Antibody-drug conjugate

Abstract

An antibody-drug conjugate compound comprising a linker, and a method of using the compound are provided.

Description

Technical Field

[0001] 1. Technical Field This specification provides novel proteins (e.g., drug conjugates comprising an antibody, a hydrophilic solubilizing group and / or a linker comprising a hydrophilic solubilizing group), and methods of treating diseases, disorders, and conditions, the methods comprising administering a protein-drug conjugate comprising a hydrophilic solubilizing group and / or its linker.

Background Art

[0002] 2. Background Art An antibody-drug conjugate (ADC) is an antibody operably linked to a biologically active small molecule, also known as a toxin or payload. ADCs selectively deliver a potent payload to target-expressing cells, resulting in a potential reduction of off-target side effects and / or toxicity, and an improvement in the therapeutic index. The lipophilicity of many payloads (i.e., drugs) can negatively affect the properties of the ADC to such an extent that the payload is not efficiently delivered to target cells. The low bioavailability of lipophilic payloads can narrow the therapeutic window for ADC treatment. Furthermore, the hydrophobicity of the payload can pose challenges for conjugation to the antibody, which is a reaction performed under aqueous conditions. Therefore, the development of hydrophilic linkers for protein conjugates (e.g., ADCs) remains continuously needed, which enables the possibility of conjugating lipophilic payloads, improved modulation of biological targets, improved bioavailability, and improved therapeutic window.

[0003] The therapy of monoclonal antibodies (mAbs) is gaining momentum as an adjuvant and front-line treatment for cancer. The success of mAb therapy, e.g., AVASTIN™ (anti-VEGF) for colorectal cancer, RITUXAN™ (rituximab, anti-CD20) for non-Hodgkin lymphoma, and HERCEPTIN™ (anti-Her2) for breast cancer, has demonstrated that unbound antibodies can potentially improve patient survival without the occurrence of significantly increased toxicity.

[0004] Monoclonal antibodies can bind to therapeutic agents to form antibody-drug conjugates. For example, the aforementioned HERCEPTIN (trademark) antibody was conjugated with a maytansine payload to form ADC KADCYLA (trademark). ADCs may show increased efficacy compared to unbound antibodies. The binding of the antibody to the drug can be direct or indirect via a linker. The linker can be cleavable or non-cleavable. One of the components considered important for the development of effective and well-tolerated ADCs is the composition and stability of the linker. For some types of ADCs, the linker preferably provides serum stability but selectively releases the drug within the target cell.

[0005] The attachment of the linker to the mAb can be achieved in various ways. For example, via surface lysine, via reductive coupling to oxidized carbohydrates, via cysteine residues liberated by reducing interchain disulfide bonds, etc. Various ADC conjugation systems (e.g., hydrazone, disulfide, and peptide-based conjugations) have been described in the literature. Some hydrazone- and disulfide-based linkers can be unstable in circulation, leading to unwanted release of the drug outside the target tissue. This early release of the drug is thought to potentially result in systemic or organ-specific toxicity and / or suboptimal therapeutic efficacy. Peptide-based linker strategies can provide linkers with increased stability, but due to the increased hydrophobicity of some linker conjugates, especially with highly hydrophobic drugs, aggregation can occur. Such aggregation can lead to many unwanted effects, such as precipitation of the ADC, difficulty in administration, and non-specific uptake of the ADC into non-target tissues, potentially affecting non-target toxicity and reducing 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. Due to its toxicity, it cannot be used as a drug by itself. MMAE is actually desmethyl-auristatin E. That is, the N-terminal amino group has only one methyl substituent instead of two in the case of 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 area of small molecule therapeutics, strategies have been developed to provide prodrugs of active chemical substances. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into the active compound. Such prodrug strategies can lead to an increase in the selectivity of the drug for its intended target and a reduction in adverse effects.

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

[0009] Furthermore, certain antibodies (e.g., patritumab) have visually observable aggregation during rapid buffer exchange. Both its aggregation temperature (Tagg) detected by dynamic light scattering (DLS) and its self-association tendency detected by AC-SINS assay are worse than those of a panel of normally operating mAbs. The aggregation tendency of patritumab results in aggregation of the corresponding ADC.

[0010] Accordingly, there is an unmet medical need to create an ADC with a linker system that provides increased serum stability and solubility of the linker at high levels, enabling efficient binding of hydrophobic drugs and effective intracellular delivery of the drugs. SUMMARY OF THE INVENTION

[0011] 3. Summary of the Invention Disclosed herein is an ADC with a linker system that provides increased serum stability and solubility of the linker at high levels, enabling efficient binding of hydrophobic drugs and effective intracellular delivery of the drugs.

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

Chemical formula

[0013] In another embodiment, the ADC is a compound of formula (Ia)

Chemical formula

[0014] In another embodiment, the ADC is a compound of formula (Ib) [Chemistry] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted form, or prodrug thereof, wherein the variables are defined herein.

[0015] In another embodiment, described herein is a method of treating a disease, condition, or disorder in a patient in need of treatment for the disease, condition, or disorder, the method comprising administering to the patient a compound described herein. Also provided is the use of a compound described herein for treating a disease, condition, or disorder described herein. Further provided is the use of a compound described herein for manufacturing a medicament for treating a disease, condition, or disorder described herein. In some embodiments, the compound is an antibody-drug conjugate.

[0016] In another embodiment, described herein is a method of preparing an antibody-drug conjugate, the method comprising contacting a binder with a linker-payload compound described herein under conditions suitable for forming a bond between the binder and the linker-payload compound. 4. Brief Description of the Drawings

Brief Description of the Drawings

[0017]

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DETAILED DESCRIPTION OF THE INVENTION

[0018] 5. DETAILED DESCRIPTION As used herein, compounds, compositions, ADCs, and methods useful for treating a variety of human cancers (e.g., without limitation, colorectal cancer, gastric cancer, breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, head and neck cancer, pancreatic cancer, and cervical cancer) are provided. In one embodiment, compounds, compositions, ADCs, and methods useful for treating a variety of human cancers are provided herein.

[0019] It is understood that within the scope of the present disclosure, the present disclosure is not limited to the specific methods and / or experimental conditions described, and such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0020] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All patents, applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0021] 5.1. DEFINITIONS When referring to the compounds provided in this specification, unless otherwise specified, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. When there are multiple definitions for the terms provided in this specification, these definitions shall prevail unless otherwise stated.

[0022] As used herein, when used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include a single referent along with plural referents unless the context clearly indicates otherwise.

[0023] As used herein, unless otherwise indicated, the terms "about" and "substantially", when used in connection with the amount or weight percentage of a component of a composition, mean an amount or weight percentage recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified amount or weight percentage. In certain embodiments, the terms "about" and "substantially", when used in this context, are intended to mean an amount or weight percentage within 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percentage.

[0024] As used herein, unless otherwise indicated, the terms “about” and “substantially” are used in connection with a numerical value or range of values provided to characterize a particular solid form, e.g., a particular temperature or temperature range (such as those describing a melting, dehydration, desolvation, or glass transition temperature), a mass change (such as a mass change as a function of temperature or humidity), a solvent or water content (e.g., in mass or percentage), a peak position (such as in an analysis by IR or Raman spectroscopy or XRPD), to indicate that the value or range of values may deviate to the extent considered reasonable by one of ordinary skill in the art, yet still describe the solid form. Techniques for characterizing crystalline and amorphous solids include, without limitation, 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 and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot stage optical microscopy, scanning electron microscopy (SEM), electron diffraction crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In certain embodiments, the terms “about” and “substantially” when used in this context indicate that the numerical value or range of values may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the recited value or range of values. For example, in some embodiments, the value of an XRPD peak position may vary by up to ±0.2° 2θ (or ±0.2 degrees 2θ) and still describe a particular XRPD peak.

[0025] An "alkyl" group is a saturated, partially saturated, or unsaturated straight-chain or branched-chain acyclic hydrocarbon having from 1 to 10 carbon atoms, usually from 1 to 8 carbons, and in some embodiments from 1 to 6, from 1 to 4, or from 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-hexyl, and saturated branched-chain alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Examples of unsaturated alkyl groups include, without limitation, among others, 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). The alkyl group can be substituted or unsubstituted. In certain embodiments, when the alkyl groups described herein are said to be "substituted," they can be substituted with any substituent or plurality of substituents, such as those found in the exemplary compounds and embodiments disclosed herein, as well as the following. Halogen (chloro, iodo, bromo, or fluoro); hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2, or O(alkyl)aminocarbonyl.

[0026] An "alkenyl" group is a straight-chain or branched-chain acyclic hydrocarbon having 2 to 10 carbon atoms, usually 2 to 8 carbon atoms, and containing at least one carbon-carbon double bond. Representative straight-chain and branched-chain (C2-C8) alkenyl groups include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, 2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, 2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, 3-octenyl, and the like. The double bond of the alkenyl group can be non-conjugated or conjugated with another unsaturated group. The alkenyl group can be unsubstituted or substituted.

[0027] A "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group having 3 to 10 carbon atoms, having a monocyclic ring or a plurality of fused rings or bridged rings, which may optionally be substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, by way of example, monocyclic structures (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.), or polycyclic or bridged ring structures (e.g., adamantyl, etc.). Examples of unsaturated cycloalkyl groups include, among others, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl. The cycloalkyl group can be substituted or unsubstituted. Such substituted cycloalkyl groups include, by way of example, cyclohexanone, etc.

[0028] The "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 anthryl). In some embodiments, the aryl group contains 6 to 14 carbons in the ring portion of the group, and in other embodiments, 6 to 12 or even 6 to 10 carbon atoms. Specific aryls include phenyl, biphenyl, naphthyl, etc. The aryl group can be substituted or unsubstituted. The term "aryl group" also includes groups containing fused rings, such as fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0029] The "heteroaryl" group is an aryl ring system having 1 to 4 heteroatoms as ring atoms in a heteroaromatic ring system, with the remaining part of the atoms being carbon atoms. In some embodiments, the heteroaryl group contains 5 to 6 ring atoms in the ring portion of the group, and in other embodiments, 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In one embodiment, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, without limitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diamine), indolyl, azaindolyl (e.g., pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guanylinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups, etc.

[0030] "Heterocyclyl" is an aromatic (also referred to as heteroaryl) or non-aromatic cycloalkyl in which 1 to 4 of the ring carbon atoms are independently replaced by a heteroatom from the group consisting of O, S, and N. In some embodiments, the heterocyclyl group contains 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. Heterocyclyl can also be attached to other groups at any ring atom (i.e., any carbon or heteroatom of the heterocycle). The heterocyclyl group can be substituted or unsubstituted. Heterocyclyl groups include unsaturated, partially saturated, and saturated ring systems, such as, for example, imidazolyl, imidazolinyl, and imidazolidinyl groups. The term heterocyclyl includes fused ring species (including fused aromatic and non-aromatic groups such as, for example, benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl). This term also includes bridged polycyclic ring systems containing heteroatoms, such as, without limitation, quinuclidyl. Exemplary heterocyclyl groups include, without limitation, the following.Aziridinyl, azetidinyl, pyrrolidyl, imidazolidinyl, pyrazolidinyl, thiazolinidyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidinyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl, e.g., 1H-imidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guanylinyl, quinolinyl, isoquinolinyl, quinolidinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclic groups may be mono-substituted or substituted more than twice, and are, for example, without limitation, pyridyl or morpholinyl groups. These are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted, with various substituents such as those listed below.

[0031] The "cycloalkylalkyl" group is a radical of the formula: -alkyl-cycloalkyl, where alkyl and cycloalkyl are as defined above. A substituted cycloalkylalkyl group may be substituted in the alkyl, cycloalkyl, or both the alkyl and cycloalkyl moieties of the group. Representative cycloalkylalkyl groups include, without limitation, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than twice.

[0032] The "aralkyl" group is a radical of the formula: -alkyl-aryl, where alkyl and aryl are as defined above. A substituted aralkyl group may be substituted in the alkyl, aryl, or both the alkyl and aryl moieties of the group. Representative aralkyl groups include, without limitation, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.

[0033] The "heterocyclic alkyl" group is a radical of the formula: -alkyl-heterocyclic, where alkyl and heterocyclic are as defined above. A substituted heterocyclic alkyl group may be substituted in the alkyl, heterocyclic, or both the alkyl and heterocyclic moieties of the group. Representative heterocyclic alkyl groups include, without limitation, 4-ethyl-morpholinyl, 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.

[0034] "Halogen" is chloro, iodo, bromo, or fluoro.

[0035] The "hydroxyalkyl" group is one or more hydroxy groups substituting the aforementioned alkyl group.

[0036] The "alkoxy" group is O(alkyl), where alkyl is as defined above.

[0037] The "alkoxyalkyl" group is (alkyl)O(alkyl), where alkyl is as defined above.

[0038] The "amine" group is a radical of the formula: NH2.

[0039] The "hydroxylamine" group is a radical of the formula: N(R # )OH, or NHOH, where R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic, or heterocyclic alkyl group as defined herein.

[0040] The "alkoxyamine" group is a radical of the formula: -N(R # )O-alkyl, or -NHO-alkyl, where R #is as defined above.

[0041] The "aralkoxyamine" group is a radical of the formula: N(R # )O-aryl, or NHO aryl, where R # is as defined above.

[0042] The "alkylamine" group is a radical of the formula: NH alkyl, or N(alkyl)2, where each alkyl is independently as defined above.

