Camptothecin analogs conjugated to glutamine residues in proteins and uses thereof - Patents.com

Abstract

For example, protein-drug conjugates and compositions thereof that are useful for target-specific delivery of therapeutic moieties, such as camptothecin analogs and / or derivatives, are described herein. In certain embodiments, specific and efficient methods are provided for generating protein-drug constructs (e.g., antibody-drug conjugates) utilizing a combination of transglutaminase and 1,3-cycloaddition technology. Camptothecin analogs, antibody-drug conjugates, and compositions comprising glutaminyl-modified antibodies and camptothecin analog payloads are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 298,786, filed January 12, 2022. The entire contents of this application are incorporated herein by reference in their entirety.

[0002] Field of Disclosure The present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods for treating diseases using the same. Also provided is a method for producing protein-drug conjugates utilizing a combination of transglutaminase and 1,3-cycloaddition technology. More specifically, the present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates) including pro-exatecan and exatecan. [Background technology]

[0003] Proliferative diseases are characterized by the uncontrolled growth and spread of abnormal cells. If the spread is uncontrolled, it can result in death. Abnormal proliferation, for example, cancer, is caused by both external factors (e.g., tobacco, chemicals, radiation, and infectious organisms) and internal factors (genetic mutations, immune system conditions, mutations resulting from metabolism). These causative factors may act together or sequentially to initiate or promote abnormal proliferation. Cancer is treated by surgery, radiation, chemotherapy, hormones, and immunotherapy. However, more effective anti-proliferative drugs are needed.

[0004] An ideal antiproliferative therapy would allow targeted delivery of highly cytotoxic drugs to tumor cells while leaving normal cells unaffected. Traditional chemotherapy treatments are limited due to toxic side effects resulting from the effect of the drugs on non-cancerous cells. Various approaches to targeted drug delivery have been attempted, including the use of conjugates of tumor-targeting probes (such as antibodies or growth factors) with toxins such as Pseudomonas or Diphtheria toxins, which halt protein and cell synthesis. However, side effects include immune system reactions due to the non-human components of the conjugates. Furthermore, the half-life of drug conjugates has been limited due to clearance from the circulation by renal filtration, as well as schematic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-target organs and tissues.

[0005] Another approach utilizes the high permeability of the vascular endothelium of tumor tissues using passive drug carriers such as polymers, liposomes, and polymeric micelles.Polymer drugs and macromolecules accumulate in solid tumors through mechanisms of increased permeability and retention.However, the use of such targeted delivery is hindered by the fast removal of foreign particles from blood, and the technical obstacles of obtaining a highly standardized, pharmacologic acceptable drug delivery system with the specificity and selectivity required to bind to tumor cells.

[0006] Protein conjugates, such as antibody conjugates, utilize the selective binding of a binding agent to deliver a payload to a target within a tissue of interest. The payload can be a therapeutic moiety that can take action at the target.

[0007] Several techniques are available for conjugating linker and payload to antibody.Many conjugates are prepared by non-selective covalent binding to cysteine ​​or lysine residues in antibody.This non-selective technique may result in heterogeneous mixtures of products with different conjugation sites and different conjugation numbers per antibody.Therefore, there is a need in the art for methods and techniques that provide site-selective antibody conjugation.

[0008] There is a need in the art for further safe and effective anti-tumor targeting agents that can bind to various antigens to provide enhanced treatment of diseases such as cancer for use in monotherapy and combination therapy. In certain embodiments, the present disclosure meets the need and provides other advantages.

[0009] The preceding discussion is presented solely to provide a better understanding of the nature of the problems faced in the art and should not be construed as an admission of prior art in any sense, nor should the citation of any reference herein be construed as an admission that such reference constitutes "prior art" to the instant application. Summary of the Invention

[0010] Various non-limiting aspects and embodiments of the disclosure are described below.

[0011] In one aspect, the present disclosure provides a compound of formula (A): BA-(L1-B-L2-P) n (A) The present invention provides a compound having the structure: BA is an antibody or antigen-binding fragment thereof, L1 is a first linker, B is a triazole-containing moiety, L2 is a second linker, and P is PI to P-IV.

[0012] [ka] is selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0013] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0014] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0015] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0016] [ka] where n is an integer from 1 to 12.

[0017] In one embodiment, the first linker L1 is connected to the side chain of a glutamine residue of BA.

[0018] In one embodiment, the BA is an antibody or an antigen-binding fragment thereof.

[0019] In one embodiment, the BA is an antibody or an antigen-binding fragment thereof.

[0020] In one embodiment, the BA comprises one or more glutamine residues.

[0021] In one embodiment, the glutamine residue is naturally present in the BA or is introduced into the BA by site-directed modification of one or more amino acids.

[0022] In one embodiment, the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

[0023] In one embodiment, the BA is an anti-HER2 antibody.

[0024] In one embodiment, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, and brain cancer.

[0025] In one embodiment, a glutamine residue, often designated Q, is naturally present in the CH2 or CH3 domain of the BA. In one embodiment, the glutamine residue is introduced into the BA by site-specific modification of one or more amino acids. In one embodiment, the glutamine residue is Q295 or N297Q.

[0026] In one embodiment, L is C 1-6Alkyl, phenyl, aralkyl-NH-, -C(O)-, -(CH 2 ) u -NH-C(O)-, -(CH 2 ) u -C(O)-NH-, -(CH 2 -CH 2 -O) v -, -(CH 2 ) u -(O-CH 2 -CH 2 ) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which may be -S-, -S(O 2 )-, -C(O)-, -C(O 2 )-, and -CO 2 H, and the subscripts u and v are independently integers from 1 to 8.

[0027] In some embodiments, L1 is

[0028] [Table 1] is selected from the group consisting of:

[0029] In one embodiment, L1 is

[0030] [ka] It is.

[0031] In one embodiment, L1 is

[0032] [Table 2-1]

[0033] [Table 2-2] or a pharma- ceutically acceptable salt thereof.

[0034] In one embodiment, B is

[0035] [ka] and Z is C or N.

[0036] In one embodiment, B is

[0037] [ka] It is.

[0038] In one embodiment, L2 has the formula (L2): -SP1-AA-SP2- (L2) in which SP1 is absent or is a first spacer unit, AA is absent or is a peptide unit comprising 2-4 amino acids, and SP2 is absent or is a second spacer unit covalently linked to P, with the proviso that at least one of SP1, AA, and SP2 may not be absent.

[0039] In one embodiment, SP1 is absent or

[0040] [ka] , C 1-6 Alkyl, -(CH 2 -CH 2 -O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH 2 ) u -, -NH-(CH 2 ) u -C(O)-, -NH-(CH 2 -CH 2 -O) v -, -NH-(CH2 -CH 2 -O) v -C(O)-, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -C(O)-, -(CH 2 ) u -NH-C(O)-, -NH-(CH 2 ) u -NH-C(O)-, -NH-(CH 2 ) u -C(O)-NH-, and combinations thereof, wherein the subscripts u and v are independently integers from 1 to 8.

[0041] In one embodiment, AA is a peptide unit containing 2 to 4 amino acids selected from alanine, glycine, valine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof. In one embodiment, AA is valine-citrulline, glutamic acid-valine-citrulline, glycine-glycine-glycine, or glycine-glycine-glycine-glycine.

