Cyclodextrin Protein Drug Conjugates

Abstract

To provide compounds, compositions, and conjugates for the treatment of diseases, and / or conditions such as proliferative diseases.SOLUTION: Provided is a compound, or a pharmaceutically acceptable salt thereof, comprising a binding agent linked to at least one payload moiety and linked to at least one cyclodextrin moiety via a covalent linker, the covalent linker being bonded directly or indirectly to each of the binding agent, the payload moiety, and the cyclodextrin moiety.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] (cross reference) This application is filed on May 18, 2017, the contents of which are incorporated by reference in their entirety. The benefit of U.S. Provisional Application No. 62 / 508,315 is claimed.

[0002] (Field) Provided herein are novel cyclodextrin-containing protein drug conjugates. and administering the cyclodextrin-containing protein drug conjugate. Also described herein are methods for treating various diseases, disorders, and illnesses. A method for making rhodextrin-containing protein drug conjugates. [Background technology]

[0003] (background) Antibody-drug conjugates (ADCs) use antibodies to deliver potent drugs to target-expressing cells. selective delivery, potentially reducing off-target side effects and / or toxicity, and therefore thus increasing the therapeutic index.

[0004] The lipophilic nature of many payloads (i.e., drugs) can prevent the payload from efficiently reaching target cells. The polar groups in the linker can adversely affect the properties of the ADC in that they are not delivered efficiently. Modulating ADC properties by adding Improved physicochemical properties may result in improved pharmacokinetics (PK) and therapeutic index of ADCs (See, for example, Nature Biotechnology, 2015, 33, 733-735). Modulation of the linker resulting in a change in charge allows for better retention of the payload inside the cell. , which may improve activity against multidrug resistant (MDR) cells (e.g., Cancer Res; 70(6) March 15, 2015). 010, p. 2528). Chemical moieties on the linker, e.g., polyethylene glycol (PEG), Sugar or sulfate groups (see, e.g., WO2014062697, US20100323973) are useful for conjugating ADCs. Not only does it affect the efficiency and ease of production of the metabolites, but it also often These compounds remain as ions, which in turn affect the activity and safety of ADCs. , various strategies have been utilized (e.g., Bioconjugate Chem., 2008, 19(10), pp. 1960-1 963).

[0005] Cyclodextrin (CD) is a biodegradable and soluble dietary fiber and food ingredient, and a drug carrier. It is also used as a carrier. Its safety has been proven. CD is more soluble than PEG and It does not have a charge like the sulfonate, so it is difficult to remove during processing. Furthermore, the glucose units are shared end-to-end via α-1,4 bonds. Because they are bonded together, all units have the same chemical properties and a unique hydrophilic outer surface. It should form a hydrophobic inner core.

[0006] Low bioavailability of lipophilic payloads can compromise the therapeutic window of ADC treatments The development of cyclodextrin-containing protein conjugates, e.g., ADCs, is particularly In addition, when a lipophilic payload is included in an ADC, improved modulation of biological targets, bioavailability, This would allow for improved operability and improved therapeutic window. The effective treatment of diseases using cyclodextrin protein drug conjugates has been proposed. It continues to be needed. Summary of the Invention

[0007] (overview) Provided herein are, for example, but not limited to, metabolic disorders, proliferative disorders, and These compounds are useful in the treatment of diseases, illnesses, and disorders, including but not limited to, cerebrospinal fluid disorders ... and other diseases and conditions.

[0008] In one embodiment, provided herein are at least one payload moiety-associated and linked to at least one cyclodextrin moiety via a covalent linker. wherein the covalent linker is connected to the binding agent, the payload, a compound bonded directly or indirectly to each of the cyclodextrin moieties and the cyclodextrin moiety; or a pharmaceutically acceptable salt thereof.

[0009] In one embodiment, provided herein are at least one payload moiety-associated and linked to at least one cyclodextrin moiety via a covalent linker. wherein the covalent linker is connected to the binding agent, the payload moiety, and a compound directly or indirectly attached to each of said cyclodextrin moieties. or a pharmaceutically acceptable salt thereof.

[0010] In another embodiment, provided herein is a method for preparing a medicament comprising administering to at least one payload moiety linked to at least one cyclodextrin moiety via a covalent linker a linked reactive group; wherein the covalent linker is connected to the reactive group, the payload, a compound bonded directly or indirectly to each of said cyclodextrin moieties; It is a thing.

[0011] In another embodiment, provided herein are antibodies that bind to an antibody or antigen-binding fragment thereof. and an antibody-drug conjugate having the above compound.

[0012] In another embodiment, provided herein is a method for treating a disease in a patient in need thereof, A method of treating a disease or disorder comprising administering to said patient a compound described herein. The method includes:

[0013] In another embodiment, provided herein are methods for preparing antibody-drug conjugates. The method comprises combining a binder with a linker-payload compound as described herein. and contacting the agent under conditions suitable to form a bond between the linker-payload compound. The method includes: [Brief explanation of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1A shows three different types of payload compounds.

[0015] FIG. 1B shows the synthetic process for preparing payload compounds 1h or 1i.

[0016] FIG. 1C shows the synthetic process for preparing payload compound 1d.

[0017] [Figure 2] FIG. 2 shows the synthetic process for preparing intermediate compounds 5a-c.

[0018] [Figure 3]FIG. 3 shows the synthetic process for preparing intermediate compound 6b.

[0019] [Figure 4] FIG. 4 shows the synthetic process for preparing intermediate compounds 7a and 7b.

[0020] [Figure 5] FIG. 5 shows the general synthetic process describing Procedure A for preparing compounds 3 (3e, 3f, and 3g) from 2a.

[0021] [Figure 6] FIG. 6 shows a general synthetic process describing procedure B for preparing compound 3 from compound 2 and also for making compound 8 from compound 3.

[0022] [Figure 7] FIG. 7 shows the general synthetic process describing Procedure C for making compounds 9a-L.

[0023] [Figure 8] FIG. 8 shows the general synthetic process describing Procedure D for making linker-payload.

[0024] [Figure 9] FIG. 9 shows a synthetic process describing the procedure for making the linker-payload.

[0025] [Figure 10] FIG. 10 shows a synthetic process describing the procedure for making the linker-payload of Example 38.

[0026] [Figure 11] FIG. 11 shows a synthetic process describing the procedure for making the linker-payload of Example 39.

[0027] [Figure 12] FIG. 12 shows the solubility plot of Example 49.

[0028] [Figure 13] FIG. 13 shows the general synthetic process for conjugation via Michael addition.

[0029] [Figure 14] FIG. 14 shows the general synthetic process for ADC conjugation via the [2+3] click reaction represented by Ab-Ex49.

[0030] [Figure 15] FIG. 15 shows a Coomassie stained SDS-PAGE gel of anti-Her2 antibody, anti-Her2-PEG3-N3, and anti-Her2-Ex49.

[0031] [Figure 16] FIG. 16 shows size exclusion chromatography (SEC) of anti-Her2 antibodies, anti-Her2-PEG3-N3, and anti-Her2-Ex49.

[0032] [Figure 17] FIG. 17 shows a plot of % cell survival versus Log10[M] for certain compounds tested in Example 72.

[0033] [Figure 18] FIG. 18 shows the bioactivity of LXR agonist ADCs with and without a cyclodextrin linker in a plot of relative light units (RLU) versus Log10[M].

[0034] [Figure 19] FIG. 19 shows the bioactivity of LXR agonist ADCs with and without a cyclodextrin linker in a plot of relative light units (RLU) versus Log10[M].

[0035] [Figure 20] FIG. 20 shows reagents that can be used in place of d-Lys in the methods presented herein. DETAILED DESCRIPTION OF THE INVENTION

[0036] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Provided herein are methods for treating a subject's condition, e.g., dyslipidemia, metabolic disease, inflammation, or diabetes. Compounds, compositions, and methods useful for treating degenerative diseases.

[0037] (definition) When referring to the compounds provided herein, the following terms are used interchangeably unless otherwise indicated: Unless otherwise defined, technical and scientific terms used herein have the following meanings: All terms have the same meaning as commonly understood by one of ordinary skill in the art. In the event that there are multiple definitions for terms used herein, those definitions shall prevail unless expressly stated otherwise. do.

[0038] As used herein, "alkyl" refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and may be linear, branched, or cyclic, i.e., cycloalkenyl. Alkyl can be a radical having 1 to 20 carbon atoms, That is, C 1-20 Alkyl; a radical having 1 to 12 carbon atoms, i.e., C 1-12 Al radicals having 1 to 8 carbon atoms, i.e., C 1-8 Alkyl; 1 to 6 carbon atoms radicals having the formula C 1-6 alkyl; and radicals having 1 to 3 carbon atoms, That is, C 1-3Examples of alkyl moieties include, but are not limited to, alkyl. The following are examples of methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl ethyl, pentyl moiety, hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, Pentyl moieties include, but are not limited to, n- and cyclohexyl. Examples of hexyl moieties include, but are not limited to, pentyl and i-pentyl. and n-hexyl.

[0039] As used herein, "alkylene" refers to a divalent alkyl group. Unless otherwise specified, alkylene includes, but is not limited to, 1 to 20 carbon atoms. The olefin group is optionally substituted as described herein for alkyl. In some embodiments, the alkylene is unsubstituted.

[0040] The designation of an amino acid or amino acid residue without specifying its stereochemistry refers to an L-amino acid, a D-amino acid, It is intended to include amino acids, or racemic mixtures thereof.

[0041] As used herein, "haloalkyl" refers to an alkyl group as defined above. wherein the alkyl is a halogen, such as fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include: , -CF3, -CH2CF3, -CCl2F, and -CCl3.

[0042] As used herein, "alkenyl" refers to an alkyl group having at least two carbon atoms and one or more alkyl groups. Alkenyl refers to a monovalent hydrocarbon radical moiety containing a non-aromatic carbon-carbon double bond. Alkenyl is optionally substituted and can be linear, branched, or cyclic. For example, radicals with 2 to 20 carbon atoms, i.e., C 2-20 Alkenyl; 2-12 carbons Radicals with elementary atoms, i.e., C 2-12 Alkenyl; Radicals containing 2 to 8 carbon atoms Cal, i.e., C 2-8 Alkenyl; a radical having 2 to 6 carbon atoms, i.e., C 2-6 alkenyl; and radicals having 2 to 4 carbon atoms, i.e., C 2-4 Alkenyl is mentioned. Examples of alkenyl moieties include, but are not limited to, vinyl, propenyl, Examples include, but are not limited to, butenyl and cyclohexenyl.

[0043] As used herein, "alkynyl" refers to an alkyl group having at least two carbon atoms and one or more alkyl groups. Alkynyl refers to a monovalent hydrocarbon radical moiety containing a carbon-carbon triple bond. It can be substituted and linear, branched, or cyclic. Alkynyl includes 2 Radicals with ∼20 carbon atoms, i.e., C 2-20 Alkynyl; 2 to 12 carbon atoms radicals, i.e., C 2-12 Alkynyl; a radical having 2 to 8 carbon atoms, That is, C 2-8 Alkynyl; a radical having 2 to 6 carbon atoms, i.e., C 2-6 Alkini and radicals having 2 to 4 carbon atoms, i.e., C 2-4 Alkynyl is an example Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl, and bromine. These include, but are not limited to, thionyl.

[0044] As used herein, "alkoxy" refers to a monovalent and saturated hydrocarbon radical moiety. wherein the hydrocarbon contains a single bond to an oxygen atom and the radical is For example, in the case of ethoxy, it is CH3CH2-O·. An alkoxy substituent is one that is The alkoxy substituent is attached to the compound it substitutes through this oxygen atom. and may be linear, branched, or cyclic, i.e., cycloalkoxy. The alkoxy includes those having 1 to 20 carbon atoms, i.e., C 1-20 Arco oxy; having 1 to 12 carbon atoms, i.e., C 1-12 Alkoxy; 1 to 8 carbon atoms That is, C 1-8 Alkoxy; having 1 to 6 carbon atoms, i.e., C 1-6 Alkoxy; and those having 1 to 3 carbon atoms, i.e., C 1-3 Alkoxy is mentioned Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, i-butoxy, pentoxoxy cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0045] As used herein, "haloalkoxy" refers to an alkoxy group as defined above. alkoxy refers to alkoxy, where the alkoxy is selected from halogen, e.g., F, Cl, Br, or I. It contains at least one substituent that is

[0046] As used herein, "aryl" refers to an aromatic compound in which the ring atoms are carbon atoms. Aryl refers to a monovalent moiety that is an optionally substituted, monocyclic or polycyclic Examples of aryl moieties include 6-2 those with 0 ring carbon atoms, i.e., C 6-20 Aryl; having 6 to 15 ring carbon atoms That is, C 6-15 Aryl and those having 6 to 10 ring carbon atoms, i.e., C 6-10 Examples of aryl moieties include, but are not limited to, fluoro ... phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrene Examples of suitable amines include, but are not limited to, methylaminobenzoates ...

[0047] As used herein, "arylalkyl" refers to the radical of an alkyl compound. refers to a monovalent moiety in which the alkyl compound is substituted with an aromatic substituent, i.e., That is, the aromatic compound contains a single bond to an alkyl group, and the radical is Arylalkyl groups are attached to the chemical structure shown through the alkyl group. Arylalkyl can be substituted with an alkyl group having the structure, for example, [ka] where B is an aromatic moiety, e.g., phenyl. The aryl alkyl is optionally substituted, i.e., the aryl and / or alkyl group is It can be substituted as disclosed herein. Examples of arylalkyl include: Examples include, but are not limited to, benzyl.

[0048] As used herein, "alkylaryl" is a radical of an aryl compound. refers to a monovalent moiety in which the aryl compound is substituted with alkyl substituents, i.e., That is, the aryl compound contains a single bond to an alkyl radical, and the radical is The alkylaryl group is connected to the illustrated chemical structure via the aryl group. The alkylaryl is bonded to the structure, e.g. [ka] where B is an aromatic moiety, e.g., phenyl. The alkylaryl is optionally substituted, i.e., the aryl and / or alkyl group is It can be substituted as disclosed herein. Examples of alkylaryl include: Examples of suitable amines include, but are not limited to, toluyl.

[0049] As used herein, "aryloxy" refers to an aryl group in which the ring atoms are carbon atoms and the It refers to a monovalent moiety that is a radical of an aromatic compound in which the ring is substituted with an oxygen radical, i.e. That is, the aromatic compound contains a single bond to an oxygen atom, and the radical is For example, in the case of phenoxy, [ka] The aryloxy substituent is attached to the compound it substitutes through this oxygen atom. The aryloxy is optionally substituted. The aryloxy is a group having 6 to 20 rings. Radicals with carbon atoms, i.e., C 6-20 Aryloxy; having 6 to 15 ring carbon atoms That is, C 6-15Aryloxy and those having 6 to 10 ring carbon atoms, That is, C 6-10 Aryloxy includes, but is not limited to, aryloxy. Examples of moieties include phenoxy, naphthoxy, and anthroxy. Not limited to.

[0050] As used herein, "R a R b "N-aryloxy" means that the ring atoms are carbon atoms, and the ring is at least one R a R b substituted with an N-substituent and at least one oxygen radical refers to a monovalent moiety that is a radical of an aromatic compound that is a R b The radical contains a single bond to an N-substituent and a single bond to an oxygen atom, and the radical is Located, for example, [ka] R a R b The N-aryloxy substituent is a substituent that is substituted through this oxygen atom in the compound it substitutes. Combine. R a R b N-aryloxy is optionally substituted. a R b N-aryloxy For example, those having 6 to 20 ring carbon atoms, such as C 6-20 (R a R b N) n -aryloxy, 6 those with 15 ring carbon atoms, e.g., C 6-15 (R a R b N) n -aryloxy, and 6 to 10 those having ring carbon atoms of, for example, C6-10 (R a R b N) n -aryloxy (where n is R a R b N- represents the number of substituents), but is not limited to these. a R b N-aryloxy moiety Examples of moieties include 4-(dimethylamino)-phenoxy, [ka] These include, but are not limited to:

[0051] As used herein, "arylene" refers to an aromatic ring in which the ring atoms are only carbon atoms. Arylene refers to a divalent moiety of a compound that is optionally substituted and is monocyclic or polycyclic, e.g., For example, the aryl moiety may be bicyclic or tricyclic. Examples of the aryl moiety include aryl moieties having 6 to 20 rings. Those with ring carbon atoms, i.e., C 6-20 Arylene; having 6 to 15 ring carbon atoms That is, C 6-15 arylene and those having 6 to 10 ring carbon atoms, i.e., C6 -10 Examples include, but are not limited to, arylene.

[0052] As used herein, "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by a heteroatom. As used herein, "heteroalkenyl" refers to an alkyl that is substituted. As used herein, an alkenyl group refers to an alkenyl group in which one or more carbon atoms are replaced by a heteroatom. When the term "heteroalkynyl" is used, one or more carbon atoms are replaced by a heteroatom. Suitable heteroatoms include nitrogen, oxygen, and sulfur atoms, Heteroalkyl is optionally substituted. Heteroalkyl moiety Examples of alkyl include aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties include, but are not limited to, methylamino, methylamino, Also included are, but are not limited to, methylsulfonyl and methylsulfinyl.

[0053] As used herein, "heteroaryl" refers to a group of ring atoms in which the ring atoms are carbon atoms and at least one heteroaryl. A univalent moiety that is a radical of an aromatic compound containing one oxygen, sulfur, nitrogen, or phosphorus atom Examples of heteroaryl moieties include those with 5 to 20 ring atoms; those with 5 to 15 ring atoms; and those with 5 Heteroaryl includes, but is not limited to, those having up to 10 ring atoms. , optionally substituted.

[0054] As used herein, "heteroarylene" refers to a heteroaryl group in which one or more carbon ring atoms of the aromatic ring are heterocyclic. refers to an arylene substituted with an oxygen, sulfur, nitrogen, or phosphorus atom. is optionally substituted.

[0055] As used herein, "heterocycloalkyl" refers to a group in which one or more carbon atoms are heterocyclic. Suitable heteroatoms include nitrogen, oxygen, and sulfur atoms. Heterocycloalkyls are optionally Examples of heterocycloalkyl moieties include morpholinyl, piperidinyl, and the like. , tetrahydropyranyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl Examples include lysinyl, dioxolanyl, dithiolanyl, oxanyl, or thianyl, Not limited to these.

[0056] As used herein, a "Lewis acid" refers to a molecule or ion that accepts a lone pair of electrons. The Lewis acids used in the methods described herein are other than protons. Lewis acids include non-metallic acids, metallic acids, hard Lewis acids, and soft Lewis acids. Lewis acids include, but are not limited to, aluminum, boron, iron, tin, and thiamin. Tungsten, magnesium, copper, antimony, phosphorus, silver, ytterbium, scandium, nickel Exemplary Lewis acids include, but are not limited to, Lewis acids of zinc, zinc ions, and zinc chloride. Examples include AlBr3, AlCl3, BCl3, boron trichloride methyl sulfide, BF3, boron trifluoride methyl ethene ether, boron trifluoride methyl sulfide, boron trifluoride tetrahydrofuran, dicyclohexane Silboron trifluoromethanesulfonate, iron(III) bromide, iron(III) chloride, tin(IV) Chloride, Titanium(IV) Chloride, Titanium(IV) Isopropoxide, Cu(OTf)2, CuCl2, CuBr2, Salt zinc halide, alkylaluminum halide (R n AlX 3-n where R is hydrocarbyl , X is a halide, and the subscript n is an integer from 0 to 3), Zn(OTf), ZnCl, Yb(OTf )3, Sc(OTf)3, MgBr2, NiCl2, Sn(OTf)2, Ni(OTf)2, and Mg(OTf)2, which Not limited to:

[0057] As used herein, an "N-containing heterocycloalkyl" refers to an N-containing heterocycloalkyl group having one or more carbon atoms. Cycloalkyl groups substituted with heteroatoms, at least one of which is a nitrogen atom In addition to nitrogen, suitable heteroatoms include oxygen and sulfur atoms. N-containing heterocycloalkyls include, but are not limited to, optionally substituted N- Examples of heterocycloalkyl-containing moieties include morpholinyl, piperidinyl, pyrrolidinyl, and the like. Examples of the alkyl groups include aryl, imidazolidinyl, oxazolidinyl, and thiazolidinyl. Not limited to.

[0058] As used herein, "optionally" when used to describe a radical moiety "Substituted," e.g., optionally substituted alkyl, means that such moiety has one or more substituents. Examples of such substituents include halo, cyano, Nitro, optionally substituted haloalkyl, azido, epoxy, optionally substituted heteroaromatic groups aryl, optionally substituted heterocycloalkyl; [ka] (where R A , R B , and R C independently at each occurrence, represent a hydrogen atom, an alkyl, an alkenyl, Alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, hetero aryl, or heterocycloalkyl, or R A and R B are combined together with the atoms forming a saturated or unsaturated carbocyclic ring, wherein the ring is optionally substituted and one or more ring atoms are optionally substituted with a heteroatom), In some embodiments, the radical moiety is an optionally substituted heterocyclic group. aryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or When an unsaturated carbocyclic ring is optionally substituted, the optionally substituted heteroaryl, optionally substituted heterocycloalkyl or optionally substituted saturated or unsaturated carbocyclic rings; The substituents, when substituted, are optionally further substituted with further substituents. In some embodiments, any of the groups described herein is not substituted with a substituent. When optionally substituted, the substituents attached to the group are substituted unless otherwise specified. It has not been done.

[0059] As used herein, a "binding agent" refers to a molecule that specifically binds to a given binding partner, e.g., an antigen. It refers to any molecule, such as a protein, that is capable of isomeric bonding.

[0060] As used herein, a "linker" refers to a linker that connects a binding agent to one or more of the compounds described herein. compounds, such as the payload compounds described herein and cyclodextrin groups. It refers to a divalent, trivalent, or multivalent moiety that is covalently attached.

[0061] As used herein, "amide synthesis conditions" refers to, for example, carboxylic acids, activated carboxylic acids, to achieve amide formation by reaction of carboxylic acids or acyl halides with amines. In some instances, "amide synthesis conditions" refer to suitable reaction conditions for the synthesis of a carboxylic acid and an amide. Some of these examples refer to reaction conditions suitable for achieving the formation of an amide bond between the amines. In some cases, the carboxylic acid is first converted to an activated carboxylic acid, and then the activated carboxylic acid is reacted with the activated carboxylic acid. The carboxylic acid reacts with the amine to form an amide. The conditions include, but are not limited to, dicyclohexylcarbodiimide (DCC), diisopropyl Dicarboxylic acid (DIC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphatase Sulfonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)trifluoromethyl Pyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazo (1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), Bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(benzotri Azol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU ), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoro Borate (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine Dinium 3-oxide hexafluorophosphate (HATU), N-ethoxycarbonyl-2-ethoxy hydroxy-1,2-dihydroquinoline (EEDQ), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide Imide (EDC), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (C IP), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), and carbonyldiimidazoline These include the use of reagents to effect the reaction of carboxylic acids with amines, including CDI. In some examples, the carboxylic acid is first activated. carboxylic acid ester, and then treating the activated carboxylic acid ester with an amine to give , to form an amide bond. In some embodiments, the carboxylic acid is treated with a reagent. deprotonates the carboxylic acid, followed by protonation by the deprotonated carboxylic acid. Nucleophilic attack on the protonated reagent results in a product complex with the deprotonated carboxylic acid. The carboxylic acid is activated by forming a bond. The activated carboxylic acid ester is more susceptible to nucleophilic attack by amines than before the carboxylic acid is activated. This results in amide bond formation. The phosphoric acid is described as activated. Exemplary reagents include DCC and DIC. do.

[0062] As used herein, "positional isomer," "positional isomers," or "positional isomers" refers to a group of isomers that are heterocyclic or heterocyclic. A "mixture of isomers" refers to the reaction of a suitable azide (e.g., -N3, or PE) with a suitable alkyne. 1,3-cycloaddition or strain-promoted alkyne-azide cycloaddition (S-G-N3 derivatized antibody) derived from In one embodiment, the term "PAAC" refers to the product of a click reaction. For example, positional isomers and mixtures of positional isomers can be synthesized by the click reaction products shown below. It is characterized by: [ka] In some embodiments, multiple suitable azides and multiple suitable alkynes are catalyzed toward the product. In this synthesis process, each azide -Alkyne pairs may participate in one or more independent click reactions to form regioisomeric click products For example, one skilled in the art can generate a mixture of the first preferred A suitable azide can be reacted independently with a first suitable alkyne, and a second suitable azide can be reacted with Four possible click reaction regioisomers that can react independently with a second suitable alkyne Recognize that a mixture of one or four possible click reaction regioisomers will result. It is likely that

[0063] As used herein, the term "residue" refers to the chemical residue in a compound that remains after a chemical reaction. For example, the term "amino acid residue" or "N-alkyl amino acid residue" Amide coupling of an amino acid or an N-alkyl amino acid with a suitable coupling partner refers to the product of a peptide coupling or peptide coupling; where, for example, a water molecule is present between the amino acid or after amide or peptide coupling of the N-alkyl amino acid, resulting in This results in a product having the amino acid residue or N-alkyl amino acid residue incorporated therein. .

[0064] As used herein, a "therapeutically effective amount" refers to a dose that is effective in treating or managing a disease or disorder. to provide a therapeutic benefit to a patient in or to treat one or more symptoms associated with a disease or disorder It refers to an amount (e.g., of a compound) that is sufficient to delay or minimize the effects of an allergic reaction.

[0065] Particular groups, moieties, substituents, and atoms are defined as the atoms to which they are attached. The bond or bonds are shown with a wavy line crossing them to indicate A propyl group, shown as follows: [ka] The phenyl group substituted with has the following structure: [ka] As used herein, a cyclic group (e.g., an aromatic group) is defined as having a bond between the ring atoms. , heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkanes Diagrams depicting substituents attached to a cyclic group indicate that the cyclic group is a ring-type group as described herein, unless otherwise specified. Any ring position in the cyclic group may be bonded according to techniques described or known in the art to which this disclosure pertains. It is intended to indicate that the substituent may be substituted at any position or on any ring in a fused ring group. For example, when the subscript q is an integer from 0 to 4 and the substituent R 1 The location of that is, any vertex of the bond line structure, i.e., a direct bond to a specific ring carbon atom Not yet [ka] is the substituent R 1 includes the following non-limiting example groups attached to specific ring carbon atoms: [ka] .

[0066] As used herein, the phrase "reactive linker" refers to, for example, [ka] (where RG' is a reactive group and SP is a spacer group). As described herein, a reactive linker refers to a monovalent group that includes a spacer group. The reactive group may comprise a reactive group and multiple spacer groups. The spacer group may connect the reactive group to another group. , e.g., any divalent or trivalent moiety that crosslinks to the payload or also to the binding agent. The linker (RL), together with the payload to which it is attached, forms the antibody complex described herein. Intermediates useful as synthetic precursors for the preparation of conjugates ("linker-payloads") The reactive linker can be linked to another group, e.g., an antibody, modified antibody, or the like, described herein. or antigen-binding fragments thereof, or reactive moieties on the cyclodextrin group. The reactive group (RG') is a functional group or functional moiety that can be bonded to a linking group. The moiety generated by reaction with an antibody having the nucleotide sequence, a modified antibody, or an antigen-binding fragment thereof includes the nucleotide sequence of the present invention. The "binder linker" ("BL") portion of the conjugates described herein is included. In embodiments, the "reactive group" is a cysteine ​​or lysine residue of the antibody or antigen-binding fragment thereof. a functional group or moiety (e.g., maleimide or N-hydroxysuccinimide (NH S) esters). In some embodiments, the "reactive group" undergoes a click chemistry reaction. A functional group or moiety that can be used to form a hydroxyl group is a functional group or moiety that can be used to form a hydroxyl group (e.g., click chemistry, Huisgen, Proc. Chem. Soc. 1961, Wang et al., J. Am. Chem. Soc. 2003, and Agard et al., J. Am. Chem. Soc. 2004). In some embodiments of the click chemistry reaction, the reactive group is an alkyne that can undergo 1,3 cycloaddition with an azide. Suitable reactive groups include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC). Preferred are cycloalkynes, such as cyclooctynes, benz-annulated alkynes, and copper catalysts. These alkynes are capable of undergoing 1,3 cycloaddition with alkynes in the absence of a catalyst. Suitable alkynes include, but are not limited to, dibenzoazacyclooctyne or teeth [ka] for example, [ka] , dibenzocyclooctyne or [ka] ,for example, [ka] , biarylazacyclooctynone or [ka] ,for example, [ka] , difluorinated cyclooctyne or [ka] , substituted, e.g., fluorinated alkynes, aza-cycloalkynes, bicycle[6.1.0]nonynes, or teeth [ka] (wherein R is alkyl, alkoxy, or acyl), and derivatives thereof; for example, [ka] Particularly useful alkynes include, but are not limited to: [ka] Examples include:

[0067] Linker-payloads containing such reactive groups can be used to link antibodies that are functionalized with azide groups. Such functionalized antibodies are useful for conjugating azide-polyethylene glycol (APO) compounds. Antibodies functionalized with glycol groups are included. In some embodiments, such functionalized The antibody may have at least one glutamine residue, e.g., Gln295, Asn297Gln, and / or Gln55 The antibody having the amino and azide groups is subjected to cleavage in the presence of the enzyme transglutaminase. It can be obtained by treating with a compound carrying the formula:

[0068] In some instances, the alkyne used in the bioconjugation reaction is Cu(I ) useful in click chemistry conjugation reactions. In some instances, the conjugate The alkynes used in the conjugation reaction react with 1,2-aminothiols in the CBT reaction. In some cases, the alkynes used are those with inverse electron demand in the Diels-Alder reaction. The compounds are BCN, derivatives of BCN, and trans-cyclooctene (TCO). See, for example, J. Am. Chem. See Soc.;(Article), 2012, 134(6), 2950-2953.