[0043] The "aminocarbonyl" group is a radical of the formula: -C(=O)N(R # )2, -C(=O)NH(R # ), or C(=O)NH2, where each R # is as defined above.

[0044] The "acylamino" group is a radical of the formula: NHC(=O)(R # ), or N(alkyl)C(=O)(R # ), where each alkyl and R # is independently as defined above.

[0045] The "O(alkyl)aminocarbonyl" group is a radical of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ), or -O(alkyl)C(=O)NH2, where each R # is independently as defined above.

[0046] The "N-oxide" group is a radical of the formula: -N + -O -

[0047] The "carboxy" group is a radical of the formula: C(=O)OH.

[0048] The "ketone" group is a radical of the formula: C(=O)(R # ), where R # ​is as defined above.

[0049] The "aldehyde" group is a radical of the formula: -CH(=O).

[0050] The "ester" group is a radical of the formula: C(=O)O(R # ), or OC(=O)(R # ), where R # is as defined above.

[0051] The "urea" group is a radical of the formula: -N(alkyl)C(=O)N(R # )2, -N(alkyl)C(=O)NH(R # ), -N(alkyl)C(=O)NH2, -NHC(=O)N(R # )2, -NHC(=O)NH(R # ), or NHC(=O)NH2 # , where each alkyl and R # is independently as defined above.

[0052] The "imine" group is a radical of the formula: -N=C(R # )2, or -C(R # )=N(R # ), where each R # is independently as defined above.

[0053] The "imide" group is a radical of the formula: -C(=O)N(R#)C(=O)(R # ), or N((C=O)(R # ))2, where each R # is independently as defined above.

[0054] The "urethane" group is a radical 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 # is independently as defined above.

[0055] The "amidine" group has 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 radicals, and each R # is independently as defined above.

[0056] The "guanidine" group has the formula: -N(R # )C(=N(R # ))N(R # )2、-NHC(=N(R # ))N(R # )2、-N(R # )C(=NH)N(R # )2、-N(R # )C(=N(R # ))NH(R # )、-N(R # )C(=N(R # ))NH2、-NHC(=NH)N(R # )2、-NHC(=N(R # ))NH(R # )、-NHC(=N(R # ))NH2、-NHC(=NH)NH(R # )、-NHC(=NH)NH2、-N=C(N(R # )2)2、-N=C(NH(R# )) 2, or a radical of -N=C(NH2)2, and each R # is independently as defined above.

[0057] The "enamine" group has 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), and each R # is independently as defined above.

[0058] The "oxime" group has the formula: -C(=NO(R # ))(R # ), -C(=NOH)(R # ), -CH(=NO(R # )) or -CH(=NOH), and each R # is independently as defined above.

[0059] The "hydrazide" group has the formula: -C(=O)N(R # )N(R # )2, -C(=O)NHN(R # )2, -C(=O)N(R # )NH(R # ), 、- C(=O)N(R # )NH2, -C(=O)NHNH(R # )2 or -C(=O)NHNH2, and each R #is independently as defined above.

[0060] The "hydrazine" group is a radical of the formula: -N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ) 、 -N(R # )NH2, -NHNH(R # )2, or -NHNH2, and each R # is independently as defined above.

[0061] The "hydrazone" group is a radical of the formula: -C(=N-N(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), and each R # is independently as defined above.

[0062] The "azide" group is a radical of the formula: -N3.

[0063] The "isocyanate" group is a radical of the formula: N=C=O.

[0064] The "isothiocyanate" group is a radical of the formula: N=C=S.

[0065] The "cyanate" group is a radical of the formula: OCN.

[0066] The "thiocyanate" group is a radical of the formula: SCN.

[0067] The "thioether" group is a radical of the formula: -S(R # ), and R # is as defined above.

[0068] The "thiocarbonyl" group is a radical of the formula: -C(=S)(R # ), where R # is as defined above.

[0069] The "sulfinyl" group is a radical of the formula: -S(=O)(R # ), where R # is as defined above.

[0070] The "sulfone" group is a radical of the formula: -S(=O)2(R # ), where R # is as defined above.

[0071] The "sulfonylamino" group is a radical of the formula: -NHSO2(R # ), or -N(alkyl)SO2(R # ), where each alkyl and R # is as defined above.

[0072] The "sulfonamide" group is a radical of the formula: -S(=O)2N(R # )2, or -S(=O)2NH(R # ), or -S(=O)2NH2, where each R # is independently as defined above.

[0073] The "phosphonate" group is a radical 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 # is independently as defined above.

[0074] The "phosphine" group is a radical of the formula: -P(R # )2, where each R # is independently as defined above.

[0075] When the groups described herein are said to be "substituted," except for alkyl groups, they may be substituted with any suitable substituent or substituents. Exemplary examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as the following. Halogen (chloro, iodo, bromo, or fluoro); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); B(OH)2, O(alkyl)aminocarbonyl; cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocyclyl, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl); monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl) aryloxy; aralkyloxy; heterocyclyloxy; and heterocyclylalkoxy.

[0076] As used herein, the term "pharmaceutically acceptable salt(s)" refers to salts prepared from pharmaceutically acceptable non-toxic acids or non-toxic bases (including inorganic acids and inorganic bases as well as organic acids and organic bases).

[0077] As used herein, unless otherwise indicated, the term "solvate" means a compound or a salt thereof that further includes a solvent, in stoichiometric or non-stoichiometric amounts, bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0078] As used herein, unless otherwise indicated, the term "hydrate" refers to a compound or a salt thereof that further includes water, in stoichiometric or non-stoichiometric amounts, bound by non-covalent intermolecular forces.

[0079] As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative of a compound that can react by hydrolysis, oxidation, or other means under biological conditions (in vitro or in vivo) to yield an active compound, particularly a compound. Examples of prodrugs include, without limitation, derivatives and metabolites of compounds that include a biolabile moiety (e.g., a biolabile amide, a biolabile ester, a biolabile carbamate, a biolabile carbonate, a biolabile ureido, and a biolabile phosphate analog). In certain embodiments, a prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. The carboxylic acid ester is conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can generally be prepared using well-known methods, such as those described below. Burger’s Medicinal Chemistry and Drug Discovery 6 th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

[0080] As used herein, unless otherwise specified, the term "stereoisomer" or "stereoisomerically pure" means a stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center is substantially free of the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers is substantially free of other diastereomers of that compound. Typical stereoisomerically pure compounds contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the 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 the 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 the 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 the other stereoisomers of the compound. Compounds can have chiral centers and can exist as racemic compounds, individual enantiomers or diastereomers, and mixtures thereof. All such isomers, including mixtures thereof, are included within the scope of the embodiments disclosed herein. The use of stereoisomerically pure forms of such compounds, as well as the use of mixtures of such forms, are encompassed by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be synthesized asymmetrically or resolved using standard techniques (e.g., chiral columns or chiral resolving agents).See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0081] It should also be noted that the compounds can include E isomers and Z isomers, or mixtures thereof, as well as cis isomers and trans isomers, or mixtures thereof. In certain embodiments, the compound is isolated as either the cis isomer or the trans isomer. In other embodiments, the compound is a mixture of the cis isomer and the trans isomer.

[0082] "Tautomers" refer to isomers of a compound that are in equilibrium with each other. The concentration of the isomers depends on the environment in which the compound is found and can vary, for example, depending on whether the compound is a solid or is in an organic or aqueous solution. For example, in an aqueous solution, pyrazole may exhibit the following isomers, which are said to be tautomers of each other. [Chemical formula]

[0083] As will be readily understood by those skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of a compound are within the scope of the present invention.

[0084] It should also be noted that a compound can contain unnatural proportions of atomic isotopes in one or more of its atoms. For example, a compound can be radiolabeled using a radioisotope (e.g., tritium ( 3 H), iodine-125 ( 125 I), sulfur-35 ( 35 S), or carbon-14 ( 14 C), etc.), or it can be isotopically enriched with, for example, deuterium ( 2 H), carbon-13 ( 13 C), or nitrogen-15 ( 15 N). As used herein, an "isotope substitute" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotope composition different from its natural isotope composition. "Isotopically enriched" can also refer to a compound containing at least one atom where the atom has an isotope composition different from its natural isotope composition. The term "isotope composition" refers to the amount of each isotope present for a given atom. Radiolabeled compounds and isotopically enriched compounds are useful as therapeutic agents, e.g., for treating cancer and inflammation, research reagents, e.g., for binding assay reagents, and diagnostic agents, e.g., for in vivo contrast agents. All isotope variations of the compounds described herein are intended to be encompassed within the scope of the embodiments provided herein, whether radioactive or not. In some embodiments, isotope substitutes of the compounds are provided, e.g., the isotope substitute is a deuterium, carbon-13, or nitrogen-15 enriched compound.

[0085] It should be noted that if there is a difference between the illustrated structure and the name for that structure, the illustrated structure should be given precedence.

[0086] The term "effective amount" in relation to a compound means an amount that can relieve a symptom, either wholly or in part, or can delay or halt further progression or worsening of that symptom. As will be apparent to those skilled in the art, it should be expected that the effective amount of the compounds disclosed herein can vary depending on the severity of the disorder being treated.

[0087] As used herein, "alkynyl" refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl may be optionally substituted and may be straight-chain, branched-chain, or cyclic. Alkynyl includes, without limitation, radicals having 2 to 20 carbon atoms, i.e., C 2-20 alkynyl, having 2 to 12 carbon atoms, i.e., C 2-12 alkynyl, having 2 to 8 carbon atoms, i.e., C 2-8 alkynyl, having 2 to 6 carbon atoms, i.e., C 2-6 alkynyl, and having 2 to 4 carbon atoms, i.e., C 2-4 alkynyl is included. Examples of alkynyl moieties include, without limitation, ethynyl, propynyl, and butynyl.

[0088] As used herein, "haloalkyl" refers to alkyl as defined above, wherein the alkyl contains at least one substituent selected from a halogen, for example, fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, without limitation, -CF3, -CH2CF3, -CCl2F, and -CCl3.

[0089] As used herein, "haloalkoxy" refers to alkoxy as defined above, wherein the alkoxy contains at least one substituent selected from a halogen, for example, F, Cl, Br, or I.

[0090] As used herein, "arylalkyl" refers to a monovalent moiety that is a radical of an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound contains a single bond to an alkyl group and the radical is located on the alkyl group. The arylalkyl group is attached to the illustrated chemical structure via an alkyl group. Arylalkyl can be represented by structures such as B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety (e.g., phenyl). Arylalkyl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include, without limitation, benzyl.

[0091] As used herein, "alkylaryl" refers to a monovalent moiety that is a radical of an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound contains a single bond to an alkyl group and the radical is located on the aryl group. The alkylaryl group is attached to the illustrated chemical structure via an aryl group. Alkylaryl 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, where B is an aromatic moiety (e.g., phenyl). Alkylaryl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl include, without limitation, toluyl.

[0092] 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 the ring is substituted with an oxygen radical, i.e., the aromatic compound contains a single bond to an oxygen atom and the radical is localized on the oxygen atom. For example, in the case of phenoxy, it is C6H5-O-. The aryloxy substituent is attached to the compound to which the aryloxy substituent is substituted via this oxygen atom. Aryloxy is optionally substituted. Aryloxy includes, without limitation, radicals having 6 to 20 ring carbon atoms, i.e., C 6-20 aryloxy, those having 6 to 15 ring carbon atoms, i.e., C 6-15 aryloxy, and those having 6 to 10 ring carbon atoms, i.e., C 6-10 aryloxy is included. Examples of aryloxy moieties include, without limitation, phenoxy, naphthoxy, and anthroxy.

[0093] As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the term "amino acid residue" or "N-alkylamino acid residue" refers to the product of an amide coupling or peptide coupling of an amino acid or N-alkylamino acid with a suitable coupling partner. For example, a water molecule is released after the amide or peptide coupling of an amino acid or N-alkylamino acid, and as a result, an amino acid residue or N-alkylamino acid residue is incorporated into the product.

[0094] As used herein, "sugar" or "sugar group" or "sugar residue" refers to a carbohydrate moiety that can include 3-carbon (triose) units, 4-carbon (tetrose) units, 5-carbon (pentose) units, 6-carbon (hexose) units, 7-carbon (heptose) units, or combinations thereof, and that can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. Optionally, "sugar" or "sugar group" or "sugar residue" includes furanose (e.g., ribofuranose, fructofuranose), or pyranose (e.g., glucopyranose, galactopyranose), or combinations thereof. Optionally, "sugar" or "sugar group" or "sugar residue" includes aldose or ketose, or combinations thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other "sugars" or "sugar groups" or "sugar residues" include polysaccharides and / or oligosaccharides, such as, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose. Optionally, "sugar" or "sugar group" or "sugar residue" is an amino sugar. Optionally, "sugar" or "sugar group" or "sugar residue" is a glucosamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the remainder of the molecule via its amino group to form an amide bond with the remainder of the molecule (i.e., glucamide).

[0095] As used herein, "binding agent" refers to any molecule (e.g., antibody) that can bind specifically to a given binding partner (e.g., antigen).

[0096] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain (R group) specific to each amino acid. Amino acids can be either proteinogenic or non-proteinogenic. "Proteinogenic" means that the amino acid is one of the 20 naturally occurring amino acids found in proteins. Proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteinogenic" means that the amino acid is either not naturally found in proteins or is not directly produced by cellular machinery (e.g., is a product of post-translational modification). Non-limiting examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, beta-alanine, ornithine, and citrulline.

