridgePrep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to purify the filtrate to obtain 15k (46 mg, 46.9% yield) as a white solid.

[0485] MS (ESI) m / z: 928.6 [M+H] +

[0486] Step 8

[0487] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((10S,13S)-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]indole (2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-3-oxa-5,8,11-triazapentadeca-13-yl)hexanamide (15)

[0488] To a solution of compound 15i (9.5 mg, 0.042 mmol) in DMF (1.5 mL) was added HATU (14 mg, 0.038 mmol) and DIEA (5.4 mg, 0.042 mmol). The mixture was stirred at rt for 15 min. Compound 15k (23 mg, 0.021 mmol) was added to the mixture and stirred at rt for 15 min. The mixture was filtered and prepared using HPLC (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min to purify the filtrate to obtain 15k (3.4 mg, 14.4% yield) as a white solid.

[0489] MS (ESI) m / z: 1143.5 [M+Na] +

[0490] Example 16

[0491]

[0492] Step 1

[0493] (S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamide)-N-((S)-1-((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]pyrrol-1-yl)acetamido)propionamide (((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propan-2-yl)-3-methylbutanamide (16)

[0494] Compound 16 (3.5 mg, 12.5% ​​yield) was synthesized according to the synthetic procedure of Step 8 of Example 15.

[0495] MS (ESI) m / z: 1158.5 [M+Na] +

[0496] Embodiment 17

[0497]

[0498]

[0499] Step 1

[0500] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-3-(((2-(benzyloxy)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (17b)

[0501] To 4 (150 mg, 0.25 mmol), 17a (148 mg, 0.37 mmol) and MS (800mg) was added anhydrous DCE (5mL) in the mixture and stirred for 30min at rt. AgOTf (83mg, 0.32mmol) was added under nitrogen atmosphere and stirred for 14h at rt. The reaction solution was diluted with EtOAc (10mL), filtered through diatomaceous earth, and washed with EtOAc (5mL*3). The organic phase was washed with saturated NaHCO (20mL), 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 obtain 17b (28mg, 12.2% yield) as a light yellow solid.

[0502] MS (ESI) m / z: 942.5 [M+Na] +

[0503] Step 2

[0504] (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-triazatetradec-14-oic acid (17c)

[0505] MeOH (3 mL) was added to a mixture of 17b (114 mg, 0.12 mmol) and 10% Pd / C (26 mg, 0.012 mmol) under nitrogen atmosphere. The reaction solution was stirred at rt for 1 h under H2 atmosphere. The filtered solution was concentrated under vacuum to give 17c (103 mg, 12.4% yield) as a white solid.

[0506] MS (ESI) m / z: 852.5 [M+Na] +

[0507] Step 3

[0508] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-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 (17d)

[0509] To a solution of 17c (103 mg, 0.12 mmol), 3 (64 mg, 0.12 mmol) and HATU (47 mg, 0.12 mmol) in anhydrous DMF (2 mL) was added DIPEA (61 μL, 0.36 mmol) and stirred at rt for 1 h. The reaction solution was added to H2O (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 17d (97 mg, 64% yield) as a light brown solid.

[0510] MS (ESI) m / z: 1269.5 [M+Na] +

[0511] Step 4

[0512] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutyramido)-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 (17e)

[0513] To a solution of 17d (30 mg, 0.024 mmol) in MeOH / H2O (0.5 / 0.5 mL) was added TEA (167 μL, 1.2 mmol) and stirred at rt for 9 h. The solution was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD5um 19*250 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluate was lyophilized to give 17e (15 mg, 70.6% yield) as a light yellow solid.

[0514] MS (ESI) m / z: 885.5 [M+H] +

[0515] Step 5

[0516] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-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 (17)

[0517] To a solution of 10 g (8.3 mg, 0.038 mmol) and HATU (11 mg, 0.029 mmol) in anhydrous DMF (0.3 mL) was added DIPEA (5 μL, 0.029 mmol) and stirred at rt for 15 min. The solution was added dropwise to a solution of 17e (17 mg, 0.019 mmol) in anhydrous DMF (0.7 mL) and stirred at rt 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 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to give 17 (13 mg, 63.5% yield) as a beige solid.

[0518] MS (ESI) m / z: 1078.6 [M+H] +

[0519] Embodiment 18

[0520]

[0521]

[0522] Step 1

[0523] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamide)-3-methylbutyramido)-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 (18)

[0524] Compound 18 (2.9 mg, 15.7% yield) was synthesized according to the synthetic procedure of Step 5 of Example 17.