[0042] In some embodiments, SP2 is absent, or

[0043] [ka] and combinations thereof; R c independently, in each occurrence, is absent or

[0044] [ka] is a group selected from

[0045] In one embodiment, L2 is

[0046] [Table 3-1]

[0047] [Table 3-2]

[0048] [Table 3-3] is selected from the group consisting of:

[0049] In one embodiment, n is 4. In one embodiment, n is 2. In one embodiment, n is 8.

[0050] In one embodiment, the compound, or a pharma- ceutically acceptable salt thereof, is

[0051] [Table 4-1]

[0052] [Table 4-2]

[0053] [Table 4-3] The compound has a structure selected from the group consisting of:

[0054] In another aspect, the present disclosure provides a compound of formula (Alk-L2-P): Alk-SP1-AA-SP2-P (Alk-L2-P) or a pharma- ceutically acceptable salt thereof, wherein Alk is an alkyne-containing moiety, SP1 is absent or is a first spacer unit, AA is absent or is a peptide unit containing 2 to 4 amino acids, SP2 is absent or is a second spacer unit, and P is PI to P-IV.

[0055] [ka] is selected from the group consisting of P is PI~P-IV

[0056] [ka] is selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0057] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0058] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0059] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0060] [ka] It is.

[0061] In one embodiment, the compound of formula (Alk-L2-P), or a pharma- ceutically acceptable salt thereof, is

[0062] [Table 5-1]

[0063] [Table 5-2]

[0064] [Table 5-3]

[0065] [Table 5-4] is selected from the group consisting of:

[0066] In yet another aspect, the disclosure provides a composition comprising a population of compounds according to any of the above embodiments, having a drug-to-antibody ratio (DAR) of from about 0.5 to about 12.0.

[0067] In one embodiment, the population of compounds has a DAR of about 1.0 to about 2.5. In one embodiment, the population of compounds has a DAR of about 2. In one embodiment, the population of compounds has a DAR of about 3.0 to about 4.5. In one embodiment, the population of compounds has a DAR of about 4. In one embodiment, the population of compounds has a DAR of about 6.5 to about 8.5. In one embodiment, the population of compounds has a DAR of about 8.

[0068] In yet another aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising:

[0069] [Table 6-1]

[0070] [Table 6-2] The present invention provides a compound having a structure selected from the group consisting of:

[0071] In another aspect, the disclosure provides a pharmaceutical composition comprising a compound according to any of the above embodiments and a diluent, carrier, and / or excipient.

[0072] In another aspect, the disclosure provides a method of treating a tumor and / or cancer comprising contacting the tumor and / or cancer with a compound according to any of the above embodiments.

[0073] In another aspect, the disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of the above embodiments, or a pharmaceutical composition according to any one of the above embodiments.

[0074] In one embodiment, the disease is cancer. In one embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, the disease is HER2+ breast cancer.

[0075] In another aspect, the disclosure provides a method for selectively delivering a compound to a cell, the compound being as described in any of the above embodiments.

[0076] In another aspect, the disclosure provides a method of selectively targeting an antigen on the surface of a cell with a compound, wherein the compound is as described in any of the above embodiments.

[0077] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell.

[0078] In one embodiment, the cancer cells are selected from the group consisting of breast cancer cells, ovarian cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, or brain cancer cells.

[0079] In another aspect, the present disclosure provides a compound of formula (A): BA-(L1-B-L2-P) n (A) wherein BA is an antibody or antigen-binding fragment thereof; L1 is a first linker that is covalently attached to the side chain of a glutamine residue of BA; B is a triazole-containing moiety; and L2 is a second linker that is covalently attached to P; P is PI to P-IV

[0080] [ka] an antitumor agent selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 )v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v-OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0081] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0082] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0083] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0084] [ka] and the method is a) contacting a BA containing at least one glutamine residue with a compound L1-B' in the presence of a transglutaminase; b) contacting the product of step a) with one or more equivalents of a compound B″-L2-P, wherein group B″ is capable of being covalently linked to group B′, and one of groups B′ and B″ is —N 3 and

[0085] [ka] and the other of groups B' and B'' is selected from

[0086] [ka] and Z is C or N; and c) isolating the compound of formula (A) produced.

[0087] In one embodiment, the compound of formula (A), or a pharma- ceutically acceptable salt thereof, is

[0088] [Table 7-1]

[0089] [Table 7-2]

[0090] [Table 7-3] The compound has a structure selected from the group consisting of:

[0091] These and other aspects of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following detailed description of the disclosure, including the appended claims. [Brief description of the drawings]

[0092] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] FIG. 1 is a schematic diagram of the two-step site-specific production of ADCs according to one embodiment of the present disclosure. [Diagram 2] 1 is a plot of the EC50 values ​​and % maximum killing in SK-BR-3 cells for free exatecan, the control ADC trastuzumab deltecan (DS8201a), and two ADCs described in this disclosure. [Diagram 3] 1 is a plot of the EC50 values ​​and % maximum killing in SK-BR-3 cells for free exatecan and five ADCs described in this disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] Detailed embodiments of the present disclosure are disclosed herein. However, it should be understood that the disclosed embodiments are merely exemplary of the present disclosure, which may be embodied in various forms. In addition, each of the examples given in relation to the various embodiments of the present disclosure is intended to be illustrative and not limiting. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting the present disclosure, but merely as a representative basis for teaching those skilled in the art to use the present disclosure in various ways.

[0094] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0095] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0096] The term "treating" or "treatment" of a condition, disorder, or disease includes (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or subclinical symptom of a condition, disorder, or disease in a subject who may be affected by or susceptible to the condition, disorder, or disease, but who has not yet experienced or exhibited the clinical or subclinical symptoms of the condition, disorder, or disease, or (2) inhibiting the condition, disorder, or disease, i.e., preventing, reducing, or delaying the occurrence of the disease or its recurrence, or at least one clinical or subclinical symptom thereof, or (3) relieving the disease, i.e., causing regression of the condition, disorder, or disease, or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician. In some embodiments, treatment includes a method of ablating cells in such a way that the disease is indirectly affected. In certain embodiments, the treatment involves depleting immune cells as a hematopoietic conditioning regimen prior to therapy.

[0097] As used herein, "subject" or "patient" or "individual" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0098] As used herein, the term "effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, the particular drug or drugs used, the mode of administration, etc.

[0099] The phrase "pharmaceutical acceptable salt" used in connection with the compositions of the present disclosure refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in Berge et al., "Pharmaceutical Salts", J.Pharm.Sci., 1977, 66:1, which is incorporated herein by reference. Examples of salts include, but are not limited to, acid-derived, base-derived, organic, inorganic, amine, and alkali or alkaline earth metal salts, including, but not limited to, calcium salts, magnesium salts, potassium salts, sodium salts, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. In some examples, the payload described herein (e.g., the rifamycin analog described herein) comprises a tertiary amine, and the nitrogen atom in the tertiary amine is the atom through which the payload is attached to the linker or linker spacer.In such examples, the attachment of the payload to the tertiary amine produces a quaternary amine in the linker-payload molecule.The positive charge on the quaternary amine can be balanced by a counterion (e.g., chloro, bromo, iodo, or any other appropriately charged moiety, such as those described herein).

[0100] Ranges can be expressed herein as from "about" or "approximately" one particular value, and / or to "about" or "approximately" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.