[0069] In some instances, the reactive group is an alkyne, e.g., [ka] which can be converted via click chemistry to azides, e.g. [ka] to produce a click chemistry reaction product, e.g., [ka] In some instances, the group can form a modified antibody or antigen-binding fragment thereof. In some instances, the reactive group is an alkyne, e.g., [ka] which can be converted via click chemistry to azides, e.g. [ka] to produce a click chemistry reaction product, e.g., [ka] In some instances, the reactive group can be an alkyne, e.g., [ka] which can be converted via click chemistry to azides, e.g. [ka] to produce a click chemistry reaction product, e.g., [ka] In some instances, the reactive group can be a functional group, such as [ka] which reacts with cysteine ​​residues on an antibody or antigen-binding fragment thereof to form bonds therewith. For example, [ka] (wherein Ab represents an antibody or an antigen-binding fragment thereof, and S represents the functional group via which the functional group is attached to the Ab. (representing the S atom on the cysteine ​​residue to which it is attached). In some instances, the reactive group is , functional groups, e.g. [ka] which reacts with and binds to lysine residues on an antibody or antigen-binding fragment thereof; for example, [ka] (wherein Ab represents an antibody or an antigen-binding fragment thereof, and NH represents the functional group via which the Ab binds to the antibody or antigen-binding fragment thereof.) represents the NH atom on the lysine residue that binds to

[0070] As used herein, the phrase "binder linker" or "BL" refers to a binder (e.g., For example, an antibody or antigen-binding fragment thereof may be administered to a subject in need thereof via a payload compound (e.g., a MM AE, bis-octahydrophenanthrenecarboxamide), and optionally one or more side chain compounds Refers to any divalent, trivalent, or polyvalent group or moiety that links, connects, or bonds to an entity Generally, suitable binder linkers for the antibody conjugates described herein are It is sufficiently stable to utilize its circulatory half-life in the body, while at the same time providing antigen-mediated protection for the conjugate. The linker is capable of releasing its payload after internalization. Cleavable linkers can be cleavable or non-cleavable. Cleavable linkers are those that inhibit intracellular metabolism after internalization, e.g. , hydrolysis, reduction, or enzymatic cleavage. The hydroxyl linker releases the attached payload by lysosomal degradation of the antibody after internalization. Suitable linkers include acid labile linkers, hydrolytically unstable linkers, and linkers, enzyme-cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers Suitable linkers include, but are not limited to, peptides, groups, and the like. Glucuronide, succinimide-thioether, polyethylene glycol (PEG) unit, hydra a dipeptide unit, a valine-citrulline unit, and a para-amino acid unit. Also included are, but are not limited to, those that are or contain polyaminobenzyl (PAB) units. In some embodiments, the binder linker (BL) is a reactive linker (RL). The reaction of the reactive group (RG) with the reactive portion of a binding agent, e.g., an antibody, modified antibody, or antigen-binding fragment thereof. It includes a portion formed by reaction.

[0071] In some examples, the BL comprises the following moiety: [ka] where: [ka] is a bond to a binding agent. In some examples, BL is a bond to a binding agent. [ka] where: [ka] is a bond to a binding agent. In some examples, BL is a bond to a binding agent. [ka] where: [ka] is a bond to a binding agent. In some examples, BL is a bond to a binding agent. [ka] where: [ka] is a bond to a cysteine ​​of the antibody or antigen-binding fragment thereof. The following part: [ka] where: [ka] is a bond to a lysine of an antibody or antigen-binding fragment thereof.

[0072] (Compounds and Payloads) In some examples, provided herein are methods for providing at least one payload moiety linked to at least one cyclodextrin moiety via a covalent linker a linked binding agent; wherein the covalent linker is connected to the binding agent, the payload, a compound bonded directly or indirectly to each of said cyclodextrin moieties; or a pharmaceutically acceptable salt thereof.

[0073] In some other examples, described herein are at least one payload at least one cyclodextrin linked to the moiety and via a covalent linker a protein linked to a moiety, wherein the covalent linker is a payload moiety and a cyclodextrin moiety, each of which is directly or indirectly bound to the payload moiety and the cyclodextrin moiety; or a pharmaceutically acceptable salt thereof.

[0074] As exemplified herein, in some instances, the binding agent comprises a covalent linker. The binding agent is directly attached to an anchor, for example, a lysine amino acid. This means that the amino acid is one bond position away from the covalent linker. In some cases, the covalent linker is also directly attached to the payload moiety. This means that the covalent linker may be, but is not limited to, MMAE, a steroid, or any of the compounds described herein. This means that the payload is one binding position away from any payload that is attached to the payload. In some of these examples, the covalent linker is also directly attached to the cyclodextrin moiety. This is because the covalent linker is a cyclodexamine shown herein. This means that the bond is one bond away from the cyclodextrin, such as cyclodextrin. In some of the above, the covalent linker is a lysine amino acid or a derivative thereof.

[0075] In other instances, the binding agent is indirectly attached to a covalent linker, such as means that the binding agent is more than one binding position away from the covalent linker This also means that the binding agent is attached to the covalent linker through another moiety. For example, the linking agent may be attached to a maleimide group, which is The polyethylene glycol is attached to the covalently attached phosphorus. In some of these examples, the covalent linker is attached to a payload. This is because the covalent linker is indirectly linked to the base moiety, including but not limited to, , MMAE or a steroid, or any payload described herein This means that the covalent linker It also means that the covalent linker is attached to the payload through another moiety. The anchor may be attached to a dipeptide, such as, but not limited to, Val-Ala or Val-Cit. The dipeptide may be conjugated to a PAB, and the PAB may be conjugated to a payload. In some of these examples, the covalent linker may comprise a cyclodextrin moiety. This is because the covalent linker is indirectly linked to the cyclodextrins, such as those described in the specification, which have more than one bond position This means that the covalent linker is spaced apart through another moiety. For example, the covalent linker may be a poly(hydroxybenzoate) or a hydroxybenzoate. The polyethylene glycol group may be linked to a reactive group. The reactive group may be attached to the cyclodextrin. In some examples, the covalent linker is a lysine amino acid or a derivative thereof. .

[0076] In one example, shown herein is a compound according to formula (I): [ka] In formula (I), BA is a binder, L is a trivalent linker, and CD is a cyclodextrin. residue, the subscript n is an integer from 1 to 30, and PA is a payload residue. In some instances, multiple trivalent linkers L may be present. In some embodiments, n is 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In some embodiments, n is 1. In some embodiments, n is 3.

[0077] In some examples, the compound according to Formula (I) is a compound according to Formula (Ia): [ka] In formula (Ia), BA is a binding agent, LL is a trivalent linker, and RG is a reactive linker residue. is a group, SP is, in each occurrence, independently, absent or a spacer group, and the subscript n is and PA is a payload. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In an embodiment, n is 1. In some embodiments, n is 3.

[0078] In some examples, the compound according to Formula (I) is a compound according to Formula (Ib) or (Ic): [ka] In either formula (Ib) or (Ic), BA is a binding agent and LL is a trivalent linker. SP, in each occurrence, is independently absent or a spacer, and ring A is a triazo fused to a cycloalkyl, cycloalkenyl, heterocycloalkyl, and heterocycloalkenyl, wherein cycloalkyl, cyclo Alkenyl, heterocycloalkyl, and heterocycloalkenyl are alkyl, OH, or is NR a R b where R a and R b is alkyl or H, and n is an integer from 1 to 30, and PA is a payload. In some examples, ring A is In some examples, ring A is an optionally substituted cycloalkyl. In some examples, ring A is an optionally substituted cycloalkenyl. In some examples, ring A is an optionally substituted heterocycloalkynyl. In some examples, ring A is an optionally substituted heterocycloalkynyl. In some examples, either Formula (Ib) or (Ic) includes a compound represented by Formula (Ib) or (Ic). (Ic) are positional isomers, stereoisomers, and mixtures thereof of any of the compounds.

[0079] In some examples, the compound according to formula (I) is a compound according to formula (Ib). In some examples, the compound according to formula (Ib) is selected from the group consisting of formulas (Ib1), (Ib2), (Ib3), (Ib4), (Ib5) or (Ib6). In some of these examples, compounds according to formula (Ib) is a compound according to formula (Ib1): [ka] In formula (Ib1), BA is a binding agent, LL is a trivalent linker, and SP is, in each case, independently, n is absent or a spacer, n is an integer from 1 to 30, and PA is a phenyl group. Formula (Ib1) also includes positional isomers (e.g., triazole bonds). In some embodiments, n is 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In an embodiment, n is 1. In some embodiments, n is 3.

[0080] In some of these examples, the counterpart to formula (Ib) is a compound according to formula (Ib2): [ka] In formula (Ib2), BA is a binding agent, LL is a trivalent linker, and SP is, in each case, independently, n is absent or a spacer, n is an integer from 1 to 30, and PA is a phenyl group. Formula (Ib2) also includes positional isomers (e.g., triazole bond isomers). In some embodiments, n is 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In an embodiment, n is 1. In some embodiments, n is 3.

[0081] In some of these examples, the counterpart to formula (Ib) is a compound according to formula (Ib3): [ka] In formula (Ib3), BA is a binding agent, LL is a trivalent linker, and n is an integer of 1 to 30. and PA is the payload. Also included in formula (Ib3) are positional isomers (e.g., with respect to the triazole bond), stereoisomers, and mixtures thereof. In some embodiments, n is 1 to 4. In some embodiments, n is 4. , n is 2. In some embodiments, n is 1. In some embodiments, n is 3 .

[0082] In some of these examples, the counterpart to formula (Ib) is a compound according to formula (Ib4): [ka] In formula (Ib4), BA is a binding agent, LL is a trivalent linker, and n is an integer of 1 to 30. and PA is the payload. Also included in formula (Ib4) are positional isomers (e.g., with respect to the triazole bond), stereoisomers, and mixtures thereof. In some embodiments, n is 1 to 4. In some embodiments, n is 4. , n is 2. In some embodiments, n is 1. In some embodiments, n is 3 .

[0083] In some of these examples, the counterpart to formula (Ib) is a compound according to formula (Ib5): [ka] In formula (Ib5), BA is a binding agent, LL is a trivalent linker, and e is an integer of 0 to 4. where n is an integer of 1 to 30, and PA is a payload. The term "isomers" refers to positional isomers (e.g., with respect to the traizole bond), stereoisomers, and In some embodiments, n is a mixture of 1 to 4. In some embodiments, n is a mixture of 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In some embodiments, n is 1. In some embodiments, n is 3.

[0084] In some of these examples, the counterpart to formula (Ib) is a compound according to formula (Ib6): [ka] In formula (Ib6), BA is a binding agent, LL is a trivalent linker, and e is an integer of 0 to 4. where n is an integer of 1 to 30, and PA is a payload. The term "isomers" refers to positional isomers (e.g., with respect to the traizole bond), stereoisomers, and In some embodiments, n is a mixture of 1 to 4. In some embodiments, n is a mixture of 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In some embodiments, n is 1. In some embodiments, n is 3.

[0085] In some examples, the compound according to formula (I) is a compound according to formula (Ic). In some examples, the compound according to formula (Ic) is a compound according to formula (Ic1): [ka] In formula (Ic1), BA is a binding agent, LL is a trivalent linker, and n is an integer of 1 to 30. and PA is the payload. Formula (Ic1) also includes its positional isomers, isomers, and In some embodiments, n is 1 to 4. In some embodiments, n is 4. In some embodiments, n is 2. In some embodiments, n is 1. In some embodiments, n is 3.

[0086] In some examples, provided herein are compounds according to formula (Id): [ka] In formula (Id), BA is a binder; RG is a reactive group residue; SP 1 and SP 2 In each case , each independently being absent or a spacer group residue, and SP 1 comprises a trivalent linker; A A 1 is a trivalent linker comprising amino acid residues; AA 2 is a dipeptide residue; PEG is is a polyethylene glycol residue; PAB is [ka] where: [ka] indicates the atom at which the PAB is attached to the adjacent group in the formula, and CD, in each occurrence, independently and wherein at least one CD is present and absent or a cyclodextrin residue. where the subscript n is an integer from 1 to 30; the subscript m is an integer from 0 to 5; and the subscript p is 0 or 1; and PA is the payload portion. In these examples, the subscript m is 0 , 1, 2, 3, 4, or 5. In some instances, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In some instances, the subscript p is 1. In some instances, AA 1 or AA 2 Noi one of which, in each occurrence, independently is alanine, valine, leucine, isoleucine, methyl Thionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine Insulin, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, An amino acid selected from arginine, histidine, or citrulline, or a derivative thereof or a combination thereof. In some embodiments, AA 1 are alanine, valine, and leucine. sucrine, isoleucine, methionine, tryptophan, phenylalanine, proline, Synthin, serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparagine Select from glutamic acid, glutamic acid, lysine, arginine, histidine, or citrulline In one embodiment, the amino acid is selected from the group consisting of: , A.A. 1 is lysine. In some embodiments, AA 1 is lysine or a derivative of lysine In one embodiment, AA 2 is valine-citrulline. AA 2 is citrulline-valine. In some embodiments, AA 2 Ha, ballin- In some embodiments, AA is alanine. 2 is alanine-valine. In some embodiments, AA 2 is valine-glycine. In some embodiments, AA 2 is glycine-valine. In some embodiments, AA 1 -AA 2 is glutamine- In some embodiments, AA is valine-citrulline. 1 -AA 2 Glutamine-Bali In some embodiments, AA is citrulline. 1 -AA 2 Lysine-valine-alanine In some embodiments, AA 1 -AA 2 is lysine-valine-citrulline In some embodiments, AA 1 -AA 2 The structure is glutamine-valine-citrulline. In some embodiments, the lysine is L-lysine. In some instances, SP 1 is, in each case independently, C 1-6 Alkylene , -NH-, -C(O)-, (-CH2-CH2-O)e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O )-, -C(O)-NH-(CH2) v -, and combinations thereof, wherein the subscript e is an integer from 0 to 4, the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. In some cases, SP 2 is, in each case independently, C 1-6 Alkylene, -NH -, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O)-, - C(O)-NH-(CH2) v -, -, and combinations thereof, wherein the subscript e is The subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. do.

[0087] In some examples of formula (Id), CD has the structure: [ka] wherein RG is, independently in each occurrence, a reactive linker residue; SP 3 is, in each occurrence, independently absent or a spacer group residue; C 1-6 Alkire -NH-, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C (O)-, -C(O)-NH-(CH2) v-, and combinations thereof, wherein the subscript The letter e is an integer from 0 to 4, the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. is an integer; q is an integer from 0 to 5, and [ka] is a group in which the modified CD group is SP 1 or AA 1 represents an atom bonded to

[0088] In some examples, provided herein are compounds according to formula (Ie): [ka] In formula (Ie), BA is a binding agent; RG is, independently in each occurrence, a reactive linker residue. Yes; SP 2 is, in each occurrence, independently absent or a spacer group residue; AA 2 Is Jipe PEG is a polyethylene glycol residue; PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula, and CD represents the cyclodextrin the subscript n is an integer from 1 to 30; the subscript m is an integer from 0 to 5; the subscript p is 0 or 1; and PA is the payload moiety. In these examples, the subscript m is 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In some examples, the subscript p is 1. In some examples, AA 1 or AA 2 any one of, in each occurrence, independently, alanine, valine, leucine, isoleucine , methionine, tryptophan, phenylalanine, proline, glycine, serine, threonine Onine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutami an amino acid selected from phosphate, lysine, arginine, histidine, or citrulline; In one embodiment, AA 2 Is Balin-shi In some embodiments, AA 2 is citrulline-valine. In some embodiments, AA 2 is valine-alanine. , A.A. 2 is alanine-valine. In some embodiments, AA 2 Valin-Grisi In some embodiments, AA 2 The amino acid sequence is glycine-valine. In the embodiment, AA 2 is glutamine-valine-citrulline. Hey, AA 2 is glutamine-valine-citrulline. In some embodiments, A A 2 is lysine-valine-alanine. In some embodiments, AA 2 Lysine-Ba In some embodiments, AA2 is glutamine-valine-cytosine In some instances, SP 2 In each case, C 1-6 Alkylene, -NH-, - C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O)-, -C(O) -NH-(CH2) v -, and combinations thereof, wherein the subscript e is selected from the group consisting of 0 to 4 where the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8.

[0089] In some examples, the compound according to formula (1e) is a compound according to formula (Ie1): [ka] In formula (1e1), BA is a binder; RG is a reactive linker residue; SP 2 is in each place are independently absent or a spacer group residue; 2 is a dipeptide residue; PEG is a polyethylene glycol residue; PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula; CD represents cyclodextrin the subscript n is an integer from 1 to 30, the subscript m is an integer from 0 to 5, and the subscript The letter p is 0 or 1, and PA is the payload portion. The letter m is 0, 1, 2, 3, 4, or 5. In some instances, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In some examples, the subscript p is 1. In some examples, AA 2 teeth , alanine, valine, leucine, isoleucine, methionine, tryptophan, phenyl Alanine, proline, glycine, serine, threonine, cysteine, tyrosine, asparagus Gin, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or a combination thereof. In one embodiment, AA 2 is valine-citrulline. AA 2 is citrulline-valine. In some embodiments, AA 2 Ha, ballin- In some embodiments, AA is alanine. 2 is alanine-valine. In some embodiments, AA 2 is valine-glycine. In some embodiments, AA 2 is glycine-valine.

[0090] In some examples, the compound according to formula (1e) is a compound according to formula (Ie2): [ka] In formula (1e2), BA is a binder; RG is a reactive linker residue; SP2 is in each place are independently absent or a spacer group residue; 2 is a dipeptide residue; PEG is a polyethylene glycol residue; PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula; CD represents cyclodextrin the subscript n is an integer from 1 to 30; the subscript m is an integer from 0 to 5; the subscript p is 0 or 1; and PA is the payload moiety. In these examples, the subscript m is 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In some examples, the subscript p is 1. In some examples, AA 2 Is, A Leucine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine Nine, proline, glycine, serine, threonine, cysteine, tyrosine, asparagine , glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, if and citrulline, a derivative thereof, or a combination thereof. In one embodiment, AA 2 is valine-citrulline. , A.A. 2 is citrulline-valine. In some embodiments, AA 2 Is, Balin-Ala In some embodiments, AA 2 is alanine-valine. In an embodiment, AA 2 is valine-glycine. In some embodiments, AA 2 is glycine-valine.

[0091] In some examples, presented herein are compounds of formula (If): [ka] In formula (If), BA is a binder; RG is, independently in each occurrence, a reactive linker residue. Yes, SP 1 , SP 2 , and SP 3 is, in each occurrence, independently, absent or a spacer group residue AA 2 is a dipeptide residue; PEG is a polyethylene glycol residue; PA B is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula; CD represents cyclodextrin the subscript n is an integer from 1 to 30, the subscript m is an integer from 0 to 5, and the subscript The letter p is 0 or 1, the subscript q is an integer from 0 to 5; and PA is a payload moiety. In some examples of formula (If), SP 1 is non-existent and SP 2In each case, Independently, C 1-6 Alkylene, -NH-, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C (O)-, -C(O)-(CH2) u -C(O)-, -C(O)-NH-(CH2) v -, and combinations thereof where the subscript e is an integer from 0 to 4, and the subscript u is an integer from 1 to 8, and The subscript v is an integer from 1 to 8. In some examples, AA 2 Alanine, valine, Isoleucine, methionine, tryptophan, phenylalanine, proline, Lysine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparagine From paraben, glutamic acid, lysine, arginine, histidine, or citrulline In one embodiment, the amino acid sequence comprises a selected amino acid, a derivative thereof, or a combination thereof. AA 2 is valine-citrulline. In some embodiments, AA 2 Citrulli In some embodiments, AA 2 is valine-alanine. In some embodiments, AA 2 is alanine-valine. In some embodiments , A.A. 2 is valine-glycine. In some embodiments, AA 2 Glycine-Bali It is.

[0092] In some examples, including any of the foregoing, the compound of formula (I) comprises at least one In a specific example, the compound of formula (I) comprises two CD groups. , Formula (Ie1), or Formula (Ie2), and containing one CD group. Thus, a compound of formula (I) is a compound of formula (If) as described herein and contains one CD group. nothing.

[0093] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: In some instances, the CD is an α-cyclodextrin. In some examples, the CD is β-cyclodextrin. In any of these examples, α-, β-, Or the gamma-cyclodextrin is optionally substituted.

[0094] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: Composed of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin In some examples, the CD is α-cyclodextrin. In some instances, the CD is β-cyclodextrin. , γ-cyclodextrin.

[0095] In certain examples, including any of the foregoing, CD may, in each instance, independently select: [ka] is selected from, where: [ka] indicates the atom to which the CD is attached to the adjacent group in the formula. [ka] In one example, the CD is [ka] is.

[0096] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: [ka] In some examples, CD is selected from: [ka] In some examples, CD is [ka] In some examples, CD is [ka] In some examples, CD is [ka] In some examples, CD is [ka] In some examples, CD is [ka] is.

[0097] In certain examples, including any of the foregoing, RG, in each occurrence, independently represents: In some examples, including any of the foregoing, RG is a click chemistry residue. In each case, independently, it comprises a triazole or a fused triazole.

[0098] In certain examples, including any of the foregoing, RG, in each occurrence, independently represents: [ka] wherein: [ka] indicates the atom to which the RG is attached to the adjacent group in the formula. and RG is, in each occurrence independently, [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] is.

[0099] In some examples, including any of the foregoing, compounds of formula (I) may comprise a group [ka] which includes: [ka] TIFF0007617974000103.tif247170TIFF0007617974000104.tif249170TIFF0007617974000105.tif236170TIFF0007617974000106.tif160170, wherein [ka] indicates binding to a binding agent, e.g., an antibody or antigen-binding fragment thereof, and wherein R 9 -C H3 or -(CH2)3N(H)C(O)NH2. [ka] indicates a bond to cyclodextrin. [ka] indicates binding to the payload. In one example, R 9 is -CH3. In one example, R 9 is —(CH)N(H)C(O)NH. In these examples, the antibody or antigen-binding fragment thereof is , [ka] In some examples, the antibody or antigen-binding fragment thereof [ka] When the antibody or antigen-binding fragment thereof is indirectly bound to a group that is bound to [ka] In some instances, [ka] teeth, [ka] where the subscript x is an integer from 1 to 4. In some examples, the subscript x is In some examples, the subscript x is 2. In some examples, the subscript The subscript x is 3. In some instances, the subscript x is 4. As used herein, Ab refers to: In some instances, the depicted PEG group, i.e., Wow, [ka] is bound to a glutamine on the Ab. In some examples described or shown in The lysine derivatives are derived from D-lysine or D-lysine. In some examples described or shown in the above, the steric orientation of the lysine side chains in the linker The chemical structure is (R). In some examples shown, the stereochemistry of the lysine side chain in the linker is (S). .

[0100] In some examples, including any of the foregoing, compounds of formula (I) may comprise a group [ka] which includes [ka] TIFF0007617974000118.tif249170 TIFF0007617974000119.tif217170 TIFF0007617974000120.tif183170 TIFF0007617974000121.tif244170 TIFF0007617974000122.tif176170.

[0101] Also included in these examples are pharmaceutically acceptable salts, solvates, or isoforms thereof. isomeric forms, positional isomers thereof, or mixtures of positional isomers thereof, wherein Each [ka] is a bond with the binding agent; each [ka] is the bond to the payload; and R 9 is -CH3 or -(CH2)3N(H)C(O)NH2. In some instances, R 9 is -CH3. In some examples, R 9 is -(CH2)3N(H)C(O)N In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] In some examples, including any of the foregoing, [ka] teeth, [ka] is.

[0102] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: In some instances, the CD is an α-cyclodextrin. In some examples, the CD is β-cyclodextrin. In any of these examples, α-, β-, Or the gamma-cyclodextrin is optionally substituted.

[0103] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: Composed of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin In some examples, the CD is α-cyclodextrin. In some instances, the CD is β-cyclodextrin. , γ-cyclodextrin.

[0104] In certain examples, including any of the foregoing, CD may, in each instance, independently select: [ka] is selected from, where: [ka] indicates the atom to which the CD is attached to the adjacent group in the formula. [ka] In one example, the CD is [ka] is.

[0105] In some examples, including any of the foregoing, the compound of formula (I) is [ka] TIFF0007617974000190.tif241170TIFF0007617974000191.tif210170TIFF000761 7974000192.tif201170TIFF0007617974000193.tif242170TIFF0007617974000194 .tif248170TIFF0007617974000195.tif216170TIFF0007617974000196.tif224170TIFF0007617974000197.tif205170TIFF0007617974000198.tif81170. Also included in these examples are pharmaceutically acceptable salts thereof. , solvates, stereoisomeric forms, positional isomers thereof, or mixtures of positional isomers thereof. where each [ka] is a bond with the binding agent; and each [ka] is the binding with the payload.

[0106] In some examples, including any of the foregoing, -LL- is according to formula (LL1): be: [ka] . In formula (LL1), R AA1 , R AA2 , and R AA3 are each independently an amino acid side chain, At least one is directly or indirectly linked to -SP-RG-SP-CD. In some instances, the SP may be non-existent. In some instances, the SP is not non-existent. In some examples, in formula (LL1), R AA1 , R AA2 , and R AA3are each independently an amino acid side chains, at least one of which is directly or indirectly bound to -RG-CD. In some instances, R AA1 is a lysine amino acid side chain. In some instances, R A A1 is the L-lysine amino acid side chain. AA1 is a D-lysine amino acid In some instances, R AA2 is the valine amino acid side chain. Some examples In R AA3 is an alanine amino acid side chain. In some examples, R AA3 Ha, Shito In some instances, R AA1 binds directly or indirectly to CD The amino acid side chain of lysine is R AA2 and R AA3 independently, on the valine and alanine amino acid sides. In some instances, R AA1 is a lysine amino acid bound directly or indirectly to CD side chain, R AA2 and R AA3 are independently the valine and citrulline amino acid side chains.

[0107] In some instances, LL is [ka] In some examples, LL includes: [ka] Includes.

[0108] In some instances, LL is [ka] In some examples, LL includes: [ka] Includes:

[0109] In some instances, SP, SP 1 , SP 2 , or a combination thereof, is a spacer group. In some examples, SP, in each instance, independently represents C 1-6 Alkylene, -NH-, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O)-, -C(O)-NH-(CH 2) v -, ... where the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. In that example, SP 1 is, in each case independently, C 1-6 Alkylene, -NH-, -C(O)-, (-CH2 -CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O)-, -C(O)-NH-(CH2) v - and combinations thereof, wherein the subscript e is an integer from 0 to 4. where the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. In the example, SP 2 In each case, C 1-6 Alkylene, -NH-, -C(O)-, (-CH2-CH2-O) e , -N H-CH2-CH2-(-O-CH2-CH2) e-C(O)-, -C(O)-(CH2) u -C(O)-, -C(O)-NH-(CH2) v - and these where the subscript e is an integer from 0 to 4 and the subscript u is selected from the group consisting of is an integer from 1 to 8, and the subscript v is an integer from 1 to 8.

[0110] In certain examples, including any of the foregoing, the subscript m, q, or e is 1. In some examples, including any of the foregoing, the subscript m is 1. In some examples, including any of the above, the subscript q is 1. In some examples, including

[0111] In certain examples, including any of the foregoing, the subscript m, q, or e is 2. In some examples, including any of the foregoing, the subscript m is 2. In some examples, including any of the foregoing, the subscript q is 2. In some examples, including:

[0112] In certain examples, including any of the foregoing, the subscript m, q, or e is 3. In some examples, including any of the foregoing, the subscript m is 3. In some examples, including any of the foregoing, the subscript q is 3. In some examples, including:

[0113] In certain examples, including any of the foregoing, the subscript m, q, or e is 4. In some examples, including any of the foregoing, the subscript m is 4. In some examples, including any of the foregoing, the subscript q is 4. In some examples, including:

[0114] In some examples, including any of the foregoing, the subscript p is 1.

[0115] In certain examples, including any of the foregoing, the subscript n is 2-4.

[0116] In some examples, including any of the foregoing, the subscript n is 2.

[0117] In some examples, including any of the foregoing, the subscript n is 3.

[0118] In some examples, including any of the foregoing, the subscript n is 4.

[0119] In some examples, including any of the foregoing, the binding agent (BA) may be any polypeptide. Exemplary polypeptides include naturally occurring and non-naturally occurring polypeptides. Exemplary polypeptides include, but are not limited to, genetic These include, but are not limited to, those produced from genetically modified organisms.

[0120] Polypeptides herein include receptors, proteins, enzymes, binding agents, milk peptides, These include ribosomal peptides, nonribosomal peptides, peptones, and peptide fragments, Polypeptides herein include, but are not limited to, antimicrobial peptides, tachykinins, Peptides, vasoactive intestinal peptide, pancreatic polypeptide-related peptide, opioid peptide The peptides included in this disclosure include, but are not limited to, ribonucleotides, ribonucleotides, and calcitonin peptides. Other peptides that may be involved include B-type natriuretic peptide (BNP), lactotripeptide, and neuropeptide. These include, but are not limited to, peptides, lipopeptides, proteases, or hormones. I can't.

[0121] Polypeptides further include short chains of amino acids containing two or more amino acids joined together. Polypeptides include, but are not limited to, two amino acids linked together. Dipeptides (Val-Cit), tripeptides, and tetrapeptides with one, three, or four amino acids Polypeptides include, but are not limited to, ribonucleotides (e.g., Val-Gly-Ser-Ala). A peptide includes a longer chain containing five or more amino acids. A polypeptide includes a chain containing 50 or more amino acids. It includes long chains containing amino acids (e.g., proteins). The polypeptides in this document include dipeptides, tripeptides, tetrapeptides, pentapeptides, Hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, unpeptides It is selected from decapeptides and icosapeptides.

[0122] Polypeptides include proteins. In some instances, proteins include: Only natural amino acids are included. In some instances, proteins contain unnatural amino acids. Further included are:

[0123] In some examples, including any of the foregoing, the binding agent (BA) is an antibody or In some instances, the binding agent is an antigen-binding fragment of AXL, BAFFR, BCMA, BCR-like compound. element, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10 or f54, CCR1, CCR3, CC R4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19 , CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30 , CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD46, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2 , CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2 , CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, FELD1, Fire, GITR, HER2, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12 R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21 R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, integrin, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1 , NKG2D, NKp30, NKp46, PDCD1, PRLR, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGI RR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec Lec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list component / association, PTCRA, T CRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYR An antibody selective for an antigen selected from the group consisting of O, VLDLR, VSIG4, and VTCN1, or an antigen thereof It is a binding fragment.

[0124] In some instances, including any of those previously mentioned, the PA may be a dolastatin, auric acid, or the like. Taxines, maytansinoids, plant alkaloids, taxanes, vinca alkaloids, sterols In some cases, the compound is a residue of a group selected from the group consisting of: an LXR modulator; In some cases, the PA is a dolastatin. In some cases, the PA is an auristatin. In some cases, the PA is a maytansinoid. In some cases, the PA is a plant alkaloid. In some cases, the PA is a taxane. In some cases, the PA is a vinca alkaloid. In some cases, the PA is a steroid. In some cases, the PA is an LXR modulator. In some cases, the LXR modulator is an LXR agonist. In an embodiment, the LXR modulator is an LXR antagonist. The polymers are highly hydrophobic, e.g., those that are difficult to bind to Ab conjugates due to their hydrophobic nature. Those that are not suitable for application conditions, such as pyrrolobenzodiazepines (PBDs), SN38 (7-ethyl-10- In some embodiments, the payload may be a hydroxy-camptothecin. Therefore, provided herein are antibody-drug conjugates in which PA is a hydrophobic payload moiety. It is a route.

[0125] In some examples, the PA is any compound shown in any one of FIGS.