[0525] MS (ESI) m / z: 1093.5 [M+H] +

[0526] Embodiment 19

[0527]

[0528]

[0529] Step 1

[0530] (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 (19c)

[0531] To 19a (834 mg, 2 mmol), 19b (950 mg, 2.4 mmol) and MS (3000mg) was added to a mixture of anhydrous DCM (30mL), stirred at rt for 30min. AgOTf (617mg, 2.4mmol) was added under a nitrogen atmosphere and stirred at rt for 16h. The reaction solution was diluted with EtOAc (30mL), filtered through diatomaceous earth, and washed with EtOAc (20mL*3). The organic phase was washed with saturated NaHCO (20mL), concentrated and purified by flash column chromatography (eluent: petroleum ether / EtOAc=70 / 30 to 0 / 100) to obtain 19c (470mg, 32.1% yield) as a light yellow solid.

[0532] MS (ESI) m / z: 756.3 [M+Na] +

[0533] Step 2

[0534] (2R,3R,4S,5S,6S)-2-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19d)

[0535] To a solution of 19c (470 mg, 0.64 mmol) in DMF (5 mL) was added Et2N (1420 uL, 1011 mg, 12.8 mmol). The mixture was stirred at rt for 0.5 h. After the reaction was completed, the mixture was concentrated under vacuum to give 19d (327 mg, crude) as a yellow solid.

[0536] MS (ESI) m / z: 512.4 [M+H] +

[0537] Step 3

[0538] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19f)

[0539] HATU (291 mg, 0.76 mmol) and DIPEA (223 μL, 165 mg, 1.27 mmol) were added to a solution of 19d (327 mg) in DMF (5 mL). The resulting yellow solution was stirred at rt for 5 min, and then 19e (239 mg, 0.70 mmol) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. 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 OBD5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluted fraction was lyophilized to obtain 19f (300 mg, 56.2% yield) as a white solid.

[0540] MS (ESI) m / z: 833.4 [M+H] +

[0541] Step 4

[0542] 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 (19 g)

[0543] To a solution of 19f (300 mg, 0.36 mmol) in MeOH (10 mL) and THF (8 mL) was added wet Pd / C (30 mg), then purged with H2 balloon 3 times and stirred at rt for 2 h. After the reaction was completed, the mixture was filtered through a syringe filter with Pd / C and the filtrate was concentrated under vacuum to give 19g (265 mg, crude product) as a white solid.

[0544] MS (ESI) m / z: 765.3 [M+Na] +

[0545] Step 5

[0546] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-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 (19i)

[0547] To a solution of 19g (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 rt for 5 min, and then 19h (227 mg, 0.39 mmol) was added. The mixture was stirred at rt for 60 min. After the reaction was completed. The solvent was evaporated, and the residue was purified by flash column chromatography (eluent: DCM / MeOH=95 / 5 to 90 / 10) to give 19i (280 mg, 59.6% yield) as a brown solid.

[0548] MS (ESI) m / z: 1326.4 [M+Na] +

[0549] Step 6

[0550] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-amino-3-methylbutyramido)-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 (19j)

[0551] To a solution of 19i (280 mg, 0.21 mmol) in DMF (5 mL) was added Et2N (478 uL, 340 mg, 4.29 mmol). The mixture was stirred at rt for 0.5 h. After the reaction was completed, the mixture was concentrated under vacuum to give 19j (220 mg, crude) as a white solid.

[0552] MS (ESI) m / z: 1082.4 [M+H] +

[0553] Step 7

[0554] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutyramido)-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 (19k)

[0555] To a solution of 19j (220 mg) in MeOH / H2O (5 / 5 mL) was added Na2CO3 (151 mg, 1.42 mmol) and stirred at rt for 9 h. The solution was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluate was lyophilized to give 19k (45 mg, 23.8% yield) as a white solid.

[0556] MS (ESI) m / z: 942.3 [M+H] +

[0557] Step 8

[0558] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyramido)-3-((2-(((2-(((1R,9R)-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 (19)

[0559] To a solution of 19l (5.76 mg, 0.026 mmol) and HATU (9.576 mg, 0.025 mmol) in anhydrous DMF (1 mL) was added DIPEA (7.3 μL, 5.4 mg, 0.042 mmol) and stirred at rt for 15 min. The solution was added dropwise to a solution of 19k (20 mg, 0.021 mmol) in anhydrous DMF (1 mL) and stirred at rt for 15 min. The solution was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the eluate was lyophilized to give 19 (7.4 mg, 31.0% yield) as a white solid.