[0101] "Comprising" or "containing" or "including" means that at least the named compound, element, particle, or method step is present in a composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if other such compounds, materials, particles, or method steps have the same function as the one named.

[0102] The compounds of the present disclosure include those generally described herein, and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito:1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.:Smith, MB and March, J., John Wiley & Sons, New York:2001, the contents of which are all incorporated herein by reference.

[0103] As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl group has about 1-20 carbon atoms in its backbone (e.g., C for straight chain). 1 -C 20 , C for branched chains 2 -C 20 ), alternatively having about 1-10 carbon atoms, or about 1-6 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure (such rings are monocyclic or bicyclic), alternatively having about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group can be a lower alkyl group, which lower alkyl group contains 1-4 carbon atoms (e.g., the C of a straight chain lower alkyl). 1 -C 4 ).

[0104] As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0105] As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds.

[0106] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon, the quaternized form of any basic nitrogen, and a substitutable nitrogen of a heterocyclic ring).

[0107] The term "halogen" means F, Cl, Br, or I, and the term "halide" refers to a halogen radical or substituent, i.e., -F, -Cl, -Br, or -I.

[0108] The term "adduct" in this disclosure, e.g., an "adduct of group B'", includes any moiety that comprises the product of an addition reaction, e.g., an addition reaction of group B', regardless of the synthetic steps performed to produce said moiety.

[0109] The term "covalent bond" refers to the formation of a covalent bond, i.e., a chemical bond involving the sharing of one or more electron pairs between two atoms. Covalent bonds can include different interactions, including, but not limited to, σ-bonds, π-bonds, metal-metal bonds, agostic interactions, bent bonds, and three-center two-electron bonds. When a first group is said to be "capable of being covalently bonded" to a second group, this means that the first group can form a covalent bond with the second group, either directly or indirectly, for example, through the use of a catalyst or under certain reaction conditions. Non-limiting examples of groups that can be covalently bonded to each other can include, for example, amines and carboxylic acids (forming amide bonds), dienes and dienophiles (via the Diels-Alder reaction), and azides and alkynes (forming triazoles via 1,3-cycloaddition reactions).

[0110] As described herein, the compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. The combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to a compound that is substantially unchanged when subjected to conditions that allow for its production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein.

[0111] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, e.g., the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the disclosure.

[0112] Unless otherwise stated, all tautomers of the compounds of the present disclosure are within the scope of the present disclosure.

[0113] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen with deuterium or tritium, or 11 C or 13 C or 14 Compounds having this structure, except for the replacement of a carbon with a C-rich carbon, are within the scope of this disclosure.

[0114] It should further be understood that a reference to one or more method steps does not preclude the presence of additional or intervening method steps between the steps explicitly identified. Similarly, it should be understood that a reference to one or more components in a device or system does not preclude the presence of additional or intervening components between the components explicitly identified.

[0115] Unless otherwise specified, all crystalline forms of the compounds of the present disclosure and their salts are also within the scope of the present disclosure.The compounds of the present disclosure may be isolated in various amorphous and crystalline forms, including, but not limited to, anhydrous, hydrated, non-solvated, or solvated forms.Examples of hydrates include hemihydrate, monohydrate, dihydrate, etc.In some embodiments, the compounds of the present disclosure are anhydrous and non-solvated.By "anhydrous" is meant that the crystalline form of the compound essentially does not contain bound water in the crystal lattice structure, i.e., the compound does not form crystalline hydrates.

[0116] As used herein, "crystal form" is meant to refer to a particular lattice configuration of a crystalline substance. Different crystal forms of the same substance typically have different crystal lattices (e.g., unit cells) due to the different physical properties characteristic of each of the crystal forms. In some instances, different lattice configurations have different water or solvent contents. Different crystal lattices can be identified by solid-state characterization methods such as X-ray powder diffraction (PXRD). Other characterization methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid-state NMR, etc., further aid in the identification of crystal forms, as well as the determination of stability and solvent / water content.

[0117] Crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. Hydrated forms are crystalline forms that contain water in the crystal lattice. Hydrated forms may be stoichiometric hydrates, where water is present in the lattice at a specific water / molecule ratio, such as hemihydrate, monohydrate, dihydrate, etc. Hydrated forms may also be non-stoichiometric, where the water content is variable and dependent on external conditions such as humidity.

[0118] In some embodiments, the compounds of the present disclosure are substantially isolated. "Substantially isolated" means that a particular compound is at least partially isolated from impurities. For example, in some embodiments, the compounds of the present disclosure contain less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% impurities. Impurities generally include anything that is not a substantially isolated compound, including, for example, other crystal forms and other substances.

[0119] Certain groups, moieties, substituents, and atoms are depicted with wavy lines. The wavy lines may cross or cap a bond or bonds. The wavy lines indicate the atoms through which the group, moiety, substituent, or atom is attached. For example,

[0120] [ka] The phenyl group substituted with a propyl group, represented as follows, has the following structure:

[0121] [ka] has.

[0122] The term "HER2" or "human epidermal growth factor receptor 2" refers to a member of the human epidermal growth factor receptor family. This protein is also known as NEU, NGL, HER2, TKR1, CD340, HER-2, MLN19, and HER-2 / neu. HER 2can refer to the amino acid sequence set forth in NCBI accession number NP_004439.2. Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain grades of breast cancer. In recent years, this protein has become an important biomarker and target of treatment for approximately 30% of breast cancer patients. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless expressly specified as being derived from a non-human species. Thus, the term "HER2" refers to human HER2, unless specified as being derived from a non-human species, e.g., "mouse HER2," "monkey HER2," etc.

[0123] The phrase "antibody that binds to HER2" or "anti-HER2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize HER2.

[0124] The phrase "anti-HER2 / HER2" antibodies, e.g., "anti-HER2 / HER2 bispecific antibodies," includes antibodies and antigen-binding fragments thereof that specifically recognize two different HER2 epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise a first antigen-binding domain (D1) that specifically binds to a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds to a second epitope of human HER2.

[0125] As used herein, the expression "STEAP2" refers to six-transmembrane epithelial antigen of prostate 2. STEAP2 is an endogenous six-transmembrane protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer; for example, STEAP2 was found to be expressed at significant levels in LNCaP prostate cell line (Porkka, et al. Lab Invest 2002,82:1573-1582). STEAP2 (UniProtKB / Swiss-Prot:Q8NFT2.3) is a 490 amino acid protein that is encoded by the STEAP2 gene located in chromosome region 7q21 in humans.

[0126] As used herein, an "antibody that binds to STEAP2" or an "anti-STEAP2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize STEAP2.

[0127] An "antibody that binds MET" or an "anti-MET antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize MET. As used herein, the terms "MET," "c-MET," and the like refer to the human transmembrane receptor tyrosine kinase.

[0128] The phrase "anti-MET / MET" antibodies, e.g., "anti-MET / MET bispecific antibodies," includes antibodies and antigen-binding fragments thereof that specifically recognize two different MET epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise a first antigen-binding domain (D1) that specifically binds to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET.

[0129] All amino acid abbreviations used in this disclosure are those accepted by the U.S. Patent and Trademark Office as set forth in 37 C.FR § 1.822(B)(J).

[0130] The term "protein" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).