[0097] As used herein, "peptide" is defined in its broadest sense in its various grammatical forms and refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits can be linked by peptide bonds or by other bonds (e.g., esters, ethers, etc.). As used herein, the term "amino acid" refers to natural and / or non-natural, proteinogenic or non-proteinogenic, or synthetic amino acids, such as glycine and both D- and L-enantiomers, as well as amino acid analogs and peptidomimetics. When the peptide chain is short (e.g., two, three or more amino acids), it is generally referred to as an oligopeptide. When the peptide chain is longer, the peptide is usually referred to as a polypeptide or a protein. Full-length proteins, analogs, mutants, and fragments thereof are included by definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation reactions, etc. Furthermore, because ionizable amino and carboxyl groups are present within the molecule, certain peptides can be obtained as acidic or basic salts or in neutral form. Peptides may be obtained directly from the source organism or produced recombinantly or synthetically.

[0098] The amino acid sequences of antibodies can be numbered using any known numbering scheme, such as those described below. Kabat et al., (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme). Unless otherwise indicated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply differences in sequences where they do not exist, and one of ordinary skill in the art can readily confirm sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, the "EU numbering scheme" is generally used when referring to residues within the constant region of the antibody heavy chain (e.g., as reported in Kabat et al. (supra)).

[0099] As used herein, the term "anti-HER2 antibody" refers to an antibody that selectively binds to the HER2 receptor (e.g., trastuzumab). In one embodiment, trastuzumab can be made and used as described in US6407213 and US5821337. The entire disclosure of these documents is hereby incorporated by reference into this specification.

[0100] As used herein, the term "anti-HER3 antibody" refers to an antibody that selectively binds to the HER3 receptor (e.g., patritumab). In one embodiment, patritumab can be made and used as described in US7705130. The entire disclosure of this document is hereby incorporated by reference into this specification.

[0101] As used herein, the term "anti-PTK7 antibody" refers to an antibody that selectively binds to the PTK7 receptor (e.g., ceftobiprole). In one embodiment, ceftobiprole can be made and used as described in US9777070. The entire disclosure of this document is hereby incorporated by reference into this specification.

[0102] As used herein, the term "ifinatamab" refers to an antibody that selectively binds to the B7H3 receptor. In one embodiment, ifinatamab can be made and used as described in US10117952 or WO2022102695. The entire disclosures of these documents are hereby incorporated by reference into this specification.

[0103] The terms "cancer" and "cancerous" generally refer to or describe a physiological state in a mammal characterized by uncontrolled cell growth. A "tumor" contains one or more cancerous cells. Examples of cancers include, without limitation, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinomas (e.g., epidermoid squamous cell carcinomas), lung cancers, such as small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, e.g., gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland adenocarcinoma, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0104] As used herein, the term "cytotoxic activity" refers to the activity that reduces or decreases the cell viability of a tested cell line.

[0105] In the appended claims and the foregoing description of the invention, except where the context requires otherwise by express language or necessary implication, the term "comprise", or variations such as "comprises" or "comprising", is used in an inclusive sense, that is, to specify the presence of the stated features, but not to exclude the presence or addition of further features in various embodiments of the invention.

[0106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods, devices, and materials are described herein.

[0107] 5.2. Conjugate 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 linked to at least one hydrophilic moiety via a covalent linker, wherein the covalent linker is directly or indirectly bonded to each of the protein, the payload moiety, and the hydrophilic moiety, which is an ADC or a pharmaceutically acceptable salt thereof. In some embodiments, the protein is an antibody or an antigen-binding fragment thereof.

[0108] As shown herein, in some examples, the linker is directly attached to a covalent linker (e.g., a linker described herein). This means that the linker is separated from the covalent linker described herein by only one bond position. In some of these examples, the covalent linker is also directly attached to the payload moiety. This means that the covalent linker is separated from the payload (e.g., without limitation, Dxd, MMAE, or a stereoisomer thereof, or any payload described herein) by only one bond position. In some of these examples, the covalent linker is also directly attached to the hydrophilic moiety. This means that the covalent linker is separated from the hydrophilic residue (e.g., a hydrophilic residue described herein) by only one bond position. In some of these examples, the covalent linker is the covalent linker described herein.

[0109] In other examples, the linker is indirectly attached to the covalent linker. This means that the linker is separated from the covalent linker by two or more bond positions.

[0110] This also means that the linker is covalently linked to the payload via another moiety. For example, the linker may be attached to a maleimide group, which is attached to a polyethylene glycol group, which is attached to the covalent linker. In some of these examples, the covalent linker is also indirectly attached to the payload moiety. This means that the covalent linker is separated from the payload (e.g., without limitation, Dxd, MMAE, or its stereoisomers, or any payload described herein) by more than two bond positions. This also means that the covalent linker is attached to the payload via another moiety. For example, the covalent linker may be attached to a dipeptide (e.g., without limitation, Val-Ala or Val-Cit), which may be attached to PAB, which may be attached to the payload. In some of these examples, the covalent linker is also indirectly attached to the hydrophilic moiety. This means that the covalent linker is separated from the hydrophilic moiety (e.g., the hydrophilic residues described herein) by more than two bond positions. This also means that the covalent linker is attached to the hydrophilic moiety via another moiety.

[0111] In certain situations, the hydrophilic residue includes a terminal hydrophilic group. In some cases, the hydrophilic residue includes at least one sugar residue. In some cases, the hydrophilic residue includes a sugar residue. In some cases, the hydrophilic residue includes a terminal sugar residue. In further examples, the hydrophilic residue includes more than two sugar residues. In some cases, the hydrophilic residue includes more than two terminal sugar residues.

[0112] In another embodiment, the payload provided herein is a chromophore functional group, and the compounds provided herein can be used to detect, monitor, or study the interaction between a cell-binding molecule and a target cell. A chromophore functional group is a functional group having the ability to absorb certain light (e.g., UV light, fluorescent light, IR light, near-IR light, visible light). The chromophore functional group is a functional group selected from the following: classes or subclasses of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores; classes or subclasses of fluorophore molecules, which are fluorescent compounds that absorb light and re-emit light; classes or subclasses of visual phototransduction molecules; classes or subclasses of photophore molecules; classes or subclasses of luminescent molecules; and classes or subclasses of luciferin compounds.

[0113] 5.2.1. Aspect 1 Described herein is a compound according to formula (I),

Chemical formula

[0114] In one example, BA is an antibody. In one example, the antibody is a humanized, chimeric, or human antibody, or an antigen-binding fragment of an antibody. In one example, 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 example, the antibody is a monoclonal antibody.

[0115] In one example, BA is an antibody. In one example, the antibody is a humanized, chimeric, or human antibody, or an antigen-binding fragment of ifinatamab, covexitumab, patritumab, or trastuzumab. In one example, the antibody is a humanized, chimeric, or human antibody, or an antigen-binding fragment of ifinatamab. In one example, the antibody is ifinatamab, covexitumab, patritumab, or trastuzumab, or an antigen-binding fragment of ifinatamab, covexitumab, patritumab, or trastuzumab. In one example, the antibody is ifinatamab or an antigen-binding fragment of ifinatamab.

[0116] In certain embodiments, the antibodies described herein bind to one or more of the 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, p-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, ROR1 MSLN, FRa, CLDN18.2, CLDN6, PTK7, and Axl. In certain embodiments, the antibodies described herein bind to one or more of the receptors selected from the group consisting of B7H3, MUC1, FGFR2b, CLL1, CCR7, GPC1, and GPC3.

[0117] In certain embodiments, the antibodies described herein bind to the CEA receptor. In certain embodiments, the antibodies described herein bind to one or more of the receptors selected from the group consisting of CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21 receptors.

[0118] In certain embodiments, the antibodies described herein bind to the CEACAM5 receptor.

[0119] In certain embodiments, the antibodies described herein bind to the CEACAM6 receptor.

[0120] In certain embodiments, the antibodies described herein are bispecific antibodies.

[0121] In certain embodiments, RG is

Chemical formula

Chemical formula

[0122] In certain embodiments, RG is

Chemical formula

[0123] In one embodiment, RG is [Chemical Formula] .

[0124] In one embodiment, RG is [Chemical Formula] and EWG is an electron-withdrawing group selected from -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.

[0125] In one embodiment, 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)-, where each of n independently represents an integer from 1 to 8.

[0126] In one embodiment, 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.

[0127] In one embodiment, RG is [Chemical Formula] and SP is -(CH2)n -C(=O)-, where n is an integer of 1, 2, 3, 4, or 5, preferably, n is an integer of 1 or 2.

[0128] In certain embodiments, HG is selected from the group consisting of saccharides, phosphate esters, sulfate esters, phosphodiesters, and phosphonates.

[0129] In certain embodiments, the saccharide is selected from the group consisting of: β-D-galactose, N-acetyl-P-D-galactosamine, N-acetyl-a-D-galactosamine, N-acetyl-P-D-glucosamine, β-D-glucuronic acid, a-L-iduronic acid, a-D-galactose, a-D-glucose, β-D-glucose, a-D-mannose, β-D-mannose, a-L-fucose, β-D-xylose, neuraminic acid or any analog or modification thereof, or its sulfate, phosphate, carboxyl, amino, or O-acetyl modification, preferably β-D-galactose and b-glucuronic acid.

[0130] In certain embodiments, HG is

Chemical formula

[0131] In certain embodiments, HG is

Chemical formula

[0132] In certain embodiments, each PA independently represents a chromophore functional group.

[0133] In one embodiment, each chromophore functional group is independently a functional group selected from the following: classes or subclasses of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores; classes or subclasses of fluorophore molecules, which are fluorescent compounds that receive light and re-emit light; classes or subclasses of visual light-transmitting molecules; classes or subclasses of photophore molecules; classes or subclasses of luminescent molecules; and classes or subclasses of luciferin compounds.

[0134] In one embodiment, each PA is independently a cytotoxic agent, and the cytotoxic agent contains at least one hydroxyl group. Examples of cytotoxic agents include, without limitation, the following. 10-Deacetyl-7-xylosylpaclitaxel, 17-AAG, 17-AEP-GA, 17-AH-geldanamycin, 17-DMAP-GA, 17-GMB-APA-GA, 4-Methyl-aether-purwinaphysin F, 9-Hydroxyellipticine, hydrochloride, Actinomycin X2, Aeroplysinin 1, Aeruginosin 865, Agrocherin A, Agrocherin B, Alpha-amanitin, Alpha-amanitin - of fungal fermentation origin, Ansamycin P-3, Ansatriene B, Aphidicolin, Apoptolidin, Auristatin E, Auristatin F, AZD4547, free radical salt, AZD8055, free radical salt, Bafilomycin A1, Beta-amanitin, Boc-Nme-Val-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Doe, Boc-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Phe-Ome, Calicheamicin, Camptothecin, Caetocin, Ketoglobosin, Ketoglobosin C13, Clamidocin, Cinerubin B, Colchicine, Combretastatin-A4, Compound CL0485, Cordycepin, Cryptophycin, Cucurbitacin B, Cucurbitacin E, Curcumin, Cyclopamine, free radical salt, D8-MMAE, Down 02, Daunorubicin, Daunorubicin HCl, Daunorubicin hydrochloride, DGN462, DL-Dithiothreitol / DTT, DL-Dithiothreitol / DTT - biotech grade, DL-Dithiothreitol / DTT - pure grade, DM1, DM4, Drastatin 10, Drastatin 15, DOXO-EMCH, Doxorubicin, Doxorubicin hydrochloride, Duocarmycin DM, Duocarmycin MA, Duocarmycin TM, Englerin A, Epothilone A, Epothilone B, Epsilon-amanitin, Ferulic acid, Fumagillin, Gamma-amanitin, Geldanamycin, Glucopiericidin A, Gramicidin A, Herboxidiene, HL-100-AL1-R01(H-3137), Hygrolidin, Hypotensive mycin, Irimaquinone, Isatropolone A, Isophistularin-3, Ixabepilone, free radical salt,JW55, lactacystin, luisol A, maytansine DM3, maytansinoid AP-3, maytansinol, mechercharmycin A, mensacarcin, methotrexate, microcolin B, microcystin LR, MMAD, MMAD hydrochloride, MMAF, MMAF hydrochloride, monomethyl auristatin E, monomethyl auristatin F methyl ester, mascotoxin A, myoseverin, mitotoxin B, N-acetyl calicheamicin g1, nemorubicin, nogalamycin A, oxalactomycin, oligomycin B, paclitaxel, PF-06380101, phalarisidin, phalloidin, phytosphingosine, piericidin A, pyronetin, PNU-159682, podophyllotoxin, polyketomycin, pseudolaric acid B, shuromycin A, pwinaphysin F, pyrrolobenzodiazepine dimer, quinadpeptin, rakermycin, rebeccamycin, Ro 5-3335, saframycin B, sandramycin, sanguinarine, saporin, seco-duocarmycin SA, sinefungin, taltobulin, taltobulin hydrochloride, taltobulin trifluoroacetate, telomestatin, thiocortisone, tritoxin, tripolin A, tryptolide, tubastatin A HCl, 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 taken up and released into tumor cells. In certain embodiments, each PA is a cytotoxic agent designed to induce target cell death after being taken up into tumor cells. In certain embodiments, each PA is a small molecule drug with high systemic toxicity.,

[0135] 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 (maytansinoid DM1 / DM4), paclitaxel, docetaxel, epothilone B, epothilone A, CYT997, auristatin tyramine phosphate, auristatin aminoquinoline, halocombstatin, calicheamicin θ, 7-ethyl-10-hydroxy-camptothecin (SN-38), pyrrolobenzodiazepine (PBD), pancratistatin, cyclophosphate, cribrostatin-6, kitastatin, turbostatins 1-4, halocombstatin, eribulin, hemiasterlin, PNU, and silastatin.