[0126] In certain embodiments, the PA is an auristatin. Auristatin E, auristatin F, monomethylauristatin-D (MMAD), monomethylauristatin monomethylauristatin-E (MMAE), or monomethylauristatin F (MMAF). [ka]

[0127] In certain embodiments, the PA is a maytansinoid. Maytansinoids can be administered by one skilled in the art. The payload can be any maytansinoid deemed suitable by Published in U.S. Patent Application Publication No. 2016 / 0375147, "Maytansinoid Derivatives, and MAYTANSINOID DERIVATIVES, CONJUGATES THEREOF, AND METHODS OF USE U.S. Nonprovisional Application No. 15 / 081,759, filed March 25, 2016, entitled "METHODS OF USE" and U.S. Patent No. 9,950,076, entitled "Maytansinoid Derivatives, Their Conjugates, and Their Use in Combinations." MAYTANSINOID DERIVATIVES, CONJUGATES THEREOF, AND METHODS OF USE No. 15 / 414,537, filed January 24, 2017, entitled "DEVICES OF USE" The maytansinoid payloads described are incorporated herein by reference. In some embodiments, the PA is DM1, DM3, or DM4. [ka] In one embodiment, the PA is [ka] wherein A is an optionally substituted arylene or heteroarylene. In an embodiment, the PA is [ka] In certain embodiments, the wavy line indicates a bond to LL.

[0128] In certain embodiments, the payload in the compound of Formula (I) is a glycoconjugate having the structure of Formula (A): A corticoid or a pharmaceutically acceptable stereoisomer or derivative thereof: [ka] (In the formula: R 1 and R 2 is independently —H, alkyl, alkyl-C(O)—O—, —OH, or halo; or R 1 and R 2 together, [ka] Forming where R 4 is an alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl It is a kill, wherein the alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl is, in each occurrence independently, -NR Aa R Ab optionally substituted with; R 3 is -OH, R Z -C(O)-X-, heteroalkyl, piperidinyl, -NR Aa R Ab, -oxyaryl -NR Aa R Ab , or -Z-A'(R P ) t and; R 5 is independently, at each occurrence, —OH, halo, alkyl, or arylalkyl; R Z is alkyl; X is O or NR Aa and; Z is S, S(O), S(O)2, SO2NR Aa , O, C(O)NR Aa , C(O), or NR Aa and; A' is aryl, arylalkyl, or heteroaryl; R P , in each occurrence independently, halo, optionally substituted alkyl, —OH, or —NR Aa R Ab in can be; R Aa and R Ab is, in each occurrence, independently -H, optionally substituted alkyl, or optionally substituted substituted aryl; the subscript a is an integer from 0 to 19; and t is an integer from 1 to 3; however: (1)(a)R 1 is —OH, or (b) R 1 and R 2 But together, [ka] (where R 4 is C 1-9 Alkyl or [ka] ) is formed, R 3 is not -OH, and (2)R 3 teeth, [ka] isn't it.

[0129] In some embodiments, R 3 is NH2. In some such embodiments, R 3 teeth, [ka] where: [ka] is R 3 indicates the atom bonded to the adjacent group in formula (I).

[0130] In some embodiments, the PA is a steroid. [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In certain embodiments according to any of 10 to 1140, R 3 is -O-aryl, -NR Aa R Ab , -Al Kiren-NR Aa R Ab , -X-arylene-Y-NR Aa R Ab , -X-heteroarylene-Y-NR Aa R Ab , or N-containing wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH-; and R 4 is alkyl, aryl, alkylaryl, or arylalkyl. In certain embodiments, R 3 is -O-arylene-NRAa R Ab ,- O-Heteroarylene-NR Aa R Ab wherein aryl or heteroaryl is halogen , deuterium, hydroxyl, or methoxyl. In the same way, R 3 is -O-phenyl-NR Aa R Ab , -O-heteroarylene-NR Aa R Ab where , phenyl or heteroaryl optionally substituted with halogen or deuterium In some embodiments, R 4 is n-propyl. In certain embodiments, R Aa and R Ab are each independently hydrogen or alkyl. In certain embodiments, R Aa and R Ab Of In one embodiment, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In this, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In this, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In this, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In this, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In this, PA is [ka] or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In an embodiment, the wavy line indicates a bond to LL.

[0131] Shown herein is the following structure: [ka] TIFF0007617974000225.tif249170TIFF0007617974000226.tif88170 or a pharmaceutically acceptable stereoisomer thereof. In embodiments, the conjugate is linked to a primary or secondary amine residue of the payload. The payload is linked to a linker via a bond. "STEROIDS AND PROTEIN-CONJUGATES THEREOF," November 7, 2017 The steroid payloads disclosed in U.S. Non-provisional Application No. 15 / 806,197, filed on It is incorporated herein by reference.

[0132] In certain embodiments, the PA is a liver X receptor (LXR) modulator. wherein PA is according to Formula B or a pharmaceutically acceptable salt, solvate, or steric form thereof. It is an isomeric form: [ka] (In the formula, W is -CH2-, -N(H)-, or -O-; R B1 is -H, -OH, -NH2, alkyl, or -OP(O)(OR 6 )2; R B2 -H, -OH, -CH2NH2, R B3 , R B4 , R B5 , or -OR B5 where R B1 and R B2 teeth , and at the same time, -H; R B3 is -N(R 6 )2; R B4 is -XYZ; X is selected from the group consisting of -O- and -N(H)-; Y is alkylene, substituted alkylene (including but not limited to oxo-substituted, i.e., =O). heteroalkylene, and substituted heteroalkylene (including but not limited to oxo-substituted (i.e. i.e., ═O); Z is selected from the group consisting of -OH and -NH; R B5 is alkyl, heterocycloalkyl, or substituted heterocycloalkyl, wherein each heterocycloalkyl or substituted heterocycloalkyl is selected from nitrogen and oxygen; containing one, two, or three selected heteroatoms and at least one of -OH and -CHOH containing a substituent, or at least one primary or secondary nitrogen, e.g., O-glucose; Each R 6 represents in each case -H, an amino acid residue, an N-alkyl amino acid residue, a peptide, or alkyl; and Each R 7 are independently halo, C 1-6 Alkyl, C 1-6 Alkoxy, -CN, O-glucose, O- Amino acid residues and O-PEG b where each subscript b is an integer from 0 to 3. In certain embodiments, R B1 or R B2 is substituted with a bond to LL. And the PA: [ka] In certain embodiments, the wavy line indicates a bond to LL.

[0133] In some embodiments, the payload of formula (B) is: [ka] TIFF0007617974000230.tif46170 or a pharmaceutically acceptable stereoisomeric form thereof. wherein the conjugate is attached to a primary or secondary amine residue of the payload via binding to the primary or secondary amine residue of the payload. The payload is linked to a linker via a "bis-octahydrophenanthrene copolymer." Carboxamide and its protein conjugate (bis-octahydrophenanthrene carboxamide) A U.S. non-provisional application filed on May 18, 2017, entitled "(I) METHODS AND PROTEIN CONJUGATES THEREOF" The LXR modulator payloads disclosed in application Ser. No. 62 / 508,327 are incorporated herein by reference. The present invention is incorporated herein by reference.

[0134] Also contemplated within the scope of the embodiments presented herein are U.S. Patent Application Publication No. Published under the title "Biologically active molecules, their conjugates, and therapeutic uses" in the Japanese Patent Application No. 2016 / 0030591. (BIOLOGICALLY ACTIVE MOLECULES, CONJUGATES THEREOF, AND THERAPEUTIC USES) No. 14 / 776,668, filed September 14, 2015, entitled "Compounds for a Component of ... "Pharmaceutical composition containing macrolide diastereomers" published as Patent Publication No. 2016 / 0354482 Pharmaceutical Compositions Comprising Macrolids, Methods for Their Synthesis, and Therapeutic Use "E DIASTEREOOMERS, METHODS OF THEIR SYNTHESIS AND THERAPEUTIC USES" 201 The payload is disclosed in U.S. Non-provisional Application No. 14 / 913,965, filed February 23, 2016. , the disclosure of which payload is incorporated herein by reference.

[0135] In some examples, the compound of formula (I) is: [ka] TIFF0007617974000232.tif174170TIFF0007617974000233.tif177170TIFF0007617974000 234.tif236170TIFF0007617974000235.tif193170TIFF0007617974000236.tif130170TIFF 0007617974000237.tif226170TIFF0007617974000238.tif203170TIFF0007617974000239.tif192170TIFF0007617974000240.tif76170 or positional isomers, stereoisomeric forms, pharmaceutically acceptable salts, solvates, or the like thereof. is selected from these mixtures, where Ab is an antibody or an antigen-binding fragment thereof. In these examples, the Ab may be directly or indirectly bound to the linked payload. In some instances, when the Ab is indirectly attached to the linker-payload, [ka] teeth, [ka] where the subscript x is an integer from 1 to 4. In some examples, the subscript x is In some examples, the subscript x is 2. In some examples, the subscript The subscript x is 3. In some instances, the subscript x is 4. As used herein, Ab refers to: In some instances, the depicted PEG group, i.e., Wow, [ka] is attached to a glutamine on the Ab. In certain embodiments, n is 1 to 30, e.g., 1 is an integer between 1 and 4, for example, 1, 2, 3, or 4.

[0136] In some examples, the compound of formula (I) is: [ka] TIFF0007617974000245.tif243170TIFF0007617974000246.tif215170TIFF0007617974000247.tif206170TIFF0007617974000248.tif 246170TIFF0007617974000249.tif207170TIFF0007617974000250.tif202170TIFF0007617974000251.tif200170TIFF000761797400025 2.tif245170TIFF0007617974000253.tif224170TIFF0007617974000254.tif192170TIFF0007617974000255.tif227170TIFF0007617974000256.tif220170TIFF0007617974000257.tif196170TIFF0007617974000258.tif66170 or positional isomers, stereoisomeric forms, pharmaceutically acceptable salts, solvates, or the like thereof is selected from these mixtures, where Ab is an antibody or an antigen-binding fragment thereof. In these examples, the Ab may be directly or indirectly bound to the linked payload. In some instances, when the Ab is indirectly attached to the linker-payload, [ka] teeth, [ka] where the subscript x is an integer from 1 to 4. In some examples, the subscript x is In some examples, the subscript x is 2. In some examples, the subscript The subscript x is 3. In some instances, the subscript x is 4. As used herein, Ab refers to: In some instances, the depicted PEG group, i.e., Wow, [ka] is attached to a glutamine on the Ab. In certain embodiments, n is 1 to 30, e.g., 1 is an integer between 1 and 4, for example, 1, 2, 3, or 4.

[0137] In some examples, the compound of formula (I) is: [ka] TIFF0007617974000263.tif216170TIFF0007617974000264.tif200170TIFF0007617974000265.tif239170TIFF0007617974000 266.tif207170TIFF0007617974000267.tif203170TIFF0007617974000268.tif204170TIFF0007617974000269.tif216170TIFF0 007617974000270.tif236170TIFF0007617974000271.tif248170TIFF0007617974000272.tif232170TIFF0007617974000273.tif240170TIFF0007617974000274.tif143170TIFF0007617974000275.tif130170, or a positional isomer or stereoisomeric form thereof, or a pharmaceutically acceptable salt thereof, or These solvates are selected from, where Ab is an antibody or an antigen-binding fragment thereof.

[0138] In some instances, provided herein are methods for treating rheumatoid arthritis, including the use of any of the aforementioned compounds. The compounds described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts thereof A pharmaceutical composition comprising an excipient.

[0139] Those skilled in the art will appreciate that amino acid residues may be achiral or chiral, e.g., L-amino acids or D-amino acids. It will be recognized that an amino acid typically includes an amino acid side chain. The chain can be the side chain of any amino acid known to those of skill in the art. The side chain is selected from the group consisting of histidine, alanine, isoleucine, arginine, leucine, and asparagine. lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamine Acid, threonine, glutamine, tryptophan, valine, ornithine, selenocysteine , serine, glycine, homoglycine (e.g., β-homoglycine), or tyrosine side chains Those skilled in the art will appreciate that the peptide may be prepared using, for example, racemic DL-amino acids or non-racemic D-amino acids. Achiral or chiral amino acids, including L-amino acids and their diastereomeric mixtures. It will be recognized that the side chains of the peptides may be related to the amino acids listed above. Those skilled in the art will recognize that an N-alkyl amino acid residue is present at the end of the amino acid. The terminal amino group or the terminal amino group of the peptide is provided with an alkyl substituent as defined above. You will be aware that this includes

[0140] In certain examples, including any of the foregoing, the subscript n is an integer from 0 to 30. In some examples, including any of the foregoing, the subscript n is 0. In some instances, including any of the foregoing, the subscript n is 1. In some examples, including any of the foregoing, the subscript n is 2. In some instances, the subscript n is 3. In this example, the subscript n is 4. In some examples, including any of the above, In some examples, including any of the above, the subscript n is The subscript n is 6. In some examples, including any of the above, the subscript n is 7. In some examples, including any of the foregoing, the subscript n is 8. In some examples, including any of the foregoing, the subscript n is 9. In some examples, including any of the foregoing, the subscript n is 10. In some examples, the subscript n is 11. In some examples, the subscript n is 12. Some examples, including any of the above, where the subscript n is 13. In some examples, including any of the above , the subscript n is 14. In some examples, including any of the foregoing, the subscript The subscript n is 15. In some examples, including any of the foregoing, the subscript n is 16. In some examples, including any of the foregoing, the subscript n is 17. In certain examples, including any of the foregoing, the subscript n is 18. In some examples, the subscript n is 19, including any of the foregoing. In some examples, the subscript n is 20, including any of the foregoing. In some instances, the subscript n is 21. In some examples, the subscript n is 22. In the above, the subscript n is 23. In some examples, including any of the above, The subscript n is 24. In some examples, including any of the foregoing, the subscript n In some examples, including any of the above, the subscript n is 26. In some examples, including any of the foregoing, the subscript n is 27. In some examples, including any of the above, the subscript n is 28. In some examples, including any of the foregoing, the subscript n is 29. In some examples, the subscript n is 30, including:

[0141] (binder) Suitable binding agents for any of the conjugates provided in this disclosure include These include antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell-binding or peptide-binding molecule or substance, Suitable binding agents also include, but are not limited to, polypeptides.

[0142] In some examples, including any of the foregoing, the binding agent (BA) may be any polypeptide. Exemplary polypeptides include naturally occurring and non-naturally occurring polypeptides. Exemplary polypeptides include, but are not limited to, genetic These include, but are not limited to, those produced from genetically modified organisms.

[0143] In some examples, including any of the foregoing, the BA is a receptor, a cytokine , proteins, enzymes, binders, milk peptides, ribosomal peptides, non-ribosomal peptides , peptones, and peptide fragments. In some embodiments, the BA is an antimicrobial peptide, a tachykinin peptide, a vasoactive intestinal peptide, or , pancreatic polypeptide-related peptides, opioid peptides, and calcitonin peptides In some examples, including any of the foregoing, the BA is selected from the group consisting of B type. Natriuretic peptides (BNP), lactotripeptides, neuropeptides, lipopeptides , a protease, or a hormone.

[0144] In some examples, including any of the foregoing, the BA may comprise two or more molecules linked together. A short amino acid chain containing any of the above amino acids is selected from the group consisting of: In some examples, the BA is a sequence of two, three, or four amino acids linked together, respectively. Dipeptides (Val-Cit), tripeptides, and tetrapeptides (e.g., Val-Gly- In some examples, including any of the foregoing, the BA is selected from the group consisting of: , dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, hep Tapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, and isopeptides Cosapeptides are selected from:

[0145] In some examples, including any of the foregoing, the BA may be derived from any protein. In some instances, the protein contains only natural amino acids. In some instances, the protein further comprises an unnatural amino acid. In the present invention, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody may be any antibody known to those skilled in the art. As used herein, the term "antibody" refers to a specific At least one complementarity determinant that specifically binds to or interacts specifically with an antigen The term "antibody" refers to any antigen-binding molecule or molecular complex containing a CDR. It consists of four polypeptides: two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each of these includes immunoglobulin molecules containing a polypeptide chain, and multimers thereof (e.g., IgM). The heavy chain comprises a heavy chain variable region (HCVR or V H The heavy chain constant region is abbreviated as . The common region consists of three domains: C H 1. C H 2, and C H Each light chain comprises a light chain variable region ( In the specification, LCVR or V L The light chain constant region comprises one domain, (C L 1) V H and V L The more conserved regions are called framework regions (FR). These can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with Each V H and V L It consists of three CDRs and four FRs, and The CDRs are arranged in the following order towards the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present invention, antibodies (or antibodies thereof) suitable for the compounds herein are The FRs of the original binding moiety may be identical to the human germline sequences or may be derived from natural or artificial The amino acid consensus sequence may be based on a comparative analysis of two or more CDRs. As used herein, the term "antibody" refers to an intact antibody molecule. As used herein, the term "antigen-binding portion" of an antibody, "antibody" of an antibody, and "antigen-binding fragment" of an antibody also include the term "antigen-binding portion" of an antibody. The terms "antigen-binding fragment" and the like refer to any naturally occurring antigen-binding fragment that specifically binds to an antigen to form a complex. existing, enzymatically obtained, synthetic or genetically modified polypeptides or glycoproteins Antigen-binding fragments of antibodies can be obtained using any suitable standard technique, e.g., proteolysis. or the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA fragments can be derived from intact antibody molecules using recombinant genetic engineering techniques. A may be known and / or obtained from, for example, a commercial source, a DNA library (e.g., a phage -antibody libraries) or can be synthesized. A is sequenced and engineered chemically or by using molecular biology techniques to For example, by arranging one or more variable and / or constant domains in a suitable arrangement, or introducing amino acids, generating cysteine ​​residues, modifying, adding, or deleting amino acids Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) antibody superpolymers. Isolation of a minimal recognition unit (e.g., CDR3 peptide) consisting of amino acid residues that mimic the variable region Other modifications include a restricted complementarity determining region (CDR), or a restricted FR3-CDR3-FR4 peptide. Molecules such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies , CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies ( (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapeutics (SMIPs), and and shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein. An antigen-binding fragment of an antibody typically contains at least one variable domain. The amino acid may be of any size or amino acid composition and typically contains one or more framework amino acids. at least one CDR adjacent to or in-frame with the sequence .V H Domain is V L In the antigen-binding fragment associated with the domain, V H Domains and V L Domain The domains may be in any suitable configuration relative to each other. For example, the variable region may be a dimer. , and V H -V H , V H -V L , or V L -V L Alternatively, the antibody may contain a dimer. The fragment is a monomeric V H or V L In one embodiment, the antibody An antigen-binding fragment of the present invention comprises at least one variable domain covalently linked to at least one constant domain. The variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention may also be included. Non-limiting exemplary arrangements of domains include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)VL -C H 1;(ix)V L -C H 2;(x) V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L is mentioned Any arrangement of variable and constant domains, including any of the exemplary arrangements above, may be used. In this configuration, the variable and constant domains may be directly linked to each other or may be completely They may be linked by a full or partial hinge or linker region. A domain region is a region between adjacent variable and / or constant domains in a single polypeptide molecule. At least two (e.g., five, ten, fifteen, twenty) flexible or semi-flexible linkages The fragment may consist of 10, 40, 60, or more amino acids. Similarly, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains. wherein each variable domain is specific for a separate antigen or for a different epitope on the same antigen. Exemplary bispecific antibody formats disclosed herein include: Any multispecific antibody format, including the multispecific antibody format, can be synthesized using routine techniques available in the art. The methods can be used and adapted for use in connection with antigen-binding fragments of antibodies of the invention. In one embodiment of the invention, the antibody of the present invention is a human antibody. The term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention are intended to include antibodies having CDRs, particularly CDRs 3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by random or site-directed mutagenesis in vitro or in vivo However, as used herein, the term "mutation" may include mutations introduced by somatic mutations. The term "human antibody" as used herein refers to an antibody derived from the germline of another mammalian species, such as a mouse. It is not intended to include antibodies in which the CDR sequences of interest have been grafted onto human framework sequences. The term "human antibody" generally refers to a naturally occurring antibody without modification or human intervention / manipulation. The antibodies of the present invention do not include naturally occurring molecules present in modified organisms. In some embodiments, the antibody may be a recombinant human antibody. The term "antibody" refers to an organism prepared, expressed, produced, or isolated by recombinant means. All human antibodies, e.g., can be produced using recombinant expression vectors transfected into host cells. and antibodies expressed in a recombinant combinatorial human antibody library (described further below). antibodies isolated from the human (described further below), Antibodies isolated from animals (e.g., mice) that are transgenic (e.g., the literature described by Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or human immunoglobulin gene sequences. prepared and expressed by any other means involving splicing of the vector into other DNA sequences; The term "human antibody" is intended to include antibodies that are produced or isolated by recombinant human methods. have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, such recombinant human antibodies are prepared by in vitro mutagenesis. (or in vivo cellular proliferation when using animals transgenic for human Ig sequences) and thus the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L Derived from and related to sequences, but not in vivo human antibody germline Human antibodies are sequences that may not naturally occur within the antibody repertoire. They can exist in two forms, which are related to gender. In one form, the immunoglobulin molecule , a stable four-chain dimer of approximately 150-160 kDa linked by interchain heavy chain disulfide bonds In the second form, the dimers are not linked by interchain disulfide bonds and are covalently linked. A molecule of approximately 75-80 kDa is formed, consisting of covalently linked light and heavy chains (half of an antibody). These forms are extremely difficult to separate, even after affinity purification. The frequency of the second form in human IgG isotypes is not limited to, but can be found in the hinge region of the antibody. This is due to structural differences associated with the region isotype. A single amino acid substitution in the region significantly reduced the cleavage rate to the level typically observed with human IgG1 hinges. The occurrence of the second form can be significantly reduced (Angal et al., 1993, Molecular Immunology ology 30:105). The present invention is a hinge, C H 2 or C HAntibodies with one or more mutations in three regions The mutations include, for example, to improve the yield of a desired antibody form in production. The antibody of the present invention may be an isolated antibody. An "isolated antibody" as used herein is an antibody that has been identified and removed from at least one component of its natural environment. It refers to an antibody that has been separated and / or recovered from an element, e.g., at least one component of an organism. isolated or isolated from a source or from tissues or cells in which the antibody naturally occurs or is naturally produced An antibody from which the fragments have been removed is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. In one embodiment, the isolated antibody is an antibody that has been subjected to a purification or isolation process. The term "cellulosic acid" as used herein may be substantially free of cellular material and / or chemicals. The antibody to be produced has a variable heavy and light chain domain compared to the corresponding germline sequence from which the antibody is derived. one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the Such mutations can be used to modify the amino acid sequences disclosed herein, e.g. For example, by comparing with germline sequences available from public antibody sequence databases, The present invention provides a method for identifying a nucleotide sequence within one or more framework and / or CDR regions. or a sequence of amino acids that are identical to the corresponding residues in the germline sequence from which the antibody is derived, or to another human germline sequence. mutated to the corresponding residue in the corresponding germline sequence or to a conservative amino acid substitution of the corresponding germline residue. (Such sequence changes are collectively referred to herein as "germline mutations"). and antibodies derived from any of the amino acid sequences disclosed herein and their antigen binding. Those skilled in the art can easily create fragments of the heavy and light chain variable regions starting from the sequences disclosed herein. and a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In one embodiment, V H and / or V L Everything in the domain framework and / or CDR residues of mutated back to the residue found in the germline sequence from which the antibody was derived. In this manner, only certain residues, e.g., the first eight amino acids of FR1 or the last eight amino acids of FR4, are Mutated residues found in only one amino acid or in only one amino acid found in CDR1, CDR2, or CDR3 Only the mutation residues that are present are mutated back to the original germline sequence. In this way, one or more of the framework and / or CDR residues can be modified to a different germline sequence (i.e., mutated to the corresponding residue in a germline sequence (different from the germline sequence from which it was originally derived) Furthermore, antibodies of the invention may be used in combination with other antibodies, e.g., antibodies in which certain individual residues match the corresponding residues of a particular germline sequence. While the nucleotide sequence has been mutated to residues that differ from the original germline sequence, certain other residues The fragments are either maintained or mutated to the corresponding residues in a different germline sequence. may contain any combination of two or more germline mutations within the framework and / or CDR regions Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be For example, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic activity. facilitate the development of one or more desired properties, such as dynamic biological properties (optionally), reduced immunogenicity, etc. The antibodies and antigen-binding fragments obtained by this general method can be used in the Antibodies useful in the compounds herein include those with one or more conservative substitutions. and variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. The term "epitope" refers to the portion of an antibody molecule known as a paratope. The term refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region. An antigen may have multiple epitopes. Thus, different antibodies may bind to different parts of the antigen. and may have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are spatially distinct epitopes derived from different segments of a linear polypeptide chain. Linear epitopes are formed by juxtaposed amino acids. In some situations, epitopes are formed by saccharide residues on an antigen. The alkyl group may contain a methyl group, ...

[0146] In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a κ In certain embodiments, the light chain is a λ light chain. The antibody comprises a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the The heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2.

[0147] In some embodiments, the antibody is an antibody fragment. The antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv (sFv ) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment.

[0148] In some embodiments, the antibody is a monoclonal antibody. In embodiments, the antibody is a polyclonal antibody.

[0149] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. is.

[0150] The antibody may have binding specificity for any antigen deemed suitable by one of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include molecules such as renin; human growth hormone and bovine growth hormone. growth hormones, including growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone Lipoproteins; α1-antitrypsin; insulin A chain; insulin B chain; proinsulin follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors, e.g., v factor mc, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulants, e.g., protease inhibitors; Protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activator, For example, urokinase or human urinary or tissue-type plasminogen activator (t-PA); Thrombin; Hematopoietic growth factors; Tumor necrosis factors-α and -β; Enkephalinase; RANTES (expressed and secreted by normal T cells, regulated upon activation); human macrophage inflammatory protein Protein (MIP-I-α); serum albumin, e.g., human serum albumin; Mullerian n) inhibitors; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide peptides; microbial proteins, e.g., β-lactamase; DNase; 19E; cytotoxic T lymphocytes CTLA-associated antigens (CTLA), e.g., CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; proteins A or D; rheumatoid factors; neurotrophic factors, e.g. For example, bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4) , NT-5, or NT-6), or nerve growth factor, e.g., NGF-β; platelet-derived growth factor (PDGF) fibroblast growth factors, e.g., aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epithelial Growth factors (EGF); transforming growth factors (TGF), such as TGF-α and TGF-β1, TGF-β2, TGF-β3 TGF-β, including TGF-β, TGF-β4, or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(I-3)-IGF-1 (brain IGF-1), insulin-like growth factor binding protein, EpCAM, GD3, FLT3 , PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptors, EphB receptors, folate receptors , FOLRI, mesothelin, crypt, αvβ6, integrin, VEGF, VEGFR, EGFR, trans ferrin receptors, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins, e.g., CD2, CD 3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD3 0, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80 , CD81, CD103, CD105, CD134, CD137, CD138, CD152, or U.S. Publication No. 2008 / 0171040. or U.S. Publication No. 2008 / 0305044, which are incorporated by reference in their entirety. Antibodies that bind to one or more tumor-associated antigens or cell surface receptors; erythropoietin; bone-derived Inducers; immunotoxins; bone morphogenetic proteins (BMPs); interferons, e.g., interferon -α, -β, and -γ; colony-stimulating factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukin-1 (IL-1)-1 (IL-2)-1 (IL-3)-1 (IL-4)-1 (IL-5)-1 (IL-6)-1 (IL-7)-1 (IL-8)-1 (IL-9)-1 (IL-10)-1 (IL-11)-1 (IL Leukins (IL), e.g., IL-1 to IL-10; superoxide dismutase; T cell receptor; membrane proteins; decay-accelerating factors; viral antigens, such as parts of the HIV envelope; transport Proteins; homing receptors; addressins; regulatory proteins; integrins, e.g., CD11 a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; tumor-associated antigens, e.g., AFP, ALK, B7H 4. BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic acid anhydrase) dorase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, C TLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, endoglin, Epcam, EphA2, ErbB2 / H er2, ErbB3 / Her3, ErbB4 / Her4, ETV6-AML, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE -1, -2), GD2, GD3, globoH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / EBNA1 , HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE- 1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MU M1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PA X5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRA ME, PSCA, PSGR, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, Ste ap-1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TR P-2, tyrosinase, and uroplakin-3, and fragments of any of the above polypeptides In some embodiments, the antigen is a tumor antigens specific to a particular type of tumor or shared or overexpressed on a particular type of tumor, or tumor antigens, including antigens that are modified. Examples include: α-actinib for lung cancer; ARTC1 in melanoma, BCR-ABL fusion protein in chronic myeloid leukemia, B-RAF, CLPP, or Cdc27 for squamous cell carcinoma, CASP-8 for squamous cell carcinoma, and renal cell hsp70-2 in relation to cancer, as well as the following common tumor-specific antigens, e.g., BAGE-1, GAGE, GnTV , KK-LC-1, MAGE-A2, NA88-A, and TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. The antibody is an anti-PRLR or anti-HER2 antibody.

[0151] The binder linker binds the antibody or antigen-binding molecule via a bond at a specific amino acid. The antibody or antigen-binding molecule can be attached to the antibody or antigen-binding molecule. Exemplary amino acid linkages that can be used in the linkage include, for example, lysine (e.g., See, for example, US 5,208,020; US 2010 / 0129314; Hollander et al., Bioconjugate Chem., 20 08, 19:358-361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / 0585592), cysteine ​​(see, e.g., US 2007 / 0258987; WO 2013 / 055993; WO 201 3 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 010154 No. 6; and US Pat. No. 7,750,116), selenocysteine ​​(see, e.g., WO 2008 / 122039; and Hof er et al., Proc. Natl. Acad. Sci., USA, 2008, 105: 12451-12456), formyl Glycine (e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Pro Tocols, 2012, 10:1052-1067), unnatural amino acids (see, for example, WO 2013 / 068874 and WO 2012 / 166559), as well as acidic amino acids (see, for example, WO 2012 / 05982). The linker is conjugated to the antigen-binding protein via a bond to the carbohydrate. (See, e.g., US 2008 / 0305497, WO 2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13: 127-130).

[0152] In some instances, the binding agent is an antibody or an antigen-binding molecule, and the antibody is a lysine In some embodiments, the antibody or antigen-binding The molecule is attached to the linker via a cysteine ​​residue.