[0131] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless expressly specified as being derived from a non-human species. Thus, the term "STEAP2" refers to human STEAP2, unless specified as being derived from a non-human species, e.g., "mouse STEAP2", "monkey STEAP2", etc.

[0132] Amino acid sequences of antibodies can be numbered using known numbering schemes, including those described in Kabat et al., ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply sequence differences where none exist, and one of skill in the art can readily ascertain sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra).

[0133] The term "glutaminyl-modified antibody" refers to an antibody having at least one covalent bond from a glutamine side chain to a primary amine compound of the present disclosure. In certain embodiments, the primary amine compound is linked via an amide bond on the glutamine side chain. In certain embodiments, the glutamine is an endogenous glutamine. In other embodiments, the glutamine is an endogenous glutamine that has been made reactive by polypeptide engineering (e.g., via deletion, insertion, substitution, or mutation of an amino acid on the polypeptide). In further embodiments, the glutamine is a polypeptide engineered with an acyl donor glutamine-containing tag (e.g., a glutamine-containing peptide tag, a Q tag, or a TGase recognition tag).

[0134] The term "TGase recognition tag" refers to a sequence of amino acids that includes an acceptor glutamine residue, and when incorporated (e.g., added) into a polypeptide sequence, is recognized by TGase under appropriate conditions and results in cross-linking by TGase through a reaction between an amino acid side chain in the sequence of amino acids and a reaction partner. The recognition tag can be a peptide sequence that does not naturally occur in a polypeptide that includes a TGase recognition tag. In some embodiments, the TGase recognition tag includes at least one Gln. In some embodiments, the TGase recognition tag includes the amino acid sequence XXQX (SEQ ID NO: 1935), where X is any amino acid (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or a non-conventional amino acid). In some embodiments, the acyl donor glutamine-containing tag is LLQGG (SEQ ID NO: 1936), LLQG (SEQ ID NO: 1937), LSLSQG (SEQ ID NO: 1938), gGGLLQGG (SEQ ID NO: 1939), gLLQG (SEQ ID NO: 1940), LLQ, gSPLAQSHGG (SEQ ID NO: 1941), gLLQGGG (SEQ ID NO: 1942), gLLQGG (SEQ ID NO: 1943), gLLQ (SEQ ID NO: 1944), LL The amino acid sequence may be selected from the group consisting of QLLQGA (SEQ ID NO: 1945), LLQGA (SEQ ID NO: 1946), LLQYQGA (SEQ ID NO: 1947), LLQGSG (SEQ ID NO: 1948), LLQYQG (SEQ ID NO: 1949), LLQLLQG (SEQ ID NO: 1950), SLLQG (SEQ ID NO: 1951), LLQLQ (SEQ ID NO: 1952), LLQLLQ (SEQ ID NO: 1953), and LLQGR (SEQ ID NO: 1954). See, e.g., WO2012059882, the contents of which are incorporated herein in their entirety.

[0135] The term "antibody" as used herein means any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules with four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of an antibody (or an antigen-binding portion thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on the parallel analysis of two or more CDRs.

[0136] The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate position, or to introduce codons, to generate cysteine ​​residues, to modify, add, or delete amino acids, and the like.

[0137] 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, (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered 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 immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0138] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains can be of any size or amino acid composition, and generally contain at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments that have a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable configuration. For example, variable regions can be dimers and contain VH-VH, VH-VL, or VL-VL dimers.

[0139] Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0140] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody herein include (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (V) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-CL, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed herein, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0141] Furthermore, antigen-binding fragments of the antibodies herein can comprise homodimers or heterodimers of any of the variable and constant domain configurations enumerated herein (or other dimers) non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0142] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies herein using routine techniques available in the art.

[0143] The antibodies herein can function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies herein in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC are well known in the art and can be measured using available assays. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0144] In certain embodiments, the antibody herein, e.g., an anti-HER2 antibody, or an anti-HER2 / HER2 bispecific antibody, or an anti-MET antibody, or an anti-MET / MET bispecific antibody, or an anti-STEAP2 antibody, is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody herein may include amino acid residues, e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0145] The antibody may be a recombinant human antibody in some embodiments. As used herein, the term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0146] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds and form approximately 75-80 kDa molecules composed of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification. The frequency of occurrence of the second form in the various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. Single amino acid substitutions in the hinge region of human IgG4 hinges can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using human IgG1 hinges. The present specification encompasses antibodies with one or more mutations in the hinge, CH2, or CH3 regions that may be desirable in production, for example, to improve the yield of the desired antibody form.

[0147] The antibody herein may be an isolated or purified antibody. As used herein, an "isolated antibody" or a "purified antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for the purposes of this specification. For example, an antibody that has been purified from at least one component of a reaction or reaction sequence is a "purified antibody" or is derived from the purification of an antibody. An isolated antibody also includes an antibody in situ in a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated or purified antibody can be substantially free of other cellular material and / or chemicals.

[0148] The antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibodies are derived, or to the corresponding residues in another human germline sequence, or to the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).

[0149] Furthermore, the antibodies herein can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residues of a certain germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, improved drug-to-antibody ratio (DAR) of antibody-drug conjugates, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed herein.

[0150] The term "aglycosylated antibody" refers to an antibody that does not contain a glycosylation sequence that may interfere with transglutamination reactions, for example, an antibody that does not have a sugar group at N297 on one or more heavy chains. In certain embodiments, the antibody heavy chain has an N297 mutation. In other words, the antibody is mutated so that it no longer has an asparagine residue at position 297 according to the EU numbering system disclosed by Kabat et al. In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence, or by site-directed mutagenesis to insert a glutamine residue at a site away from any interfering glycosylation sites or any other interfering structures. Such antibodies can also be isolated from natural or artificial sources. Aglycosylated antibodies further include antibodies that contain T299 or S298P or other mutations, or combinations of mutations that result in the absence of glycosylation.

[0151] The term "deglycosylated antibody" refers to an antibody that removes sugar groups to facilitate transglutaminase-mediated conjugation. Sugars include, but are not limited to, N-linked glycan sugars. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of sugar groups is accomplished enzymatically, including but not limited to, via PNGase.

[0152] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids of different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes can include portions of sugars, phosphoryl groups, or sulfonyl groups on an antigen.

[0153] The term "conjugated protein" or "conjugated antibody" as used herein refers to a protein or antibody covalently bound to one or more chemical moieties. The chemical moieties can include the amine compounds of the present disclosure. Linkers (L) and payloads (D) suitable for use with the present disclosure are described in detail herein. In certain embodiments, the conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also called an antibody-payload conjugate or an antibody-linker-payload conjugate.

[0154] The term "drug-to-antibody ratio" or (DAR) is the average number of therapeutic moieties, e.g., drugs, conjugated to a binding agent of the disclosure.

[0155] The term "linker-antibody ratio" or (LAR), also referred to in some embodiments as lowercase l, is the average number of reactive primary amine compounds conjugated to a binding agent of the present disclosure. Such binding agents, e.g., antibodies, can be conjugated, for example, with a suitable azide- or alkyne-containing primary amine compound. The resulting binding agent can be functionalized with an azide or alkyne and then reacted with the corresponding azide- or alkyne-containing therapeutic moiety via a 1,3-cycloaddition reaction.