[0560] MS (ESI) m / z: 1135.4 [M+H] +

[0561] Embodiment 20

[0562]

[0563] Step 1

[0564] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamide)-3-methylbutyramido)-3-((2-(((2-(((1R,9R)-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 (20)

[0565] Compound 20 (12 mg, 50.1% yield) was synthesized according to the synthetic procedure of Step 8 of Example 19.

[0566] MS (ESI) m / z: 1172.4 [M+Na] +

[0567] ADC preparation and characterization

[0568] Antibody Drug Conjugate Preparation

[0569] Preparation of DAR8 Antibody Drug Conjugates

[0570] The binding buffer containing the antibody (concentration 0.5-25 mg / mL, PBS buffer pH 6.0-8.5) is incubated at a reducing temperature (0-40°C) for 10 minutes, and 8-15 equivalents of TECP solution (5 mM stock solution in PBS buffer) are added to the reaction mixture, and the reduction reaction is carried out at the reducing temperature for 1-8 hours. After the reduction mixture is cooled to 0-25°C, an organic solvent (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and a linker-payload stock solution (10-25 equivalents, 10 mM stock solution in an organic solvent) are gradually added. The binding solution is placed at 0-25°C for 1-3 hours, and the reaction can be quenched with N-acetylcysteine ​​(1 mM stock solution). The solution is buffer exchanged (spin desalting column, ultrafiltration and dialysis) into storage buffer (eg: pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, tween 20, 60, 80).

[0571] Maleimide hydrolysis after conjugation

[0572] After the binding step, the ADC is buffer exchanged into a ring opening buffer (pH 8.0-9.0, borate or tris buffer) and the solution is placed at 22 or 37°C for 5h-48h. The ring opening process is monitored by reduction LCMS. Once the bound maleimide hydrolysis is complete, the resulting ADC is buffer exchanged into a basic Tris pH 8.0-8.5 buffer or an acidic histidine-acetate pH 5.0-6.5 buffer by dialysis.

[0573] ADC Characterization

[0574] ADC examples were prepared by following the above procedure with DAR 8 curves. All ADCs were characterized by the following analytical methods.

[0575] The drug-to-antibody ratio (DAR) of ADC was determined by LCMS method or HIC method.

[0576] The SEC purity of the manufactured ADCs was all >95% pure.

[0577] Drug to Antibody Ratio (DAR) Determination

[0578] LCMS method

[0579] LC-MS analysis was performed under the following measurement conditions:

[0580] LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer

[0581] Column: MAbPac TM RP, 2.1*50mm, 4μm, Thermo Scientific TM

[0582] Column temperature: 80°C

[0583] Mobile phase A: 0.1% formic acid (FA) in water

[0584] Mobile phase B: acetonitrile solution containing 0.1% formic acid (FA)

[0585] Gradient program: 25% B-25% B (0 min-2 min), 25% B-50% B (2 min-18 min), 50% B-90% B (18 min-18.1 min), 90% B-90% B (18.1 min-20 min), 90% B-25% B (20 min-20.1 min), 25% B-25% B (20.1 min-25 min)

[0586] Injection volume: 1 μg

[0587] MS parameters: Intact and denaturing MS data were acquired in HMR mode at a setting of R = 15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in ThermoScientific™ BioPharma Finder™ 4.0 software.

[0588] HIC Method

[0589] HPLC analysis was performed under the following measurement conditions:

[0590] HPLC system: Waters ACQUITY ARC HPLC system

[0591] Detector: Measurement wavelength: 280nm

[0592] Column: Tosoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl non-porous resin column Column temperature: 25℃

[0593] Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0

[0594] Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0

[0595] Gradient program: 0% B-0% B (0 min-2 min), 0% B-100% B (2 min-15 min), 100% B-100% B (15 min-16 min), 100% B-0% B (16 min-17 min), 0% B-0% B (17 min-20 min)

[0596] Injection volume: 20 μg

[0597] ADC purity

[0598] SEC Methodology

[0599] HPLC analysis was performed under the following measurement conditions:

[0600] HPLC system: Waters H-Class UPLC system

[0601] Detector: Measurement wavelength: 280nm

[0602] Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6×150mm, Waters

[0603] Column temperature: room temperature

[0604] Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, pH 7.0

[0605] Gradient program: 10 min isocratic elution, flow rate 0.3 mL / min

[0606] Injection volume: 20 μg

[0607] ADC hydrophobicity assessment

[0608] In HIC (hydrophobic interaction column) chromatography, a more hydrophobic ADC will appear at a later retention time. The DAR8 (antibody with drug loading of 8) peak of the example ADC was used for this comparison.