[0153] The linker is conjugated via transglutaminase-based chemoenzymatic conjugation. Alternatively, the hydroxylase may be conjugated to one or more glutamine residues (see, e.g., Dennler et al., (See Bioconjugate Chem. 2014, 25, 569-578). For example, coupling one or more glutamine residues of the antibody to a primary amine compound in the presence of Primary amine compounds can be synthesized via transglutaminase-mediated coupling. to directly provide the antibody drug conjugate, payload or linker-payload The primary amine compounds include those which are further used for the synthesis of antibody drug conjugates. Linkers and spacers that are functionalized with reactive groups that can react with compounds that are Antibodies containing glutamine residues may be isolated from natural sources or may be prepared by cleaving one or more glutamine residues. The antibody polypeptide chain can be modified to contain glutamine residues (Glu Techniques for artificially producing tamoxifen-modified antibodies or antigen-binding molecules are within the capabilities of those skilled in the art. In certain embodiments, the antibody is aglycosylated.

[0154] In certain embodiments, the antibody or glutaminyl-modified antibody or antigen-binding molecule At least one polypeptide chain sequence contains at least one glutamine residue. In an embodiment, the antibody or glutaminyl-modified antibody or antigen-binding molecule each has one G In a further embodiment, the antibody or The glutaminyl-modified antibody or antigen-binding molecule has one or more glutaminyl groups at a site other than Gln 295 in the heavy chain. Included herein are those carrying the N297Q mutation described herein. Briefly, in some embodiments, the antibody contains a glutamine residue. The antibody containing the nucleotides is transglutaminase-containing, as described in more detail below, in the presence of the enzyme transglutaminase. In some embodiments, the Asn297Gln (N297Q) adenosine is treated with a primary amine compound. The antibody containing the mutation is then subjected to a step of cleavage in the presence of the enzyme transglutaminase, as described in more detail below. In some embodiments, the amino acid sequence of Gln295 (Q295) is treated with a primary amine compound, as described above. The antibody containing the residue is transglutaminase-containing, as described in more detail below, in the presence of the enzyme transglutaminase. In some embodiments, the Gln55 (Q55) residue is treated with a primary amine compound, such as those described above. The antibody containing the group is reacted with the enzyme transglutaminase in the presence of the enzyme transglutaminase, as described in more detail below. For example, in some embodiments, such Such antibodies can be prepared by site-directed mutagenesis to remove or disable sequences or can insert a glutamine residue at a site away from any interfering structures. Antibodies can also be isolated from natural or artificial sources.

[0155] (Primary amine compounds) Transglutaminase-mediated coupling of glutamine-containing antibodies (or antigen-binding compounds) The primary amine compounds useful in the method include any primary amine compounds that are deemed useful by those skilled in the art. Typically, the primary amine compound has the formula HN-R, where R is It can be any group that is compatible with the antibody and reaction conditions. In some embodiments, R is , alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl.

[0156] In some embodiments, the primary amine compound has a reactive group or a protected reactive group. Useful reactive groups include azides, alkynes, cycloalkynes, thiols, alcohols, and the like. Alkyl, ketone, aldehyde, acid, ester, hydrozide, analine, In some embodiments, the reactive group is an azide, an alkyne, a sulfonyl group, or an amine. selected from the group consisting of hydryl, cycloalkyne, aldehyde, and carboxyl .

[0157] In certain embodiments, the primary amine compound is according to the formula HN-LL-X, wherein: LL is a divalent spacer and X is a reactive group or a protected reactive group. In some embodiments, LL is a divalent polyethylene glycol (PEG) group. , X is selected from the group consisting of -SH, -N3, alkyne, aldehyde, and tetrazole. In certain embodiments, X is -N3.

[0158] In certain embodiments, the primary amine compound is according to one of the following formulas: R: H2N-(CH2) n -X; H2N-(CH2CH2O) n -(CH2) p -X; H2N-(CH2) n -N(H)C(O)-(CH2) m -X; H2N-(CH2CH2O) n -N(H)C(O)-(CH2CHO) m -(CH2) p -X; H2N-(CH2) n -C(O)N(H)-(CH2) m -X; H2N-(CH2CH2O) n -C(O)N(H)-(CH2CH2O) m -(CH2) p -X; H2N-(CH2) n -N(H)C(O)-(CH2CHO) m -(CH2) p -X; H2N-(CH2CH2O) n -N(H)C(O)-(CH2) m -X; H2N-(CH2) n -C(O)N(H)-(CH2CH2O) m -(CH2)p -X; and H2N-(CH2CH2O) n -C(O)N(H)-(CH2) m -X; (wherein n is an integer selected from 1 to 12; m is an integer selected from 0 to 12; p is an integer selected from 0 to 2; and X is -SH, -N3, -C≡CH, -C(O)H, tetrazole, and [ka] (selected from the group consisting of:

[0159] In the above, either the alkyl or alkylene (i.e., -CH2-) group can be, for example, , C 1-8 It may be optionally substituted with alkyl, methylformyl, or -SO3H. In embodiments, the alkyl group is unsubstituted.

[0160] In some embodiments, the primary amine compound is: [ka] is selected from the group consisting of:

[0161] In certain embodiments, the primary amine compound is [ka] Exemplary conditions for the above reaction are provided in the Examples below.

[0162] (Linker) The linker LL portion of the conjugates described herein connects the binding agent to the conjugates described herein. In another example, the compound is a moiety, e.g., a bivalent moiety, that covalently attaches the compound to a payload compound. The linker LL is a trivalent linker that covalently attaches the binding agent to the payload compound described herein. Suitable linkers are, for example, those described herein, the contents of each of which are fully incorporated by reference. Antibody-Drug Conjugates and Immunotoxins, as incorporated herein by reference, es and Immunotoxins); Phillips, GL (ed.); Springer Verlag: New York, 2013; Antibody-Drugs Antibody-Drug Conjugates; edited by Ducry, L.; Humana Press, 2013; Antibodies - Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, This can be found in JL (ed.); Springer International Publishing, 2015. Payload The compounds include the compounds of FIG. 1 and their derivatives after attachment to or incorporation by the linker LL. Those skilled in the art will appreciate that a particular functional group on the payload moiety may be a linker and / or a linking group. It will be appreciated that these groups are convenient for linking or bonding to an amino group. These include amines, hydroxyls, phosphates, and sugars.

[0163] In certain embodiments, the linker is stable under physiological conditions. Thus, the linker is cleavable, e.g., in the presence of an enzyme or at a particular pH range or value. In some embodiments, the receptor can release at least the payload portion. The anchor comprises an enzyme-cleavable moiety. Exemplary enzyme-cleavable moieties include a peptide bond, an endonucleaser, an enantiomer, an amino acid ... These include, but are not limited to, ester linkages, hydrazone linkages, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0164] In some embodiments, the linker comprises a non-cleavable moiety. In the embodiment, the non-cleavable linker is [ka] or a residue thereof. In some embodiments, the non-cleavable linker-payload Do is [ka] or a positional isomer thereof. In some embodiments, the non-cleavable linker is [ka] or a residue thereof. In some embodiments, the non-cleavable linker-payload Do is [ka] or a positional isomer thereof. In one embodiment, the linker is maleimidocyclohexyl cyclohexanecarboxylate or 4-(N-maleimidomethyl)cyclohexanecarboxylic acid (MCC) In the structure, [ka] represents a bond with a binder. In some examples, [ka] shows, for example, click chemistry residues obtained by reaction of a binder with a linker payload. vinegar.

[0165] In some embodiments, suitable linkers include a single binding agent, e.g., an antibody. These include, but are not limited to, those chemically linked to two cysteine ​​residues in Such linkers do not bind to antibodies that are destroyed as a result of the conjugation process. The disulfide bonds in the hydroxylase may act to mimic the disulfide bonds in the hydroxylase.

[0166] In some embodiments, the linker comprises one or more amino acids. Acids include natural, unnatural, standard, non-standard, proteinogenic, and non-proteinogenic. and L- or D-α-amino acids. In some embodiments, the linker is , alanine, valine, leucine, isoleucine, methionine, tryptophan, phenyl Alanine, proline, glycine, serine, threonine, cysteine, tyrosine, asparagus Gin, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or a combination thereof. In some embodiments, one or more side chains of the amino acid are linked to a side chain group as described below. In some embodiments, the linker comprises valine and citrulline. The anchors include lysine, valine, and citrulline. The anchors include lysine, valine, and alanine. Car contains valine and alanine.

[0167] In some embodiments, the linker comprises a self-immolative group. In certain embodiments, the self-immolative group is p- p-aminobenzyl (PAB) or its derivatives. Useful derivatives include p-aminobenzyl Those skilled in the art will appreciate that the self-immolative group can separate the payload from the remainder of the linker. You will be aware that chemical reactions can occur that release atoms.

[0168] In some embodiments, the reactive group RG' is capable of forming one or more bonds with a binding agent. The reactive group RG' can be any reactive group known to those skilled in the art to be capable of reacting with a bond. React with the combination (e.g., antibody) at its cysteine ​​or lysine residues, or at the azide moiety , for example, by reacting with a PEG-N3 functionalized antibody at one or more glutamine residues to form a compound of formula A, Aa, or A The compound b is a compound containing a moiety capable of forming a compound of formula b in its structure. After jugation, the reactive group becomes a reactive group (RG) or a linker residue. Exemplary reactive groups are: Examples include haloacetyls, isothiocyanates, succinyl groups, etc., which can react with the coupling agent. Examples include those containing an imide, N-hydroxysuccinimide, or maleimide moiety. Not limited to these.

[0169] In certain embodiments, reactive groups include, but are not limited to, alkynes. In certain embodiments, the alkyne undergoes 1,3-cycloaddition with an alkyne in the absence of a copper catalyst. An alkyne that can undergo a silylating reaction, such as a strained alkyne. Strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, e.g., cyclooctyne, and benz-annulated alkynes. Suitable alkynes include dibenzoazacyclooxo Kuching or [ka] , dibenzocyclooctyne or [ka] , biarylazacyclooctynone or [ka] , difluorinated cyclooctyne or [ka] , substituted, for example, fluorinated alkynes, aza-cycloalkynes, bicycle[6.1.0]nonynes or [ka] Particularly useful alkynes include, but are not limited to, Well, [ka] Examples include:

[0170] In some embodiments, the binding agent is directly bound to RG. In certain embodiments, the binding agent is attached to RG via a spacer. , is attached to RG via a PEG spacer. In some embodiments, the binding agent comprises one or more Each azide group is reacted with RG' to form RG. In certain embodiments, the binding agent is linked to a glutamine residue. Exemplary -N3 derivatized binders, their preparation methods, and their association with RG' are described below. Methods for its use in reactions are provided herein. In certain embodiments, RG' is an alkyne suitable for participating in a 1,3-cycloaddition, and RG′ is a coupling agent between RG′ and an azide-functionalized linking agent. As a further example, in one embodiment, In this case, RG is [ka] or a mixture of each positional isomer. As described herein.

[0171] In the formulas herein, each AA 1 is an amino acid. In some instances, each AA 2 is an amino acid. In the example, each AA 2 is a dipeptide. Some examples of the formulas herein In each AA 2 is a tripeptide. Each AA 1 or AA 2 As a preferred amino acid These include natural, non-natural, canonical, non-canonical, proteinogenic, non-proteinogenic, and In some embodiments, the linker is an L- or D-amino acid. , valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, Proline, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamic acid, thamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or cysteine In some embodiments, the amino acid sequence includes thiamin, thiamin, a derivative thereof, or a combination thereof. One or more side chains of the AA acid are linked to the following side chain groups: 2 teeth , valine-citrulline. In some embodiments, AA 2 Citrulline-Valine In some embodiments, AA 2 is valine-alanine. In an embodiment, AA2 is alanine-valine. In some embodiments, AA 2 teeth, In some embodiments, AA is valine-glycine. 2 is glycine-valine In some embodiments, AA 2 is valine-citrulline. In AA 2 is citrulline-valine. In some embodiments, AA 2 is glutamic acid-valine-citrulline. In some embodiments, AA 2 is glutami In some embodiments, AA is valine-citrulline. 2 is a lysine-valine-amino acid In some embodiments, AA 2 is lysine-valine-citrulline In some embodiments, AA 2 is glutamic acid-valine-citrulline. In some embodiments, AA 2 is glutamine-valine-citrulline-PABC.

[0172] Those skilled in the art will recognize PABC as having the following structure: [ka] The PABC residue is a p-aminobenzyloxycarbonyl residue. Groups have been shown to facilitate cleavage of certain linkers in vitro and in vivo.

[0173] In some instances, the linker is MC-Suc-PEG. m -vc-PAB. The subscript m is 0 to In some examples, the subscript m is an integer between 0 and 4. In some examples, the subscript m is 0. The subscript m is 1. In some examples, the subscript m is 2. In some examples, In some examples, subscript m is 3. In some examples, subscript m is 4.

[0174] In some instances, the linker is DIBAC-suc-PEG. m -vc-PAB. The subscript m is is an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, , the subscript m is 1. In some examples, the subscript m is 2. In some examples, subscript m is 3. In some examples, subscript m is 4.

[0175] In some instances, the linker is MAL-PEG m -Lys(COT-CD)-vc-PAB. The letter m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the linker is MAL-PEG. m -N(suc-DIBAC-CD)Lys-vc-PAB. The letter m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4.

[0176] In some examples, the linker is DIBAC-suc-Lys(mc-pip-CD)-vc-PAB. The subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, subscript m is 3. In some examples, subscript m is 4.

[0177] In some instances, the linker is DIBAC-Suc-PEG m -N(CD-COT)Lys-vc-PAB. The subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. .

[0178] In some instances, the linker is BCN-PEG m -Lys(COT-CD)-vc-PAB. The letter m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4.

[0179] In some instances, the linker is DIBAC-Suc-PEG m -d-Lys(PEG4-COT-CD)-vc-PAB The subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. is.

[0180] In some instances, the linker is BCN-PEG-d-Lys-(PEG m -COT-CD)-vc-PAB The subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. do.

[0181] In some instances, the linker is DIBAC-suc-PEG. m -Val-Ala. The subscript m is , an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4.

[0182] In some instances, the linker is PEG m -d-Lys-Val-Ala. The subscript m is 0 to In some examples, the subscript m is an integer between 0 and 4. In some examples, the subscript m is 0. The subscript m is 1. In some examples, the subscript m is 2. In some examples, In some examples, subscript m is 3. In some examples, subscript m is 4.

[0183] In some instances, the linker is DIBAC-suc-PEG. m -d-Lys(PEG m )-vcPAB. The subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, subscript m is 3. In some examples, subscript m is 4.

[0184] In some instances, the linker is BCN-PEG m -d-Lys(PEG m )-Val-Ala. The letter m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4.

[0185] In some instances, the linker is BCN-PEG m -d-Lys(PEG m )-vcPAB. Subscript m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, subscript m is 3. In some examples, subscript m is 4.

[0186] In some examples, the linker is BCN-PEG-d-Lys-Val-Ala. , an integer from 0 to 4. In some examples, the subscript m is 0. In some examples, In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4.

[0187] In some instances, the linker is DIBAC-suc-PEG. m-d-Lys-vcPAB. m is an integer from 0 to 4. In some examples, the subscript m is 0. In some instances, the subscript m is 1. In some instances, the subscript m is 2. In some examples, subscript m is 3. In some examples, subscript m is 4.

[0188] In some examples, the linker is MC-Suc-PEG4-vc-PAB.

[0189] In some examples, the linker is DIBAC-suc-PEG4-vc-PAB.

[0190] In some examples, the linker is MAL-Lys(suc-DIBAC-CD)-vc-PAB.

[0191] In some examples, the linker is MAL-PEG4-Lys(COT-CD)-vc-PAB.

[0192] In some examples, the linker is MAL-PEG4-N(suc-DIBAC-CD)Lys-vc-PAB.

[0193] In some examples, the linker is DIBAC-suc-Lys(mc-pip-CD)-vc-PAB.

[0194] In some examples, the linker is DIBAC-Suc-PEG4-N(CD-COT)Lys-vc-PAB.

[0195] In some examples, the linker is BCN-PEG4-Lys(COT-CD)-vc-PAB.

[0196] In some examples, the linker is DIBAC-Suc-PEG4-d-Lys(PEG4-COT-CD)-vc-PAB. be.

[0197] In some examples, the linker is BCN-PEG-d-Lys(PEG-COT-CD)-vc-PAB.

[0198] In some examples, the linker is DIBAC-suc-PEG4-Val-Ala.

[0199] In some examples, the linker is DIBAC-suc-PEG4-d-Lys(COT-CD)-Val-Ala .

[0200] In some examples, the linker is DIBAC-suc-PEG4-d-Lys(COT-CD)-Val-Ala .

[0201] In some examples, the linker is DIBAC-suc-PEG4-Val-Ala.

[0202] In some examples, the linker is DIBAC-suc-PEG4-d-Lys(COT-CD)-Val-Ala .

[0203] In some examples, the linker is DIBAC-suc-PEG4-d-Lys(PEG4-COT-CD)-vcPAB. be.

[0204] In some examples, the linker is BCN-PEG-d-Lys(PEG-COT-CD)-Val-Ala .

[0205] In some examples, the linker is BCN-PEG-d-Lys(PEG-COT-CD)-vcPAB.

[0206] In some examples, the linker is DIBAC-suc-PEG-d-Lys(PEG-COT-CD)-Val-Ala is.

[0207] In some examples, the linker is BCN-PEG4-d-Lys(COT-CD)-Val-Ala.

[0208] In some examples, the linker is DIBAC-suc-PEG4-d-Lys(COT-CD)-vc-PAB .

[0209] (Reactive Linker-Payload) The conjugates provided herein comprise a reactive linker-peptide having the reactive group RG′ described above. The reactive linker payload can be prepared from erode according to the following method: and can be linked to the cyclodextrin groups and / or binding agents described herein. can.

[0210] In some embodiments, provided herein are at least one payload at least one linked to a moiety and linked to a reactive group via a covalent linker a reactive linker-payload comprising a cyclodextrin moiety, wherein the covalent bond The reactive linker is directly connected to each of the reactive group, the payload moiety, and the cyclodextrin moiety. or indirectly bound to

[0211] As provided herein, in some instances, the reactive group is a lysine amino acid. This is because the reactive group is directly attached to the covalent linker, such as In some of these examples, the The covalent linker is also directly attached to the payload moiety. The hydroxyl linker may be, but is not limited to, MMAE, a steroid, or any of the payloads described herein. This means that the payload is one binding position away from the load. In some cases, the covalent linker is also directly attached to the cyclodextrin moiety. This is because the covalent linker is a cyclodextrin, such as those shown herein. , meaning one bond position away from the cyclodextrin. wherein the covalent linker is a lysine amino acid or a derivative thereof.

[0212] In other instances, the reactive group is indirectly attached to a covalent linker, such as means that the reactive group is more than one bond position away from the covalent linker This also means that the reactive group is attached to the covalent linker via another moiety. For example, the reactive group may be a polyethylene glycol bonded to the covalent linker. In some of these examples, the covalent linker may be attached to a hydroxyl group. The covalent linker is also indirectly linked to the payload moiety. However, payloads such as MMAE, steroids, or any payloads set forth herein may be used. This means that the covalent linker is more than one bond position away from the bond. It also means that the covalent bond is attached to the payload via another moiety. The hydroxyl linker is bonded to a dipeptide, such as, but not limited to, Val-Ala or Val-Cit. The dipeptide may be conjugated to a PAB, and the PAB may be conjugated to the payload. In some of these examples, the covalent linker may be a cyclodextrin. The covalent linker is also indirectly linked to the phosphorus moiety. cyclodextrins, such as cyclodextrins with This means that the covalent linker is attached to the cyclodextrin via another moiety. For example, the covalent linker may be a polyethylene glycol. The polyethylene glycol group may be attached to a polyethylene glycol group which is attached to a reactive group. The reactive group may be attached to the cyclodextrin. In some, the covalent linker is a lysine amino acid or a derivative thereof.

[0213] In some embodiments, provided herein are reactive linkers according to formula (II): -Payload is: [ka] In formula (II), RG' is a reactive group, L is a trivalent linker, and CD is a cyclodextrin. and PA is the payload residue.

[0214] In some examples, the compound according to Formula (II) is a compound according to Formula (IIa): [ka] In formula (IIa), RG' is a reactive group, RG is a reactive linker residue, and LL is a trivalent phosphorus SP is, in each occurrence, independently, absent or a spacer group; and PA is The payload.

[0215] In some instances, the compound according to formula (II) is a compound according to formula (IIb) or (IIc): In some examples, the compound according to formula (II) is a compound according to formula (IIb): [ka] In either formula (IIb) or (IIc), RG' is a reactive group and LL is a trivalent linker. SP, in each occurrence, is independently absent or a spacer, and ring A is a tricyclic ring. fused to an azole and a cycloalkyl, cycloalkenyl, cycloalkynyl, Consists of heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl is selected from the group consisting of cycloalkyl, cycloalkenyl, cycloalkynyl, hetero Heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are alkyl , OH, or NR a R b where R a and R b Each of is alkyl or H and PA is a payload. In some examples, ring A is an optionally substituted cycloalkyl group. In some examples, ring A is an optionally substituted cycloalkenyl. In some examples, ring A is an optionally substituted cycloalkynyl. In some examples, ring A is an optionally substituted heterocycloalkyl. In some examples, ring A is an optionally substituted heterocycloalkynyl. In either formula (IIb) or (IIc), the compound represented by either formula (IIb) or (IIc) is Regioisomers (e.g., with respect to the triazole linkage), stereoisomers, and mixtures thereof of the compounds It is a mixture.

[0216] In some examples, the compound according to Formula (II) is a compound according to Formula (IIc): [ka] In formula (IIc), RG' is a reactive group and LL is a trivalent linker. SP is absent or or a spacer, and PA is a payload. In some examples, Included in formula (IIc) are positional isomers (e.g., with respect to the succinimide linkage), stereoisomers, and the like. and mixtures thereof.

[0217] In some examples, the compound according to formula (II) is represented by formula (IIb1), (IIb2), (IIb3), (IIb4), In some examples, the compound according to formula (II) is , a compound according to formula (IIb1): [ka] In formula (IIb1), RG' is a reactive group, LL is a trivalent linker, and SP is absent. or a spacer, and PA is a payload. Included are positional isomers (e.g., with respect to the triazole linkage) of compounds of formula (IIb1), isomers and mixtures thereof.

[0218] In some examples, the compound according to Formula (II) is a compound according to Formula (IIb2): [ka] In formula (IIb2), RG' is a reactive group, LL is a trivalent linker, and SP is absent. or a spacer, and PA is a payload. Included are positional isomers (e.g., with respect to the triazole linkage) of compounds of formula (IIb2), isomers and mixtures thereof.

[0219] In some examples, the compound according to Formula (II) is a compound according to Formula (IIb3): [ka] In formula (IIb3), RG' is a reactive group, LL is a trivalent linker, and PA is a payload. In some instances, Formula (IIb3) includes positional isomers of the compound of Formula (IIb3). (e.g., with respect to the triazole linkage), stereoisomers, and mixtures thereof.

[0220] In some examples, the compound according to Formula (II) is a compound according to Formula (IIb4): [ka] In formula (IIb4), RG' is a reactive group, LL is a trivalent linker, and PA is a payload. In some instances, Formula (IIb4) includes positional isomers of the compound of Formula (IIb4). (e.g., with respect to the triazole linkage), stereoisomers, and mixtures thereof.

[0221] In some examples, the compound according to Formula (II) is a compound according to Formula (IIb5): [ka] In formula (IIb5), RG' is a reactive group, LL is a trivalent linker, and ne is an integer of 0 to 4. and PA is the payload. In some examples, e is 0. In some examples, In some instances, e is 1. In some instances, e is 2. In some instances, e is 3. In some examples, e is 4. In some examples, the compound represented by formula (IIb5) is are positional isomers (e.g., with respect to the triazole linkage), stereoisomers of compounds of formula (IIb5) , and mixtures thereof.

[0222] In some examples, the compound according to Formula (II) is a compound according to Formula (IIb6): [ka] In formula (IIb6), RG' is a reactive group, LL is a trivalent linker, and ne is an integer of 0 to 4. and PA is the payload. In some examples, e is 0. In some examples, In some instances, e is 1. In some instances, e is 2. In some instances, e is 3. In some examples, e is 4. In some examples, the compound represented by formula (IIb6) is are positional isomers (e.g., with respect to the triazole linkage), stereoisomers of compounds of formula (IIb6) , and mixtures thereof.

[0223] In some examples, the compound according to Formula (II) is a compound according to Formula (IIc1): [ka] In formula (IIc1), RG' is a binder, LL is a trivalent linker, and PA is a payload. Formula (IIc1) also includes its positional isomers (e.g., succinimide linkages). ), stereoisomers, and mixtures.

[0224] In some examples, the compound according to Formula (II) is a compound according to Formula (IId): [ka] In formula (IId), RG' is a reactive group, and SP 1 and SP 2 is, in each case independently, absent or a spacer group residue, where SP 1 comprises a trivalent linker, and AA 1 is an amino acid residue is a trivalent linker comprising a group; 2 is a dipeptide residue, and PEG is polyethylene glycol. is the chol residue, and PAB is [ka] where: [ka] indicates the atom at which the PAB is attached to the adjacent group in the formula, and CD, in each occurrence, independently and wherein at least one CD is present and absent or a cyclodextrin residue. where subscript m is an integer from 0 to 5, subscript p is 0 or 1; and PA is Payload. In these examples, the subscript m is 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In some examples, the subscript The letter p is 1.

[0225] In some examples of formula (IId), CD has the structure: [ka] wherein RG is, independently in each occurrence, a reactive linker residue; It is the base, SP 3 is, in each occurrence, independently absent or a spacer group residue; C 1-6 a alkylene, -NH-, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(C H2) u -C(O)-, -C(O)-NH-(CH2) v -, and combinations thereof, wherein The subscript e is an integer from 0 to 4, the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. q is an integer from 0 to 5; and [ka] is a group in which the modified CD group is SP 1 or AA 1 represents an atom bonded to

[0226] In some examples, the compound according to Formula (II) is a compound according to Formula (IIe): [ka] In formula (IIe), RG′ is a reactive group, RG is a reactive group residue; SP 2 In each case, independently absent or a spacer group residue, where AA 2 is a dipeptide residue, and P EG is a polyethylene glycol residue and PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula, and CD represents absence or cyclode. a kistrin residue, where subscript m is an integer from 0 to 5, and subscript p is 0 or 1; and PA is the payload portion. In these examples, the subscript m can be 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is The subscript m is 1. In some examples, the subscript m is 2. In some examples, , the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In this case, the subscript p is 1.

[0227] In some examples, the compound according to formula (II) is a compound according to formula (IIe1): [ka] In formula (IIe1), RG′ is a reactive group, and SP 2 is, in each occurrence independently, absent or a space a sir group residue, where AA 2 is a dipeptide residue, and PEG is polyethylene glycol. PAB is a hydroxyl residue, [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula, and CD represents absence or cyclode. a kistrin residue, where subscript m is an integer from 0 to 5, and subscript p is 0 or 1; and PA is the payload portion. In these examples, the subscript m can be 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is The subscript m is 1. In some examples, the subscript m is 2. In some examples, , the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, the subscript p is 0. In this case, the subscript p is 1.

[0228] In some examples, the compound according to formula (II) is a compound according to formula (IIe2): [ka] In formula (IIe), RG' is a reactive group, and SP 2 is, in each occurrence independently, absent or a space A AA 2 is a dipeptide residue, and PEG is a polyethylene glycol residue and PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula, and CD represents the cyclodextrin residue, the subscript m is an integer from 0 to 5, the subscript p is 0 or 1, and PA is In these examples, the subscript m is 0, 1, 2, 3, 4, or 5. In some examples, the subscript m is 0. In some examples, the subscript m is 1. In some examples, the subscript m is 2. In some examples, the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript The letter m is 5. In some instances, the subscript p is 0. In some instances, The subscript p is 1.

[0229] In some examples, the compound according to formula (II) is a compound according to formula (IIf): [ka] In formula (IIf), RG' is a binder, RG is a reactive group residue, and SP 1 , SP 2 , and SP 3 teeth, in each instance, independently, absent or a spacer group residue; AA 2 is a dipeptide residue where PEG is a polyethylene glycol residue and PAB is [ka] where: [ka] represents the atom where the PAB is bonded to the adjacent group in the formula, and CD represents the cyclodextrin residue, the subscript q is an integer from 0 to 5, the subscript m is an integer from 0 to 5, and the subscript The letter p is 0 or 1; and PA is a payload moiety. In some examples, m is 0. In some examples, m is 1. In some examples, m is 2. In some examples, m is 3. In some examples, m is 4. In some examples, In some examples, m is 5. In some examples, p is 0. In some examples, p is 1. In some examples, q is 0. In some examples, q is 1. In some examples, q is 2. In some examples, q is 3. In some examples, , q is 4. In some examples, q is 5. In some examples of formula (IIf), S P 1 is non-existent, and SP 2 In each case, C 1-6 Alkylene, -NH-, -C(O)-, (-CH2-CH2 -O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-, -C(O)-(CH2) u -C(O)-, -C(O)-NH-(CH2) v -, and and combinations thereof, wherein the subscript e is an integer from 0 to 4; The subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8.

[0230] In some examples, including any of the foregoing, the compound of formula (II) comprises at least In a specific example, the compound of formula (I) comprises one CD group. ), Formula (IIe1), or Formula (IIe2), and containing one CD group. wherein the compound of formula (II) is a compound of formula (IIf) as described herein and contains one CD group Includes.

[0231] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: In some instances, the CD is an α-cyclodextrin. In some examples, the CD is β-cyclodextrin. In any of these examples, α-, β-, Or the gamma-cyclodextrin is optionally substituted.

[0232] In certain examples, including any of the foregoing, CD may, in each instance, independently select: [ka] is selected from, where: [ka] indicates the atom to which the CD is attached to the adjacent group in the formula. [ka] In one example, the CD is [ka] is.

[0233] In certain examples, including any of the foregoing, RG, in each occurrence, independently represents: Residues derived from click chemistry reactions. In some examples, including any of the above, and RG, in each occurrence, independently comprises a triazole or a fused triazole.

[0234] In certain instances, including any of the foregoing, RG′, in each instance, independently represents , [ka] wherein: [ka] indicates the atom to which the RG′ is bonded to the adjacent group in the formula. wherein RG' is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] In some of these examples, RG′ is, independently in each occurrence: [ka] is.

[0235] In certain examples, including any of the foregoing, RG, in each occurrence, independently represents: [ka] wherein: [ka] indicates the atom to which the RG is attached to the adjacent group in the formula. and RG is, in each occurrence independently, [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] In some of these examples, RG is, independently in each occurrence: [ka] is.

[0236] In some instances, [ka] teeth, [ka] TIFF0007617974000355.tif248170TIFF0007617974000356.tif125170. Also included are salts, solvates, stereoisomeric forms, and their corresponding positional units. isomers, or a mixture of these positional isomers, wherein each [ka] is the bond to the payload; and R 9 is -CH3 or -(CH2)3N(H)C(O)NH2. In some instances, R 9 is -CH3. In some examples, R 9 is -(CH2)3N(H)C(O)N It is H2.