[0136] In certain embodiments, each PA independently represents formula (VI),

Chemical formula

[0137] In one embodiment, R 2 and R 3 are hydrogen.

[0138] In one embodiment, R 2 and R 3 are methyl.

[0139] In one embodiment, R 2 is methyl and R 3 is F.

[0140] In one embodiment, the carbon to which R 2 and R 3 are attached has the S configuration.

[0141] In one embodiment, the carbon to which R 2 and R 3 are attached has the R configuration.

[0142] In one embodiment, each PA is independently

Chemical formula

[0143] In one embodiment, each PA is independently

Chemical formula

[0144] In one embodiment, the subscript x is from 2 to 12. In one embodiment, the subscript x is from 4 to 12. In one embodiment, the subscript x is from 6 to 12. In one embodiment, the subscript x is from 8 to 12. In one embodiment, the subscript x is from 9 to 11.

[0145] 5.2.2. Aspect 2 Described herein is a compound according to formula (Ia)

Chemical formula

[0146] In one embodiment, BA, RG, SP, HG, PA, and x are as provided herein.

[0147] 5.2.3. Aspect 3 Described herein is a compound according to formula (Ib)

Chemical formula

[0148] In certain embodiments, BA, RG, SP, HG, PA, and x are as provided herein.

[0149] 5.2.4. Aspect 4 Described herein is a compound of formula (II)

Chemical formula

[0150] In certain embodiments, RG is

Chemical formula

Chemical formula

[0151] In certain embodiments, RG is

Chemical formula

[0152] In certain embodiments, RG is

Chemical formula

[0153] In certain embodiments, RG is

Chemical formula

[0154] In certain embodiments, SP, A, B, C, HG, PAB, PA, a, c, and p are as provided herein.

[0155] 5.2.5. Aspect 5 Described herein are compounds of formula (IIa)

Chemical formula

[0156] In certain embodiments, RG is

Chemical formula

[0157] In certain embodiments, RG, SP, HG, and PA are as provided herein.

[0158] 5.2.6. Aspect 6 Described herein are compounds of formula (IIb)

Chemical formula

[0159] In one embodiment, RG is

Chemical Formula

[0160] In one embodiment, RG, SP, HG, and PA are as provided herein.

[0161] 5.2.7. Aspect 17 Described herein is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the ligand-drug conjugate comprising a structure of formula (III),

Chemical Formula

[0162] In some embodiments, the ligand-drug conjugate comprises a structure of formula (E2),

Chemical Formula

[0163] In some embodiments, the antibody is infliximab, pertuzumab, ceftobiprole, or trastuzumab. In one embodiment, the antibody is infliximab.

[0164] In one embodiment, R 2 and R 3 are hydrogen.

[0165] In one embodiment, R 2 and R 3 are methyl.

[0166] In one embodiment, R 2 is methyl and R 3 is F.

[0167] In one embodiment, the carbon to which R 2 and R 3 are attached has the S configuration.

[0168] In one embodiment, the carbon to which R 2 and R 3 are attached has the R configuration.

[0169] In one embodiment, the covalent linker L has the structure of formula (I-L).

Chemical formula

[0170] In one embodiment, the covalent linker L has the structure of formula (Ia-L).

Chemical formula

[0171] In one embodiment, the covalent linker L has the structure of formula (Ib-L).

Chemical formula

[0172] In certain embodiments, RG, SP, HG, PAB, A, B, C, a, c, and p are as provided herein.

[0173] 5.2.8. Aspect 18 Described herein are compounds having formula (IV)

Chemical formula

[0174] In one embodiment, R 2 and R 3 are hydrogen.

[0175] In one embodiment, R 2 and R 3 are methyl.

[0176] In one embodiment, R 2 is methyl and R 3 is F.

[0177] In one embodiment, the carbon to which R 2 and R 3 are attached has an S configuration.

[0178] In one embodiment, the carbon to which R 2 and R 3 are attached has an R configuration.

[0179] In one embodiment, the covalent linker L has a structure of formula (II-L).

Chemical formula

[0180] In one embodiment, the covalent linker L has a structure of formula (IIa-L).

Chemical formula

[0181] In one embodiment, the covalent linker L has a structure of formula (IIb-L).

Chemical formula

[0182] In certain embodiments, RG, SP, HG, PAB, A, B, C, a, c, and p are as provided herein.

[0183] 5.3. Payload Provided herein is a compound, or a pharmaceutically acceptable solvate, stereoisomer, or derivative thereof, the compound having the compound formula (V),

Chemical formula

[0184] In one embodiment, R 2 and R 3 are hydrogen.

[0185] In one embodiment, R 2 and R 3 are methyl.

[0186] In one embodiment, R 2 is methyl and R 3 is F.

[0187] In one embodiment, the carbon to which R 2 and R 3 are attached is in the S configuration.

[0188] In one embodiment, the carbon to which R 2 and R 3 are attached is in the R configuration.

[0189] 5.4. Method or process for making the complex In some embodiments, described herein is a method of preparing an antibody-drug conjugate, the method comprising contacting a binder with a linker-payload compound under conditions suitable to form a bond between the binder and the linker-payload compound. Also provided is a method of preparing a compound of formula (I), formula (Ia), or formula (Ib) under conditions suitable to form a bond between the binder and the linker-payload compound.

[0190] In certain embodiments, an antibody is reacted or treated with a reactive linker-payload to form an antibody-payload conjugate. The reaction can proceed under conditions that are considered suitable by those skilled in the art. In certain embodiments, the antibody is contacted with a reactive linker-payload compound under conditions suitable to form a bond between the antibody and the linker-payload compound. Suitable reaction conditions are known to those skilled in the art.

[0191] Examples of such reactions are shown in the following examples.

[0192] In some embodiments, described herein is a method of making a conjugate, the method comprising treating or contacting a compound with a binder under coupling conditions, the compound comprising a reactive linker attached to at least one payload moiety, and the compound that reacts with the binder is a compound of formula (II), formula (IIa), or formula (IIb), or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof.

[0193] 5.5. Pharmaceutical Compositions Provided herein are the pharmaceutical compositions, compounds, or pharmaceutically acceptable salts thereof described herein, and pharmaceutically acceptable excipients.

[0194] 5.6. Methods of Use In some embodiments, described herein is a method of treating a disease or disorder 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 to be administered is an antibody-drug conjugate described herein.

[0195] 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 immune disorder, an autoimmune disease, an inflammatory disorder, a skin disease, a metabolic disorder, a cardiovascular disease, and a gastrointestinal disease, the method comprising administering to a subject an effective treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound to be administered is an antibody-drug conjugate described herein.

[0196] In some embodiments, described herein is a method of treating a proliferative disease, a metabolic disease, an inflammation, or a neurodegenerative disease in a subject, the method comprising administering to the subject an effective treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, described herein is a method of treating a proliferative disease in a subject, the method comprising administering to the subject an effective treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound to be administered is an antibody-drug conjugate described herein.

[0197] 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 treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound to be administered is an antibody-drug conjugate described herein.

[0198] In some embodiments, described herein is a method of treating inflammation in a subject, the method comprising administering to the subject an effective treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound to be administered is an antibody-drug conjugate described herein.

[0199] In some embodiments, described herein is a method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject an effective treatment amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound to be administered is an antibody-drug conjugate described herein.

[0200] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All patents, applications, and non-patent publications referred to herein are incorporated herein by reference in their entirety.

[0201] 6. Examples The following examples are merely intended to be exemplary and should in no way be construed as limiting. Unless otherwise indicated, the experimental methods in the examples described below are conventional methods. Unless otherwise indicated, all reagents and materials are commercially available. All solvents and chemicals used are of analytical grade or chemical purity. All solvents are redistilled before use. All anhydrous solvents are prepared according to standard methods or reference methods. Silica gel (100 - 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, and unless otherwise indicated, all are eluted with petroleum ether (60 - 90 °C) / ethyl acetate (v / v) and visualized with an ethanol solution of iodine or phosphomolybdic acid. Unless otherwise indicated, all extraction solvents are dried over anhydrous Na2SO4.1 1H NMR spectra were recorded on Bruck-400 and Varian 400MR nuclear magnetic resonance spectrometers using TMS (tetramethylsilane) as the internal standard. Coupling constants were expressed in Hertz. Peaks were reported as singlet (s), doublet (d), triplet (t), quartet (q), quintet (p), sextet (h), septet (hept), multiplet (m), or combinations thereof. Br means broad. LC / MS data were recorded using an Agilent 1100, 1200 high-performance liquid chromatography ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) that detects at 214 nm and 254 nm and an ion trap (ESI source). All compound names except reagents were generated by ChemDraw® 18.0.

[0202] For the purpose of brevity, certain abbreviations are used in this specification. One example is the single-letter abbreviations representing amino acid residues. Amino acids and their corresponding three-letter and one-letter abbreviations are as follows.

[0203] List of abbreviations for amino acids [Table 1]

[0204] In the following examples, the following abbreviations are used.

[0205] List of abbreviations [Table 2-1] [Table 2-2]

[0206] Example 1

[0207] [Chemical formula] [Chemistry]

[0208] Step 1

[0209] Benzyl(((3aR,4R,6S,6aS)-6-(2-amino-2-oxoethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)carbamate (1b)

[0210] To a suspension of 1a (2.01 g, 5.47 mmol) in DMF (20 mL) were added 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). The resulting brown suspension was stirred at 50 °C overnight. After the reaction was complete, the mixture was quenched with water (20 mL) and extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL * 3), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a residue. This 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 pale yellow solid.

[0211] MS(ESI) m / z: 365.4 [M+H] +

[0212] Step 2

[0213] 2-((3aS,4S,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)acetamide (1c)

[0214] 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-based, 10%, 450 mg) was added. The resulting mixture was stirred at 25 °C for 3 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 1c as a gray syrup (1.14 g, crude). The crude product was used directly in the next step without purification.

[0215] MS(ESI) m / z: 231.4 [M+H] +

[0216] Step 3

[0217] Benzyl 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-glutamate (1e)

[0218] A mixture of 1c (900 mg, crude) and 1d (2.18 g, 3.91 mmol) in THF (20 mL) was added with saturated aqueous NaHCO3 solution (5 mL). The mixture was stirred at r.t. for 1 hour. After the reaction was completed, the mixture was diluted with water (20 mL) and 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 obtain a brown oil. This was purified by flash column (DCM / MeOH = 100 / 0~85 / 15) to obtain 1e as a white solid (2.15 g, 82.1% yield).

[0219] MS(ESI) m / z: 672.6 [M+H] +

[0220] Step 4

[0221] 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)

[0222] 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 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 1f (1.87 g, crude) as a white solid. This was used directly in the next step without purification.

[0223] MS(ESI) m / z: 582.5 [M+H] +

[0224] Step 5

[0225] 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)

[0226] 1f (1.80 g, 3.09 mmol) was dissolved in DCM (20 mL), and after the solution was cooled to 0 °C, a mixed solvent of TFA / H2O (20 mL, v / v = 9:1) was added dropwise. The resulting mixture was further stirred at 0 °C for 1 hour. After the reaction was completed, the reaction mixture was purified by prep-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 as a white solid (630 mg, 37.6%).

[0227] MS(ESI) m / z: 542.5 [M+H] +

[0228] Example 2

[0229]

Chem.

[0230] 2 was synthesized according to the standard protocol of solid-phase peptide synthesis (Muriel et al., Methods and protocols of modern solid phase peptide synthesis, Mol. Biotechnol., 2006, 33, 239-254) using readily available commercial starting materials.

[0231] Example 3

[0232]

Chem.

[0233] 3 was purchased from ShangHai HaoYuan MedChemExpress CO.LTD.

[0234] Example 4

[0235]

Chem.

[0236] 4 was synthesized according to the 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 available commercial starting materials.

[0237] Example 5

[0238]

Chem.

[0239] 5 was purchased from ShangHai HaoYuan MedChemExpress CO.LTD.

[0240] Other readily available commercial reactants or reagents are not specifically listed and are not numbered.

[0241] Example 6

[0242]

Chem.

Chem.

[0243] Step 1

[0244] (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanylglycylglycine (6b)

[0245] To a mixture of 2 (1.60 g, 3.05 mmol) and Cu(OAc)2 (221 mg, 1.22 mmol) in DMF (20 mL) was added Pb(OAc)4 (1.62 g, 3.66 mmol) and HOAc (348 μL, 6.10 mmol). The resulting black mixture was purged three times with an N2 balloon and then stirred at 70 °C for 1 h. The black mixture turned into a dark blue mixture. At the completion of the reaction, the mixture was diluted with EtOAc (100 mL), then washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to give 6b (1.40 g, 85.2% yield) as a white solid.