[0609] By comparing HIC D8 RT, it can be clearly seen that the order of ADC hydrophilicity is ADC-C4>ADC-C5>ADC-C2>ADC-C3>ADC-C1. ADC-C1 is the most hydrophobic in this group.

[0610] HIC Method

[0611] Method 1

[0612] HPLC analysis was performed under the following measurement conditions:

[0613] HPLC system: Waters ACQUITY ARC HPLC system

[0614] Detector: Measurement wavelength: 280nm

[0615] Column: Tosoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl non-porous resin column Column temperature: 25℃

[0616] Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0

[0617] Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0

[0618] Gradient program: 0% B-0% B (0 min-2 min), 0% B-100% B (2 min-15 min), 100% B-100% B (15 min-16 min), 100% B-0% B (16 min-17 min), 0% B-0% B (17 min-20 min)

[0619] Injection volume: 20 μg

[0620] Method 2

[0621] HPLC analysis was performed under the following measurement conditions:

[0622] HPLC system: Waters ACQUITY ARC HPLC system

[0623] Detector: Measurement wavelength: 280nm

[0624] Column: MABPac HIC-10, 5μm, 4.6×10mm (Thermo)

[0625] Column temperature: 25°C

[0626] Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0

[0627] Mobile phase B: 50 mM sodium phosphate, pH 7.0

[0628] Gradient program: 20% B-20% B (0 min-1 min), 0% B-0% B (1 min-35 min), 20% B-20% B (35 min-40 min)

[0629] Flow rate: 0.5mL / min

[0630] Sample preparation: Dilute the sample to 0.5 mg / mL with the initial mobile phase.

[0631] Table 1. ADC Examples (with Structure)

[0632]

[0633]

[0634]

[0635] Based on the DAR8 species retention time in the HIC curves, all ADCs appeared to be more hydrophilic than ADC-C1.

[0636] Primary sequence of ifenatumomab

[0637] Heavy Chain:

[0638] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0639] Light chain:

[0640] EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0641] ADC cell killing assay

[0642] Cell line information

[0643] cell resource TAA Levels H358 ATCC high H441 ATCC middle H1048 ATCC Medium to low MDA-MB-453 SIBS burden

[0644] Determination method

[0645] Cells H358 (1E3 / well), H441 (1E3 / well), H1048 (1E3 / well) and MDA-MB-453 (1E3 / well) were seeded into 3D-96-well plates (Corning: 4520), 80ul / well, and incubated overnight at 37°C, 5% CO2.

[0646] Fresh growth medium containing different concentrations of ADC was added to H358, H441, H1048 and MDA-MB-453 at 40ul / well.

[0647] Incubate at 37°C, 5% CO2 for 6 days.

[0648] The viability of H358, H441, H1048 and MDA-MB-453 cells was detected by 3D reagent (Promega, G9683), 100ul / well of 3D reagent.

[0649] The 3D-plate was incubated at room temperature for 30 minutes to stabilize the luminescent signal.

[0650] Analyze using a microplate reader.

[0651] Cell killing results

[0652] Dataset 1

[0653]

[0654] Using the tandem release linker design, ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 appear to be similar to ADC-C1 in terms of cell killing, indicating that the payload is effectively released inside the cell ( Figure 2 , Figure 3 , Figure 4 and Figure 5 ).

[0655] Dataset 2

[0656]

[0657] Using the tandem release linker design, ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 appear to kill cells similarly to ADC-C1, indicating that the payload is effectively released inside the cell ( Figure 6 , Figure 7 , Figure 8 and Fig. 9 ).

[0658] Dataset 3

[0659]

[0660] Using the tandem release linker design, ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 appear to kill cells similarly to ADC-C1, indicating that the payload is effectively released inside the cell ( Fig.10 , Fig.11 , Fig.12 and Fig.13 ).

[0661] ADC enzymatic release study

[0662]

[0663] Assay protocol

[0664] 1. Prepare 50 mM sodium acetate solution (pH 5.0), then add TCEP to make the concentration of TCEP 2 mM to obtain a reaction solution

[0665] 2. Use the reaction solution to dilute the ADC and lysosomes, so that the final concentration of ADC is 1uM and the final concentration of lysosomes is 0.125mg / mL (or cathepsin B is 20 units / mL). Prepare 5 samples in parallel for each ADC, with a final volume of 100uL

[0666] 3. Incubate the samples prepared in parallel at 37°C and add 200uL of cold acetonitrile (including internal standard) at 0h, 0.5h, 1h, 2h and 6h to terminate the reaction.