[0237] In some examples, the compound of formula (II) is [ka] TIFF0007617974000359.tif213170TIFF0007617974000360.tif242170TIFF0007617974000361.tif206170TIFF0007617974000362.tif248170, or a salt thereof, a solvate thereof, a stereoisomeric form thereof, or a positional isomer thereof. or a mixture of these positional isomers, wherein each [ka] is the binding with the payload.

[0238] In some examples, -LL- is a group represented by the formula (LL1): [ka] wherein R AA1 , R AA2 , and R AA3 are each independently an amino acid side chain; At least one of them is directly or indirectly bound to -SP-RG-SP-CD. In some instances, R AA1 is a lysine amino acid side chain. In some instances, R AA2 teeth , the valine amino acid side chain. AA3 is the alanine amino acid side chain In some instances, R AA3 is the citrulline amino acid side chain. , R AA1 is a lysine amino acid side chain directly or indirectly attached to CD, and R AA2 and R AA3 are independently valine and alanine amino acid side chains. AA1 Directly on CD directly or indirectly attached lysine amino acid side chain, R AA2 and R AA3 independently, Bali and citrulline amino acid side chains.

[0239] In some instances, including any of the foregoing, R AA1 directly or indirectly Lysine side chain bound to CD, R AA2 and R AA3 are lysine and valine or valine, respectively. The amino acid sequence is either the amino acid sequence or the lysine side chain.

[0240] In certain examples, including any of the foregoing, CD, in each occurrence, independently represents: In some instances, the CD is an α-cyclodextrin. In some examples, the CD is β-cyclodextrin. In any of these examples, α-, β-, Or the gamma-cyclodextrin is optionally substituted.

[0241] In certain examples, including any of the foregoing, CD may, in each instance, independently select: [ka] is selected from, where: [ka] indicates the atom to which the CD is attached to the adjacent group in the formula. [ka] In one example, the CD is [ka] is.

[0242] In some examples, including any of the foregoing, -AA 2 -teeth, [ka] is.

[0243] In some examples, including any of the foregoing, the spacer group in each instance is , independently, C 1-6 Alkylene, -NH-, -C(O)-, (-CH2-CH2-O) e , -NH-CH2-CH2-(-O-CH2-CH2) e -C(O)-.

[0244] In certain examples, including any of the foregoing, the subscript m, q, or e is 1. In some examples, including any of the foregoing, the subscript m is 1. In some examples, including any of the above, the subscript q is 1. In some examples, including

[0245] In certain examples, including any of the foregoing, the subscript m, q, or e is 2. In some examples, including any of the foregoing, the subscript m is 2. In some examples, including any of the foregoing, the subscript q is 2. In some examples, including:

[0246] In certain examples, including any of the foregoing, the subscript m, q, or e is 3. In some examples, including any of the foregoing, the subscript m is 3. In some examples, including any of the foregoing, the subscript q is 3. In some examples, including:

[0247] In certain examples, including any of the foregoing, the subscript m, q, or e is 4. In some examples, including any of the foregoing, the subscript m is 4. In some examples, including any of the foregoing, the subscript q is 4. In some examples, including:

[0248] In some examples, including any of the foregoing, the subscript p is 1.

[0249] In some instances, including any of those previously mentioned, the PA may be a dolastatin, auric acid, or the like. From taxanes, maytansinoids, plant alkaloids, taxanes, and vinca alkaloids In some examples, PA is a residue of a group selected from the group consisting of: It is an interesting compound.

[0250] In some examples, the compound of formula (II) is: [ka] TIFF0007617974000371.tif239170TIFF0007617974000372.tif214170TIFF0007617974000373.tif205170TIFF0007617974000374.tif220170TIFF0007617974000375.tif216170TIFF0007617974000376.tif234170 or a stereoisomeric form thereof, or a positional isomer thereof, or a mixture of positional isomers thereof. can be.

[0251] In some instances, provided herein are compounds comprising the compounds provided herein. anchor-payload.

[0252] In some instances, provided herein is any compound described herein. a linker comprising a compound as set forth herein attached to an oxygen or primary or secondary nitrogen of The payload.

[0253] In some instances, provided herein are antibodies that bind to an antibody or antigen-binding fragment thereof. and antibody-drug-conjugates comprising the compounds described herein or linker-payloads. is.

[0254] (Method for preparing compounds) The compounds provided herein may be prepared and isolated by any method apparent to one of skill in the art. Exemplary preparation methods are described in detail in the Examples below. In certain embodiments, the compounds provided herein can be prepared according to Figures 1-11 and 13-14. It can be prepared.

[0255] The conjugates described herein may comprise a linker-payload as described herein. is coupled to a binding agent, e.g., an antibody, under standard conjugation conditions. (See, for example, the references herein incorporated by reference in their entirety.) (See Doronina et al., Nature Biotechnology 2003, 21, 7, 778). the antibody is linked to the linker- The linker-payload can be coupled to the antibody. The antibody may be subjected to a reducing agent, such as dithiotheritol, to reduce the disulfides of the antibody. The peptide bond is cleaved and the reduced antibody is purified, for example, by gel filtration. The antibody is treated with a suitable reactive moiety, e.g., a linker-payload containing a maleimide group. By using a solvent such as cyclohexane, the cysteine ​​residue can be coupled to the cysteine ​​residue. , water, DMA, DMF, and DMSO. The linker-payload containing a functionalized ester or acid halide group is linked to the lysine residue of the antibody. Suitable solvents include water, DMA, DMF, and DMSO. Conjugates can be used, for example, by size exclusion chromatography. The protein may be purified using known protein techniques, including filtration, dialysis, and ultrafiltration / diafiltration. This can be done. Binding agents, e.g., antibodies, can also be conjugated via click chemistry reactions In some embodiments of the click chemistry reaction, the linker-payload is an azido Reactive groups that can undergo 1,3-cycloaddition reactions with alkyl groups include, for example, alkynes. Such suitable reactive groups are described above. The antibody may contain one or more azide groups. Such antibodies include, for example, antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such functionalized antibodies include antibodies having at least one Antibodies having glutamine residues, for example, heavy chain Gln295 and / or Gln55, are subjected to the enzyme transglutaminase (TGT). It is induced by treatment with a primary amine compound in the presence of glutaminase. Such antibodies may also include an Asn297Gln (N297Q) mutation. Such functionalized antibodies have at least two glutamine residues, e.g., heavy chain Gln295 and heavy chain Gln306. The antibody having the chain Gln297 is subjected to primary amino acid conversion in the presence of the enzyme transglutaminase. Such antibodies include those with the Asn297Gln (N297Q) mutation. In certain embodiments, the antibody contains a total of two or a total of four glutamine residues. For example, the conjugate may have two heavy chains as described in this paragraph. The antibody to be grafted is accessible to transglutaminase and therefore , one or more additional naturally occurring glutamine residues that can be conjugated, e.g., G In these cases, conjugation by transglutaminase is possible. The antibody after conjugation may have a DAR value greater than 4. Such antibodies may include those having a residue at Asn297 In some embodiments, the antibody comprises a total of two or more Gln(N297Q) mutations. For a total of four or a total of five or six glutamine residues, It has a heavy chain of

[0256] In certain embodiments, the antibody comprises a group A at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295 or Gln295 or Q295 Those skilled in the art will recognize that this is the conserved glutamine residue in the wild-type sequence of many antibodies. In other useful embodiments, the antibody is The antibody sequence can be modified to include glutamine residues. Techniques for doing so are within the capabilities of those skilled in the art (see, for example, Ausubel et al., Current Protocols. Molecular Biology, 1999). l. Biol.).

[0257] In certain embodiments, the antibody contains two glutamine residues, one in each heavy chain. In certain embodiments, the antibody comprises a Q295 residue in each heavy chain. The antibody may contain one, two, three, four, five, six, seven, eight, or more glutamic acid residues. These glutamine residues may be present in the heavy chain, the light chain, or both the heavy and light chains. These glutamine residues can be wild-type or modified residues. The antibody can be prepared according to standard techniques. In some embodiments, the antibody The entity comprises or further comprises a Q55 residue.

[0258] Those skilled in the art will recognize that the antibody is glycosylated at residue N297, which is near residue Q295 in the heavy chain sequence. Glycosylation at residue N297 is associated with a glycosylation at residue Q295. This can interfere with transglutaminase (Dennler et al., supra). Thus, in a preferred embodiment, the antibody is aglycosylated. Thus, the antibody is aglycosylated or aglycosylated. In other words, the antibody has an N297 mutation at position 297. It has been mutated so that it no longer has a paragine residue. In the antibody heavy chain, the antibody heavy chain has a N297Q mutation. Such antibodies also have glycosylation sequences removed. or by site-directed mutagenesis to remove or disable any interfering glycosylation sites. site-specific insertion of glutamine residues at positions away from any other interfering structures Such antibodies can be prepared by selective mutagenesis. It can also be isolated from

[0259] The antibody, which does not have interfering glycosylation, is then treated with a primary amine compound. In embodiments, an aglycosylated antibody is treated with a primary amine compound to form a glutaminyl-modified antibody. In one embodiment, the deglycosylated antibody is treated with a primary amine compound. to generate glutaminyl-modified antibodies.

[0260] Primary amines form covalent bonds with glutamine residues in the presence of transglutaminase The primary amine can be any primary amine capable of reacting with the amine described herein. Transglutaminase is considered suitable by those skilled in the art. It can be any transglutaminase. The enzyme reacts with the free amine group on the primary amine compound and the acyl group on the side chain of the glutamine residue. Transglutaminase is an enzyme that catalyzes the formation of isopeptide bonds between amino acid residues. Also known as protein-glutamine-γ-glutamyltransferase. In embodiments, the transglutaminase is classified under EC 2.3.2.13. The glutaminase can be from any source deemed suitable. In some embodiments, the transglutaminase is microbial. Insuglutaminase is derived from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces g Streptomyces griseo-carneum, Streptomyces lavendulae It has been isolated from Streptomyces lavendulae and Bacillus subtilis. Non-microbial transglutaminases, including animal transglutaminases, can be used. In one embodiment, the transglutaminase can be produced by any technique. It may be obtained from any source found in the art or deemed suitable by one skilled in the art. In certain embodiments, transglutaminase is obtained from a commercial source. .

[0261] In certain embodiments, the primary amine compound is further In these embodiments, the glutaminyl-modified antibody comprises a reactive group capable of reacting with the React with or react with a reactive payload compound or reactive linker-payload compound These can be treated to form antibody-payload conjugates. In embodiments, the primary amine compound comprises an azide as described herein.

[0262] In one embodiment, the glutaminyl-modified antibody is reacted with a reactive linker-payload. The antibody is then reacted with or treated with a hydroxybenzoate to form the antibody-payload conjugate. The reaction may proceed under conditions deemed suitable by one skilled in the art. In the method, a glutaminyl-modified antibody is combined with the glutaminyl-modified antibody and a linker-payload. and the reactive linker-payload compound under conditions suitable to form a bond between the compound. Suitable reaction conditions are well known to those skilled in the art.

[0263] Exemplary reactions are provided in the Examples below.

[0264] In some examples, provided herein are methods of making conjugates. Thus, the compound is treated or contacted with a coupling agent under coupling conditions. wherein the compound is linked to at least one payload moiety and covalently bound a reactive linker linked to at least one cyclodextrin moiety via a reactive linker; wherein the covalent linker is a covalent linker comprising the reactive linker, the payload moiety, the synaptone moiety, the The method is such that the cyclodextrin moiety is directly or indirectly bonded to each of the cyclodextrin moieties. In some instances, the compound that reacts with the binder is a compound according to formula (II): [ka] In formula (II), RG' is a reactive group, L is a trivalent linker, and CD is a cyclodextrin. and PA is the payload residue.

[0265] In some instances, the binding agent reactive with the compound of formula (II) is an antibody or an antigen-binding fragment thereof. It is one piece.

[0266] In some examples herein, d-Lys can be replaced with the reagents shown in Figure 20. can be done.

[0267] In some instances, the cyclodextrin-containing moiety is a [3+2] click reaction, Selected from Ruth-Alder reaction, reductive amination, photoclick reaction, or other reactions It can react in a bioorthogonal reaction.

[0268] Other useful bioorthogonal reactions suitable for use with the methods herein include Schwann-Schwarz- Page 11 Page 12 Taudinger ligation, click reaction, tetrazine ligation, and photocatalysis These include, but are not limited to, click reactions.

[0269] The following references provide exemplary reactions that may be used in conjunction with the bioorthogonal reactions presented herein: and reagents: Zheng, Mengmeng et al., Molecules 2015, 20, 3190-3205 (this (The contents of which are incorporated herein by reference in their entirety for all purposes).

[0270] Pharmaceutical Compositions and Methods of Treatment Provided herein are methods for treating and preventing a disease, illness, or disorder, comprising: One or more of the compounds disclosed herein, for example, one or more of the compounds of the formula provided herein Those skilled in the art will readily understand the nature of the disease, disorder, and and / or diseases, including but not limited to those associated with the antigens listed herein. You will understand that this will be

[0271] The compounds described herein may be administered alone or in combination with one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered immediately prior to the administration of the compounds described herein. , or can be administered simultaneously with or immediately after the administration of the same. in combination with one or more additional therapeutic agents; and methods of treatment comprising administering such combinations to a subject in need thereof. .

[0272] Suitable additional therapeutic agents include: a second glucocorticoid, an autoimmune agent, a hormone These include, but are not limited to, anti-cancer drugs, biologics, or monoclonal antibodies. Suitable therapeutic agents include any pharmaceutically acceptable salts of the compounds described herein. These include, but are not limited to, salts, acids, or derivatives thereof.

[0273] In some embodiments of the methods described herein, multiple doses of the methods described herein are administered. a compound described herein (or a compound described herein and an additional therapeutic agent referred to herein) administering to a subject a pharmaceutical composition comprising any combination of The method according to this aspect of the disclosure can involve administering to a subject multiple doses of a compound described herein. As used herein, "sequentially administering" includes administering each compound sequentially. Different doses of the compound may be administered at different times, e.g., at predetermined intervals (e.g., hours, days, weeks, or The present disclosure provides a method for administering a single dose to a patient on different days separated by a single month. one initial dose of a compound described herein, followed by one or more secondary doses of the compound, and and optionally thereafter sequentially administering one or more tertiary doses of the compound.

[0274] The terms "primary dose," "secondary dose," and "tertiary dose" are used herein. The "initial dose" refers to the time sequence of administration of a compound. Thus, the "initial dose" refers to the dose administered at the beginning of a treatment regimen. a "secondary dose" is a dose given after the initial dose (also known as the "baseline dose"); a "tertiary dose" is a dose administered after the secondary dose. The first, second, and third doses may all contain the same amount of a compound described herein. In some embodiments, the initial dose, secondary dose, and secondary doses may differ from each other, typically with respect to frequency of administration. The amounts of compounds contained in the doses, and / or tertiary doses may vary relative to one another during the course of treatment (e.g. In some embodiments, two or more (e.g., For example, doses 2, 3, 4, or 5) are administered as "loading doses" at the start of a treatment regimen. and then as subsequent doses (e.g., "maintenance doses") administered less frequently. can be.

[0275] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered in a dose that is greater than or equal to the dose immediately preceding the immediately preceding dose. doses from 1 to 26 weeks (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7 , 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2 , 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 2 2, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2 weeks or longer) As used herein, the phrase "immediately preceding dose" refers to a series of multiple doses. In the administration of a compound, a compound administered to a patient before the administration of the very next dose in a sequence with no intervening doses is The compound dose is referred to as the dose of the compound.

[0276] The method according to this aspect of the disclosure can include administering to the patient any number of secondary and / or tertiary doses of a compound. For example, in some embodiments, only a single secondary dose may be administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) Similarly, in some embodiments, a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7) doses are administered to the patient. , 8, or more) tertiary doses are administered to the patient. The dosing regimen may be tailored to the specific subject. Lifelong, indefinitely, or until such treatment is no longer therapeutically necessary can be carried out until it is no longer beneficial.

[0277] In embodiments comprising multiple secondary doses, each secondary dose is administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to a patient 1 to 2 weeks or more after the immediately preceding dose. may be administered 1 to 2 months later. Similarly, in embodiments involving multiple tertiary doses, each Each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose The dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In this case, the frequency with which secondary and / or tertiary doses are administered to a patient may vary over the course of the treatment regimen. The frequency of administration may also vary depending on the individual patient's needs after clinical testing, as determined by the physician. may be adjusted during the course of treatment.

[0278] The present disclosure provides that 2-6 loading doses are administered at a first frequency (e.g., once weekly, once every two weeks, once every 3 weeks, once a month, once every 2 months, etc.) and then administered to patients in two or more maintenance doses. For example, this aspect of the disclosure includes a dosing regimen in which a patient receives a less frequent dose of According to the study, if the loading dose is administered once a month, the maintenance dose is administered once every six weeks. It may be administered to the patient once every two months, once every three months, etc.

[0279] The present disclosure relates to compounds and / or conjugates described herein, e.g., compounds of Formula I, Ia, Pharmaceutical compositions of compounds of formula Ib, such as the compounds described herein, their salts, stereoisomers, and compositions comprising a pharmaceutically acceptable carrier, diluent, and / or excipient. Examples of suitable carriers, diluents, and excipients include: buffers to maintain the appropriate composition pH; buffers (e.g., citrate buffer, succinate buffer, acetate buffer, phosphorus acid salt buffer, lactate buffer, oxalate buffer, etc.), carrier proteins (e.g. human serum albumin), saline, polyols (e.g., trehalose, sucrose, sorbitol, etc.), surfactants (e.g., polysorbate 20, polysorbate antimicrobials, and antioxidants. I can't.

[0280] In some instances, provided herein are methods for treating a disease, disorder, or illness. a method for treating a patient with the disorder, the method comprising administering to the patient a compound represented by formula (I), (Ia), (Ib), (Ib1), (Ib2), (Ib3), (Ib4) , (Ib5), (Ib6), (Ic), (Ic1), (Id), (Ie), (Ie1), (1e2), (If), or a compound thereof administering a therapeutically effective amount of the pharmaceutical composition.

[0281] In some instances, provided herein are methods for preventing a disease, disorder, or condition. a method for treating a patient with the disorder, the method comprising administering to the patient a compound represented by formula (I), (Ia), (Ib), (Ib1), (Ib2), (Ib3), (Ib4) , (Ib5), (Ib6), (Ic), (Ic1), (Id), (Ie), (Ie1), (1e2), (If), or a compound thereof administering a prophylactically effective amount of a pharmaceutical composition.

[0282] In some examples, provided herein are methods for treating proliferative disorders, neurodegenerative disorders, immunological disorders, and the like. disorders, autoimmune diseases, inflammatory disorders, skin diseases, metabolic diseases, cardiovascular diseases, and gastrointestinal diseases A method for treating or preventing a disease, disorder, or condition selected from the group consisting of:

[0283] Provided herein are methods for detecting LDLR (low density lipoprotein receptor) protein in cells. A method for modulating protein expression or cholesterol efflux, comprising administering to the cell a drug conjugate ... and contacting the cells with an antibody-derived cell counter (ADC), wherein the ADC contains an antibody and a cyclodeoxynucleotide that targets the cells. and an LXR agonist.

[0284] The proliferative disorder can be any proliferative disorder known to those of skill in the art. In this context, proliferative disorders include, but are not limited to, oncology disorders. wherein the oncological disorder can be any cancer disorder known to those skilled in the art. In embodiments, provided herein are methods of treating or preventing melanoma. In certain embodiments, provided herein are methods for treating or preventing metastatic melanoma. In certain embodiments, provided herein are methods for treating or preventing lung cancer. In one embodiment, provided herein is a method for preventing EGFR-tyrosine In one embodiment, the present invention provides a method for treating or preventing kinase inhibitor-resistant lung cancer. Provided herein are methods for treating or preventing oral cancer. Provided herein are methods for treating or preventing oral squamous cell carcinoma. In certain embodiments, provided herein are methods for treating or preventing prostate cancer. In some embodiments, provided herein are methods for treating or preventing Hodgkin's lymphoma. In some embodiments, provided herein are methods for treating or preventing breast cancer. is a preventative method.

[0285] The neurodegenerative disorder can be any neurodegenerative disorder known to those of skill in the art. In accordance with the present invention, provided herein are methods for treating or preventing Alzheimer's disease. In certain embodiments, provided herein are methods for treating or preventing Parkinson's disease. In some embodiments, provided herein are methods for treating Huntington's disease. In one embodiment, provided herein are methods for treating or preventing muscle atrophy. In one embodiment, the method comprises administering to a subject a compound provided herein, the compound being administered to a subject ... Provided are methods for treating or preventing myelin gene expression. Provided herein are methods for treating or preventing diseases, disorders, or disorders of myelination and remyelination. It is a preventative method.

[0286] The immunological disorder can be any immunological disorder known to those of skill in the art. In accordance with the present invention, provided herein are methods for treating or preventing inflammatory bowel disease. In certain embodiments, provided herein are methods for treating or preventing ulcerative colitis. In certain embodiments, provided herein are methods for treating or preventing Crohn's disease. It is a way to prevent this.

[0287] The inflammatory disorder can be any inflammatory disorder known to those of skill in the art. In one embodiment, provided herein is a method for treating or preventing arthritis. In accordance with the present invention, provided herein are methods for treating or preventing rheumatoid arthritis.

[0288] The metabolic disorder can be any metabolic disorder known to those of skill in the art. In this study, dyslipidemia includes hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, and hyperlipidemia. Polyproteinemia, HDL deficiency, ApoA-I deficiency, and cardiovascular diseases, e.g., coronary artery disease (e.g., including the treatment and prevention of angina, myocardial infarction, and sudden cardiac death; atherosclerosis and restenosis (e.g., treatment and prevention of vascular diseases, including atherosclerosis); Healing atherosclerotic plaques that develop as a result of medical procedures such as angioplasty In one embodiment, the compounds provided herein are selected from the group consisting of: What is discussed is a method for treating or preventing diabetes.

[0289] The cardiovascular disease can be any cardiovascular disease known to those of skill in the art. Provided herein is a method for treating or preventing atherosclerosis, In certain embodiments, provided herein are methods for treating abnormal macrophage processes. The present invention is a method for treating or preventing atherosclerosis resulting from vasoconstriction. In the present invention, a method for treating oxLDL in macrophages is provided. Atherosclerosis resulting from the formation of oxidized low-density lipoprotein (oxLDL) that cannot be oxidized In one embodiment, provided herein is a method for treating or preventing a deficiency. In one embodiment, the method for treating or preventing ischemic heart disease is a method for treating or preventing ischemic heart disease. In one embodiment, the method provided herein is for treating or preventing stroke. Contemplated is a method for treating or preventing hypertensive heart disease. Provided herein are methods for treating or preventing aortic aneurysms. Provided herein are methods for treating or preventing endocarditis. In accordance with the present invention, provided herein is a method for treating or preventing peripheral arterial disease. In embodiments, provided herein are methods for treating any of the diseases provided in this paragraph. The present invention is a method for treating or preventing any of the above-mentioned combinations.

[0290] In some instances, provided herein are methods for modulating the function of a nuclear receptor, By way of non-limiting example, the function may be to inhibit the production of inflammatory mediators (e.g., cytokines, chemokine expression / secretion, cholesterol regulation, cholesterol uptake, cholesterol flux Egress, cholesterol oxidation, migration, chemotaxis, apoptosis and necrosis, inflammatory activity, lipid regulation, The effect may be selected from apoptosis, migration, chemotaxis, gene transcription, and protein expression.

[0291] In some instances, provided herein are methods for treating a disease or condition in a patient in need thereof. A method of treating a disorder comprising administering to the patient a compound or pharmaceutical composition described herein. In some instances, the compound administered is a compound described herein. The antibody-drug conjugates shown in the document are

[0292] In some examples, provided herein are methods for combining a binding agent with a linker-payload complex. and contacting the compound with the binding agent under conditions suitable to form a bond between the binding agent and the compound. The method for antibody-drug conjugates includes the steps of:

[0293] In some examples, provided herein are methods for treating a proliferative disorder, metabolic disorder, inflammatory disease, or the like in a subject. a method for treating a neurodegenerative disease, or a neurodegenerative disorder, comprising administering to the subject a compound or compound(s) described herein. is a method comprising administering an effective therapeutic amount of a pharmaceutical composition. The compound administered is an antibody-drug conjugate as described herein.

[0294] In some instances, provided herein are methods for treating a disease, disorder, or illness in a subject. The method comprises administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition provided herein. In some instances, the compound administered is a compound described herein. The antibody-drug conjugates shown in the document are

[0295] In some instances, provided herein are methods for treating a proliferative disorder in a subject. by administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition provided herein. In some instances, the compound administered is a compound described herein. It is an antibody-drug conjugate.

[0296] In some examples, provided herein are methods for treating a metabolic disorder in a subject. by administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition provided herein. In some instances, the compound administered is a compound described herein. It is an antibody-drug conjugate.

[0297] In some examples, provided herein are methods of treating inflammation in a subject, comprising: administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some examples, the administered compound is an antibody described herein. -drug conjugates.

[0298] In some examples, provided herein are methods of treating a neurodegenerative disease in a subject. administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition provided herein. In some instances, the compound administered is a compound described herein. It is an antibody-drug conjugate that can be used in combination with other drugs. [Example]

[0299] (Example) Presented in this example are schemes that link antibodies to at least three types of payloads. Chlodextrin-(PEG) n The linker is an anti-HER2 antibody and an anti-PRLR (anti-PRLR Ab) (see Figure 1). Two antibodies, maleimide- The azide functionality can be added either directly to the linker-payload (see Figure 13) or via a [3+2] click reaction. The payload was linked to the DIBAC-linker via a carboxyl group (see Figure 14).

[0300] Compared to those without the cyclodextrin moiety, cyclodextrin-(PEG) n Phosphorus Conjugates with carboxyl groups exhibited lower calculated LogP / LogD values ​​and shorter retention times on reverse HPLC. These new compounds showed better aqueous solubility and hydrophilicity (Table 8). The linker-payload was also cleavable in the cathepsin B assay (Table 8). These ADCs showed bioactivity equivalent to that of those without the CD moiety in in vitro assays ( Tables 10, 11, 12).

[0301] As used herein, the symbols and conventions used in the processes and examples herein Examples are taken from contemporary scientific literature, e.g., the Journal, unless otherwise specified. of the American Chemical Society or the Journal of Biological Chemistry Specifically, but not exclusively, the following abbreviations are used in the examples and this specification: Can be used throughout the specification: [Table 1] TIFF0007617974000379.tif245170TIFF0007617974000380.tif242170TIFF0007617974000381.tif180170

[0302] Certain embodiments of the present invention are illustrated by the following non-limiting examples.

[0303] Unless otherwise expressly stated, reagents and solvents were obtained from commercial sources, e.g., Sinopharm Chemical Reagent Co. (SCRC), Sigma-Aldrich, Alfa, or other suppliers.

[0304] 1 1 H NMR and other NMR spectra were recorded on a Bruker AVIII 400 or Bruker AVIII 500. The data were processed using Nuts software or MestReNova software, and the proton shifts were calculated internally. Measured in parts per million (ppm) downfield from a standard (e.g., tetramethylsilane (TMS)) did.

[0305] HPLC-MS measurements were performed on an Agilent 1200 HPLC / 6100 SQ system using the following conditions: .

[0306] Method A for HPLC-MS measurement included the following mobile phase: A: Water (0.01% trifluoroacetic acid (TFA)) B: acetonitrile (0.01% TFA); gradient phase: 5% B increased to 95% B within 15 minutes (min). Flow rate: 1.0 mL / min; Column: SunFire C18, 4.6 x 50 mm, 3.5 μm; Column temperature: 50°C; Detection Detectors: Analog-to-digital converter (ADC), evaporative light scattering detector (ELSD), diode array detector (D AD) (214 nm and 254 nm), electrospray ionization-air ionization (ES-API).

[0307] Method B for HPLC-MS measurement included the following mobile phases: A: water (10 mM NH4HCO3), B: acetonitrile Gradient phase: 5% to 95% B in 15 min; Flow rate: 1.0 mL / min; Column: XBridge C18, 4.6 x 50 m m, 3.5 μm; Column temperature: 50°C; Detector: ADC ELSD, DAD (214 nm and 254 nm), mass selective detection The detector (MSD) (ES-API) was included.

[0308] LC-MS measurements were performed on an Agilent 1200 HPLC / 6100 SQ system using the following conditions:

[0309] Method A for LC-MS measurement includes: WATERS 2767; Column: Two columns connected in series. Shimadzu Shim-Pack, PRC-ODS, 20 x 250 mm, 15 μm; Mobile phase: A: water (0.01% TFA), B: Acetonitrile (0.01% TFA); Gradient phase: 5% B increased to 95% B within 3 min; Flow rate: 1 0.8~2.3mL / min; Column: SunFire C18, 4.6×50mm, 3.5μm; Column temperature: 50℃; Detector: AD C ELSD, DAD (214 nm and 254 nm), and ES-API were included.

[0310] Method B for LC-MS measurement: Equipment: Gilson GX-281; ​​Column: Xbridge Prep C18 10 μm OBD, 19 × 250 mm; Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile; Gradient phase: ≥ 3 min 5% to 95% B in the column; Flow rate: 1.8-2.3 mL / min; Column: XBridge C18, 4.6 x 50 mm, 3.5 μm; Ram temperature: 50°C, detectors: ADC ELSD, DAD (214 nm and 254 nm), MSD (ES-API) were included.

[0311] Preparative high-pressure liquid chromatography (HPLC) in an acidic solvent system (Method A) or a basic solvent system (Method B) Preparative HPLC was performed on a Gilson GX-281 instrument. For acidic solvent systems, a Waters SunFire 10 μm C18 column was used. A column (100 Å, 250 × 19 mm) was used. Solvent A for preparative HPLC was water / 0.05% TFA, and solvent B was water / 0.05% TFA. The solvent was acetonitrile. The elution conditions were 5% to 10% over 20 minutes at a flow rate of 30 mL / min. The basic solvent system was a linear gradient increase of solvent B to 0%. The solvent A for preparative HPLC was water / 10 mM ammonium bicarbonate (N The solvent B was acetonitrile. The elution conditions were 20 mL / min at a flow rate of 30 mL / min. The gradient was a linear increase of solvent B from 5% to 100% over 2 min.