[0246] MS(ESI) m / z: 561.5 [M+Na] +

[0247] Step 2

[0248] Benzyl (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazapentadecane-17-oate (6d)

[0249] To a solution of 6b (0.60 g, 1.12 mmol) and 4 Å molecular sieves in THF (6 mL) were added 6c (0.56 g, 3.34 mmol) and Sc(OTf)3 (0.55 g, 1.12 mmol). The resulting yellow suspension was stirred at r.t. overnight. At the completion of the reaction, the reaction mixture was filtered and the cake was washed with THF. The combined THF phases were then quenched by the addition of saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (100 mL). After separation, the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue. This was purified by silica column gel chromatography (eluent: DCM / MeOH = 100 / 0 to 85 / 15) to give 6d (600 mg, 83.5% yield) as a yellow solid.

[0250] MS(ESI) m / z: 667.5 [M+Na] +

[0251] Step 3

[0252] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazapentadecanoic acid (6e)

[0253] To a solution of 6d (215 mg, 0.33 mmol) in MeOH was added wet Pd / C (20 mg). The mixture was then purged three times with an H2 balloon and stirred at r.t. for 2 h. At the completion of the reaction, the mixture was filtered through a syringe filter head and the filtrate was concentrated in vacuo to give 6e (185 mg, crude) as a white solid.

[0254] MS(ESI) m / z: 578.4 [M+Na] +

[0255] Step 4

[0256] (9H-Fluoren-9-yl)methyl ((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[d]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-triazapentadecane-13-yl)carbamate (6 g)

[0257] To a solution of 6e (185 mg, 0.33 mmol) in DMF (2 mL), HBTU (190 mg, 0.5 mmol) and DIPEA (170 μL, 129 mg, 1 mmol) were added. The resulting yellow solution was stirred at r.t. for 5 min, then 3 (195 mg, 0.37 mmol) was added. The mixture was stirred at r.t. for 60 min. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 6g (150 mg, 46.3% yield) as a white powder.

[0258] MS(ESI) m / z: 972.5 [M+H] +

[0259] Step 5

[0260] (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[d]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)

[0261] To a solution of 6g (30 mg, 0.03 mmol) in DMF (2 mL), Et2N (30 μL, 22.59 mg, 0.31 mmol) was added. The mixture was stirred at r.t. for 1 hour. At the completion of the reaction, the mixture was concentrated under vacuum to give 6h (24 mg, crude) as a yellow oil.

[0262] Step 6

[0263] 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[d]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-triazapentadecane-13-yl)hexanamide (6)

[0264] To a solution of 6i (13 mg) in DMF (2 mL) were added HBTU (23 mg, 0.06 mmol) and DIPEA (16 μL, 12 mg, 0.09 mmol). The resulting yellow solution was stirred at r.t. for 5 minutes and then 6h (24 mg) was added. The mixture was stirred at r.t. for 60 minutes. Upon completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to obtain 6 (18 mg, 61.5% yield) as a white powder.

[0265] MS(ESI) m / z: 943.6 [M + H] +

[0266] Example 7

[0267]

Chemical formula

Chemical formula

[0268] Step 1

[0269] (S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-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-[d]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)

[0270] To a solution of 7a (19 mg) in DMF (2 mL) were added HBTU (31 mg, 0.08 mmol) and DIPEA (22 μL, 16 mg, 0.12 mmol). The resulting yellow solution was stirred at r.t. for 5 min, then 6h (30 mg) was added. The mixture was stirred at r.t. for 60 min. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 7 (20 mg, 50.7% yield) as a white powder.

[0271] MS(ESI) m / z: 958.5[M + H] +

[0272] Example 8

[0273]

Chemical formula

Chemical formula

[0274] Step 1

[0275] Benzyl (10S,13S)-13-amino-10,14-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazapentadecanoate (8b)

[0276] 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 r.t. for 1 h. At the completion of the reaction, the mixture was concentrated under vacuum to give 8b (395 mg, crude) as a yellow solid.

[0277] Step 2

[0278] Benzyl (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-pentaazacosane-20-oate (8d)

[0279] To a solution of 1 (604 mg, 1.12 mmol) in THF (5 mL) were added EDCI (214 mg, 1.12 mmol), HOBt (151 mg, 1.12 mmol) and DIPEA (238 μL, 180 mg, 1.4 mmol). The resulting yellow solution was stirred at r.t. for 15 min and then 8b (395 mg, 0.93 mmol) was added. The mixture was stirred at r.t. for 16 h. Upon completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 8d (350 mg, 40.8% yield) as a white powder.

[0280] MS(ESI) m / z: 968.7 [M+Na] +

[0281] Step 3

[0282] (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-pentaazacosane-20-oic acid (8e)

[0283] To a solution of 8d (190 mg, 0.33 mmol) in MeOH / THF, wet Pd / C (20 mg) was added, then purged three times with an H2 balloon and stirred at r.t. for 3 h. At the completion of the reaction, the mixture was filtered through a syringe filter head to remove Pd / C, and the filtrate was concentrated under vacuum to give 8e (172 mg, crude) as a white solid.

[0284] Step 4

[0285] (9H-Fluoren-9-yl)methyl ((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[d]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-pentaazapentacosane-6-yl)carbamate (8g)

[0286] To a solution of 8e (172 mg, 0.33 mmol) in DMF (2 mL), HBTU (109.75 mg, 0.29 mmol) and DIPEA (68.5 uL, 51.95 mg, 0.4 mmol) were added. The resulting yellow solution was stirred at r.t. for 5 min, then 3 (128 mg, 0.24 mmol) was added. The mixture was stirred at r.t. for 60 min. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 8g (100 mg, 39.1% yield) as a white powder.

[0287] MS(ESI) m / z: 1272.51 [M+Na] +

[0288] Step 5

[0289] (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[d]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-triazapentadecane-13-yl)pentaanediamide (8h)

[0290] 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 r.t. for 1 hour. At the completion of the reaction, the mixture was concentrated under vacuum to give 8h (25 mg, crude) as a yellow oil.

[0291] Step 6

[0292] (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)propanamide)-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[d]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-triazapentadecane-13-yl)pentaanediamide (8)

[0293] To a solution of 8i (11 mg) in DMF (2 mL) were added HBTU (18 mg, 0.05 mmol) and DIPEA (12 μL, 9.1 mg, 0.07 mmol). The resulting yellow solution was stirred at r.t. for 5 min, then 8h (25 mg) was added. The mixture was stirred at r.t. for 60 min. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 8 (8.8 mg, 29.7% yield) as a white powder.

[0294] MS(ESI) m / z: 1281.8 [M+Na] +

[0295] Example 9

[0296]

Chemical formula

Chemical formula

[0297] Step 1

[0298] (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[d]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-triazapentadecane-13-yl)penta diamide (9)

[0299] To a solution of 9b (13 mg) in DMF (2 mL) were added HBTU (24 mg, 0.06 mmol) and DIPEA (16 μL, 12 mg, 0.09 mmol). The resulting yellow solution was stirred at r.t. for 5 minutes, then 9a (40 mg) was added. The mixture was stirred at r.t. for 60 minutes. Upon completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to obtain 9 (18 mg, 45.8% yield) as a white powder.

[0300] MS(ESI) m / z: 1266.8 [M+Na] +

[0301] Example 10

[0302]

Chemical formula

Chemical formula

[0303] Step 1

[0304] (9H-Fluoren-9-yl)methyl ((10S,13S)-1-hydroxy-10,14-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazapentadecan-13-yl)carbamate (10b)

[0305] 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 hour, then quenched with H2O (100 μL) and filtered through celite. The filtrate was concentrated under vacuum. The crude product was purified by silica column gel chromatography (Eluent: DCM / MeOH = 100 / 0 - 90 / 10) to obtain 10b as a white solid (120 mg, 89.6% yield).

[0306] MS(ESI) m / z: 563.4 [M+Na] +

[0307] Step 2

[0308] (9H-Fluoren-9-yl)methyl ((10S,13S)-10,14-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)carbamate (10c)

[0309] To a solution of 10b (120 mg, 0.22 mmol) in anhydrous DMSO (157 μL, 172 mg, 2.2 mmol) was added oxalyl chloride (95 μL, 140 mg, 1.1 mmol) at -78 °C. The reaction mixture was stirred at -78 °C for 30 minutes, and then Et3N (457 μL, 334 mg, 3.3 mmol) was added. The mixture was stirred at -78 °C for 30 minutes, then warmed to r.t. and diluted with DCM (10 mL). The organic solution was then washed with saturated NaHCO3 (1 mL), H2O (1 mL), and brine (1 mL). The organic phase was collected, dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give 10c as a pale yellow oil. The crude product was used in the next step without further purification.

[0310] MS(ESI) m / z: 561.4 [M+Na] +

[0311] Step 3

[0312] (9H-Fluoren-9-yl)methyl ((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-hexaazapentacosane-24-yl)carbamate (10d)

[0313] To a solution of 10c (20 mg, 0.037 mmol) in anhydrous MeOH (1 mL) was added 5 (26.56 mg, 0.037 mmol). The reaction mixture was stirred at r.t. for 15 minutes, and then NaBH3CN (12 mg, 0.19 mmol) was added. The mixture was stirred at r.t. for 12 hours. At the completion of the reaction, the mixture was quenched with H2O and then purified by prep-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 fractions were lyophilized to give 10d (11 mg, 24.5% yield) as a white powder.

[0314] MS(ESI) m / z: 1242.1 [M+H] +

[0315] Step 4

[0316] (S)-2-((2S,13S,16S)-16-Amino-2-isopropyl-3,13,17-trimethyl-9,12,15-trioxo-6-oxa-3,8,11,14-tetraazaoctadecanamide)-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-oxoheptan-4-yl)-N,3-dimethylbutanamide (10e)

[0317] To a solution of 10d (10 mg, 0.008 mmol) in DMF (0.5 mL) was added Et2NH (8.4 μL, 5.8 mg, 0.08 mmol). The mixture was stirred at r.t. for 0.5 h. At the completion of the reaction, the mixture was concentrated under vacuum to give 10e (10 mg) as a pale yellow solid. This was used directly in the next step without further purification.

[0318] MS(ESI) m / z: 1018.8 [M+H] +

[0319] Step 5

[0320] 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-hexaazaoctacosane-24-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (10)

[0321] To a solution of 10e (8.15 mg, 0.008 mmol) in DMF (0.5 mL), HBTU (6.07 mg, 0.016 mmol) and DIPEA (4.19 μL, 3.11 mg, 0.024 mmol) were added, and then 10f (3.38 mg, 0.016 mm) was added. The mixture was stirred at r.t. for 30 minutes. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to obtain 10 (5 mg, 51.5% yield) as a white powder.

[0322] MS(ESI) m / z: 1212.6 [M + H] +

[0323] Example 11

[0324]

Chemical formula

Chemical formula

[0325] Step 1

[0326] (5R,8S)-1-(9H-fluoren-9-yl)-8-(hydroxymethyl)-5-isopropyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecanoic acid (11f)

[0327] To a solution of 4 (100 mg, 0.17 mmol) in MeOH, wet Pd / C (10 mg) was added, then purged three times with an H2 balloon and stirred at r.t. for 2 hours. At the completion of the reaction, the mixture was filtered through a syringe filter head to remove Pd / C, and the filtrate was concentrated under vacuum to obtain 11f (90 mg, crude) as a white solid.

[0328] MS(ESI) m / z: 536.4 [M + Na]+

[0329] Step 2

[0330] 2-(((S)-2-((R)-2-Amino-3-methylbutanamide)-3-hydroxypropanamide)methoxy)acetic acid (11 g)

[0331] 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 r.t. for 0.5 h. At the completion of the reaction, the mixture was concentrated under vacuum to give 11g (57 mg, crude) as a yellow solid.

[0332] MS (ESI) m / z: 292.4 [M+H] +

[0333] Step 3

[0334] (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-triazapentadecanoic acid (11i)

[0335] To a solution of 11g (29 mg, 0.098 mmol) and 11h (45 mg, 0.15 mmol) in DMF (2 mL) was added DIPEA (24 uL, 19 mg, 0.15 mmol). The mixture was stirred at r.t. for 1.5 h. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 11i (18 mg, 25.3% yield) as a white powder.

[0336] MS (ESI) m / z: 507.4 [M+Na] +

[0337] Step 4

[0338] 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[d]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)

[0339] To a solution of 11i (18 mg, 0.037 mmol) in DMF (2 mL) were added HATU (15 mg, 0.039 mmol) and DIPEA (12 μL, 9.6 mg, 0.074 mmol). The resulting yellow solution was stirred at r.t. for 5 minutes and then 3 (20 mg, 0.037 mmol) was added. The mixture was stirred at r.t. for 60 minutes. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 11 (29 mg, 78.4% yield) as a white powder.

[0340] MS(ESI) m / z: 902.6 [M+H] +

[0341] Example 12

[0342]

Chemical formula

Chemical formula

[0343] Step 1

[0344] (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-tetraazapentadecanoic acid (12b)

[0345] To a DMF solution of 11g (29 mg, 0.098 mmol) and 12a (33 mg, 0.15 mmol) were added TSTU (41 mg, 0.14 mmol) and DIPEA (24 uL, 19 mg, 0.15 mmol). The mixture was stirred at room temperature for 1.5 h. At the completion of the reaction, the mixture was purified by prep-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 fractions were lyophilized to give 12b (23 mg, 31.4% yield) as a white powder.

[0346] MS(ESI) m / z: 522.4 [M+Na] +

[0347] Step 2

[0348] (S)-2-(3-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-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[d]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)

[0349] Compound 12 (13 mg, 32.9% yield) was synthesized according to the synthesis procedure of Step 6 of Example 11.