[0667] 4. After centrifugation, 200uL of the supernatant was taken for LC-MS analysis to detect the free payload and calculate the payload release rate.

[0668] Effective load release rate (%) = (concentration of DXd in supernatant / molecular weight of DXd) / (concentration of ADC / molecular weight of ADC)*DAR*100

[0669] result

[0670] Group 1

[0671] Payload release upon lysosomal processing:

[0672]

[0673] The payload release curves of ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under lysosomal processing can be referred to Fig.14 .

[0674] The payload release rates of ADC-C14 and ADC-C1 in lysosomes were faster than that of ADC-C13 and much faster than that of ADC-C10 and ADC-C11.

[0675] Payload release under cathepsin B treatment:

[0676]

[0677] The payload release curves of ADC-C1, ADC-C10, ADC-C11, ADC-C13 and ADC-C14 under cathepsin B treatment can be referred to Fig.15 .

[0678] The payload release rate of ADC-C1 under protease B treatment was faster than that of ADC-C14, and much faster than that of ADC-C13, ADC-C10, and ADC-C11. Figure 1 The serial release mode of ADC is shown.

[0679] Group 2

[0680] Payload release upon lysosomal processing:

[0681]

[0682] The payload release curves of ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under lysosomal processing can be referred to Fig.16 .

[0683] The payload release rates of ADC-C1 and ADC-C3 under lysosomal treatment were faster than ADC-C5 and much faster than ADC-C4 and C6. It is suspected that debinding events of ADC-C3 occur under this lysosomal assay, and the debound linker-payload is more susceptible to enzymatic cleavage than the bound linker-payload. Therefore, the payload release observed in ADC-C3 is greater than that observed in ADC-C5. Although ADC-C4 and ADC-C6 exhibited slower DXd payload release rates under this assay condition, both ADCs exhibited comparable cell killing activity, and all ADCs indicate that the payload is indeed effectively released in the in vitro cellular environment. Figure 8The serial release mode of ADC is shown.

[0684] Payload release under cathepsin B treatment:

[0685]

[0686] The payload release curves of ADC-C1, ADC-C3, ADC-C4, ADC-C5 and ADC-C6 under cathepsin B treatment can be referred to Fig.17 .

[0687] The effective load release rate of ADC-C1 under cathepsin B treatment is faster than that of ADC-C3 and much faster than that of ADC-C5, ADC-C4 and C6. There is abundant b-galactosidase in lysosomes, and it can recognize b-galactose moieties, thereby initiating the cleavage of glycan CO bonds (J. Med. Chem. 2006, 49, 6290-6297, Chem. Commun. 2021 Spring DR et al.). Figure 8 The serial release mode of ADC is shown.

[0688] Group 3

[0689] Payload release upon lysosomal processing:

[0690]

[0691] The payload release curves of ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under lysosomal processing can be referred to Fig.18 .

[0692] The effective load release rate of ADC-C1 under lysosomal treatment is faster than that of ADC-C7 and much faster than that of ADC-C8, ADC-C9, and C12. It is suspected that debinding events of ADC-C7 will occur under this lysosomal assay, and the debound linker-payload is more susceptible to enzymatic cleavage than the bound linker-payload. Therefore, the effective load release observed in ADC-C7 is greater than that observed in ADC-C8. Although ADC-C8, ADC-C9, and ADC-C12 showed slower DXd effective load release rates under this assay condition, both ADCs showed comparable cell killing activity, and all ADCs showed that the effective load was indeed effectively released in the in vitro cell environment. Figure 1 The serial release mode of ADC is shown.

[0693] Payload release under cathepsin B treatment:

[0694]

[0695] The payload release curves of ADC-C1, ADC-C7, ADC-C8, ADC-C9 and ADC-C12 under cathepsin B treatment can be referred to Fig.19 .

[0696] The effective load release rate of ADC-C1 under cathepsin B treatment is much faster than that of ADC-C7, ADC-C8, ADC-C9 and ADC-C12. Glucuronidase is abundant in lysosomes, and under acidic conditions, it can recognize the b-galactose moiety, thereby initiating the cleavage of the glycan CO bond (Euro. J. Med. Chem. 2014 Papot S. et al.; Mol. Can. Ther. 2016 Lyon RP et al.). Figure 8 The serial release mode of ADC is shown.