[0312] Flash chromatography was performed using Agela Flash Column Silica-CS cartridges. reversed-phase flash chromatography was performed on a Boston ODS or Agel a Performed on a Biotage instrument using a C18 cartridge. Table 1. Linker-Payload List [Table 2] Table 2A. Linker-payload structures [Table 3] TIFF0007617974000384.tif249170TIFF0007617974000385.tif239170TIFF0007617974000386.tif245170TIFF0007617974000387.tif196170TIFF0007617974000388.tif236170TIFF0007617974000389.tif244170TIFF0007617974000390.tif118170Table 2B. Linker-Payload cLogP [Table 4] +≦-6; -6<++≦-3; -3<+++≦1; 1<++++≦5; 5<+++++≦9.5 Table 3A. Linker-payload analysis results [Table 5] Table 3B. Linker-payload analysis results [Table 6] TIFF0007617974000394.tif54170

[0313] Examples 1-6 include, among other things, linkers, linker-payloads, and / or the preparation of specific compounds useful as synthetic intermediates for the synthesis of conjugates. It has been done.

[0314] Example 1 Preparation of Intermediate 5a (see Figure 2) ((2R)-6-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4,6,8,13,15-hexaene -10-yn-2-yl}-4-oxobutanamido)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole] (1-yl)hexanamido]hexanoic acid (5a) To a mixture of HD-Lys(Boc)-OH (11a, 0.25 g, 1.0 mmol) in DMF (10 mL) was added 6-maleimidocaproic acid. Sulfonic acid-NHS (10a, 0.31 g, 1.0 mmol) and DIPEA (0.26 g, 2.0 mmol) were added sequentially at room temperature. The reaction was stirred overnight at room temperature until compound 11a was completely consumed, which was monitored by LCMS. The mixture was purified by reversed-phase flash chromatography (0-100% TFA in water (0.05% TFA) Direct separation with acetonitrile gave intermediate Boc-12a (ESI m / z: 440 (M+H) + ) is colorless This was obtained as an oil, which was dissolved in DCM (5 mL). To the solution was added TFA (0.5 mL) dropwise at 0°C. The reaction was stirred at room temperature for 1 hour. The volatiles were removed in vacuo to give crude 12a (ESI m / z: 340 (M / z) +H) + ) was obtained, which was used in the next step without further purification. A mixture of crude compound 12a (0.21 g, 0.62 mmol) was treated with activated ester 10b (0.20 g, 0.50 mmol) and DI PEA (50 mg, 0.39 mmol) was added sequentially at room temperature. The reaction mixture was stirred overnight at room temperature, and then the compound The product 12a was completely consumed, which was monitored by LCMS. Separation was performed directly by phase flash chromatography (0-100% acetonitrile in water). This gave compound 5a (0.10 g, 20% yield over three steps from 10a) as a white solid. ESI m / z : 627(M+H) + . [ka]

[0315] Example 2 Preparation of Intermediate 5b (see Figure 2) ((2S)-6-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4,6,8,13,15-hexaene -10-yn-2-yl}-4-oxobutanamido)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole] (1-yl)hexanamido]hexanoic acid (5b) Intermediate 5a, except that HL-Lys(Boc)-OH (11b) was used instead of HD-Lys(Boc)-OH (11a). Following the procedure of Example 1 for 11b, intermediate 5b was obtained as a white solid (0.16 g from 11b, 19% yield). ESI m / z: 627(M+H) + . [ka]

[0316] Example 3 Preparation of Intermediate 5c (See Figure 2) ((2R)-2-amino-6-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4,6,8,13,15- Hexaen-10-yn-2-yl}-4-oxobutanamido)hexanoic acid (12c) To a mixture of commercially available HD-Lys(Fmoc)-OH (11c, 0.55 g, 1.5 mmol) in anhydrous DMF (3.0 mL), DIPEA (0.38 g, 3.0 mmol) and compound 10b (see Accounts of Chemical Research, 2011, 805-815 ) (0.60 g, 1.5 mmol) were added sequentially at room temperature. Compound 11c was confirmed to be completely synthesized by LCMS. The resulting solution was stirred at room temperature for 2 hours until complete consumption. Purification by flash chromatography (0–80% acetonitrile in water) gave intermediate Fmo c-12c (0.48 g, 49% yield) was obtained as a white solid. ESI m / z: 656.3 (M+H) + DMF (3 mL) To a mixture of intermediate Fmoc-12c (0.15 g, 0.23 mmol) in HCl was added diethylamine (0.1 mL). The mixture was stirred at room temperature for 1 hour, and LCMS showed that Fmoc-12c was completely consumed. The mixture was filtered and the filtrate was purified by preparative HPLC (Method B) to give compound 12c (42 mg, 4 2% yield) was obtained as a white solid. ESI m / z: 434 (M+H) + . [ka]

[0317] ((2R)-6-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4,6,8,13,15-hexaene -10-yn-2-yl}-4-oxobutanamido)-2-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrole] (1-yl)-3,6,9,12-tetraoxapentadecan-15-amido]hexanoic acid (5c) A mixture of compound 12c (40 mg, 92 μmol) in DMF (2 mL) was treated with compound 4d (41 mg, 0.11 mmol) in DMF. ) and TEA (18 mg, 0.18 mmol) were added successively at room temperature. The mixture was stirred at room temperature for 1 hour, and then When monitored by S, most of the compound 4d was consumed. The mixture was subjected to reverse-phase flash chromatography (0–100% acetonitrile in water) to determine its Immediate isolation afforded intermediate 5c (33 mg, 47% yield) as a pale yellow oil. ESI m / z: 761(M+H) + . [ka]

[0318] Example 4 Preparation of Intermediate 6b (see Figure 3) ([31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis (Hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2 .2.2 3,6 .28 ,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl 4-methylbenzene Benzene-1-sulfonate (14) 2 ) A solution of α-cyclodextrin (13, 10 g, 10 mmol) in anhydrous pyridine (Py) (0.32 L) was added to 4-chloride. Toluenesulfonyl (TsCl) (20 g, 0.10 mol) was added at room temperature, and the mixture was stirred at this temperature for 15 minutes. The reaction mixture was stirred and immediately quenched with water (60 mL). The reaction mixture was concentrated under reduced pressure to remove the volatiles ( The residue was purified by reversed-phase flash chromatography (0-25% in water). Purification by hexane (acetonitrile) gave product 14 (1.1 g, 10% yield) as a white solid. ESI m / z: 1127(M+H) + .

[0319] (5,10,15,20,25-pentakis(hydroxymethyl)-30-(piperazin-1-ylmethyl)-2,4,7,9 ,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .28 ,11 .2 13,16 .2 18,2 1 .2 23,26 ]dotetracontane-31,32,33,34,35,36,37,38,39,40,41,42-dodecol (6b)) A mixture of compound 14 (1.2 g, 1.0 mmol) in piperazine (1.7 g, 20 mmol) was sealed and heated to 120 °C for 16 h. After the reaction mixture was cooled to room temperature, the mixture was diluted with DMF (3 mL) and acetonitrile (30 mL). The precipitate was collected by filtration and the crude product was then purified by preparative HPLC (Method B). This gave intermediate 6b (0.32 g, 30% yield) as a white solid. ESI m / z: 1041 (M+H) + .

[0320] Example 5 Preparation of Intermediate 7a (see Figure 4) ((2R)-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]-2-{[(9H-fluorene-9- (Iylmethoxy)carbonyl]amino}hexanoic acid (7a) To a mixture of commercially available compound 17 (65 mg, 0.23 mmol, CAS: 1425803-45-7) in DMF (2 mL) was added Fmoc-D- Lys-OH (15a, 85 mg, 0.23 mmol) and triethylamine (52 mg, 0.51 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was purified by reversed-phase flash chromatography (water (0.05 Direct isolation using 0-100% acetonitrile in 100% TFA gave intermediate 7a (85 mg, 70% yield). %) was obtained as a white solid. ESI m / z: 533 (M+H) + . [ka]

[0321] Example 6 Preparation of Intermediate 7b (see Figure 4) ((R)-26-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2,2-dimethyl-4,20-di Oxo-3,8,11,14,17-pentaoxa-5,21-diazaheptacosane-27-oic acid (16) To a mixture of compound 10c (4.6 g, 10 mmol, CAS: 859230-20-9) in DMF (10 mL) was added 15a (3.6 g, 10 The reaction mixture was stirred for 1 hour at 4°C for 1 hour at 4°C for 1 hour at 4°C for 1 minute ... The reaction mixture was stirred at 25°C for 1 hour and monitored by TLC. The mixture was diluted with MgCl and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate and purified by evaporation. The residue was purified by silica gel column chromatography (0-5% methanol in DCM). ) to give 16 (5.5 g, 77% yield) as a colorless oil. ESI m / z: 716. 2(M+H) + . [ka]

[0322] ((24R)-24-(((9H-fluoren-9-yl)methoxy)carbonylamino)-1-(cycloocta-2- (ynyloxy)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazapentacosane-25-oic acid (7b)) To a solution of compound 16 (0.60 g, 0.84 mmol) in DCM (10 mL) was added trifluoroacetic acid (2.0 mL) at 0°C. The reaction solution was warmed and stirred at 25°C overnight until compound 16 was completely consumed, which was analyzed by LCMS. (ESI m / z: 616.3 (M+H) + The volatiles were removed in vacuo to give a crude 15b was obtained and used directly in the next step.

[0323] Following the procedure of Example 5 to make intermediate 7a, except using 15b instead of 15a After purification by silica gel column chromatography (0-5% methanol in DCM), The title compound 7b was obtained as a colorless oil (0.38 g, 49% yield). ESI m / z: 780.3 (M+H) + . [ka]

[0324] Example 7 (General Procedure A (see Figure 5):) Fmoc-vc-PAB-PNP (2a, 0.68-1.0 equivalents) and compound 1 (1e, 1f, 1g, 1h, 1i, or 1m, 1.0 equivalents) To a solution of 100% ethanol (100%) in DMF, DIPEA (5 equiv.) was added via syringe at RT. The mixture was then heated at 25°C for 16-24 hours. After stirring for 1 hour, most of 2a was consumed by LCMS. The resulting mixture was treated with piperidine (0.1 ~1 mL, excess) was added and stirred at 25°C for 2 hours until the Fmoc was completely removed. was monitored by LCMS. After filtration through a membrane, the filtrate was purified by preparative HPLC (Method B). Direct purification gave compound 3 (32-73% yield) as a white solid. Table 4 shows the Specific reaction conditions for making compounds 3e-3g, 3H, and 3I are provided. Table 4 [Table 7] Pip. * = piperidine; purified * :Preparative HPLC method; ** :Contains an equivalent amount of HOBt

[0325] Example 8 (General Procedure B (see Figure 6):) Acid (Fmoc-Val-Ala-OH(2b), Fmoc-dLys(COT)-OH(7a), or Fmoc-dLys(PEG4-COT)-OH(7b) To a solution of 8a in DMF (or DCM / THF in the case of 8b), HATU (1.0-2.8 equiv.) and DIPEA or TEA (2.0-5.0 equiv.) was added at 25°C. The mixture was stirred at 25°C for 30 min, and then the amine (1b- A solution of 1.0 equiv. (1d, 1i, or 3a-I) in DMF (1 mL) was added via syringe. The resulting mixture was stirred at 25°C for 2 to 24 hours until the amine was almost consumed. To the mixture, piperidine or diethylamine (excess) was added and the reaction was continued until the Fmoc was completely removed. The mixture was stirred at 25°C for 1-20 hours and monitored by LCMS. is filtered through a membrane and the filtrate is subjected to preparative HPLC (Method B) or reversed-phase flash chromatography. Direct purification of the compounds 3b-d or 8a-J (23-80% yield) gave white solids. It was. Table 5. Reaction conditions for the synthesis of compounds 3B-D, 3J, and 8A-L following general procedure B [Table 8] Regarding purification - B: Preparative HPLC method B; A: Preparative HPLC method A; RP: RP-flash; RP-B: RP-flash Flash Method B; RP-A: RP-Flash Method A. S ** : DCM / THF=4 / 8(mL) * : 40% of the product from step 1 was used in step 32.

[0326] Example 9 Preparation of Compound 3a (see Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentane amido]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1 S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methyl ethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carbohydrate bamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate ( 3a)) (See WO2012 / 166560, which is incorporated herein by reference in its entirety.) In a 25 mL round-bottom flask, add Fmoc-vc-PAB-PNP (2a) (0.33 g, 0.43 mmol) and anhydrous DMF (5 mL). To the solution, MMAE (1a, 0.21 g, 0.21 mmol), HOBt (38 mg, 0.28 mmol), and DIPEA (74 mg , 0.57 mmol) was added sequentially at 25° C. The mixture was stirred for 2 hours until MMAE was completely consumed by LCMS. The reaction mixture was stirred at 5-30°C for 16 hours. The reaction mixture was filtered through a membrane and the filtrate was analyzed by preparative HPLC (Method A). After further purification, Fmoc-3a was obtained as a white solid, which was dissolved in DMF (5 mL). Diethylamine (0.3 mL) was added to the reaction mixture until the Fmoc was removed by LCMS. The mixture was stirred for 1 hour at 30° C. The reaction mixture was filtered through a membrane and the filtrate was subjected to preparative HPLC (Method A). Further purification gave vcPAB-MMAE (3a) (0.20 g, 58% yield) as a white solid. / z: 562.5(M / 2+H) + .

[0327] Example 10 Preparation of Compound 3b (see Figure 6 and Table 5) ((2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13 -Dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ] Icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamoyl]ethyl]-3-methyl Chilbutanamide (3b) Following general procedure B, compound 3b (60 mg, 23% yield) was obtained as a white solid. ESI m / z : 692(M+H) + . [ka]

[0328] Example 11 Preparation of Compound 3c (see Figure 6 and Table 5) ((2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-9 ,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13, 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamoyl]ethyl]-3- Methylbutanamide (3c) Following general procedure B, compound 3c (36 mg, 28% yield) was obtained as a white solid. ESI m / z : 692(M+H) + . [ka]

[0329] Example 12 Preparation of Compound 3d (see Figure 6 and Table 5) ((1S,4aS,10aR)-6-((S)-2-((S)-2-amino-3-methylbutanamido)propanamide)-N-((1 S,4aS,10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenane (1-carbonyl)-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene Benzene-1-carboxamide (3d) Following general procedure B, compound 3d (0.88 g, 67% yield) was obtained as a white solid. z: 699(M+H) + . [ka]

[0330] Example 13 Preparation of Compound 3e (see Figure 5 and Table 4) (4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl 2-((4bS,8S,8aR)-8-((1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a- Octahydrophenanthrene-1-carbonylcarbamoyl)-4b,8-dimethyl-4b,5,6,7,8,8a ,9,10-Octahydrophenanthrene-3-yloxy)ethyl carbamate (3e) Following general procedure A, compound 3e (14 mg, 68% yield) was obtained as a white solid. ESI m / z : 978(M+1) + . [ka]

[0331] Example 14 Preparation of Compound 3f (See Figure 5 and Table 4) ({4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentane Amido]phenyl}methyl N-({[(4bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydroxy-1,4a-di Methyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl]carbamoyl} -4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]carbamoyl }methyl) carbamate (3f) Following general procedure A, compound 3f (85 mg, 32% yield) was obtained as a white solid. ESI m / z : 991(M+1) + . [ka]

[0332] Example 15 Preparation of Compound 3g (See Figure 5 and Table 4) ({4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentane Amido]phenyl}methyl N-[(1S)-1-{[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy- 1,4a-Dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide }carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl] Carbamoyl}-2-hydroxyethyl]carbamate (3g) Following general procedure A, compound 3g (22 mg, 40% yield) was obtained as a white solid. ESI m / z : 1021(M+1) + . [ka]

[0333] (Example 15-A) Preparation of Compound 3H - See Figure 5 and Table 4 ({4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentane Amido]phenyl}methyl N-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-1 1-Hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0. 0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethyl}carbamate (3H) [ka] Following the general procedure described in Example 7, compound 3H (127 mg, 73% yield) was obtained as a white solid. ESI m / z: 871(M+1) + .

[0334] (Example 15-B) Preparation of Compound 3I - See Figure 5 and Table 4 ({4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentane Amido]phenyl}methyl N-(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro 11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10. 8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl) carbamazepine Bamate (3I) [ka] Following the general procedure described in Example 7, compound 3I (80 mg, 64% yield) was obtained as a white solid. ESI m / z: 963(M+1) + . [ka]

[0335] (Example 15-C) (Preparation of Compound 3J - See Figure 6) ((2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro 11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10. 8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl) carbamazepine Bamoyl]ethyl]-3-methylbutanamide TFA salt (3J) [ka] To a solution of Boc-Val-Ala-OH (2d, 0.31 g, 1.1 mmol) in DMF (15 mL) was added HATU (0.48 g, 1.3 mmol) and D IPEA (0.35 g, 2.7 mmol) was added at 10° C. The mixture was stirred at 10° C. for 30 min, and then the amine (1i , 0.50 g, 0.90 mmol) was added to the mixture. The reaction mixture was stirred until the amine was completely consumed. The mixture was stirred at 10° C. for 1 hour. The mixture was quenched with water and extracted with ethyl acetate. The organic solution was washed with brine, dried over sodium sulfate, and concentrated in vacuo. The product was purified by silica gel column chromatography (50% ethyl acetate in petroleum ether). This gave Boc-3J, which was dissolved in DCM (9 mL). To the solution was added TFA (3 mL) at 10° C. The reaction mixture was stirred at 10° C. for 2 hours and then concentrated in vacuo to give compound 3J (0.53 g). (84% yield) was obtained as the TFA salt, which was used in the next step without further purification. ESI m / z: 728(M+1) + .

[0336] Example 16 Preparation of Compound 8a (see Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetate Amido]hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido do]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R )-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methylethyl ]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carbamo yl}-2-methylpropyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate (8a) General procedure B was followed by reversed-phase flash chromatography (0-100% TFA in water (0.05% TFA) After purification with acetonitrile, compound 8a (17 mg, 35% yield) was obtained as a white solid. ESI m / z: 1415(M+H) + . [ka]

[0337] Example 17 Preparation of Compound 8b (see Figure 6 and Table 5) ((2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]-N-[(1S)-1-{[ (1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo So-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]Icosa-14,17-Jie phenyl)carbamoyl]ethyl]carbamoyl}-2-methyl Propyl]hexanamide (8b) Following general procedure B, compound 8b (50 mg, 49% yield) was obtained as a white solid. ESI m / z : 984(M+1) + .

[0338] Example 18 Preparation of Compound 8c (see Figure 6 and Table 5) ((2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]-N-[(1S)-1-{[ (1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-o Dibenzo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]Icosa-14,17-di Ethoxy-2-phenyl)carbamoyl]ethyl]carbamoyl}-2-methyl [propyl]hexanamide (8c) Following general procedure B, compound 8c (80 mg, 56% yield) was obtained as a white solid. ESI m / z : 984(M+1) + . [ka]

[0339] Example 19 Preparation of Compound 8d (see Figure 6 and Table 5) ((1S,4aS,10aR)-6-((2S)-2-((2S)-2-((2R)-2-amino-6-(2-(cyclooct-2-ynyl) Acetamido)hexanamido)-3-methylbutanamido)propanamido)-N-((1S,4aS, 10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene- 1-carbonyl)-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl Ruboxamide (8d) Following general procedure B, compound 8d (30 mg, 47% yield) was obtained as a white solid. ESI m / z : 991(M+1) + . [ka]

[0340] Example 20 Preparation of Compound 8e (see Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetate Amido]hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido do]phenyl}methyl N-({[(4bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl {1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl]carbamoyl}-4b, 8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]carbamoyl}methyl ethyl) carbamate (8e) Following general procedure B, compound 8e (65 mg, 62% yield) was obtained as a white solid. ESI m / z : 1283(M+1) + . [ka]

[0341] Example 21 Preparation of Compound 8f (See Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetate Amido]hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido do]phenyl}methyl N-[(1S)-1-{[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a -dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide (carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]ca {rubamoyl}-2-hydroxyethyl]carbamate (8f) Following general procedure B, compound 8f (15 mg, 33% yield) was obtained as a white solid. ESI m / z : 1313.6(M+H) + . [ka]

[0342] Example 22 Preparation of Compound 8g (See Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{1-[2-(cyclooct-2-yn-1-yloxy)a cetoamido]-3,6,9,12-tetraoxapentadecan-15-amido}hexanamido]-3-methyl butanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-[(1S)-1-{[( 1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropanol phenyl-2-yl]carbamoyl}-1-methoxy-2-methylethyl]pyrrolidin-1-yl]-3-methoxy- 5-methyl-1-oxoheptan-4-yl](methyl)carbamoyl}-2-methylpropyl]carbamo {2-methylpropyl}-N-methylcarbamate (8g) Following general procedure B, compound 8g (17 mg, 43% yield) was obtained as a white solid. ESI m / z : 832.2(M / 2+H) + . [ka]

[0343] Example 23 Preparation of Compound 8h (See Figure 6 and Table 5) (N-[(5R)-5-amino-5-{[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydro 1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl] carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl ]carbamoyl}ethyl]carbamoyl}-2-methylpropyl]carbamoyl}pentyl]-1-[2- (Cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca phen-15-amide (8h) Following general procedure B, compound 8h (54 mg, 50% yield) was obtained as a white solid. ESI m / z : 620(M / 2+1) + . [ka]

[0344] Example 24 Preparation of Compound 8i (see Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{1-[2-(cyclooct-2-yn-1-yloxy)a cetoamido]-3,6,9,12-tetraoxapentadecan-15-amido}hexanamido]-3-methyl butanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-(2-{[(4bS,8 S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octa Hydrophenanthrene-1-yl]formamide}carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a ,9,10-Octahydrophenanthrene-3-yl]oxy}ethyl)carbamate (8i) Following general procedure B, compound 8i (40 mg, 30% yield) was obtained as a white solid. ESI m / z : 759(M / 2+1) + . [ka]

[0345] Example 24A Preparation of Compound 8J - (See Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetate Amido]hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido phenyl}methyl N-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-phenyl hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 , 9 .04 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethyl}carbamate (8J) [ka] Following the general procedure described in Example 8, compound 8J (120 mg, 52% yield) was obtained as a white solid. ESI m / z: 1163(M+1) + . [ka]

[0346] Example 24B Preparation of Compound 8K - (See Figure 6 and Table 5) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetate Amido]hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido do]phenyl}methyl N-(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11 -Hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamate (8K) [ka] Following the general procedure described in Example 8, compound 8K (192 mg, 80% yield) was obtained as a white solid. ESI m / z: 1256(M+1)+ .

[0347] Example 24C (Preparation of Compound 8L - See Figure 6 and Table 5) ((2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]-N-[(1S)-1-{[ (1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9 ,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13, 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamoyl]ethyl]ca {rubamoyl}-2-methylpropyl]hexanamide (8L) [ka] Following the general procedure described in Example 8, compound 8L (0.21 g, 46% yield) was obtained as a white solid. ESI m / z: 1021(M+1) + ESI m / z: 1021.5(M+H) + . [ka]

[0348] Example 25 General Procedure C for Making Compounds 9a-i and J-L (See Figure 7) To a solution of alkynes 8a–i, J–L (1.0 equiv.) in the corresponding solvent, α-cyclodextrin- Dido6a (Synth. Commun., 2002, 32(21), 3367-3372; J. Am. Chem. Soc., 2012, 134(46) , 19108-19117; J. Med. Chem., 1997, 40(17), 2755-2761; J. Am. Chem. Soc., 1993, 115(12), 5035-5040) (1.5-3.0 equivalents) was added. The resulting mixture was stirred until compound 8 was almost consumed and the desired intermediate mass was detected. The mixture was stirred at 20 to 30°C for 16 hours to 3 days. After filtration, the resulting mixture was analyzed by preparative HPLC. When purified (or used as is), compounds 9 (9a-i, J-L) (16-78% yield) were obtained as white solids. was obtained as a solid (containing the triazole regioisomer). Table 6. Reaction conditions for the synthesis of compounds 9a-i according to general procedure c [Table 9] * : 9bA and 9bB are two triazole positional isomers. Regarding purification - B: Preparative HPLC method B; A: Preparative HPLC method A; RP: RP-flash; RP-B: RP-flash Flash Method B; RP-A: RP-Flash Method A.

[0349] Example 26 Preparation of Compound 9a (see Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ] dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]trimethyl azole-4-yl)oxy]acetamido}hexanamido]-3-methylbutanamido]-5-(caprolactone) (1S)-1-{[(3R,4S,5S)- 1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl {1-methoxy-2-methylethyl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxohe butan-4-yl](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl pyri]-N-methylcarbamate (9a) Following general procedure C, compound 9a (9.6 mg, 25% yield) was obtained as a white solid. z: 1207(M / 2+H) + . [ka]

[0350] Example 27 Preparation of Compound 9b (see Figure 7 and Table 6) ((2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-1 0,15,20,25,30-Pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodeca Caoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontane-5- 1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxa Acetamido}-N-[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydro Roxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamoyl]e ethyl]carbamoyl}-2-methylpropyl]hexanamide (9b) General procedure C was followed to give triazole regioisomer 9bA (first peak in LCMS, 13 mg, 2 2% yield) and 9bB (second peak in LCMS, 19 mg, 32% yield) were isolated as white solids. ESI m / z for both compounds: 991 (M+H) + .

[0351] Example 28 Preparation of Compound 9c (see Figure 7 and Table 6) ((2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-1 0,15,20,25,30-Pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodeca Caoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontane-5- 1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxa Acetamide}-N-[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11- Hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 ,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamoyl {ethyl]carbamoyl}-2-methylpropyl]hexanamide (9c) Following general procedure C, compound 9c (80 mg, 40% yield) was obtained as a white solid. ESI m / z : 991(M+H) + . [ka]

[0352] Example 29 Preparation of Compound 9d (See Figure 7 and Table 6) ((1S,4aS,10aR)-N-{[(1S,4aS,10aR)-6-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31, 32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydrogen) (hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 ,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H, 9H-Cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamide}hexanamide] -3-methylbutanamido]propanamido]-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydroxy phenanthrene-1-yl]carbonyl}-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10 ,10a-Octahydrophenanthrene-1-carboxamide (9d) Following general procedure C, compound 9d (0.38 g, 60% yield) was obtained as a white solid. z: 995(M / 2+1) + . [ka]

[0353] Example 30 Preparation of Compound 9e (See Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ] dotetracontan-5-yl]methyl}-1H,2H,3H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3 ]triazol-4-yl)oxy]acetamide}hexanamide]-3-methylbutanamide]-5 -(carbamoylamino)pentanamido]phenyl}methyl N-({[(4bS,8S,8aR)-8-{[(1S,4a S,10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene {4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophen-1-carbonyl]carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophen [Nantren-3-yl]carbamoyl}methyl)carbamate (9e) Following general procedure C, compound 9e (67 mg, 64% yield) was obtained as a white solid. ESI m / z : 1141(M / 2+1) + . [ka]

[0354] Example 31 Preparation of Compound 9f (See Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ] dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]trimethyl azole-4-yl)oxy]acetamido}hexanamido]-3-methylbutanamido]-5-(caprolactone) rubamoylamino)pentanamido]phenyl}methyl N-[(1S)-1-{[(4bS,8S,8aR)-8-({[(1 S,4aS,10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenane [Tren-1-yl]formamide}carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydroxybenzoate 2-hydroxyethyl]carbamate (9f) Following general procedure C, compound 9f (20 mg, 57% yield) was obtained as a white solid. ESI m / z : 1156.0(M / 2+1) + .

[0355] Example 32 Preparation of Compound 9g (See Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,4 1,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14, 17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,2 6 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]to riazol-4-yl)oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido] phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1- Hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methylethyl]pi [roridin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carbamoyl methyl}-2-propyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate (9g) Following general procedure C, compound 9g (46 mg, 58% yield) was obtained as a white solid. ESI m / z : 887.9(M / 3+H) + . [ka]

[0356] Example 33 Preparation of Compound 9h (See Figure 7 and Table 6) (N-[(5R)-5-amino-5-{[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydro 1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl] carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl ]carbamoyl}ethyl]carbamoyl}-2-methylpropyl]carbamoyl}pentyl]-1-{2- [(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-penta[ Kis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[ 26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl}-1H,4H,5H, 6H,7H,8H,9H-Cycloocta[d][1,2,3]triazol-9-yl)oxy]acetamide}-3,6,9 ,12-tetraoxapentadecan-15-amide (9h) Following general procedure C, compound 9h (30 mg, 72% yield) was obtained as a white solid. ESI m / z : 1118(M / 2+1)+ . [ka]

[0357] Example 34 Preparation of Compound 9i (see Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,4 1,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14, 17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,2 6 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]to thiazol-9-yl)oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido] phenyl}methyl N-(2-{[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl- 1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide}carbonyl)-4 b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]oxy}ethyl) Carbamate (9i) Following general procedure C, compound 9i (51 mg, 78% yield) was obtained as a white solid. ESI m / z : 1258(M / 2+1) + . [ka]

[0358] Example 34A Preparation of Compound 9J (See Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ] dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]trimethyl azole-4-yl)oxy]acetamido}hexanamido]-3-methylbutanamido]-5-(caprolactone) N-{2-[(1S,2S,4R,8S,9S,11S,12R,13 S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-di Oxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxo Ethyl}carbamate (9J) [ka] Following the general procedure described in Example 25, compound 9J (0.11 g, 51% yield) was obtained as a white solid. ESI m / z: 1081 (M / 2+1) + . [ka]

[0359] Example 34B Preparation of Compound 9K (See Figure 7 and Table 6) ({4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ] dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]trimethyl azole-4-yl)oxy]acetamido}hexanamido]-3-methylbutanamido]-5-(caprolactone) N-(4-[(1S,2S,4R,8S,9S,11S,12R,13R,14R,15R,16R,17R,18R,19R,20R,21R,22R,23R,24R,25R,26R,27R,28R,29R,30R,31R,32R,33R,34R,35R,36R,3 ,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7- Dioxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 ,18 ]icosa-14,17-dien-8-yl]-2-oxo (ethoxy)phenyl)carbamate (9K) [ka] Following the general procedure described in Example 25, compound 9K (0.11 g, 58% yield) was obtained as a white solid. ESI m / z: 751 (M / 3+1) + .

[0360] (Example 34C) Preparation of Compound 9L (See Figure 7 and Table 6) ((2R)-2-amino-N-[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19 -Difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapenta Cyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phen 31,32,33,34, 35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl) ethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cyclohexyl Octa[d][1,2,3]triazol-4-yl)oxy]acetamide}hexanamide (9L) [ka] Following the general procedure described in Example 25, compound 9L (40 mg, 34% yield) was obtained as a white solid. ESI m / z: 1009.5 (M / 2+1) + .

[0361] Example 35 Specific linker-payloads were synthesized following the procedure below (General Procedure D (see Figure 8)).