[0156] The phrase "pharmacologically acceptable amount" refers to an amount effective or sufficient to treat, alleviate, relieve, or modulate the effects or symptoms of at least one health problem in a subject in need thereof. For example, a pharma- ceutically acceptable amount of an antibody or antibody-drug conjugate is an amount effective to modulate a biological target using an antibody or antibody-drug conjugate provided herein. Suitable pharma- ceutically acceptable amounts include, but are not limited to, from about 0.001% up to about 10%, and any amount therebetween, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of an antibody or antibody-drug conjugate provided herein.

[0157] The phrase "reaction pH" refers to the pH of the reaction after all reaction components or reactants have been added.

[0158] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0159] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by programs gAP or BESTFIT using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, the conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0160] Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0161] The sequence similarity of polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, gCG software includes programs such as gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, gCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in gCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence (Pearson (2000), supra). Another specific algorithm for comparing a written sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0162] Protein-drug conjugate compounds In accordance with the foregoing and other objects, the present disclosure provides protein-drug conjugate compounds, such as antibody-drug conjugate compounds, as well as precursors and intermediates thereof, pharmaceutical compositions, and methods for treating certain diseases in subjects in need of such treatment. In accordance with the present disclosure, the protein-drug conjugate compounds provided herein include a glutaminyl-modified binding agent conjugated to a therapeutic moiety, such as a primary amine compound bound to an exatecan or proexatecan moiety, as described herein.

[0163] In one aspect, the present disclosure provides a compound comprising a binding agent (e.g., an antibody or fragment thereof) according to the present disclosure, having one or more glutamine residues conjugated to one or more compounds (e.g., exatecan or proexatecan) via a first linker, a triazole-containing unit, and a second linker. Illustrative non-limiting examples include Formula (A) described herein. In certain embodiments of the protein-drug conjugate according to the present disclosure, where the binding agent is an antibody (e.g., a monoclonal antibody), the term "antibody drug conjugate" or ADC is optionally used.

[0164] In one aspect, the present disclosure provides a compound of formula (A): BA-(L1-B-L2-P) n (A) The present invention provides a compound having the structure: BA is an antibody or an antigen-binding fragment thereof, L1 is the first linker, B is a triazole-containing moiety; L2 is a second linker, P is PI~P-IV

[0165] [ka] is selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 )v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0166] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0167] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v-NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0168] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0169] [ka] where n is an integer from 1 to 12.

[0170] In one embodiment, -L1-B-L2-P of the compound of formula (A) has the structure

[0171] [ka] has.

[0172] In one embodiment, -L1-B-L2-P of the compound of formula (A) has the structure

[0173] [Table 8-1]

[0174] [Table 8-2] has.

[0175] In one embodiment, the compound of the present disclosure, or a pharma- ceutically acceptable salt thereof, is

[0176] [Table 9-1]

[0177] [Table 9-2] The compound has a structure selected from the group consisting of:

[0178] Linker L1 In certain embodiments, the linker L1 is covalently attached to the amine of a glutamine residue of the binding agent BA.

[0179] In certain embodiments, the linker L is alkyl (e.g., C 1-12 Alkyl, or C 1-12 Alkyl, or C 1-6 alkyl), phenyl, aralkyl-NH-, -C(O)-, -(CH 2 ) u -NH-C(O)-, -(CH 2 ) u-C(O)-NH-, -(CH 2 -CH 2 -O) v -, -(CH 2 ) u -(O-CH 2 -CH 2 ) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which may be -S-, -S(O 2 )-, -C(O)-, -C(O 2 )- or -CO 2 H, and the subscripts u and v are independently integers from 1 to 8.

[0180] In certain embodiments, the free (unconjugated) linker L1 comprises a primary amine for attachment to a glutamine residue via a transglutamation reaction.

[0181] In one embodiment, the linker L1 comprises one or more polyethylene glycol (PEG) units. In one embodiment, L1 comprises 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 PEG units.

[0182] In one embodiment, the linker L1 comprises a disulfide (-SS-) bond.

[0183] In one embodiment, the linker L1 is -S(O 2 )-part.

[0184] In one embodiment, one or more carbons on the linker L1 is -CO 2 It is replaced by H.

[0185] In one embodiment, the linker L1 comprises a peptide unit comprising 2 to 4 amino acids, or a peptide unit comprising 2 amino acids, or a peptide unit comprising 3 amino acids, or a peptide unit comprising 4 amino acids.

[0186] In one embodiment, the linker L1 comprises a peptide unit comprising two amino acids selected from alanine, glycine, valine, phenylalanine, proline, glutamic acid, and citrulline, and combinations thereof. In one particular embodiment, the linker L1 comprises a valine-citrulline unit.

[0187] In some embodiments, the linker L1 is

[0188] [ka] wherein R A is a group including an alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, lipid residue, and the subscripts x, n, p, and q are independently integers from 0 to 12, and combinations thereof.

[0189] In some embodiments, the linker L1 is

[0190] [Table 10] is selected from the group consisting of:

[0191] In one embodiment, L1 is

[0192] [ka] It is.

[0193] In another embodiment, L1 is

[0194] [Table 11] or a pharma- ceutically acceptable salt thereof.

[0195] Triazole-containing portion B In one embodiment, B is a triazole (

[0196] [ka] ) is a compound containing

[0197] In some embodiments, B is

[0198] [ka] and Z is C or N.

[0199] In one embodiment, B is

[0200] [ka] In another embodiment, B is

[0201] [ka] It is.

[0202] Linker L2 In certain embodiments of the present disclosure, the linker L2 has the formula (L2): -SP1-AA-SP2- (L2) wherein: SP1 is absent or is the first spacer unit, AA is absent or is a peptide unit containing 2-4 amino acids; SP2 is absent or is a second spacer unit covalently linked to P, with the proviso that at least one of SP1, AA, and SP2 is not absent.

[0203] In one embodiment, SP1 is absent.

[0204] In one embodiment, SP1 is

[0205] [ka] , C 1-6 Alkyl, -(CH 2 -CH 2 -O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH 2 ) u -, -NH-(CH 2 ) u -C(O)-, -NH-(CH 2 -CH 2 -O) v -, -NH-(CH 2 -CH 2 -O) v -C(O)-, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -C(O)-, -(CH 2 ) u -NH-C(O)-, -NH-(CH 2 ) u -NH-C(O)-, -NH-(CH 2 ) u -C(O)-NH-, and combinations thereof, wherein the subscripts u and v are independently integers from 1 to 8.

[0206] In one embodiment, AA is absent.

[0207] In one embodiment, AA is a peptide unit comprising two to four amino acids selected from alanine, glycine, valine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0208] In one embodiment, AA is valine-citrulline, glutamic acid-valine-citrulline, glycine-glycine-glycine, or glycine-glycine-glycine-glycine.

[0209] In one embodiment, AA is valine-citrulline.

[0210] In one embodiment, AA is glutamic acid-valine-citrulline.

[0211] In one embodiment, AA is glycine-glycine-glycine.

[0212] In one embodiment, AA is glycine-glycine-glycine-glycine.

[0213] In one embodiment, SP2 is absent.

[0214] In some embodiments, SP2 is

[0215] [ka] and combinations thereof; R c independently, in each occurrence, is absent or

[0216] [ka] is a group selected from

[0217] In certain embodiments, L2 is

[0218] [Table 12-1]

[0219] [Table 12-2] is selected from the group consisting of:

[0220] payload In certain embodiments, the payloads of the present disclosure are camptothecin analogs and / or derivatives.