[0697] ADC plasma stability

[0698] Cultivation of ADC and plasma

[0699] Dilute ADC into mouse or human plasma to obtain a final solution of 100 μg / mL ADC in plasma

[0700] The samples were incubated at 37°C

[0701] Aliquots (100 μL) were taken at four time points (0, 4, 24, 72, 96, or 168 hours)

[0702] Samples were frozen at –80°C until analysis.

[0703] The plasma payload concentration was measured under the following conditions:

[0704] Instrument: LC-MS / MS (Triple Quad 5500)

[0705] Monitoring: MRM

[0706] Column: Advanced Materials Technology, HALO AQ-C18 2.7μm 50*2.1mm

[0707] Column temperature: 40°C

[0708] Mobile phase A: H2O-0.1% FA

[0709] Mobile phase B: ACN-0.1% FA

[0710] Gradient program of DXd: 20% B-20% B (0 min-0.2 min), 20% B-80% B (0.2 min-1.5 min), 80% B-80% B (1.5 min-2.2 min), 80% B-20% B (2.20 min-2.21 min), 20% B-20% B (2.21 min-3.0 min); Gradient program of DXd: 2% B-2% B (0 min-0.2 min), 2% B-98% B (0.2 min-1.2 min), 98% B-98% B (1.2 min-2.0 min), 98% B-2% B (2.0 min-2.01 min), 2% B-2% B (2.01 min-4.0 min);

[0711] Injection volume: 10 μL

[0712] Plasma ADC and total Ab (Tab) concentrations were measured under the following conditions:

[0713] Assay: Ligand Binding Assay (ELISA)

[0714] Capture reagent: B7H3 ECD

[0715] Detection reagents: anti-payload Ab for ADC and anti-human IgG polyclonal Ab for total Ab.

[0716] result

[0717] Payload release in human plasma

[0718] Group 1

[0719]

[0720] The effective load release curves of ADC-C1, ADC-C10, ADC-C11, ADC-C13 and ADC-C14 can be referenced Fig. 20 .

[0721] All ADCs showed comparable and acceptable payload release rates (<5% after 168 h).

[0722] Group 2

[0723]

[0724] The effective load release curves of ADC-C1, ADC-C3, ADC-C4, ADC-C5 and ADC-C6 can be referenced Fig.21 .

[0725] All ADCs showed comparable and acceptable payload release rates (<5% after 168 h).

[0726] Group 3

[0727]

[0728] The effective load release curves of ADC-C1, ADC-C7, ADC-C8, ADC-C9 and ADC-C12 can be referenced Fig. 22 .

[0729] All ADCs showed comparable and acceptable payload release rates (<5% after 168 h).

[0730] Efficacy Methods in the H1975 Model Update Model

[0731] 5×10 6 H1975 cells / 200 μL PBS / Matrigel were implanted subcutaneously on the right flank of female BALB / c nude mice. After inoculation, tumor volume was measured twice a week using calipers in two dimensions and expressed in mm using the following formula: 3 Meaning: V = 0.5 (a × b 2 ), where a and b are the long and short diameters of the tumor, respectively. When the tumor size reaches about 100-200 mm 3 When the mean volume of the tumor reached 14 mm, the mice were randomized and treated intravenously with vehicle or ADC at 3 mg / kg or 10 mg / kg once a week. Partial regression (PR) was defined as tumor volume less than 50% of the starting tumor volume on the first day of dosing in three consecutive measurements, and complete regression (CR) was defined as tumor volume less than 14 mm in three consecutive measurements. 3 Data are presented as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula.

[0732]

[0733] Treatment t = volume of tumor treated at time t

[0734] Treatment t0 = volume of tumor treated at time 0

[0735] Placebo t = Placebo tumor volume at time t

[0736] Placebo t0 = Placebo tumor volume at time 0

[0737] Efficacy Methods in the H1650 Model

[0738] 3×10 6 H1650 cells / 200 μL PBS / Matrigel were implanted subcutaneously on the right side of female BALB / c nude mice. When the tumor size reached approximately 100-200 mm 3When the mean volume of ADC was 1.04 μg / kg, mice were randomized into groups and treated intravenously with vehicle once a week at 10 mg / kg of ADC.

[0739] Efficacy Methods in the H441 Model

[0740] 5×10 6 H441 cells / 300 μL PBS / Matrigel were implanted subcutaneously on the right flank of female NOG mice. When the tumor size reached approximately 100-200 mm 3 When the mean volume of ADC was 1.04 μg / kg, mice were randomized into groups and treated intravenously with vehicle once a week at 10 mg / kg of ADC.