[0362] General Procedure D: To a solution of acid 4a or 4c (1.0-2.5 equiv.) in DMF (or DCM / DMF) was added DIPEA (1.5-10 equiv.). The resulting mixture was stirred at this temperature for 0.5-1 After stirring for 1 hour, an amine (3a, 3c, 3d, 3H, 3I, or 9a-i, 9J-L) (1.0 equivalent) was added. The reaction mixture was stirred at room temperature for 2 hours until the amine was completely consumed, as monitored by LCMS. The reaction mixture was filtered through a membrane and the filtrate was then analyzed by preparative HPLC. Upon separation, the example compounds (20-69% yield) were obtained as white solids. Table 7. Reaction conditions for EX.36 and 41-61 - General procedure. [Table 10] TIFF0007617974000440.tif32170 Purification -B: Preparative HPLC Method B. Example 36 Preparation of Linker-MMAE (see Figure 8 and Table 7) ({4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5, 7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapeptide Pentadecane-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamide ]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)- 1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methylethyl] [pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carbamo yl}-2-methylpropyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate [ka] ESI m / z: 829.7(M / 2+H) + (100%), 1659.7(M+H) + (20%). [ka] Analytical HPLC: 98%, retention time: 8.25 minutes (Method B). Solubility: <0.1 mg per mL of water.

[0363] Example 37 This example provides representative synthetic routes for making specific linker-payloads. (See Figure 9.)

[0364] ({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)hexanamide]-6-[4-(3-{[31,32,33,34,35,36,37,38,39,40,41,4 2-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17, 19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ] dotetracontan-5-yl]methyl}-3,4,5,13-tetraazatetracyclo[13.4.0.0 2,6 .0 7,1 2 ]nonadeca-1(19),2(6),4,7(12),8,10,15,17-octaen-13-yl)-4-oxobutanamine N-[(1S)-1-methyl-3-butanamido-3-methylbutanamido-3-phenyl]pentanamido-phenyl}methyl -{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropionate propan-2-yl]carbamoyl}-1-methoxy-2-methylethyl]pyrrolidin-1-yl]-3-meth [(5-methyl-1-oxoheptan-4-yl)(methyl)carbamoyl}-2-methylpropyl] {rubamoyl}-2-methylpropyl]-N-methylcarbamate) A solution of Mc-(L)D-Lys(suc-DIBAC)-OH (5b, 2.0 mg, 3.1 μmol) in anhydrous THF (1 mL) was treated with HATU (1.8 mg , 4.7 μmol) and DIPEA (0.8 mg, 6.2 μmol) were added sequentially at room temperature. The mixture was stirred at room temperature for 30 minutes. After stirring, a solution of compound 3a (3.6 mg, 3.1 μmol) in anhydrous THF (1 mL) was added to the reaction mixture. The resulting mixture was stirred at room temperature for 16 hours until the reaction was deemed complete by LCMS. The volatiles were removed in vacuo to give residue 8j (ESI m / z: 866.3 (M / 2+H) + ) in acetonitrile (1 mL To the solution was added a solution of αCD-azide 6a (4.6 mg, 4.7 μmol) in water (1 mL). The reaction mixture was stirred at room temperature for 16 hours and monitored by LCMS. When purified directly by method A), the linker-payload compounds described in this example was obtained as a white solid (2 mg, 25% yield). ESI m / z: 910.8(M / 3+H) + (100%), 1365.4(M / 2+H) + (20%). Analytical HPLC: >99.9%, retention time: 6.97 minutes (Method B).

[0365] Example 38 ({4-[(2S)-2-[(2S)-2-[(2R)-6-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4, 6,8,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-2-{6-[3-(4-{[31,32,33 ,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxy) Dimethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8 ,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl}piperazin-1-yl)-2,5- Dioxopyrrolidin-1-yl]hexanamide}hexanamide]-3-methylbutanamide]-5 -(carbamoylamino)pentanamido]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S, 5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carba 1-(2-methyl-1-oxo-2-methylethyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo Soheptan-4-yl](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methyl Preparation of [propyl]-N-methylcarbamate (see Figure 10). A mixture of intermediate 5a (15 mg, 24 μmol) and HATU (15 mg, 39 μmol) in THF (1 mL) was stirred at room temperature for 30 min. Next, vc-PAB-MMAE (1, 26 mg, 23 μmol) was dissolved in DMF (2 mL) and DIPEA (8.9 mg, 69 μmol). l) solution was added later. The mixture was stirred at room temperature overnight. vc-PAB-MMAE was consumed and the desired The reaction was monitored by LCMS until the compound was detected. The volatiles were removed in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in water). Purification afforded Ex38-Int (18 mg, 26% yield) as a white solid. ESI m / z: 1732 (M+ H) + . [ka] The above intermediate was dissolved in DMF (0.7 mL). To the solution, TEA (0.5 mg, 5.0 μmol) and αCD-pip 6b (9.6 mg, 9.4 μmol) was added successively at room temperature. The mixture was stirred at room temperature for 16 hours and directly purified by preparative HPLC (Method B). The linker-payload compound described in this example (3 mg, 23% yield) was obtained as a white solid. This was obtained.

[0366] ESI m / z: 925.1(M / 3+H)+(100%), 1387(M / 2+H)+(10%).

[0367] [ka] Analytical HPLC: 96%, retention time: 7.22 minutes (Method B). Solubility: 0.25 mg per mL of water.

[0368] Example 39 This example provides representative synthetic routes for making specific linker-payloads. (See Figure 11).

[0369] ({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[(2R)-2-[1-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido]-6-{2-[(1-{[31, 32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydrogen) (hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 ,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H, 9H-Cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamide}hexanamide] -3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S ,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carboxamide bamoyl}-1-methoxy-2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1- 4-Hexoheptan-4-yl](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methyl (N-methylpropyl)carbamate To a mixture of compound 8a (10 mg, 7.1 μmol) and compound 4d (6.2 mg, 14 μmol) in DMF (0.5 mL) TEA (2.1 mg, 21 μmol) was added via syringe at 25° C. Monitoring by LCMS revealed that 8 The mixture was stirred at 20-25°C for 16 hours until most of a was consumed. Flash chromatography (0–100% acetonitrile in water (containing 0.03% TFA)) Purification by HCl gave a white solid (ESI m / z: 1743 (M+H) + ) was obtained, which was dissolved in DMF (1 mL) and water (1 mL) To the solution, azide 6a (3.0 mg, 3.0 μmol) was added at 25° C., and the mixture was The mixture was stirred at room temperature for 3 days, after which the reaction was deemed complete by LCMS. The material was removed in vacuo and the residue was purified by preparative HPLC (Method A) to give the compound described in this example. The resulting linker-payload compound (1.5 mg, 9% yield) was obtained as a white solid.

[0370] ESI m / z: 1371(M / 2+H) + .

[0371] [ka]

[0372] Analytical HPLC: >99.9%, retention time: 5.78 min (Method A).

[0373] Example 40 ({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[(2R)-2-[1-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido]-6-[4-(3-{[31,3 2,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydrogen (hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 ,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]dotetracontan-5-yl]methyl}-3,4,5,13-tetraa Zatetracyclo[13.4.0.0 2,6 .0 7,12 ]nonadeca-1(19),2(6),4,7(12),8,10,15,17-octa (Hexan-13-yl)-4-oxobutanamido]hexanamido]-3-methylbutanamido]pentana mido]phenyl}methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S, 2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methylethyl 1-[(2-methyl-1-pyrrolidinyl)-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carba of]carbamoyl}-2-methylpropyl]-N-methylcarbamate Preparation (see Figure 9) [ka] See the synthetic route in Example 37. Following the procedure of Example 37, the linker-payload compound described in this example (8.0 mg, 36% yield) was obtained as a white solid.

[0374] ESI m / z: 1432(M / 2+H) + .

[0375] [ka]

[0376] Analytical HPLC: >99.9%, retention time: 6.84 minutes (Method B).

[0377] Solubility: 0.33 mg per mL of water.

[0378] Example 41 ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-de Decahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,2 2,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .28,11 .2 13,16 .2 18,21 .2 23,26 ]Dote cycloocta[d][1,2,3]triazo[1H,4H,5H,6H,7H,8H,9H- ... hexanamido]-3-methylbutanamido]-5-(carbamoyl-4-yl)oxy]acetamido} ... methyl N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[( 2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}- 1-Methoxy-2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxohepta methyl-4-yl](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl] Preparation of N-methylcarbamate (see Figure 8 and Table 7) [ka] ESI m / z: 983.4(M / 3+H) + (100%), 1475(M / 2+H) + (5%).

[0379] [ka]

[0380] Analytical HPLC: >99.9%, retention time: 7.29 minutes (Method B). Solubility: 0.25 mg per mL of water.

[0381] Example 42 ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1R)-1-{[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R, 2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy- 2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]( methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl](methyl)carbamoyl carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropion pyryl]carbamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy C10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29- Dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetracontane -5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl) Oxy]acetamido}pentyl]carbamoyl}-3,6,9,12-tetraoxatetradecane-1- Preparation of (C1H-yl)carbamates (see Figure 8 and Table 7) [ka] ESI m / z: 946.3(M / 3+H) + (100%), 1418.9(M / 2+H) + (20%).

[0382] [ka] Analytical HPLC: >99.9%, retention time: 7.33 minutes (Method B). Solubility: 1.0 mg per mL of water.

[0383] Example 43 ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42- Dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19 ,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Do tetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol- (4-yl)oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amide Hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phen N-[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1- {hydroxy-1-phenylpropan-2-yl}carbamoyl}-1-methoxy-2-methylethyl]pyrro Lysin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)carbamoyl} Preparation of {-2-methylpropyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate (Figure 8) See Table 7) [ka] ESI m / z: 1065.9(M / 3+H) + (100%), 1598.4(M / 2+H) + (20%).

[0384] [ka]

[0385] Analytical HPLC: 98.2%, retention time: 5.79 minutes (Method B).

[0386] Solubility: 0.5 mg per mL of water.

[0387] Example 44 ([(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl]methyl N-(14-{[(1R)-1-{[(1S)-1-{[ (1S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[(1S)-1-{[(3R,4S,5S)-1-[(2S)-2-[(1 (R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1-methoxy [2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl ](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl](methyl) carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropyl 31,32,33,34,35,36,37,38,39,40,41,42-dodecahydro[4-(1-[(2-propyl]carbamoyl)-5-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydro[4-(1-propyl]carbamoyl)-5-(1-[(2 ... Droxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,2 7,29-Dodecaoxaheptacyclo[26.2.2.2 3,6 .28 ,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetrako methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazole-4- yl)oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentyl] Preparation of {carbamoyl}-3,6,9,12-tetraoxatetradecan-1-yl)carbamate (Figure 8 and See Table 7) [ka] ESI m / z: 1028.7(M / 3+H) + (100%), 1541.5(M / 2+H) + (5%).

[0388] [ka]

[0389] Analytical HPLC: >99.9%, retention time: 7.02 minutes (Method B).

[0390] Solubility: 1.0 mg per mL of water.

[0391] Example 45 This example provides the synthesis of linker-glucocorticoid (GC) steroids (Figure 8 and Table 7). (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S, 9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapeptide Ntacyclo [10.8.0.0 2,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy} (phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxo Sapentadecane-15-amide) [ka] ESI m / z: 1227(M+H) + . [ka]

[0392] Analytical HPLC: 99%, retention time: 8.55 minutes (Method B).

[0393] Solubility: <0.1 mg per mL in water; 0.06 mg per mL in 20% DMSO in water; 0.06 mg per mL in 30% DMSO in water 0.07mg.

[0394] Example 46 (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-5-{2-[(1-{[31,32,33,34,35,36,37,38, 39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9 ,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,2 1 .2 23,26 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1, 2,3]triazol-4-yl)oxy]acetamide}-1-{[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S, 4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-diol Xapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-8-yl]-2-oxoe (2-methylpropyl)carbamoyl} Preparation of [pentyl]-3,6,9,12-tetraoxapentadecan-15-amide (see Figure 8 and Table 7) [ka] ESI m / z: 1259(M / 2+H) + .

[0395] [ka]

[0396] Analytical HPLC: 97%, retention time: 6.62 and 6.67 minutes (Method B).

[0397] Example 47 (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-5-{2-[(1-{[31,32,33,34,35,36,37,38, 39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9 ,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,2 1 .2 23,26 ]dotetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1, 2,3]triazol-4-yl)oxy]acetamide}-1-{[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S, 4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-di Oxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-8-yl]-2-oxo Ethoxy}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]carbamoyl Preparation of [[[(2-methyl-1,2-diyl)-2,4-diyl]-3,6,9,12-tetraoxapentadecan-15-amide]] (see Figure 8 and Table 7) ) [ka] ESI m / z: 839.5(M / 3+H) + , 1259(M / 2+H) + (60%).

[0398] [ka]

[0399] Analytical HPLC: 98%, retention time: 6.61 (59%) and 6.73 (39%) min (method B).

[0400] Solubility: 0.1 mg per mL of 10% DMSO in water.

[0401] Example 48 (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)-8-{[(1S,4 aS,10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene {4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophen-1-carbonyl]carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophen anthren-3-yl]carbamoyl}ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12- Preparation of Tetraoxapentadecan-15-amide (see Figure 8 and Table 7) [ka] ESI m / z: 1234(M+H) + .

[0402] [ka]

[0403] Analytical HPLC: >99%, retention time: 9.21 min (Method B).

[0404] Solubility: <0.1 mg per mL of water.

[0405] Example 49 (1-(4-{2-azatricyclo[10.4.0.0 4,9]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-1-{[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)- 8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrofuran phenanthrene-1-yl]formamide}carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-o hexahydrophenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methylpropyl 31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxycarbamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy -10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-do Decaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetracontane-5 -yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)o Preparation of [oxy]acetamido}pentyl]-3,6,9,12-tetraoxapentadecan-15-amide (Figure 8 and Table 7) [ka] ESI m / z: 1261(M / 2+H) + .

[0406] [ka]

[0407] Analytical HPLC: 99%, retention time: 6.55 minutes (Method B).

[0408] Solubility: 0.05 mg per mL of water.

[0409] Example 50 ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42- Dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19 ,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Do tetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol- (3,6,9,12-tetraoxapentadecan-15-amide) Hexanamido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phen Nyl}methyl N-(2-{[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1, 2,3,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide}carbonyl)-4b, 8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]oxy}ethyl)ca Preparation of Carbamates (see Figure 8 and Table 7) [ka] Compound 9i (25 mg, 9.9 μmol) and DIBAC-Suc-PEG-OSu 4b (6.8 mg, 12 μmol) were dissolved in DMF (1 mL). To the solution, triethylamine (2.0 mg, 20 μmol) was added, and the mixture was stirred at 20-25°C for 16 hours. After this time, the reaction was deemed complete by LCMS. Most of the volatiles were removed in vacuo. The residue was purified by preparative HPLC (Method B) to give (12 mg, 39% yield) as a white solid. This was obtained.

[0410] ESI m / z: 1017(M / 3+H) + .

[0411] [ka]

[0412] Analytical HPLC: 96%, retention time: 6.68 minutes (Method B).

[0413] Solubility: 0.33 mg per mL of water.

[0414] Example 51 ({bicyclo[6.1.0]non-4-yn-9-yl}methyl N-(14-{[(1R)-1-{[(1S)-1-{[(4 bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octyl {4b,8-dimethyl-4b,5,6,7,8,8a,9,(trihydrophenanthrene-1-carbonyl)carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9, 10-Octahydrophenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methyl propyl]carbamoyl}-5-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodeca Hydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24 ,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetra contan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazole- 9-yl)oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentyl Preparation of ]carbamoyl}-3,6,9,12-tetraoxatetradecan-1-yl)carbamate (Figure 8 and See Table 7) [ka] ESI m / z: 1330(M / 2+H) + .

[0415] [ka]

[0416] Analytical HPLC: >99%, retention time: 6.33 minutes (Method B).

[0417] Solubility: 0.33 mg per mL of water.

[0418] Example 52 Prepared according to Figure 8 and Table 7. ({bicyclo[6.1.0]non-4-yn-9-yl}methyl N-(14-{[(1R)-1-{[(1S)-1-{[(1S)-1-{[4- ({[(2-{[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9, 10,10a-octahydrophenanthrene-1-yl]formamide}carbonyl)-4b,8-dimethyl- 4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]oxy}ethyl)carbamoyl] oxy}methyl)phenyl]carbamoyl}-4-(carbamoylamino)butyl]carbamoyl}- 2-methylpropyl]carbamoyl}-5-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42 -dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,1 9,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Do tetracontan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol- (3,6,9,12-tetraoxapentadecan-15-amide) Preparation of pentyl]carbamoyl}-3,6,9,12-tetraoxatetradecan-1-yl)carbamate (See Figure 8 and Table 7) [ka]

[0419] ESI m / z: 1470(M / 2+H) + .

[0420] [ka]

[0421] Analytical HPLC: >99%, retention time: 6.62 minutes (Method B).

[0422] Solubility: 0.29 mg per mL of water.

[0423] Example 53 (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-1-{[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)- 8-{[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrofuran {phenanthrene-1-carbonyl]carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octa Hydrophenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methylpropyl] carbamoyl}-5-(1-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy -10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-do Decaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetracontane-5 -yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-9-yl)o oxy]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentyl]-3,6,9,1 Preparation of 2-tetraoxapentadecan-15-amide (see Figure 8 and Table 7) [ka] ESI m / z: 1385.9(M / 2+H) + .

[0424] [ka]

[0425] Analytical HPLC: 98%, retention time: 6.42 minutes (Method B).

[0426] Solubility: 0.18 mg per mL of water.

[0427] Example 54 ({bicyclo[6.1.0]non-4-yn-9-yl}methyl N-(14-{[(1R)-1-{[(1S)-1-{[(4 bS,8S,8aR)-8-{[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octyl {4b,8-dimethyl-4b,5,6,7,8,8a,9,(trihydrophenanthrene-1-carbonyl)carbamoyl}-4b,8-dimethyl-4b,5,6,7,8,8a,9, 10-Octahydrophenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methyl propyl]carbamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydr Roxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27, 29-Dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dotetracon 1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazole-4-yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazole-4-yl phenyl)oxy]acetamido}pentyl]carbamoyl}-3,6,9,12-tetraoxatetradecane- Preparation of (1-yl)carbamates (see Figure 8 and Table 7) [ka] ESI m / z: 1207(M / 2+H) + .

[0428] [ka]

[0429] Analytical HPLC: >99%, retention time: 6.37 minutes (Method B).

[0430] Example 55 (1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-1-{[(1S)-1-{[(1S)-1-{[(4bS,8S,8aR)- 8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrofuran phenanthrene-1-yl]formamide}carbonyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-o hexahydrophenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methylpropyl 31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxycarbamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy -10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-do Decaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16.2 18,21 .2 23,26 ]Dotetracontane-5 -yl]methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)o Preparation of [oxy]acetamido}pentyl]-3,6,9,12-tetraoxapentadecan-15-amide (Figure 8 and Table 7) [ka] ESI m / z: 1409(M / 2+H) + .

[0431] [ka]

[0432] Analytical HPLC: 99%, retention time: 6.33 and 6.44 minutes (Method B).

[0433] Example 56 ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-de Decahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,2 2,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6 .2 8,11 .2 13,16 .2 18,21 .2 23,26 ]Dote cycloocta[d][1,2,3]triazo[1H,4H,5H,6H,7H,8H,9H- ... hexanamido]-3-methylbutanamido]-5-(carbamoyl-4-yl)oxy]acetamido} ... N-[(1S)-1-{[(4bS,8S,8aR)-8-({[(1S,4a S,10aR)-6-Hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene 4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydro- ... Preparation of {phenanthren-3-yl}carbamoyl}-2-hydroxyethyl}carbamate (Figure 8 and See Table 7) [ka] ESI m / z: 1424.3(M / 2+H) + . [ka]

[0434] Analytical HPLC: >99%, retention time: 6.11 and 6.21 min (Method B).

[0435] Solubility: <0.1 mg per mL of water.

[0436] Example 57 (1-(4-{2-azatricyclo[10.4.0.0+]hexadeca-1(12),4(9),5,7,13,15-hexaene-10- yn-2-yl}-4-oxobutanamido)-N-[(1R)-1-{[(1S)-1-{[(4bS,8S,8aR)-8- ({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophen 4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octanol [Hydrogenanthrene-3-yl]carbamoyl}ethyl]carbamoyl}-2-methylpropyl ]carbamoyl}-5-{2-[(1-{[41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56-hexa Decahydroxy-10,15,20,25,30,35,40-heptakis(hydroxymethyl)-2,4,7,9,12,14,1 7,19,22,24,27,29,32,34,37,39-Hexadecaoxanonacyclo[36.2.2.2 3,6 .28 ,11 .2 13,16 .2 18,21 .2 23,26 .2 28,31 .2 33,36 ]hexapentacontan-5-yl]methyl}-1H,4H,5H,6H,7H, 8H,9H-Cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}pentyl]-3, Preparation of 6,9,12-tetraoxapentadecan-15-amide (see Figure 9 and Table 7) [ka] C 136 H 195 N 11 O 54 , exact mass: 2846.3

[0437] ESI m / z: 1424.2(M / 2+H) + . [ka]

[0438] Example 58 Prepared according to Figure 8 and Table 7. ({4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5, 7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapeptide Pentadecane-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamide ]phenyl}methyl N-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydro Roxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethyl}carbamate) [ka] C 74 H 94 F2N8O 17 , exact mass: 1404.7

[0439] ESI m / z: 1406(M+H) + . [ka]

[0440] Analytical HPLC: 100%, retention time: 7.40 minutes (Method B).

[0441] Solubility: 0.02 mg per mL of water.

[0442] Example 59 Prepared according to Figure 8 and Table 7. ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-de Decahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,2 2,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ]Dote cycloocta[d][1,2,3]triazo[1H,4H,5H,6H,7H,8H,9H- ... hexanamido]-3-methylbutanamido]-5-(carbamoyl-4-yl)oxy]acetamido} ... methyl N-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19 S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-diox Sapentacyclo [10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethyl (carbamate) [ka] C 126 H 177 F2N 13 O 49 , exact mass: 2694.2

[0443] ESI m / z: 1349(M+H) + . [ka]

[0444] Analytical HPLC: 100%, retention time: 6.23 minutes (Method B).

[0445] Solubility: 0.026 mg per mL of water.

[0446] Example 60 Prepared according to Figure 8 and Table 7. ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1R)-1-{[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-di Fluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacycl RO [10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethyl}carbamoyl (phenyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl] rubamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,1 5,20,25,30-Pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaio Xaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ]dotetracontan-5-yl] Methyl}-1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxy]a cetoamido}pentyl]carbamoyl}-3,6,9,12-tetraoxatetradecan-1-yl)carbamoyl Bamate) [ka] C 118 H 176 F2N 12 O 49 , exact mass: 2583.2

[0447] ESI m / z: 1292.8(M / 2+1) + . [ka]

[0448] Analytical HPLC (contains triazole region-isomers): 48%, retention time: 7.31 min and 52%, retention time: 7.41 min (Method B).

[0449] Example 61 Prepared according to Figure 8 and Table 7. ({4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5, 7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapeptide Pentadecane-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamide ]phenyl}methyl N-(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11- Hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamate to) [ka] C 80 H 98 F2N8O 18 , exact mass: 1496.7

[0450] ESI m / z: 1499(M+H) + . [ka]

[0451] Analytical HPLC: 100%, retention time: 7.99 minutes (Method B).

[0452] Solubility: <0.01 mg per mL of water.

[0453] Example 62 Prepared according to Figure 8 and Table 7. ({4-[(2S)-2-[(2S)-2-[(2R)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12) ,4(9),5,7,13,15-Hexaen-10-yn-2-yl}-4-oxobutanamide)-3,6,9,12-tetramethyl Oxapentadecan-15-amido]-6-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-de Decahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,2 2,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ]Dote cycloocta[d][1,2,3]triazo[1H,4H,5H,6H,7H,8H,9H- ... hexanamido]-3-methylbutanamido]-5-(carbamoyl-4-yl)oxy]acetamido} ... methyl N-(4-[(1S,2S,4R,8S,9S,11S,12R,13S ,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-diol Xapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]icosa-14,17-dien-8-yl]-2-oxoe (phenyl)carbamate [ka] C 132 H 181 F2N 13 O 50 , exact mass: 2786.2

[0454] ESI m / z: 1394(M / 2+H) + . [ka]

[0455] Analytical HPLC (contains triazole region-isomers): 82%, retention time: 7.93 min ; 18%, retention time: 8.02 minutes (Method B).

[0456] Solubility: 0.02 mg per mL of water.

[0457] Example 63 Prepared according to Figure 8 and Table 7. (1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaene-1 0-yn-2-yl}-4-oxobutanamide)-N-[(1R)-1-{[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S, 4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo -6-propyl-5,7-dioxapentacyclo[10.8.0.0 2 , 9 .0 4 , 8 .0 13 , 18 ]Icosa-14,17-diene -8-yl]-2-oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylprop propyl]carbamoyl}-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydro Oxy-10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29 -Dodecaoxaheptacyclo[26.2.2.2 3 , 6 .2 8 , 11 .2 13 , 16 .2 18 , 21 .2 23 , 26 ]Dotetrakonta 1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl )oxy]acetamido}pentyl]-3,6,9,12-tetraoxapentadecan-15-amide) [ka] A solution of compound 9L (19 mg, 9.4 μmol) in DMF (2 mL) was diluted with DIBAC-Suc-PEG-OSu 4b (7.0 mg, 11 μmol) ) and DIPEA (2.3 mg, 18 μmol) were added. After stirring overnight at room temperature, the resulting mixture was collected and Direct purification by HPLC (Method B) gave Ex105 (7 mg, 29% yield) as a white solid. It was.

[0458] C 121 H 168 F2N 10 O 47 , exact mass: 2551.1

[0459] ESI m / z: 1276.8(M / 2+H) + . [ka]

[0460] Analytical HPLC: 100%, retention time: 7.76 minutes (Method B).

[0461] Example 64 (Synthesis of payloads 1h and 1i, see Figure 1B) The steroidal payloads 1h and 1i were administered using commercially available fluocinolone acetonide P2 (CAS: 67-73- 2) was prepared according to Scheme 1. Compound P3 obtained from P2 was converted to mesylate P4 by aryl exchange, and then the mesylate group was Substitution of P5 with an azide moiety gave P5, which was further reduced to amine 1h. In the case of P4, the mesylate moiety in P4 can also be replaced by 4-aminophenol, resulting in aniline 1 i was obtained.

[0462] ((1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-8-(2-hydroxy Acetyl)-9,13-dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13 ,18 ]Icosa-14,17-dien-16-one (P3)) [ka]

[0463] Fluocinolone acetonide (P2, 0.90 g, 2.0 mmol) and silica gel (1 To the mixture of 8 g of butylaldehyde (0.27 mL, 3.0 mmol) was added at 10°C, and the suspension was heated at 10 to 20°C. The mixture was stirred at RT for 10 min. To the mixture was added perchloric acid (70%, 0.68 mL, 8.3 mmol) dropwise at 0°C. The reaction mixture was then stirred overnight at 10-20°C. TLC and LCMS confirmed that fluocinolone acetate was Most of the ionized P2 was consumed. The reaction mixture was diluted with petroleum ether and washed with saturated aqueous Na2CO3. The suspension was filtered and the solid was washed with DCM / methanol (v / v=1). The solution was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (petroleum ether). Compound P3 (0.15 g, 16% yield) was obtained as a white solid. Obtained as a solid. ESI m / z: 467.1 (M+H) + .

[0464] (2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl 16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ] Echo- 14,17-dien-8-yl]-2-oxoethyl methanesulfonate (P4) [ka]

[0465] Compound P3 (0.28 g, 0.65 mmol) and triethylamine (0.13 g, 1.3 mmol) in DCM (3 mL) Methanesulfonyl chloride (89 mg, 0.78 mmol) was added at 0° C. After stirring at 0° C. for 0.5 hours, The reaction mixture was diluted with DCM (20 mL), washed with H2O (20 mL x 2), and concentrated over anhydrous Na2SO4 The residue was purified by silica gel column chromatography on silica gel. Purification by HPLC (0-50% ethyl acetate in petroleum ether) gave compound P4 (0.26 g , >99% yield) was obtained as a white solid. ESI m / z: 545 (M+H) + .

[0466] ((1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-(2-azidoacetyl)-12,19-difluoro-11-hydroxybenzoate Roxy-9,13-dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 2,9 .0 4,8 .0 13,18 ]Icosa-14,17-dien-16-one (P5)) [ka] Compound P4 (1.0 g, 1.8 mmol) and sodium azide (1.2 g, 18 mmol) suspended in acetone (15 mL) The mixture was stirred at 50° C. overnight. The mixture was cooled to RT and poured into water (80 mL). The aqueous mixture was diluted with vinegar The combined organic layer was washed with brine (30 mL) and extracted with anhydrous Na2SO4 After drying over 200°C, filtering and concentrating in vacuo, crude compound P5 (0.90 g, >99% yield) was obtained as a yellow solid. Obtained as a solid, which was used in the next step without further purification. ESI m / z: 492(M+H) + .

[0467] (Payload 1h, (1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-(2-aminoacetyl)-12,19-di Fluoro-11-hydroxy-9,13-dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0. 0 2,9 .0 4,8 .0 13,18 ]Icosa-14,17-dien-16-one; Trifluoroacetate (1h) [ka]

[0468] To a solution of compound P5 (0.85 g, 1.7 mmol) in THF (20 mL) was added aqueous hydrochloric acid (1 N, 10 mL). Stir at 28-32°C until clear, then add triphenylphosphine (0.68g , 2.6 mmol) was added. The resulting yellow clear solution was stirred at RT for 18 h. The mixture was evaporated in vacuo The residue was purified by reversed-phase flash chromatography (0-50% aqueous TFA (0.05%)). Compound 1h (0.56 g, 57% yield, TFA salt) was purified with acetonitrile to give an off-white ESI m / z: 466(M+H) + . [ka] Analytical HPLC: 100%, retention time: 6.86 minutes (Method A).

[0469] (Payload 1i, (1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-[2-(4-aminophenoxy)acetyl]

[0043] -12,19-difluoro-11-hydroxy-9,13-dimethyl-6-propyl-5,7-dioxapentasilyl Kuro [10.8.0.0 2,9 .0 4,8 .0 13,18 ]icosa-14,17-dien-16-one (1i)) [ka]

[0470] Compound P4 (93 mg, 0.17 mmol), 4-aminophenol (37 mg, 0.34 mmol) in acetone (0.5 mL) A mixture of 1,2-dimethyl-3,4-trimethyl-2,4-trimethyl-1 ... The mixture was cooled and diluted with H2O (10 mL). The mixture was extracted with ethyl acetate (10 mL x 3). The combined organic layer The residue was washed with water (20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC to give payload 1i (6.0 mg, 6.3% yield) as a white solid. ESI m / z: 298 (M / 2+H) + , 558(M+H) + (10%). [ka] Analytical HPLC: 97.4% purity, retention time: 7.55 minutes (Method B).