[0221] [ka]

[0222] Camptothecin (CPT), shown above, is a topoisomerase poison. It was discovered in 1966 by ME Wall and MC Wani in a systematic screening of natural products for anticancer drugs. It is isolated from the bark and stems of Camptotheca acuminata (Camptotheca, Happy tree), a tree endemic to China used as a cancer treatment in traditional Chinese medicine. Camptothecin has shown significant anticancer activity in preliminary clinical trials. However, due to its poor solubility, synthetic and medicinal chemists have developed many syntheses of camptothecin and various derivatives to increase the profitability of the chemical with good results. Four camptothecin analogs: topotecan, irinotecan, belotecan, and deruxtecan (Dxd) have been approved and are used in cancer chemotherapy today.

[0223] Trastuzumab deruxtecan (T-Dxd, also known as DS8201a) is an antibody-drug conjugate that contains the human epidermal growth factor receptor 2 (HER2)-directed antibody trastuzumab and the topoisomerase I inhibitor conjugate deruxtecan (Dxd, a derivative of exatecan). It was approved for use in the United States in February 2019.

[0224] Exatecan, shown below, is a camptothecin analog.

[0225] [ka]

[0226] In one embodiment, the payload of the present disclosure is exatecan.

[0227] In certain embodiments, the payload of the present disclosure is a compound having the structure P2(pro-Exatecan), or a pharma- ceutically acceptable salt thereof:

[0228] [ka] It is.

[0229] In certain embodiments, the present disclosure provides P'-I through P'-IV

[0230] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v-C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0231] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0232] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0233] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0234] [ka] It is.

[0235] In certain embodiments, the payload of the present disclosure is

[0236] [Table 13-1]

[0237] [Table 13-2] or a pharma- ceutically acceptable salt thereof.

[0238] The present disclosure further relates to pharmaceutical compositions comprising a therapeutically effective amount of the above-described compound, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0239] Linker-Payload (L2-P) In another aspect, the present disclosure provides a compound of formula (Alk-L2-P): Alk-SP1-AA-SP2-P (Alk-L2-P) wherein Alk is the alkyne-containing moiety SP1 is absent or is the first spacer unit, AA is absent or is a peptide unit containing 2-4 amino acids; SP2 is absent or is a second spacer unit, P is PI~P-IV

[0240] [ka] is selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0241] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0242] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0243] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0244] [ka] It is.

[0245] In certain embodiments, Alk is

[0246] [ka] is selected from the group consisting of:

[0247] In some embodiments, SP1 is absent.

[0248] In some embodiments, AA is absent.

[0249] In some embodiments, SP2 is absent.

[0250] In some embodiments, SP1 is present and is a moiety as described above.

[0251] In some embodiments, AA is present and is a moiety as described above.

[0252] In some embodiments, SP2 is present and is a moiety as described above.

[0253] In certain embodiments, the compound of formula (Alk-L2-P), or a pharma- ceutically acceptable salt thereof, is

[0254] [Table 14-1]

[0255] [Table 14-2]

[0256] [Table 14-3]

[0257] [Table 14-4] is selected from the group consisting of:

[0258] Binder In one embodiment, the effectiveness of the embodiments of the protein-drug conjugates described herein depends on the selectivity with which the binding agent binds to its binding partner.In one embodiment of the present disclosure, the binding agent is any molecule that can bind to a given binding partner with some degree of specificity.In one embodiment, the binding agent is in a mammal, where the interaction can lead to therapeutic applications.In an alternative embodiment, the binding agent is in vitro, where the interaction can lead to diagnostic applications.In some aspects, the binding agent can bind to a cell or cell population.

[0259] Suitable binding agents of the present disclosure include proteins that bind to binding partners, where the binding agent comprises one or more glutamine residues. Suitable binding agents include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell- or peptide-binding molecules or substances.

[0260] In one embodiment, the binding agent is an antibody. In certain embodiments, the antibody is selected from monoclonal antibodies, polyclonal antibodies, antibody fragments (Fab, Fab', and F(ab)2, minibodies, diabodies, triabodies, etc.). The antibodies herein can be humanized using the methods described in U.S. Patent No. 6,596,541 and U.S. Application Publication No. 2012 / 0096572, each of which is incorporated by reference in its entirety. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, the BA is a humanized monoclonal antibody. For example, the BA can be a monoclonal antibody that binds to HER2, MET, or STEAP2. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, the BA is a bispecific antibody, such as an anti-HER2 / HER2 bispecific antibody, or an anti-MET / MET bispecific antibody.

[0261] In the present disclosure, the antibody may be any antibody that would be suitable for a person skilled in the art. In some embodiments, the antibody comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody comprises one or more gln295 residues. In certain embodiments, the antibody comprises two heavy chain polypeptides, each having one gln295 residue. In further embodiments, the antibody comprises one or more glutamine residues at a site other than heavy chain 295. Such antibodies may be isolated from natural sources or may be engineered to include one or more glutamine residues. Techniques for engineering glutamine residues into antibody polypeptide chains are within the skill of the art. In certain embodiments, the antibody is non-glycosylated.

[0262] The antibody may be in any form known to those of skill in the art. In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain.

[0263] In certain embodiments, 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 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.

[0264] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab')2 fragment. In some aspects, the antibody fragment is a Fab' fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

[0265] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0266] 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.

[0267] The antibody can have binding specificity for any antigen deemed appropriate 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, but are not limited to, molecules such as renin, growth hormones including human growth hormone and bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, lipoproteins, alpha 1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, clotting factors, e.g., factor vmc, factor IX, tissue factor (TF), and von Willebrand factor, anticoagulants such as protein C, atrial natriuretic factor, pulmonary surfactant, plasminogen activators such as urokinase or human urinary or tissue plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factors, tumor necrosis factors alpha and beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted (regulated upon activation and expressed and secreted in normal T cells), human macrophage inflammatory protein (MIP-I-α), serum albumins such as human serum albumin, Müllerian inhibitory factor, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, microbial proteins such as β-lactamase, DNase, 19E, cytotoxic T lymphocyte-associated antigen (CTLA), e.g., CTLA-4, inhibin, activin, vascular endothelial growth factor (VEGF), receptors for hormones or growth factors, protein A or is D, rheumatoid factor, neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF), transforming growth factors (TGFs) such as TGFα and TGFβ (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5),Insulin-like growth factors-1 and -2 (IGF-1 and IGF-2), des(I-3)-IGF-1 (brain IGF-2), insulin-like growth factor binding protein, EpCAM, gD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, lRTAI, lRTA2, lRTA3, lRTA4, lRTA 5, CD proteins, e.g., CD2, CD3, CD4, CD5, CD6, CD8, CDII, CDI4, CDI9, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CDI52, or any of the CD proteins described in U.S. Patent Publication No. 2008 / 0171040 or U.S. Patent Publication No. 2008 / 0171040, which are incorporated by reference in their entirety. Antibodies that bind to one or more tumor associated antigens or cell surface receptors disclosed in US Pat. No. 8 / 0305044, erythropoietin, bone morphogenetic factors, immunotoxins, bone morphogenetic proteins (BMPs), interferons, e.g., interferon alpha, interferon beta, and interferon gamma, colony stimulating factors (CSFs), e.g., M-CSF, gM-CSF, and g-CSF, interleukins (ILs), e.g., IL-1 to IL-10, superoxide dismutase, T cell receptors, surface membrane proteins, decay accelerating factors, viral antigens, e.g., HIV Parts of the envelope, carrier proteins, homing receptors, addressins, regulatory proteins, integrins, e.g., CDlla, CDllb, CDllc, CDI8, ICAM, VLA-4, and VCAM, tumor associated antigens, e.g., AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR,EGFRVIII, endoglin, Epcam, EphA2, ErbB2 / HER2, 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), MET, MUM1, NA17, NGEP, N Y-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, as well as fragments of any of the polypeptides set forth herein.