[0741] PK studies in mice

[0742] Blood samples were collected from A375 tumor-bearing mice at 0, 2, 4, 8, 24, 72, and 168 hours after intravenous administration of 10 mg / kg of ADC, and then centrifuged (4°C, 3000×g, 7 min) to separate plasma. The concentrations of ADC and total Ab were measured by the Meso Scale Discovery (MSD) ligand binding method developed in-house. Briefly, His-tagged B7H3 extracellular domain fusion protein was used as a capture reagent, and biotin-labeled anti-DXd Ab or goat anti-human κ Ab was used as a detection reagent for ADC or total Ab measurement, respectively.

[0743] The antibodies used herein are prepared according to conventional methods, for example, vector construction, eukaryotic cell transfection such as HEK293 cells (Life Technologies catalog number 11625019), purification and expression. The sequences of the antibodies used herein can be found herein.

[0744] Table 2. Platform examples

[0745]

[0746]

[0747]

[0748]

[0749] The present invention is generally disclosed herein, and numerous embodiments are described using affirmative language. The present invention also specifically includes embodiments that exclude, in whole or in part, specific subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays, or analyses. Therefore, although it is not generally expressed herein that the present invention does not include, aspects that are not explicitly included in the present invention are still disclosed herein.

[0750] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clear understanding, it is obvious to those skilled in the art that some minor changes and modifications will be implemented. Therefore, the description and examples should not be construed as limiting the scope of the invention.

[0751] It will be appreciated that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in any country.

[0752] The disclosures of all publications, patents, patent applications, and published patent applications referred to herein by identification are hereby incorporated by reference in their entirety.

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding fragment thereof; RG is a reactive group residue; SP is a spacer group residue; each of A, B and C is independently an amino acid residue in each case; HG is a hydrophilic residue or hydrogen; the subscripts a, c and p are independently 0 or 1 in each case; PA is a payload residue; the subscript x is from 1 to 15; and PAB represents -NH-CH2-O-.

2. The compound according to claim 1, wherein the compound is a compound of formula (Ia): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

3. The compound according to claim 1, wherein the compound is a compound of formula (Ib): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

4. The compound of any one of claims 1 to 3, wherein the antibody is ifenatumomab, cofiutuzumab, pertrastuzumab or trastuzumab, or an antigen-binding fragment of ifenatumomab, cofiutuzumab, pertrastuzumab or trastuzumab.

5. The compound of any one of claims 1 to 3, wherein the antibody is a humanized, chimeric or human antibody or an antigen binding fragment thereof that binds to one or more receptors selected from the group consisting of HER2, HER3, CD7, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD46, CD70, CD71, CD74, CD79b, CD123, CD138, CD142, CD166, CD205, CD228, CCR2, CA6, placental cadherin, CEA, CEACAM5, C 4.4a, DLL3, EGFR, EGFRVIII, ENPP3, EphA2, EphrinA, FLOR1, FGFR2, GCC, cKIT, LIV1, LY6E, MSLN, MUC16, NaPi2b, Nectin4, gpNMB, PS MA, SLITRK6, STEAP1, TROP2, 5T4, SSEA4, GloboH, Gb5, STn, Tn, B7H3, BCMA, MUC1, cMet, ROR1MSLN, FRa, CLDN18.2, CLDN6, PTK7 and Axl.

6. The compound of any one of claims 1 to 3, wherein the antibody is a humanized, chimeric or human antibody or antigen-binding fragment thereof that binds to one or more receptors selected from the group consisting of: B7H3, MUC1, FGFR2b, CLL1, CCR7, GPC1 and GPC3.

7. A compound as claimed in any one of claims 1 to 6, wherein RG is -(succinimidyl-3-yl-N)—, 8. A compound as claimed in any one of claims 1 to 6, wherein RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 electron-withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

9. The compound of any one of claims 1 to 6, wherein RG is 10. The compound of any one of claims 1 to 6, wherein RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 electron-withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

11. A compound of formula (II): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, wherein RG is a reactive group residue; SP is a spacer group residue; each of A, B and C is independently an amino acid residue in each case; HG is a hydrophilic residue or hydrogen; the subscripts a, c and p are independently 0 or 1 in each case; PA is a payload residue; and PAB stands for -NH-CH2-O.

12. The compound of claim 11, wherein the compound is a compound of formula (IIa): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

13. The compound of claim 11, wherein the compound is a compound of formula (IIb): or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof.