[0471] (Synthesis of payload 1d, see Figure 1C) (Methyl(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro Phenanthrene-1-carboxylate (P7) [ka]

[0472] A solution of podocarpic acid (P6, 90 g, 0.33 mol) in methanol (200 mL) and toluene (600 mL) was (Trimethylsilyl)diazomethane (2M in hexane, 200 mL) was added. The reaction mixture was allowed to stand at room temperature. The mixture was stirred at rt for 2 h, after which time the podocarpic acid was completely consumed by LCMS. The solids were removed in vacuo and the residue was triturated from petroleum ether (2 L) to give compound P7 (91 g , 96% yield) was obtained as a white solid. ESI m / z: 289 (M+H) + . [ka]

[0473] (Methyl(1S,4aS,10aR)-1,4a-dimethyl-6-(trifluoromethanesulfonyloxy)-1,2,3, 4,4a,9,10,10a-Octahydrophenanthrene-1-carboxylate (P8) [ka]

[0474] A solution of compound P7 (10 g, 35 mmol) in methylene chloride (200 mL) was added with pyridine (3.3 g, 42 mmol) and DMSO. AP (0.84 g, 6.9 mmol) was added under a nitrogen atmosphere. The mixture was cooled to -78°C and trifluoroacetate was added. Methanesulfonic anhydride (12 g, 42 mmol) was added and the resulting mixture was allowed to warm to 25°C. The mixture was stirred for an additional 4 hours at 25° C. The reaction mixture was diluted with DCM (500 mL) and diluted with water (100 mL), aqueous hydrochloric acid (1N, 150 mL), and brine (100 mL), dried over sodium sulfate, and concentrated in vacuo. Concentration afforded crude compound P8 (14 g, 97% crude yield) as a viscous oil, which was subsequently purified by The crude compound P8 was purified by flash chromatography (petroleum ether). Purification by ethanol (0-10% ethyl acetate in ethanol) afforded the pure product as a viscous oil. ESI m / z: 421.2 (M+1) + . [ka]

[0475] (Methyl(1S,4aS,10aR)-6-((tert-butoxycarbonyl)amino)-1,4a-dimethyl-1,2,3,4,4 a,9,10,10a-Octahydrophenanthrene-1-carboxylate (P9) [ka]

[0476] Compound P8 (14 g, 34 mmol) and tert-butyl carbamate (BocNH2, 7.9 g, 68 mmol) In a solution of butanol (100 mL), cesium carbonate (22 g, 68 mmol), tris(dibenzylidene acetone) Dipalladium(0) (Pd2(dba)3, 1.8 g, 2.0 mmol) and X-Phos (1.8 g, 4.0 mmol) were added at room temperature. The mixture was degassed and backfilled with argon three times, after which compound P8 was completely consumed. The mixture was stirred overnight at 80°C under argon protection (balloon) until the reaction mixture was dissolved. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The solid was washed three times with ethyl acetate. The combined filtrate was concentrated in vacuo and the residue was evaporated onto silica gel. Purify by gel column chromatography (0-6.25% ethyl acetate in petroleum ether) Compound P9 (11 g, 82% yield) was obtained as a white solid. ESI m / z: 410 (M+23) + . [ka]

[0477] ((1S,4aS,10aR)-6-{[(tert-butoxy)carbonyl]amino}-1,4a-dimethyl-1,2,3,4,4a,9, 10,10a-Octahydrophenanthrene-1-carboxylic acid (P10) [ka]

[0478] To a DMSO solution of compound P9 (4.9 g, 13 mmol), potassium tert-butoxide (15 g, 0.13 mol) was added. The reaction mixture was heated under argon protection until the reaction was complete by LCMS. The mixture was stirred at 60° C. for 3 hours. After cooling to room temperature, the reaction mixture was poured onto ice and aqueous hydrochloric acid (0.5M ) slowly to pH 5, while not allowing the temperature to exceed 25°C. The precipitate was collected by filtration and washed several times with water. The crude product was purified by silica gel column chromatography. Further purification with distillation (0-20% ethyl acetate in petroleum ether) gave compound P10 (4.5 g, 9 3% yield) was obtained as a white solid. ESI m / z: 318 (M-55) + . [ka]

[0479] (tert-butyl N-[(4bS,8S,8aR)-8-carbamoyl-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-o octahydrophenanthrene-3-yl]carbamate (P11) [ka]

[0480] A solution of P10 (4.5 g, 12 mmol) and HATU (4.9 g, 13 mmol) in DMF (50 mL) was treated with diisopropyl ethyl acetate. Amine (20 mL, 0.12 mol) was added and the mixture was stirred at 25° C. for 1 hour. Ammonium chloride (16 g, 0.30 mol) was added and the mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, and evaporated in vacuo. The residue was purified by flash chromatography (0-20% ethyl acetate in petroleum ether). Purification by ESI m / z: 373.3(M+1) + . [ka]

[0481] (methyl(1S,4aS,10aR)-6-(benzyloxy)-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octa Hydrophenanthrene-1-carboxylate (P13) [ka]

[0482] A mixture of compound P7 (12 g, 40 mmol) and cesium carbonate (14 g, 44 mmol) in DMF (100 mL) was stirred for 20 min. The mixture was stirred for 15 minutes at 25° C. To the mixture was added benzyl bromide (7.1 mL, 60 mmol) at room temperature. After stirring at room temperature for 4 hours, the resulting mixture was poured into cold water and extracted with ethyl acetate. The combined organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (0-10% ethyl acetate in petroleum ether). Purification by ESI m / gave the title compound P13 (13 g, 89% yield) as a white solid. z: 379(M+H) + . [ka]

[0483] ((1S,4aS,10aR)-6-(benzyloxy)-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro Phenanthrene-1-carboxylic acid (P14) [ka]

[0484] Compound P13 (11 g, 29 mmol) and potassium hydroxide in DMSO (0.19 L) were dissolved in DMSO (0.19 L) until the methyl group was completely removed. A mixture of ammonium tert-butoxide (33 g, 0.29 mol) was stirred at 100° C. for 1 hour, and this was analyzed by LCMS and TL. After cooling to 25°C, the mixture was quenched with aqueous hydrochloric acid (1N) and Extract with ethyl acetate, wash the combined organic solution with brine and dry over sodium sulfate. The residue was purified by silica gel column chromatography (petroleum ether Purification by elution with 0-24% ethyl acetate afforded compound P14 (7.5 g, 71% yield) as a white solid. Obtained by ESI m / z: 365(M+H) + . [ka]

[0485] (pentafluorophenyl(1S,4aS,10aR)-6-(benzyloxy)-1,4a-dimethyl-1,2,3,4,4a ,9,10,10a-Octahydrophenanthrene-1-carboxylate (P15) [ka]

[0486] A solution of P14 (9.6 g, 26 mmol) in DMF (100 mL) was added with DIPEA (14 mL, 79 mmol) and perfluorophenyl To the mixture was added 2,2,2-trifluoroacetate (15 g, 53 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was then diluted with ether (200 mL) and the resulting mixture was stirred for 1 hour and monitored by LCMS. The organic solution was dried over sodium sulfate and washed with water (300 mL) and brine (200 mL). The residue was purified by flash chromatography (0-10% in petroleum ether). ethyl acetate) to give compound P15 (12 g, 88% yield) as a white solid. ESI m / z: 531(M+H) + . [ka]

[0487] (tert-butyl N-[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-(benzyloxy)-1,4a-dimethyl -1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide}carbonyl)- 4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]carbamate ( P16)) [ka]

[0488] A solution of compound P11 (2.3 g, 6.2 mmol) in THF (20 mL) was treated with n-BuLi (2.5 M in hexane, 5.5 mL, 14 mmol). P15 (3.0 g, 5.6 mmol) was added dropwise at -78°C. The reaction mixture was stirred at this temperature for 1 hour. l) in THF (20 mL) was added, and the resulting mixture was then stirred for 10 minutes until compound P15 was consumed. The reaction was stirred at ∼20°C overnight and monitored by LCMS. The mixture was quenched with sodium hydroxide and extracted with ethyl acetate. The combined organic solution was washed with water and brine. The residue was purified by flash chromatography. Purification by distillation with ethyl acetate (0-30% ethyl acetate in petroleum ether) gave compound P16 (1.59 g, 51% yield). (yield) was obtained as a white solid. ESI m / z: 719 (M+1) + .

[0489] (tert-butyl N-[(4bS,8S,8aR)-8-({[(1S,4aS,10aR)-6-hydroxy-1,4a-dimethyl-1,2,3 ,4,4a,9,10,10a-octahydrophenanthrene-1-yl]formamide}carbonyl)-4b,8- Dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-3-yl]carbamate (P17) [ka]

[0490] To a solution of P16 (2.0 g, 2.78 mmol) in ethyl acetate (40 mL), wet palladium on carbon (10% Pd, 0.9 g) was added. The mixture was degassed and filled with hydrogen until P16 was completely consumed. The mixture was stirred under a hydrogen balloon at room temperature overnight and monitored by LCMS. The mixture was filtered through a filter and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography. Purification by elution with 0-55% ethyl acetate in petroleum ether gave P17 (1.06 g, 61% yield). Obtained as a white solid. ESI m / z: 629(M+H) + . [ka]

[0491] (Payload 1d, (1S,4aS,10aR)-N-[(1S,4aS,10aR)-6-amino-1,4a-dimethyl-1,2,3,4,4a, 9,10,10a-Octahydrophenanthrene-1-carbonyl]-6-hydroxy-1,4a-dimethyl-1,2 ,3,4,4a,9,10,10a-Octahydrophenanthrene-1-carboxamide (1d) [ka]

[0492] To a solution of compound P17 (0.17 g, 0.27 mmol) in DCM (10 mL) was added TFA (3 mL) dropwise at room temperature. The reaction mixture was stirred at room temperature for 1 hour until Boc was removed by HCl. The volatiles were removed in vacuo. The residue was purified by preparative HPLC (Method B) to give 1d (0.10 g, 70% yield) as a white solid. This was obtained.

[0493] ESI m / z: 529.3(M+1) + .

[0494] [ka]

[0495] [ka]

[0496] [ka]

[0497] HPLC (Method B): Retention time: 8.92 min, Purity: 99.4%. Chiral HPLC: >99.9% (Columns AD, AS, OD) , and in OJ).

[0498] Optical rotation (α): +2.53° (1.7g / 100mL THF, 25℃).

[0499] Example 65 This example describes the analysis of specific linker-payloads.

[0500] A linker-payload having a cyclodextrin moiety is To compare linker-payloads without conjugation, linker-payloads were conjugated to the antibody. Prior to gating, the compounds were evaluated in several physicochemical and enzymatic assays.

[0501] The following studies were performed on linker-payloads and cyclodextrin-containing payloads. The solubility of the linker-payload without the string moiety is assayed.

[0502] Test compounds were dissolved in DMSO and a standard curve was generated using LC-MS method A or B. The test sample was dissolved in water, sonicated for 10 minutes, and then centrifuged. The solubility was calculated based on the peak area. Shown are the solubility results for Ex49. The corresponding linker-payload (Ex47) , had below detectable levels of water solubility.

[0503] See the solubility of the compound of Example 49 in FIG. [Table 11]

[0504] The detailed test procedure was as follows: 1. 0.5 mg of test compound was dissolved in 0.5 mL of DMSO. Dilute the DMSO solution (1 mg / mL, 0.5 mL, 0.5 mg) with water (4.5 mL) to form solution A (0.1 mg / mL, 5 mL, 0.5 mg). g) was obtained. 0.5 mL of solution A (0.1 mg / mL, 0.5 mL, 0.05 mg) was diluted with water (0.5 mL) and solution B (0.05 mg / mL, 2 mL) was diluted with water (0.5 mL). mL, 0.05 mg) was obtained. 0.5 mL of solution A (0.1 mg / mL, 0.5 mL, 0.05 mg) was diluted with water (1.5 mL) to obtain solution C (0.025 mg / mL, 2 mL, 0.05 mg) was obtained. Dilute 0.5 mL of solution A (0.1 mg / mL, 0.5 mL, 0.05 mg) with water (4.5 mL) and add solution D (0.01 mg / mL, 5 mL) mL, 0.05 mg) was obtained. Dilute 0.5 mL of solution D (0.01 mg / mL, 0.5 mL, 5 μg) with water (4.5 mL) and add solution E (1 μg / mL, 5 mL) , 0.5 μg) was obtained. 2. 0.05 mg of the compound was suspended in 1 mL of water and sonicated for 10 minutes. After centrifugation, the upper filtrate was The clear solution was collected to obtain a saturated sample solution. 3. Dissolve all standard solutions A, B, C, D, E, and sample solutions (using the same method and same injection volume) in L The peak areas were obtained by CMS analysis. 4. A standard curve was calculated according to the data from A to D (X = concentration, Y = peak area). 5. The concentration of the saturated sample solution was calculated according to the function obtained above.

[0505] Example 66 Linker-payload with cyclodextrin and linker-payload without cyclodextrin Cathepsin B cleavage rate of car-payload

[0506] The procedure for the cathepsin B assay is as follows: The test results are summarized in Table 8.

[0507] (Step 1:) Cathepsin B buffer: Preparation of 0.1M NaOAc / 0.01M DTT (pH 5.0) Dissolve 8.3 g / L of NaOAc and 1.54 g / L of DTT in deionized water. Adjust the pH with HCl / sodium hydroxide. Adjust to 5.0.

[0508] (Step 2:) Test Cut Rate 1. Prepare a 25 μM test sample: Add 0.5 mg of test sample to a 2 mL tube, then add 0. 2 mL of DMSO was added, and this solution was added dropwise to 0.8 mL of 0.1 M NaOAc / 0.01 M DTT buffer. The resulting mixture was sonicated for 5 minutes to obtain a clear, colorless solution. The pH value of this solution was pH The test strip said it was about 5. 2. Assay buffer: Preheat 0.1M NaOAc / 0.01M DTT (pH 5.0). 3. Spiking solution for test compounds: 25 μM spiking solution for test compounds: 2 μL of 5 mM stop The solution was added to 398 μL of 0.1 M NaOAc / 0.01 M DTT buffer (pH 5.0). 4. Preheat 0.47 μg / μL of cathepsin B in 50 mM NaOAc / 1 mM EDTA (pH 5.0). Place on ice. . 5. Sample without CA074: Add 4 μL of 0.47 μg / μL cathepsin B to 196 μL of 25 μM spiked solution. Add the solution (from step 2) and incubate the tube at 37°C. 6. Sample containing CA074: Add 4 µL of 0.47 µg / µL of cathepsin B to 4 µL of 10 mM inhibitor (CA074 ) to 196 μL of 25 μM spike solution (from step 2) and incubate the tube. To bet. 7. After quenching, shake the plate and centrifuge it at 14000 rpm. 8. 50 μL of supernatant from each well was mixed with 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC / MS analysis. ) into a 96-well sample plate containing 100 mL of PBS.

[0509] (Step 3:) Test reference compounds: 9. Assay Buffer: Prepare 100 mM Na / K phosphate, pH 6.0, containing 1.33 mM EDTA and 2 mM DTT. heat. 10. Prepare 0.024 μg / μL cathepsin B: 1 μL of 0.47 μg / μL cathepsin B stock Add to 19 μL of Assay Buffer (from step 11). 11. Add 2 μL of 0.024 μg / μL cathepsin B (from step 12) to an opaque 96-well plate Add to. 12. Add 96 μL of assay buffer to each sample. 13. Add 2 μL of 10 mM substrate Z-RR-MNA (final concentration of 200 μM). 14. For the negative control (containing inhibitor), add 2 μL of 10 mM inhibitor (CA074). : Read samples immediately in kinetic mode at 340 nm excitation / 425 nm emission (plate read every 30 seconds) (Read for 3 minutes).

[0510] (Step 4:) Cathepsin B stability in incubation samples: 15. Dissolve 5 μL of incubation sample in 93 μL of assay buffer (from step 11). and then add to 2 μL of 10 mM substrate (Z-RR-MNA). 16. Incubate the sample at 37°C for 2 minutes. 17. Read the sample at 340 nm excitation / 425 nm emission.

[0511] In general, most conjugates with a vcPAB moiety are cleaved by capB with a cleavage rate of >50%. It can be cut.

[0512] Generally, the incorporation of cyclodextrin into the linker increases the rate of cathepsin B cleavage, e.g. , increasing it from 14% (Ex47) to 62% (Ex48).

[0513] Other samples were tested and are summarized in Table 8. In general, most test compounds were detected within 6-7 minutes by HPLC. It has a retention time (RT) in the range of 7 min. If RT > 7 min, the cLogP is greater than 5 and its water solubility is It was low. Table 8. Solubility and CapB cleavage rate of linker-payloads with cyclodextrins [Table 12] * A and B: See general procedures HPLC Method A and HPLC Method B, respectively. ** : Water solubility in other solvents For the compound of Example 44: 0.06 mg / mL in 20% DMSO in water; 0.07 mg / mL in 30% DMSO in water mL. For the compound of Example 46: 0.10 mg / mL in 10% DMSO / H2O. +≦-6 cLogP -6<++≦-3 cLogP -3<+++≦1 cLogP 1<++++≦5 cLogP 5<+++++≦9.5 cLogP %≦50 CapB(%) 50<%≦60 CapB(%) 60<%%%≦70 CapB(%) 70<%%%%≦80 CapB(%) 80<%%%%% Table 8A. Linker-payload with and without cyclodextrin Comparison of solubility and CapB cleavage rate of different linker-payloads [Table 13]

[0514] Example 67 Cyclodextrin-containing linker-payload and cyclodextrin-containing linkers under various pH buffer conditions Stability of linker-payload without string

[0515] Test samples were dissolved at 0.5 mg / mL in 20% DMSO + 80% PBS buffer at pH 5.0, 7.4, or 8.0. These buffers were prepared as follows: 1. 0.05M sodium phosphate and 0.07M NaCl buffer, pH 7.4: 14.505 g / L Na2HPO4_12H2O, 1.483 g / L NaH2PO4_2H2O, and 4.095 g / L NaCl were deionized. Dissolve in water. Adjust pH to 7.40 with phosphoric acid / sodium hydroxide. 2. PBS containing 0.5% BSA: Dissolve 0.5 g of BSA in 100 mL of buffer 1.

[0516] These results demonstrate that the DIBAC-hydrophilic linker-payload is highly stable at pH 6–8. The increased hydrophilicity of the linker resulted in the linker-payoff of >95%. The water solubility of the load increases over time.

[0517] Example 68 (ADC conjugation)

[0518] Conjugation of maleimide-linker-payload to antibody (Figure 13) and [3+2] Conjugation of DIBAC-linker-payload to azide-functionalized antibodies via a crosslinking reaction The patterns (Fig. 14) are outlined in Figs. 13 and 14, respectively.

[0519] A study of non-site-specific maleimide-linker-payload conjugated to antibodies General procedure E

[0520] This example demonstrates antibody or antigen binding via Michael addition or thiol-maleimide bond formation. This paper presents a method for non-site-specific conjugation of linker-payload to a conjugate fragment. This example refers to the compounds shown in Figure 13. Antibody-Cysteine-Mediated Conjugation The jugation was performed using Adcetris®-like ADCs (see Mol. Pharm. 2015, 12(6), 1863-71). The procedure was the same as that for preparing the monoclonal antibody (Ab) in two steps. The antibody (mAb) (10 mg / mL in 50 mM HEPES, 150 mM NaCl) was diluted with 1 mM dithiothreitol (0.01 mg per mg of antibody). The antibody was reduced with 0.006 mg of TCEP (2.5 molar equivalents relative to the antibody) at 37°C for 30 minutes. After 5 minutes of ethanol (sodium acetate, pH 4.5), the compound of Example 38 was reduced to a concentration of 1-10 mg / mL in DMSO. The resulting mixture was added to the purified antibody and stirred for 3-14 hours. The DAR(UV) values ​​were calculated based on the measured absorbance of the ADC. The extinction coefficients of the antibody and the compound of Example 38 were determined. This is summarized in Table 9 below.

[0521] Conjugation via Michael addition represented by Ab-Ex.38. See Figure 13 stomach. Table 9. Examples of conjugation via Michael addition [Table 14]

[0522] Example 69 Site-specific conjugation to antibody-conjugated alkyne-linker-payload General Procedure for Application F

[0523] This example shows the overall linker-payload site on an antibody or antigen-binding fragment thereof. This example demonstrates a method for specific conjugation of the compound shown in Figure 14. It is reaching

[0524] In this example, the site-specific conjugate was produced in two steps. To antibodies bearing a small molecule Q-tag (references on Q-tags) such as dido-PEG3-amine (see above). Microbial transglutaminase (MTG)-based enzymatic conjugation of The second step is a [2+3] cycloaddition, e.g., the reaction of an azide with a cyclopentyl group. via 1,3-dipolar cycloaddition between cyclooctynes ​​(aka copper-free click chemistry) Conjugation of anchor-payloads to azide-functionalized antibodies was utilized. Baskin, JM; Prescher, JA; Laughlin, ST; Agard, NJ; Chang, PV; Miller, IA; Lo, A.; Codel See Li, JA; Bertozzi, CR, PNAS 2007, 104(43), 16793-7. Shown in 4 is a hydroxybenzoate conjugated to an azide-functionalized antibody via a [2+3] cycloaddition. This is an example of a linker-payload containing a DIBAC moiety. The stoichiometric conjugates were obtained in approximately 50-80% isolated yield.

[0525] Scheme 10. ADC conjugates via [2+3] click reaction, represented by Ab-Ex49 in Figure 14. Engagement.

[0526] (Step 1:) Preparation of azide-functionalized antibodies

[0527] Aglycosylated human antibody IgG (IgG1, IgG4, etc.) or N297Q mutation in PBS (pH 6.5-8.0) A human IgG1 isotype having ≥ 200 molar equivalents of azido-dPEG3-amine (MW = 218.26 g / mol The resulting solution was mixed with MTG (EC 2.3.2.13, manufactured by Zedira, Darmstadt, Germany), or Mo Dernist Pantry [L# 210115A] - ACTIVA TI is made with maltodextrin from Ajinomoto, Japan When mixed with 5 U MTG per mg of antibody (containing 25 U / mL of MTG), a final concentration of 0.5–5 mg / mL of antibody is obtained. The solution is then incubated at 37°C for 4 to 24 hours with gentle shaking. The reaction was monitored by ESI-MS. When the reaction was complete, excess amine and MTG was removed by SEC or Protein A column chromatography, and the azide-functionalized antibody This product was characterized by SDS-PAGE (Fig. 15) and ESI-MS (Fig. 16). Azido-dPEG3-amine attached to two sites on the antibody results in a 2DAR antibody-PEG3-amine. For the dido conjugate, this resulted in an increase of 204 Da.

[0528] In specific experiments, N-terminal Q-tag antibody ( 24 mg) in the presence of MTG (0.350 mL, 35 U, mTGase, Zedira, Darmstadt, Germany) The reaction mixture was incubated with a molar equivalent of azido-PEG3-amine (MW 218.26). The mixture was incubated overnight at 37°C with gentle mixing. The enzyme was removed by size exclusion chromatography (SEC, Superdex 200 PG, GE Healthcare). He left.

[0529] (Step 2:) Preparation of site-specific drug-antibody conjugates using click chemistry

[0530] The site-specific antibody drug conjugates of Table 10 with human IgG (IgG1, IgG4, etc.) were treated with azide. Prepared by a [2+3] click reaction between a functionalized antibody and an alkyne-containing linker-payload. The detailed conjugation procedure is as follows: The site-specific antibody conjugates are then incubated in an aqueous medium (e.g., PBS containing 5% glycerol). Dissolve mAb-PEG3-N3 (1-3 mg / mL) in PBS (HBS) or a suitable organic solvent such as DMSO, DMF, or DMA. Incubate with ≥6 molar equivalents ...

Claims

1. (i) Formula (Ib1) or (Ib2): 【Chemistry 1】 or (ii) 【Chemistry 2】 or a positional and / or stereoisomer thereof, of the formula selected from the group consisting of: (In the formula, BA is an antibody or an antigen-binding fragment thereof; LL is SP, RG, and the following: 【Chemistry 3】 is a trivalent linker comprising a structure selected from SP, at each occurrence, is independently absent or a spacer group residue selected from C 1-6 alkylene, -NH-, -C(O)-, -(CH 2 -CH 2 -O) e , -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e -C(O)-, and combinations thereof; RG is a reactive linker moiety containing succinimide, triazole, or fused triazole; e is an integer from 0 to 4; n is an integer from 1 to 30; PA is as follows: 【Chemistry 4】 (wherein the payload moiety is selected from the group consisting of:

2. The compound according to claim 1 of formula (Ib3), (Ib4), (Ib5) or (Ib6) or a positional isomer and / or stereoisomer thereof: 【Chemistry 5】 。

3. A compound of formula (Id) or a pharma- ceutically acceptable salt, solvate, or stereoisomeric form thereof, or a positional isomer thereof, or a mixture of positional isomers thereof: 【Chemistry 6】 (In the formula: 【Chemistry 7】 is below: 【Chemistry 8】 【change】 【change】 【change】 【change】 【change】 selected from the group consisting of; BA is an antibody or an antigen-binding fragment thereof; RG is a reactive group residue containing a triazole or fused triazole; SP 3 is absent or a spacer group residue selected from C 1-6 alkylene, -NH-, -C(O)-, -(CH 2 -CH 2 -O) e , -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e -C(O)-, and combinations thereof; CD-SP 3 -RG-CD 1 and; CD 1 is below: 【Chemistry 9】 is a cyclodextrin residue selected from the group consisting of: 【Chemistry 10】 represents the atom through which the cyclodextrin is attached to the adjacent groups in the formula; The subscript n is an integer from 1 to 30; PA is as follows: 【Chemistry 11】 a payload moiety selected from the group consisting of: Each 【Chemistry 12】 is the bond with BA; Each 【Chemistry 13】 is a bond to PA; and R9 is -CH3 or -(CH2)3N(H)C(O)NH2).

4. 4. The compound of any one of claims 1-3, wherein RG, at each occurrence, independently comprises a triazole or a fused triazole.

5. RG, independently in each occurrence, 【Chemistry 14】 wherein: 【Chemistry 15】 The compound according to any one of claims 1 to 3, wherein represents the atom through which said RG is bonded to the adjacent group in said formula.

6. LL, 【Chemistry 16】 3. The compound according to claim 1 or 2,

7. LL is 【Chemistry 17】 3. The compound according to claim 1 or 2,

8. The spacer group residues, in each occurrence, are independently -(CH2-CH2-O)e and -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e The compound of any one of claims 1 to 7, selected from the group consisting of -C(O)-.

9. 9. The compound of any one of claims 1 to 8, wherein subscript e is 4.

10. 10. The compound of any one of claims 1 to 9, wherein subscript n is 2 to 4.

11. A compound described in any one of claims 1 to 10, wherein BA is an antibody.

12. The BA is AXL, BAFFR, BCMA, BCR-list component, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10 or f54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19, CD20, CD209, CD209L , CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD46, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1 , DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA Class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL2 1R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integr The compound of any one of claims 1 to 10, which is an antibody or antigen-binding fragment thereof selective for an antigen selected from the group consisting of phosphodiesterase, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list component / association, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, and VTCN1.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 or a pharma- ceutical acceptable salt thereof, and a pharma- ceutical acceptable excipient.

14. 13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient, for the treatment of a disease, disorder or condition in a patient in need thereof.

15. (i) Formula (IIb1) or (IIb2): 【Chemistry 18】 or (ii) 【Chemistry 19】 or a positional isomer, stereoisomer, and / or mixture thereof. (In the formula, RG' is a reactive group comprising an alkyne or maleimide; LL is SP, RG, and the following 【Chemistry 20】 is a trivalent linker comprising a structure selected from SP, at each occurrence, is independently absent or a spacer group residue selected from C 1-6 alkylene, -NH-, -C(O)-, -(CH 2 -CH 2 -O) e , -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e -C(O)-, and combinations thereof; and e is an integer from 0 to 4; PA is as follows: 【Chemistry 21】 (wherein the payload moiety is selected from the group consisting of:

16. 16. The compound according to claim 15, of formula (IIb3), (IIb4), (IIb5), or (IIb6): or its positional isomers, stereoisomers, and / or mixtures thereof. 【Chemical 22】 (In the formula, The subscript e is an integer from 0 to 4.

17. A compound of formula (IId) or a pharma- ceutically acceptable salt, solvate, or stereoisomeric form thereof, or a positional isomer thereof, or a mixture of positional isomers thereof: 【Chemistry 23】 (In the formula: 【Chemistry 24】 is below: 【Chemistry 25】 【change】 【change】 selected from the group consisting of; Each 【Chemistry 26】 is the bond with BA; Each 【Chemical 27】 is the bond with PA; R9 is -CH3 or -(CH2)3N(H)C(O)NH2; RG' is a reactive group residue including an alkyne or maleimide; SP 3 is a spacer group residue selected from C 1-6 alkylene, -NH-, -C(O)-, -(CH 2 -CH 2 -O) e , -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e -C(O)-, and combinations thereof; CD-SP 3 -RG-CD 1 and; CD 1 is below: 【Chemistry 28】 is a cyclodextrin residue selected from the group consisting of: 【Chemical 29】 represents the atom through which the cyclodextrin is attached to the adjacent groups in the formula; RG is a reactive group residue comprising a succinimide, triazole, or fused triazole; and PA is as follows: 【Chemistry 30】 (wherein the payload moiety is selected from the group consisting of:

18. RG', independently in each occurrence, 【Chemistry 31】 wherein: 【Chemistry 32】 18. The compound of any one of claims 15 to 17, wherein represents the atom through which said RG' is bonded to the adjacent group in said formula.

19. LL, 【Chemical 33】 17. The compound according to claim 15 or 16,

20. LL is 【Chemical 34】 17. The compound according to claim 15 or 16,

21. The spacer group, in each occurrence, is independently selected from -(CH2-CH2-O)e and -NH-CH 2 -CH 2 -(-O-CH 2 -CH 2 ) e 21. The compound of any one of claims 15 to 20, selected from the group consisting of -C(O)-.

22. 17. A method for preparing a compound of claim 1, comprising contacting a binding agent with a compound of claim 15 under conditions suitable for forming a bond between the binding agent and the compound.

23. 13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 or a pharma- ceutical acceptable salt thereof, and a pharma-ceutical acceptable excipient, for treating a proliferative disease, a metabolic disease, an inflammatory disease, or a neurodegenerative disease in a subject in need thereof.

24. 24. The pharmaceutical composition of claim 23 for the treatment of a proliferative disease.

25. 24. The pharmaceutical composition of claim 23 for the treatment of a metabolic disease.

26. 24. The pharmaceutical composition of claim 23 for the treatment of inflammation.

27. 24. The pharmaceutical composition of claim 23 for the treatment of a neurodegenerative disease.

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