[0268] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, gPNMB, UPK3A, and LGR5. Exemplary antigens also include, but are not limited to, MUC16, PSMA, STEAP2, and HER2.

[0269] In some embodiments, antigens also include, but are not limited to, hematological targets, such as CD22, CD30, CD33, CD79a, and CD79b.

[0270] Some embodiments herein are target specific for therapeutic or diagnostic use. In one embodiment, the binding agent is prepared to interact with and bind to antigens defined as tumor antigens, including antigens specific to a certain type of tumor, or antigens shared, overexpressed, or modified on a certain type of tumor. Examples include α-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B-RAF, CLPP, or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma, as well as the following shared tumor specific antigens, for example: BAGE-1, gAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody binds to STEAP2, MUC16, EGFR, EGFRVIII, FGR2, or PRLR.

[0271] In some embodiments, the antigen comprises HER2. In some embodiments, the antigen comprises STEAP2. In some embodiments, the antigen comprises MET. In some embodiments, the antigen comprises EGFRVIII. In some embodiments, the antigen comprises MUC16. In some embodiments, the antigen comprises PRLR. In some embodiments, the antigen comprises PSMA. In some embodiments, the antigen comprises FGFR2.

[0272] In some embodiments, the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, or an anti-FGFR2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0273] In some embodiments, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0274] Anti-HER2 antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the antibody is trastuzumab, pertuzumab (2C4), or margetuximab (MGAH22). In some embodiments, the antibody is trastuzumab. According to certain embodiments, the protein-drug conjugate, e.g., the ADC of the present disclosure, comprises an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody may include those described in WO2019 / 212965A1.

[0275] In some embodiments, the antibody is an anti-HER2 / HER2 bispecific antibody comprising a first antigen-binding domain (D1) that specifically binds to a first epitope on human HER2 and a second antigen-binding domain (D2) that specifically binds to a second epitope on human HER2.

[0276] In certain embodiments, the D1 and D2 domains of the anti-HER2 / HER2 bispecific antibody are non-competitive with each other. The non-competitive nature of D1 and D2 for binding to HER2 means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human HER2. Exemplary antigen-binding protein competition assays are known in the art.

[0277] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on HER2.

[0278] In one non-limiting embodiment, the present disclosure provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: a first antigen-binding domain (D1), Second antigen-binding domain (D2) and a protein-drug conjugate comprising a bispecific antigen-binding molecule comprising: D1 specifically binds to the first epitope of human HER2; D2 specifically binds to a second epitope on human HER2.

[0279] Anti-HER2 / HER2 bispecific antibodies can be constructed using the antigen-binding domains of two separate monospecific anti-HER2 antibodies. For example, a collection of monoclonal monospecific anti-HER2 antibodies can be generated using standard methods known in the art. The individual antibodies thus generated can be tested pairwise against each other for cross-competition for HER2 protein. If two different anti-HER2 antibodies can bind to HER2 simultaneously (i.e., do not compete with each other), the antigen-binding domain from the first anti-HER2 antibody and the antigen-binding domain from the second non-competitive anti-HER2 antibody can be engineered into a single anti-HER2 / HER2 bispecific antibody according to the present disclosure.

[0280] According to the present disclosure, bispecific antigen-binding molecule can be a single multifunctional polypeptide, or can be a multimeric complex of two or more polypeptides that are covalently or non-covalently linked to each other.As will become clear from the present disclosure, any antigen-binding construct that has the ability to simultaneously bind to two separate non-identical epitopes of HER2 molecule is considered as a bispecific antigen-binding molecule.Any of the bispecific antigen-binding molecules described herein or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) known to those skilled in the art.

[0281] In another aspect, the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to HER2 and a pharma- ceutically acceptable carrier. In one non-limiting embodiment, the antibody can bind to two distinct epitopes on the HER2 protein, i.e., the antibody is a HER2 / HER2 bispecific antibody. In a related aspect, the disclosure features a composition that is a combination of an anti-HER2 / HER2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-HER2 / HER2 antibody. Additional combination therapies and co-formulations that include the anti-HER2 / HER2 bispecific antibody of the disclosure are disclosed elsewhere herein.

[0282] In another aspect, the present disclosure provides a therapeutic method for targeting / killing tumor cells expressing HER2 using the anti-HER2 / HER2 bispecific antibody of the present disclosure, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the anti-HER2 / HER2 antibody of the present disclosure. In some cases, the anti-HER2 / HER2 antibody (or antigen-binding fragment thereof) can be used to treat breast cancer or modified to be more cytotoxic by methods including, but not limited to, modified Fc domains to increase ADCC (see, e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods to increase the efficiency of tumor ablation.

[0283] The present disclosure further includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by HER2-expressing cells. In one aspect, the present disclosure relates to a compound comprising an anti-HER2 antibody or antigen-binding fragment, or a HER2 / HER2 bispecific antibody, as disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.

[0284] In yet another aspect, the disclosure provides bispecific anti-HER2 / HER2 antibodies for diagnostic applications, such as, for example, as imaging reagents.

[0285] Antibody Conjugation Techniques and linkers for conjugating to residues of an antibody or antigen-binding fragment are known in the art. Exemplary amino acid linkages that can be used in the context of this embodiment include, for example, lysine (see, e.g., US 5,208,020, US 2010 / 0129314, Hollander et al., Bioconjugate Chem., 2008,19:358-361, WO2005 / 089808, US5,714,586, US2013 / 0101546, and US2012 / 0585592), cysteine ​​(see, e.g., US2007 / 0258987, WO2013 / 055993, WO2013 / 055990, WO2013 / 053873, WO2013 / 053872, WO2011 / 130598, US2013 / 0101546, and US7,750,116), selenocysteine ​​(see, e.g., WO2008 / 122039, and Hofer et al. al., Proc. Natl. Acad. Sci., USA, 2008, 105: 12451-12456), formylglycine (see, 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. Protocols, 2012, 10: 1052-1067), unnatural amino acids (see, e.g., WO2013 / 068874 and WO2012 / 166559), and acidic amino acids (see, e.g., WO2012 / 05982). Lysine conjugation can also proceed via NHS (N-hydroxysuccinimide). Linkers can further be conjugated to cysteine ​​residues, including those of cleaved interchain disulfide bonds, by forming a carbon bridge between the thiols (see, for example, US Pat. No. 9,951,141 and US Pat. No. 9,950,076).Linkers can further be conjugated to antigen-binding proteins via attachment to carbohydrates (see, e.g., US2008 / 0305497, WO2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO2013 / 085925, WO2010 / 010324, WO2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Site-specific conjugation techniques can also be used to direct conjugation to specific residues of an antibody or antigen-binding protein (see, e.g., Schumacher et al. J Clin Immunol (2016) 36(Suppl 1):100). In certain embodiments, discussed in more detail below, site-specific conjugation techniques include transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0286] Payloads according to the present disclosure linked via lysine and / or cysteine, for example via maleimide or amide conjugation, are included within the scope of the present disclosure.