14. A compound as claimed in any one of claims 11 to 13, wherein RG is Succinimide-N-, 15. A compound as claimed in any one of claims 11 to 13, wherein RG is Wherein EWG is an electron withdrawing group selected from the group consisting of: -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 , and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

16. A compound as claimed in any one of claims 11 to 13, wherein RG is 17. A compound as claimed in any one of claims 11 to 13, wherein RG is Wherein EWG is selected from -CN, halogen, -CF3, -C(=O)OR 1 and -C(=O)R 1 electron-withdrawing group, and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

18. A compound as claimed in any one of claims 11 to 17, wherein SP is (CH2) 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)-, wherein each of n independently represents an integer from 1 to 8.

19. A compound as claimed in any one of claims 1 to 18, wherein SP is -(CH2) n -C(═O)- or -CH2-C(═O)-NH-(CH2) n -C(═O); and each of n independently represents an integer of 5 or 2.

20. The compound of any one of claims 1 to 7, wherein RG is SP is -(CH2) n -C(═O)-; and n is an integer of 1, 2, 3, 4 or 5; preferably n is an integer of 1 or 2.

21. A compound as claimed in any one of claims 11 to 14, wherein RG is SP is -(CH2) n -C(═O)-; and n is an integer of 1, 2, 3, 4 or 5; preferably n is an integer of 1 or 2.

22. The compound of any one of claims 1 to 21, wherein HG is selected from the group consisting of sugars, phosphates, sulfates, phosphodiesters, and phosphonates.

23. The compound of claim 22, wherein the sugar is selected from the group consisting of β-D-galactose, N-acetyl-β-D-galactosamine, N-acetyl-α-D-galactosamine, N-acetyl-α-D-glucosamine, β-D-glucuronic acid, α-L-iduronic acid, α-D-galactose, α-D-glucose, β-D-glucose, α-D-mannose, β-D-mannose, α-L-fucose, β-D-xylose, neuraminic acid, or any analog or modification thereof, or a sulfate, phosphate, carboxyl, amino, or O-acetyl modification thereof; preferably β-D-galactose and β-D-glucuronic acid, or stereoisomers, enantiomers, isotopomers, or prodrugs thereof.

24. The compound of claim 23, wherein HG is or stereoisomers, enantiomers, isotopomers or prodrugs thereof.

25. The compound of any one of claims 1 to 24, wherein each PA independently represents a chromophore functional group.

26. The compound of claim 25, wherein each chromophore functional group is independently selected from the class or subclass of xanthophophytes, erythrophophytes, iridophytes, leukophhophytes, melanophhophytes, and cyanophhophytes; the class or subclass of fluorophore molecules, which are fluorescent compounds that re-emit light upon illumination; the class or subclass of visual phototransduction molecules; the class or subclass of luminophore molecules; the class or subclass of luminescent molecules; and the class or subclass of luciferin compounds.

27. The compound of any one of claims 1 to 24, wherein each PA is independently a cytotoxic agent, wherein the cytotoxic agent contains at least one hydroxyl group.

28. The compound of any one of claims 1 to 24, wherein each PA is independently selected from the group consisting of kinesin spindle protein (KSP) inhibitors (KSPi), camptothecins (such as DXd, 7-ethyl-10-hydroxy-camptothecin (SN-38) and other analogs), eribulin, PNU-159682, anthracyclines, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) and amatoxin.

29. The compound of any one of claims 1 to 24, wherein each PA independently represents formula (VI): Where R 2 and R 3 Each of which is independently hydrogen, halogen, or substituted or unsubstituted C 1-4 alkyl.

30. A compound as described in any one of claims 1 to 24, wherein each PA independently represents 31. The compound of any one of claims 1 to 10 and 18 to 30, wherein the compound is selected from the group consisting of compounds in Table 1.

32. A compound as described in any one of claims 11 to 30, wherein the compound is selected from the group consisting of compounds in Table 2.

33. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure of formula (III): in R 2 and R 3 Each of is independently hydrogen, halogen or alkyl.

34. A pharmaceutical composition comprising a compound as described in any one of claims 1 to 10, 18 to 31 and 33, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof, and a pharmaceutically acceptable excipient.

35. A method for treating a proliferative disease, metabolic disease, inflammation or neurodegenerative disease in a subject, the method comprising administering to the subject an effective therapeutic amount of a compound as described in any one of claims 1 to 10, 18 to 31 and 33 or a pharmaceutical composition as described in claim 34.

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