[0350] MS(ESI) m / z: 917.6 [M+H] +

[0351] Example 13

[0352]

Chem.

Chem.

[0353] Step 1

[0354] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((2-(benzyloxy)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (13b)

[0355] To a solution of compound 4 (420 mg, 0.70 mmol) in DCM (12 mL) were added 13a (486 mg, 1.18 mmol) and 4 Å molecular sieves (800 mg). The mixture was stirred at r.t. for 30 minutes. AgOTf (250 mg, 0.97 mmol) was added to the mixture, and the mixture was stirred at r.t. for 4 hours. After the reaction was completed, the mixture was filtered, the filtrate was diluted with EA (200 mL), and washed with saturated NaHCO3 (50 mL * 3) and brine (50 mL * 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography (elution: PE / EA = 100 / 0 to 70 / 30) to give 13b (183 mg, 28.2% yield) as a white solid.

[0356] MS(ESI) m / z: 956.5 [M+Na]+

[0357] Step 2

[0358] (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-triazatetradecanoic acid (13c)

[0359] 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 for 4 h under a H2 atmosphere (15 psi). The mixture was filtered through a pad of celite and concentrated to give compound 13c (165 mg, crude) as a white solid.

[0360] MS (ESI) m / z: 866.4 [M+Na]+

[0361] Step 3

[0362] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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-triyltriacetate (13e)

[0363] To a solution of compound 13c (165 mg, crude) in DMF (4 mL) were added 3 (109 mg, 0.21 mmol), HATU (112 mg, 0.29 mmol), and DIEA (101 mg, 0.78 mmol). The mixture was stirred at r.t. for 30 minutes. The mixture was filtered, and the filtrate was purified using prep-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 obtain 13f (124 mg, 50.2% yield) as a white solid.

[0364] MS(ESI) m / z: 1283.6 [M+Na]+

[0365] Step 4

[0366] (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-[d]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)

[0367] To a solution of compound 13e (124 mg, 0.098 mmol) in MeOH (6 mL) was added K2CO3 (136 mg, 0.98 mmol). The mixture was stirred at r.t. for 4 hours. The mixture was filtered, and the filtrate was purified using prep-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 obtain 13f (42 mg, 49.1% yield) as a white solid.

[0368] MS(ESI) m / z: 893.5 [M+Na]+

[0369] Step 5

[0370] 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[d]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)

[0371] To a solution of compound 13g (11 mg, 0.048 mmol) in DMF (1.5 mL) were added HATU (17 mg, 0.043 mmol) and DIEA (6.2 mg, 0.048 mmol). The mixture was stirred at r.t. for 15 minutes. Compound 13f (21 mg, 0.024 mmol) was added to the mixture and stirred at r.t. for 15 minutes. The mixture was filtered and the filtrate was purified using prep-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), and 13 (6.3 mg, 24.6% yield) was obtained as a white solid.

[0372] MS(ESI) m / z: 1086.6 [M+Na] +

[0373] Example 14

[0374]

Chemical Structure

Chemical Structure

[0375] Step 1

[0376] Compound 14 (3.6 mg, 13.8% yield) was synthesized according to the synthesis procedure of Step 5 in Example 13.

[0377] MS(ESI) m / z: 1101.6 [M+Na] +

[0378] Example 15

[0379]

Chemical Structure

Chemical Structure

[0380] Step 1

[0381] (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)

[0382] To a solution of compound 15a (200 mg, 0.48 mmol) in DCM (8 mL) were added 15b (335 mg, 0.81 mmol) and 4 Å molecular sieves (400 mg). The mixture was stirred at r.t. for 30 minutes. AgOTf (172 mg, 0.67 mmol) was added to the mixture, and the mixture was stirred at r.t. for 4 hours. After the reaction was completed, the mixture was filtered, the filtrate was diluted with EA (100 mL), and washed with saturated NaHCO3 (50 mL * 3) and brine (50 mL * 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography (elution: PE / EA = 100 / 0 - 70 / 30) to obtain 15c (230 mg, 64.2% yield) as a white solid.

[0383] MS(ESI) m / z: 770.4 [M+Na] +

[0384] Step 2

[0385] (2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15d)

[0386] To a solution of compound 15c (230 mg, 0.31 mmol) in DMF (6 mL) was added Et2NH (450 mg, 602 mmol). The mixture was stirred at r.t. for 30 minutes. 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.

[0387] MS(ESI) m / z: 526.4 [M+H] +

[0388] Step 3

[0389] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(benzyloxy)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15f)

[0390] To a solution of compound 15d (162 mg, 0.31 mmol) in DMF (4 mL) were 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 r.t. for 30 minutes. The mixture was concentrated and the residue was purified by flash column chromatography (elution: PE / EA = 100 / 0 ~ 70 / 30) to give compound 15f (145 mg, 55.6% yield) as a white solid.

[0391] MS(ESI) m / z: 869.5 [M+Na] +

[0392] Step 4

[0393] 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)

[0394] 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 for 4 h under a H2 atmosphere (15 psi). The mixture was filtered through a pad of celite and concentrated to give compound 15g (95 mg, crude) as a white solid.

[0395] MS(ESI) m / z: 779.5 [M+Na] +

[0396] Step 5

[0397] (2R,3R,4S,5S,6R)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-((2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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)

[0398] To a solution of 15 g (95 mg, crude) of the compound in DMF (4 mL) were added 15 h (80 mg, 0.14 mol), HATU (72 mg, 0.19 mmol), and DIEA (49 mg, 0.38 mmol). The mixture was stirred at r.t. for 30 minutes. The mixture was filtered, and the filtrate was purified using prep-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 obtain 15i (117 mg, 70.4% yield) as a white solid.

[0399] MS(ESI) m / z: 1340.5 [M+Na] +

[0400] Step 6

[0401] (2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-((S)-2-((S)-2-Amino-3-methylbutanamido)-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[d]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)

[0402] Compound 15j (116 mg, crude) was synthesized according to the synthesis procedure of Step 2 of Example 15.

[0403] Step 7

[0404] (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[d]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)

[0405] To a solution of compound 15j (116 mg, 0.11 mmol) in MeOH (4 mL) was added K2CO3 (44 mg, 0.32 mmol). The mixture was stirred at r.t. for 3 h. The mixture was filtered and the filtrate was purified using prep-HPLC (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) to give 15k (46 mg, 46.9% yield) as a white solid.

[0406] MS(ESI) m / z: 928.6 [M+H] +

[0407] Step 8

[0408] 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[d]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-14-methyl-1,6,9,12-tetraoxo-10-((((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-3-oxa-5,8,11-triazapentadecane-13-yl)hexanamide (15)

[0409] To a solution of compound 15i (9.5 mg, 0.042 mmol) in DMF (1.5 mL) were added HATU (14 mg, 0.038 mmol) and DIEA (5.4 mg, 0.042 mmol). The mixture was stirred at r.t. for 15 minutes. Compound 15k (23 mg, 0.021 mmol) was added to the mixture and stirred at r.t. for 15 minutes. The mixture was filtered and the filtrate was purified using prep-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), and 15k (3.4 mg, 14.4% yield) was obtained as a white solid.

[0410] MS(ESI) m / z: 1143.5 [M+Na] +

[0411] Example 16

[0412]

Chemical Structure

Chemical Structure

[0413] Step 1

[0414] (S)-2-(3-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-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[d]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 (16)

[0415] Compound 16 (3.5 mg, 12.5% yield) was synthesized according to the synthesis procedure of Step 8 in Example 15.

[0416] MS(ESI) m / z: 1158.5 [M+Na] +

[0417] Example 17

[0418]

Chem.

Chem.

[0419] Step 1

[0420] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((2-(benzyloxy)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (17b)

[0421] A mixture of 4 (150 mg, 0.25 mmol), 17a (148 mg, 0.37 mmol), and 4 Å MS (800 mg) was added anhydrous DCE (5 mL), and stirred at r.t. for 30 minutes. AgOTf (83 mg, 0.32 mmol) was added under a nitrogen atmosphere, and stirred at r.t. for 14 hours. The reaction solution was diluted with EtOAc (10 mL), filtered through celite, and washed with EtOAc (5 mL * 3). The organic phase was washed with saturated NaHCO3 (20 mL), concentrated, and purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100, and DCM / MeOH = 100 / 0 to 95 / 5) to obtain 17b (28 mg, 12.2% yield) as a pale yellow solid.

[0422] MS (ESI) m / z: 942.5 [M+Na] +

[0423] Step 2

[0424] (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-triazatetradecanoic acid (17c)

[0425] To a mixture of 17b (114 mg, 0.12 mmol) and 10% Pd / C (26 mg, 0.012 mmol), MeOH (3 mL) was added under a nitrogen atmosphere. The reaction solution was stirred at r.t. under a H2 atmosphere for 1 hour. The solution was filtered and concentrated under vacuum to obtain 17c (103 mg, 12.4% yield) as a white solid.

[0426] MS (ESI) m / z: 852.5 [M+Na] +

[0427] Step 3

[0428] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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-triyltriacetate (17d)

[0429] 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 the mixture was stirred at r.t. for 1 h. The reaction mixture was added into 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 - 95 / 5) to give 17d (97 mg, 64% yield) as a light brown solid.

[0430] MS(ESI) m / z: 1269.5 [M+Na] +

[0431] Step 4

[0432] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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)

[0433] To a solution of 17d (30 mg, 0.024 mmol) in MeOH / H2O (0.5 / 0.5 mL), TEA (167 μL, 1.2 mmol) was added and stirred at r.t. for 9 h. The solution was purified by prep-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 fractions were lyophilized to give 17e (15 mg, 70.6% yield) as a pale yellow solid.

[0434] MS(ESI) m / z: 885.5 [M+H] +

[0435] Step 5

[0436] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-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[d]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)

[0437] 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), DIPEA (5 μL, 0.029 mmol) was added and stirred at r.t. for 15 minutes. This solution was added dropwise to a solution of 17e (17 mg, 0.019 mmol) in anhydrous DMF (0.7 mL), and stirred at r.t. for 15 minutes. HOAc was added to adjust the pH to 5. The solution was purified by prep-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 fraction was lyophilized to obtain 17 (13 mg, 63.5% yield) as a beige solid.

[0438] MS(ESI) m / z: 1078.6 [M + H] +

[0439] Example 18

[0440]

Chemical formula

Chemical formula

[0441] Step 1

[0442] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-3-methylbutanamido)-3-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18)

[0443] Compound 18 (2.9 mg, 15.7% yield) was synthesized according to the synthetic procedure of Step 5 in Example 17.

[0444] MS (ESI) m / z: 1093.5 [M+H] +

[0445] Example 19

[0446]

Chemical formula

Chemical formula

[0447] Step 1

[0448] (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)

[0449] To a mixture of 19a (834 mg, 2 mmol), 19b (950 mg, 2.4 mmol), and 4 Å MS (3000 mg) was added anhydrous DCM (30 mL), and the mixture was stirred at r.t. for 30 minutes. AgOTf (617 mg, 2.4 mmol) was added under a nitrogen atmosphere, and the mixture was stirred at r.t. for 16 hours. The reaction solution was diluted with EtOAc (30 mL), filtered through celite, and washed with EtOAc (20 mL * 3). The organic phase was washed with saturated NaHCO3 (20 mL), concentrated, and purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100) to obtain 19c (470 mg, 32.1% yield) as a pale yellow solid.

[0450] MS (ESI) m / z: 756.3 [M+Na] +

[0451] Step 2

[0452] (2R,3R,4S,5S,6S)-2-((S)-2-Amino-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19d)

[0453] 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 r.t. for 0.5 h. At the completion of the reaction, the mixture was concentrated under vacuum to give 19d (327 mg, crude) as a yellow solid.

[0454] MS(ESI) m / z: 512.4 [M+H] +

[0455] Step 3

[0456] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19f)

[0457] To a solution of 19d (327 mg) in DMF (5 mL) were added HATU (291 mg, 0.76 mmol) and DIPEA (223 μL, 165 mg, 1.27 mmol). The resulting yellow solution was stirred at r.t. for 5 minutes, and then 19e (239 mg, 0.70 mmol) was added. The mixture was stirred at r.t. for 60 minutes. At the completion of the reaction, the solvent was evaporated and the residue was purified by flash column chromatography (eluent: petroleum ether / EtOAc = 70 / 30 to 0 / 100) and prep-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). The fractions were lyophilized to give 19f (300 mg, 56.2% yield) as a white solid.

[0458] MS(ESI) m / z: 833.4 [M + H] +

[0459] Step 4

[0460] 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 (19g)

[0461] 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 three times with an H2 balloon and stirred at r.t. for 2 hours. At the completion of the reaction, the mixture was filtered through a syringe filter head to remove Pd / C, and the filtrate was concentrated under vacuum to give 19g (265 mg, crude) as a white solid.

[0462] MS(ESI) m / z: 765.3 [M + Na] +

[0463] Step 5

[0464] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-((2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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-triyltriacetate (19i)

[0465] To a solution of 19g (265 mg) in DMF (5 mL) were added HATU (120 mg, 0.42 mmol) and DIPEA (124 μL, 92 mg, 0.71 mmol). The resulting yellow solution was stirred at r.t. for 5 min, and then 19h (227 mg, 0.39 mmol) was added. The mixture was stirred at r.t. for 60 min. At the completion of the reaction, the solvent was evaporated and the residue was purified by flash column chromatography (eluent: DCM / MeOH = 95 / 5~90 / 10) to give 19i (280 mg, 59.6% yield) as a brown solid.

[0466] MS(ESI) m / z: 1326.4 [M+Na] +

[0467] Step 6

[0468] (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-Amino-3-methylbutanamido)-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[d]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-triyltriacetate (19j)

[0469] To a solution of 19i (280 mg, 0.21 mmol) in DMF (5 mL) was added Et2N (478 μL, 340 mg, 4.29 mmol). The mixture was stirred at r.t. for 0.5 h. At the completion of the reaction, the mixture was concentrated under vacuum to give 19j (220 mg, crude) as a white solid.

[0470] MS(ESI) m / z: 1082.4 [M+H] +

[0471] Step 7

[0472] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-Amino-3-methylbutanamido)-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[d]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)

[0473] To a solution of 19j (220 mg) in MeOH / H2O (5 / 5 mL), Na2CO3 (151 mg, 1.42 mmol) was added and stirred at r.t. for 9 h. The solution was purified by prep-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 fractions were lyophilized to give 19k (45 mg, 23.8% yield) as a white solid.

[0474] MS(ESI) m / z: 942.3 [M + H] +

[0475] Step 8

[0476] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-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-[d]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)

[0477] 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 the mixture was stirred at r.t. for 15 minutes. This solution was added dropwise to a solution of 19k (20 mg, 0.021 mmol) in anhydrous DMF (1 mL), and the mixture was stirred at r.t. for 15 minutes. The solution was purified by prep-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 fractions were lyophilized to obtain 19 (7.4 mg, 31.0% yield) as a white solid.

[0478] MS(ESI) m / z: 1135.4 [M + H] +

[0479] Example 20

[0480]

Chemical formula

Chemical formula

[0481] Step 1

[0482] (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)-3-methylbutanamido)-3-((2-(((2-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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)

[0483] Compound 20 (12 mg, 50.1% yield) was synthesized according to the synthesis procedure of Step 8 in Example 19.

[0484] MS (ESI) m / z: 1172.4 [M+Na] +

[0485] Preparation and Characterization of ADC

[0486] Preparation of Antibody-Drug Conjugate

[0487] Preparation of DAR8 Antibody-Drug Conjugate

[0488] The antibody in binding buffer (concentration 0.5 - 25 mg / mL, PBS buffer pH 6.0 - 8.5) was incubated for 10 minutes at a reduction temperature (0 - 40°C) and at 8 - 15 equivalents. A TECP solution (5 mM stock in PBS buffer) was added to the reaction mixture and the reduction reaction was left at the reduction temperature for 1 - 8 hours. After cooling the reduction mixture to 0 - 25°C, an organic solvent (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0 - 25% v / v) and a linker payload stock (10 - 25 equivalents, 10 mM stock in organic solvent) were added stepwise. The binding solution was left at 0 - 25°C for 1 - 3 hours and the reaction can be quenched with N-acetylcysteine (1 mM stock). The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) in a storage buffer (e.g., histidine acetate buffer at pH 5.5 - 6.5 with optional additives (e.g., sucrose, trehalose, Tween 20, 60, 80)).

[0489] Hydrolysis of Maleimide after Binding

[0490] After the conjugation step, the ADC was subjected to buffer exchange in an open-loop buffer (pH 8.0 - 9.0, borate or Tris buffer), and the solution was left at 22 or 37 °C for 5 - 48 hours. The open-loop process was monitored by reductive LCMS. Once the hydrolysis of the conjugated maleimide was complete, the resulting ADC was buffer-exchanged via dialysis in a basic Tris pH 8.0 - 8.5 buffer or an acidic histidine acetate pH 5.0 - 6.5 buffer.

[0491] Characterization of ADC

[0492] ADC examples were prepared by following the aforementioned procedure using the DAR8 profile. All ADCs were characterized by the following analytical methods.

[0493] The drug - antibody ratio (DAR) of the ADC was determined by LCMS method or HIC method.

[0494] The SEC purity of the prepared ADCs was all > 95% purity.

[0495] Determination of drug - antibody ratio (DAR)

[0496] LCMS method

[0497] LC - MS analysis was performed under the following measurement conditions.

[0498] LC - MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer

[0499] Column: MAbPac™ RP, 2.1 * 50 mm, 4 μm, 1,500 Å, Thermo Scientific™

[0500] Column temperature: 80 °C

[0501] Mobile phase A: 0.1% formic acid (FA) aqueous solution

[0502] Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA)

[0503] 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)

[0504] Injected sample amount: 1 μg

[0505] MS parameters: Intact and denatured MS data were acquired in HMR mode with a setting of R = 15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0506] HIC method

[0507] HPLC analysis was performed under the following measurement conditions.

[0508] HPLC system: Waters ACQUITY ARC HPLC system

[0509] Detector: Measurement wavelength: 280 nm

[0510] Column: Tosoh Bioscience 4.6 μm ID × 3.5 cm, 2.5 μm butyl non-porous resin column

[0511] Column temperature: 25 °C

[0512] Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0

[0513] Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0

[0514] 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)

[0515] Amount of injected sample: 20 μg

[0516] ADC purity

[0517] SEC method

[0518] HPLC analysis was performed under the following measurement conditions.

[0519] HPLC system: Waters H-Class UPLC system

[0520] Detector: Measurement wavelength: 280 nm

[0521] Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6x150mm, Waters

[0522] Column temperature: Room temperature

[0523] Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropyl alcohol, PH 7.0

[0524] Gradient program: Eluent with a uniform concentration for 10 minutes at a flow rate of 0.3 mL / min

[0525] Amount of injected sample: 20 μg

[0526] ADC hydrophobicity evaluation

[0527] More hydrophobic ADCs appeared as the retention time from HIC (hydrophobic interaction column) chromatography became slower. The DAR8 (antibody with a drug load of 8) peak of the ADC example for this comparison

[0528] By comparing HIC D8 RT, it is clearly found that the hydrophilicity of the ADCs is ranked as ADC-C4 > ADC-C5 > ADC-C2 > ADC-C3 > ADC-C1. ADC-C1 seems to be the most hydrophobic in this setting.

[0529] HIC method

[0530] Method 1

[0531] HPLC analysis was performed under the following measurement conditions.

[0532] HPLC system: Waters ACQUITY ARC HPLC system

[0533] Detector: Measurement wavelength: 280 nm

[0534] Column: Tosoh Bioscience 4.6 μm ID × 3.5 cm, 2.5 μm butyl non-porous resin column

[0535] Column temperature: 25 °C

[0536] Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0

[0537] Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0

[0538] 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)

[0539] Injected sample amount: 20 μg

[0540] Method 2

[0541] HPLC analysis was performed under the following measurement conditions.

[0542] HPLC System: Waters ACQUITY ARC HPLC System

[0543] Detector: Measurement wavelength: 280 nm

[0544] Column: MABPac HIC-10, 5 μm, 4.6×10 mm (Thermo)

[0545] Column temperature: 25 °C

[0546] Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0

[0547] Mobile phase B: 50 mM sodium phosphate, pH 7.0

[0548] 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)

[0549] Flow rate: 0.5 mL / min

[0550] Sample preparation: The sample was diluted to 0.5 mg / mL with the initial mobile phase.

[0551] Table 1. ADC examples (including structures)

Table 3-1

Table 3-2

Table 3-3

[0552] All ADCs appear to be more hydrophilic than ADC-C1 based on the retention times of 8 DARs from the HIC profile.

[0553] The main sequence of ifinatamab

[0554] Heavy chain: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0555] Light chain: EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0556] ADC cell killing assay

[0557] Cell line information

Table 4

[0558] Assay method

[0559] Seed cells H358 (1E3 / well), H441 (1E3 / well), H1048 (1E3 / well), and MDA-MB-453 (1E3 / well) into a 3D-96 well plate (Corning: 4520) at 80 ul / well. Incubate overnight at 37 °C, 5% CO2.

[0560] Add 40 ul / well of fresh growth medium containing ADCs at various concentrations to H358, H441, H1048, and MDA-MB-453.

[0561] Incubate at 37 °C, 5% CO2 for 6 days.

[0562] Detect the cell viability of H358, H441, H1048, and MDA-MB-453 with 100 ul / well of 3D reagent (Promega, G9683).

[0563] Incubate the 3D plate at room temperature for 30 minutes to stabilize the luminescence signal.

[0564] Analyze with a microplate reader.

[0565] Cell assay killing results

[0566] Dataset 1

Table 5

[0567] Using the tandem release linker design, the cell killing of ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 appears to be the same as that of ADC-C1, showing efficient payload release inside the cells (Figure 2, Figure 3, Figure 4, and Figure 5).

[0568] Dataset 2

Table 6

[0569] Using the tandem release linker design, the cell killing of ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 is thought to be similar to that of ADC-C1, showing efficient payload release inside the cells (Figures 6, 7, 8, and 9).

[0570] Dataset 3

Table 7

[0571] Using the tandem release linker design, the cell killing of ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 is thought to be similar to that of ADC-C1, showing efficient payload release inside the cells (Figures 10, 11, 12, and 13).

[0572] ADC Enzyme Release Investigation

Table 8

[0573] Assay Protocol

[0574] Prepare a 1.50 mM sodium acetate solution (pH 5.0), then add TCEP to make the concentration of TCEP 2 mM to obtain a reaction solution.

[0575] Dilute ADC and lysosomes using the reaction solution to a final concentration of 1 μM for ADC and 0.125 mg / mL (or 20 units / mL of cathepsin-B) for lysosomes. Five copies of the sample were prepared in parallel for each ADC at a final volume of 100 μL.

[0576] Incubate the samples prepared in parallel at 37 °C, and add 200 μL of cold acetonitrile (containing internal standard) at 0 h, 0.5 h, 1 h, 2 h, and 6 h to terminate the reaction.

[0577] After centrifugation, 200 μL of the supernatant was collected for LC-MS analysis to detect the free payload and calculate the payload release rate.

[0578] Payload release rate (%) = (Concentration of DXd in the supernatant / MW of DXd) / (Concentration of ADC / MW of ADC) * DAR * 100

[0579] Results

[0580] Group 1

[0581] Payload release under lysosome treatment: [Table 9]

[0582] The payload release profiles of ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under lysosome treatment can be referred to Figure 14.

[0583] The payload release rates of ADC-C14 and ADC-C1 are faster than ADC-C13 in lysosomes and much faster than ADC-C10 and ADC-C11.

[0584] Payload release under cathepsin B treatment: [Table 10]

[0585] The payload release profiles of ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 under cathepsin B treatment can be referred to Figure 15.

[0586] The payload release rate of ADC-C1 is faster than that of ADC-C14 and much faster than those of ADC-C13, ADC-C10, and ADC-C11 under cathepsin B treatment. The tandem release pattern of the ADCs is shown in Figure 1.

[0587] Group 2

[0588] Payload release under lysosome treatment: [Table 11]

[0589] The payload release profiles of ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under lysosome treatment can be referred to Figure 16.

[0590] The payload release rates of ADC-C1 and ADC-C3 are faster than that of ADC-C5 and much faster than those of ADC-C4 and C6 under lysosome treatment. It is suspected that the decomplexation event of ADC-C3 may occur under this lysosome assay, and the unbound linker payload is more vulnerable to enzymatic cleavage than the bound one. Therefore, more payload release was observed in ADC-C3 than in ADC-C5. ADC-C4 and ADC-C6 showed slower DXd payload release rates under this assay condition, but both ADCs showed equivalent cell killing activities, indicating that efficient payload release occurred for all ADCs in the in vitro cell environment. The tandem release pattern of the ADCs is shown in Figure 8.

[0591] Payload release under cathepsin B treatment: [Table 12]

[0592] The payload release profiles of ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 under cathepsin B treatment can be referred to Figure 17.

[0593] The payload release rate of ADC-C1 is faster than that of ADC-C3 and much faster than those of ADC-C5, ADC-C4, and ADC-C6 under cathepsin B treatment. Abundant β-galactosidase exists in lysosomes, recognizes the β-galactose moiety, and triggers glycan C-O bond cleavage (J. Med. Chem. 2006, 49, 6290 - 6297, Chem. Commun. 2021 Spring D.R. et al). The tandem release pattern of the ADCs is shown in Figure 8.

[0594] Group 3

[0595] Payload release under lysosome treatment:

Table 13

[0596] The payload release profiles of ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under lysosome treatment can be referred to Figure 18.

[0597] The payload release rate of ADC-C1 is faster than that of ADC-C7 and much faster than those of ADC-C8, ADC-C9, and C12 under lysosome treatment. It is suspected that the decomplexation event of ADC-C7 may occur under this lysosome assay, and the unbound linker payload is more vulnerable to enzymatic cleavage than the bound one. Therefore, more payload release was observed in ADC-C7 than in ADC-C8. ADC-C8, ADC-C9, and ADC-C12 showed a slower DXd payload release rate under this assay condition, but both ADCs showed equivalent cell killing activity, indicating that efficient payload release occurred for all ADCs in the in vitro cell environment. The tandem release pattern of the ADCs is shown in Figure 1.

[0598] Payload release under cathepsin B treatment:

Table 14

[0599] The payload release profiles of ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under cathepsin B treatment can be referred to Figure 19.

[0600] The payload release rate of ADC-C1 is much faster than that of ADC-C7, ADC-C8, ADC-C9, and ADC-C12 under cathepsin B treatment. Abundant glucuronidase exists in lysosomes, recognizes the b-galactose moiety, and triggers glycan C-O bond cleavage under acidic conditions (Euro. J. Med. Chem. 2014 Papot S. et al.; Mol. Can. Ther. 2016 Lyon R. P. et al.). The tandem release pattern of the ADC is shown in Figure 8.

[0601] ADC Plasma Stability

[0602] Incubation of ADC with Plasma

[0603] Dilute the ADC in mouse or human plasma to give a final solution of 100 μg / mL ADC in plasma.

[0604] Incubate the samples at 37 °C.

[0605] Aliquots (100 μL) were taken at four time points (0, 4, 24, 72, 96, or 168 h).

[0606] Freeze the samples at -80 °C until analysis.

[0607] Plasma payload concentrations were performed under the following measurement conditions.

[0608] Instrument: LC-MS / MS (triple quad 5500)

[0609] Monitor: MRM

[0610] Column: Advanced Materials Technology, HALO AQ-C18 2.7μm 90Å, 50 * 2.1mm

[0611] Column temperature: 40°C

[0612] Mobile phase A: H2O - 0.1% FA

[0613] Mobile phase B: ACN - 0.1% FA

[0614] Gradient program for 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 for 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).

[0615] Injected sample volume: 10 μL

[0616] Plasma ADC and total Ab (Tab) concentrations were measured under the following conditions.

[0617] Assay: Ligand binding assay (ELISA)

[0618] Capture reagent: B7H3 ECD

[0619] Detection reagent: Anti-payload Ab for ADC, anti-human IgG polyclonal Ab for total Ab.

[0620] Results

[0621] Payload release in human plasma

[0622] Group 1

Table 15

[0623] The payload release profiles of ADC-C1, ADC-C10, ADC-C11, ADC-C13, and ADC-C14 can be referred to Figure 20.

[0624] All ADCs showed equivalent and acceptable payload release rates (<5% after 168 h).

[0625] Group 2

Table 16

[0626] The payload release profiles of ADC-C1, ADC-C3, ADC-C4, ADC-C5, and ADC-C6 can be referred to Figure 21.

[0627] All ADCs showed equivalent and acceptable payload release rates (<5% after 168 h).

[0628] Group 3

Table 17

[0629] The payload release profiles of ADC-C1, ADC-C7, ADC-C8, ADC-C9, and ADC-C12 can be referred to Figure 22.

[0630] All ADCs showed equivalent and acceptable payload release rates (<5% after 168 h).

[0631] Method for efficacy in H1975 model, updated model

[0632] On the right flank of female BALB / c nude mice, 5×10 6 H1975 cells per 200 μL of PBS / matrigel were subcutaneously transplanted. After inoculation, the tumor volume was measured two times a week in two dimensions using calipers, and the formula: V = 0.5(a × b 2 ) was used to express it in mm 3 . Here, a and b are the major and minor diameters of the tumor, respectively. When the tumors reached an average volume of approximately 100 - 200 mm 3 , the mice were randomly assigned to groups and intravenously treated once a week with vehicle or ADC at 3 or 10 mg / kg, respectively. Partial response (PR) was defined as a tumor volume smaller than 50% of the starting tumor volume on the first day of dosing in three consecutive measurements, and complete response (CR) was defined as a tumor volume less than 14 mm 3 in three consecutive measurements. Data are shown as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula.

Equation

[0633] Treated t = Treated tumor volume at time t

[0634] Treated t0 = Treated tumor volume at time 0

[0635] Placebo t = Placebo tumor volume at time t

[0636] Placebo t0 = Placebo tumor volume at time 0

[0637] Method for efficacy in the H1650 model

[0638] On the right flank of female BALB / c nude mice, 3×10 6 H1650 cells per 200 μL of PBS / matrigel were subcutaneously transplanted. When the tumors reached an average volume of approximately 100 - 200 mm 3Once the size was reached, the mice were randomly assigned to groups and treated intravenously with 10 mg / kg of vehicle or ADC once a week.

[0639] Method for efficacy in H441 model

[0640] 5×10 6 H441 cells per 300 μL of PBS / matrigel were subcutaneously implanted into the right flank of female NOG mice. Once the tumors reached an average volume of approximately 100 - 200 mm 3 Once the size was reached, the mice were randomly assigned to groups and treated intravenously with 10 mg / kg of vehicle or ADC once a week.

[0641] PK study in mice

[0642] Blood samples were collected from mice with A375 tumors at 0, 2, 4, 8, 24, 72, and 168 h after intravenous administration of 10 mg / kg of ADC, and then plasma was separated by centrifugation (4°C, 3000×g, 7 min). The concentrations of ADC and total Ab were measured by the Meso Scale Discovery (MSD) ligand binding method developed in-house. Briefly, the His-tagged B7H3 extracellular domain fusion protein was used as the capture reagent, and biotin-labeled anti-DXd Ab or goat anti-human kappa Ab was used as the detection reagent for ADC or total Ab measurement, respectively.

[0643] The antibodies used in this specification were prepared according to conventional methods (e.g., vector construction, eukaryotic cell transfection, e.g., transfection of HEK293 cells (Life Technologies catalog number 11625019), purification, and expression). The sequences of the antibodies used in this specification are provided herein and can be found.

[0644] Table 2. Platform examples

Table 18-1

Table 18-2

Table 18-3

Table 18-4

[0645] The present invention has generally been disclosed herein using affirmative language to describe many embodiments. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, are excluded, either in whole or in part. Thus, the present invention has generally not been represented herein with respect to what it does not include, but nevertheless, aspects not explicitly included in the present invention are also disclosed herein.

[0646] The foregoing invention has been described in some detail by way of figures and examples for purposes of clarity of understanding, but it will be apparent to those skilled in the art that certain minor changes and modifications may be made. Accordingly, the description and examples should not be construed as limiting the scope of the invention.

[0647] It should be understood that any reference herein to any prior art publication does not constitute an admission that such publication forms part of the common general knowledge in the art in any country.

[0648] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein by identification reference are hereby incorporated by reference in their entirety.

Claims

1. Compound of formula (I), 【Chemistry 1】 or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope substitution, or prodrug thereof, BA is a binder selected from humanized, chimeric, or human antibodies, or their antigen-binding fragments. RG is a reactive group residue, SP is a spacer group residue, A, B, and C are each independently an amino acid residue in each case. HG is a hydrophilic residue or hydrogen, Each of the subscripts a, c, and p is independently either 0 or 1 in each case. PA is a payload residue, The subscript x is between 1 and 15. PAB is -NH-CH ２ The compound representing -O-.

2. The aforementioned compound is a compound of formula (Ia). 【Chemistry 2】 The compound according to claim 1, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted compound, or prodrug thereof.

3. The aforementioned compound is a compound of formula (Ib). 【Transformation 3】 The compound according to claim 1, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted compound, or prodrug thereof.

4. The compound according to any one of claims 1 to 3, wherein the antibody is ifinatamab, cofetuzumab, patritumab, or trastuzumab, or an antigen-binding fragment of ifinatamab, cofetuzumab, patritumab, or trastuzumab.

5. The antibodies mentioned above are 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, p-cadherin, CEA, CEACAM5, C4.4a, DLL3, and EGFR. The compound according to any one of claims 1 to 3, which 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 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, ROR1, MSLN, FRa, CLDN18.2, CLDN6, PTK7, and Axl.

6. The compound according to 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 B7H3, MUC1, FFFR2b, CLL1, CCR7, GPC1, and GPC3.

7. RG is 【Chemistry 4】 【Transformation 5】 The compound according to any one of claims 1 to 3.

8. RG is 【Transformation 6】 EWG consists of -CN, halogen, and -CF ３ , -C (=O) OR １ , and -C(=O)R １ It is an electron-withdrawing group selected from R １ The compound according to any one of claims 1 to 3, wherein is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

9. RG is 【Transformation 7】 The compound according to any one of claims 1 to 3.

10. RG is 【Transformation 8】 and EWG is -CN, halogen, -CF ３ , -C(=O)OR １ , and -C(=O)R １ is an electron-withdrawing group selected from, 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. The compound according to any one of claims 1 to 3.

11. Compound of formula (II) 【Chemistry 9】 or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope substitution, or prodrug thereof, RG is a reactive group residue, SP is a spacer group residue, A, B, and C are each independently an amino acid residue in each case. HG is a hydrophilic residue or hydrogen, Each of the subscripts a, c, and p is independently either 0 or 1 in each case. PA is a payload residue, PAB is -NH-CH ２ The compound representing -O.

12. The compound is a compound of formula (IIa). 【Chemistry 10】 The compound according to claim 11, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted compound, or prodrug thereof.

13. The aforementioned compound is a compound of formula (IIb). 【Chemistry 11】 The compound according to claim 11, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted compound, or prodrug thereof.

14. RG is 【Chemistry 12】 【Chemistry 13】 The compound according to any one of claims 11 to 13.

15. RG is 【Chemistry 14】 EWG consists of -CN, halogen, and -CF ３ , -C (=O) OR １ , and -C(=O)R １ An electron-withdrawing group selected from the group consisting of R １ The compound according to any one of claims 11 to 13, wherein is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

16. SP is - (CH ２ ) ｎ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, -(CH ２ CH ２ O) ｎ -CH ２ CH ２ -C(=O)-, -CH[-(CH ２ ) ｎ -COOH] -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-NH-(CH ２ ) ｎ -C(=O)-, or -C(=O)-(CH ２ ) ｎ The compound according to any one of claims 1 to 3 or 11 to 13, wherein -C(=O)-, and each n independently represents an integer from 1 to 8.

17. SP is - (CH ２ ) ｎ -C (=O)-, or -CH ２ -C(=O)-NH-(CH ２ ) ｎ The compound according to claim 16, wherein -C(=O)-, and each n independently represents an integer of 5 or 2.

18. RG is 【Chemistry 17】 Therefore, SP is - (CH ２ ) ｎ The compound according to any one of claims 1 to 3, wherein it is -C(=O)- and n is an integer of 1, 2, 3, 4, or 5.

19. RG is [Chemistry 18] Therefore, SP is - (CH ２ ) ｎ The compound according to any one of claims 11 to 13, wherein it is -C(=O)- and n is an integer of 1, 2, 3, 4, or 5.

20. HG is a compound selected from the group consisting of sugars, phosphate esters, sulfate esters, phosphodiesters, and phosphonates, according to any one of claims 1 to 3 or 11 to 13.

21. The compound according to claim 20, wherein HG is a sugar, and the sugar is selected from the group consisting of β-D-galactose, N-acetyl-P-D-galactosamine, N-acetyl-a-D-galactosamine, N-acetyl-P-D-glucosamine, β-D-glucuronic acid, a-L-iduronic acid, a-D-galactose, a-D-glucose, β-D-glucose, a-D-mannose, β-D-mannose, a-L-fucose, β-D-xylose, neuraminic acid or any analogue or modification thereof, or sulfate, phosphate, carboxyl, amino, or O-acetyl modification thereof.

22. HG is 【Chemistry 19】 The compound according to claim 21, or a stereoisomer, enantiomer, or isotope-substituted compound thereof.

23. Each PA independently represents a chromophore functional group, the compound according to any one of claims 1 to 3 or 11 to 13.

24. The compound according to claim 23, wherein each chromophore functional group is independently a functional group selected from the class or subclass of xanthophore, erythrophore, ylidephore, leucophore, melanophore, and cyanophore; the class or subclass of fluorophore molecules, which are fluorescent compounds that re-emit light upon receiving light; the class or subclass of visual light transmission molecules; the class or subclass of photophore molecules; the class or subclass of luminescent molecules; and the class or subclass of luciferin compounds.

25. Each PA is independently a cytotoxic agent, and the cytotoxic agent contains at least one hydroxyl group, the compound according to any one of claims 1 to 3 or 11 to 13.

26. Each PA is independently selected from the group consisting of kinesin spindle protein (KSP) inhibitors (KSPi), camptothecin (including DXd and 7-ethyl-10-hydroxy-camptothecin (SN-38)), eribulin, PNU-159682, anthracycline, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), and amatoxin, according to any one of claims 1 to 3 or 11 to 13.

27. Each PA independently corresponds to formula (III) or formula (VI) 【Chemistry 20】 Represents R ２ and R ３ Each of these can independently be hydrogen, halogen, or substituted or unsubstituted C １－４ A compound according to any one of claims 1 to 3 or 11 to 13, wherein the compound is alkyl.

28. Each PA system operates independently. 【Chemistry 21】 A compound according to any one of claims 1 to 3 or 11 to 13, which represents the compound.

29. The aforementioned compound, 【Chemistry 75】 【Transformation 76】 【Chemical Formula 77】 【Transformation 78】 【Chemistry 79】 A compound according to any one of claims 1 to 3, wherein Ab is BA and n is about 7 to about 8.

30. The aforementioned compound, 【Chemistry 80】 【Chemistry 81】 【Chemistry 82】 【Chemistry 83】 【Chemical 84】 【Chemical 85】 A compound according to any one of claims 11 to 13, selected from a pharmaceutically acceptable salt thereof.

31. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, tautomer, solvate, stereoisomer, enantiomer, isotope-substituted compound, or prodrug, and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition according to claim 31 for treating proliferative disorders, metabolic disorders, inflammation, or neurodegenerative disorders in a subject.

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