[0287] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a lysine-based linker conjugation, e.g., with an NHS-ester linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0288] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a cysteine-based linker conjugation, e.g., with a maleimide linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0289] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0290] FIG. 1 illustrates a two-step conjugation method (Step 1: Conjugating deglycosylated antibody or N297Q mutant antibody to amine-PEG 3 -Azide linker (AL-N 3 ) to the Ab-Q295 / 7 site via MTG-mediated conjugation to produce an azide-functionalized antibody (Ab(AL) 4 ), and step 2: generating Ab(AL) 4 via [3+2] cycloaddition of alkynes with 2 1 depicts structures of site-specific ADCs according to the present disclosure generated using linker-payload (P#) conjugated to the Q295 / 297 site of the antibody. All ADCs with linker-payload were conjugated to the Q295 / 297 site of the antibody.

[0291] Step 1: Transglutaminase-mediated site-specific conjugation In some embodiments, proteins (e.g., antibodies) may be modified according to known methods to provide glutaminyl-modified proteins. Techniques for conjugating antibodies with primary amine compounds are known in the art. Herein, site-specific conjugation techniques are used to specify conjugation to glutamine using transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0292] The primary amine-containing compound (e.g., linker L1) of the present disclosure can be conjugated to one or more glutamine residues of a binder (e.g., a protein, e.g., an antibody) via transglutaminase-based chemical enzymatic conjugation (see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and WO2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be coupled to a primary amine linker compound. Briefly, in some embodiments, a binder having a glutamine residue (e.g., gln295, i.e., Q295 residue) is treated with the above-mentioned primary amine-containing linker L1 in the presence of the enzyme transglutaminase. In certain embodiments, the binder is non-glycosylated. In certain embodiments, the binder is deglycosylated.

[0293] In certain embodiments, a binding agent (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, a binding agent comprises two heavy chain polypeptides, each having a gln295 residue. In further embodiments, a binding agent comprises one or more glutamine residues at a site other than 295 in the heavy chain.

[0294] In some embodiments, binding agents such as antibodies can be prepared by site-directed mutagenesis to insert glutamine residues at certain sites without impairing the function or binding of the antibody. For example, antibodies with Asn297Gln (N297Q) mutations as described herein are included herein. In certain embodiments, the binding agent comprises two heavy chain polypeptides, each with one gln295 residue and one gln297 residue.

[0295] In some embodiments, the antibody with gln295 residue and / or N297Q mutation contains one or more additional naturally occurring glutamine residues in its variable region, which may be accessible to transglutaminase and thus can be conjugated to linker or linker-payload.Exemplary naturally occurring glutamine residues can be found, for example, at Q55 of light chain.In such an example, the binding agent, for example, antibody, conjugated via transglutaminase can have a higher than expected LAR value (for example, LAR higher than 4).Any such antibody can be isolated from natural or artificial sources.

[0296] In certain embodiments of the present disclosure, the linker-antibody ratio, i.e., LAR, is 1, 2, 3, 4, 5, 6, 7, or 8 linker L1 molecules per antibody. In some embodiments, the LAR is 1-8. In some embodiments, the LAR is 1-6. In certain embodiments, the LAR is 2-4. In some cases, the LAR is 2-3. In certain cases, the LAR is 0.5-3.5. In some embodiments, the LAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the LAR is 2. In some embodiments, the LAR is 4.

[0297] Step 2: Payload conjugation reaction In certain embodiments, the linker L1 according to the present disclosure comprises at least one reactive group B' capable of further reaction after transglutamination. In these embodiments, the glutaminyl-modified protein (e.g., an antibody) can be further reacted with a reactive payload compound or a reactive linker-payload compound (e.g., L2-P as disclosed herein) to form a protein-payload conjugate. More specifically, the reactive linker-payload compound L2-P can comprise a reactive group B" capable of reacting with the reactive group B' of the linker L1. In certain embodiments, the reactive group B' according to the present disclosure comprises a moiety capable of undergoing a 1,3-cycloaddition reaction. In certain embodiments, the reactive group B' is an azide. In certain embodiments, the reactive group B" comprises an alkyne (e.g., a terminal alkyne or an internally strained alkyne). In certain embodiments of the present disclosure, the reactive group B' is compatible with the conjugating agent and the transglutamination reaction conditions.

[0298] In certain embodiments of the present disclosure, a linker L1 molecule comprises one reactive group B'. In certain embodiments of the present disclosure, a linker L1 molecule comprises more than one reactive group B'.

[0299] In certain embodiments, the reactive linker-payload L2-P comprises one payload molecule (n=1). In certain other embodiments, the reactive linker-payload L2-P comprises two or more payload molecules (n≧2). In certain embodiments, the reactive linker-payload L2-P comprises 1-12 payload molecules, or 1-10 payload molecules, or 1-8 payload molecules, or 1-6 payload molecules, or 1-4 payload molecules, or 1-2 payload molecules.

[0300] In certain embodiments, the reactive linker-payload L2-P comprises one payload molecule. Such an L2-P can be represented by BA-L1-B' (e.g., where B' is azide, BA-L1-N 3For example, if L2-P, which contains one payload molecule, is reacted with BA-L1-B', which has a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate will have a DAR of 4.

[0301] In certain embodiments, the reactive linker-payload L2-P comprises two payload molecules. When such L2-P is reacted with BA-L1-B', the DAR is about twice the LAR of BA-L1-B'. For example, when L2-P comprising two payload molecules is reacted with BA-L1-B' having a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate has a DAR of 8.

[0302] In certain embodiments of the present disclosure, the drug-antibody ratio, i.e., DAR (e.g., abbreviated as lowercase n), is about 1 to about 30, or about 1 to about 24, or about 1 to about 20, or about 1 to about 16, or about 1 to about 12, or about 1 to about 10, or about 1 to about 8, or about 1, 2, 3, 4, 5, 6, 7, or 8 payload molecules per antibody. In some embodiments, the DAR is 1 to 30. In some embodiments, the DAR is 1 to 24. In some embodiments, the DAR is 1 to 16. In some embodiments, the DAR is 1 to 8. In some embodiments, the DAR is 1 to 6. In certain embodiments, the DAR is 2 to 4. In some cases, the DAR is 2 to 3. In certain cases, the DAR is 0.5 to 3.5. In some embodiments, the DAR is 4.

[0303] In one aspect, the present disclosure provides a compound of formula (A): BA-(L1-B-L2-P) n (A) The present invention provides a method for producing a compound having the structure: BA is an antibody or an antigen-binding fragment thereof, L1 is a first linker that is covalently attached to the side chain of a glutamine residue of BA; B is a triazole-containing moiety; L2 is a second linker covalently binding to P; P is PI to P-IV

[0304] [ka] an antitumor agent selected from the group consisting of R 1 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH 2 ) v -phenyl, and -(CH 2 ) v -SO 2 CH 3 where v is an integer from 0 to 12; R 3 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -C(O)OH, -(CH2 ) v -phenyl, -(CH 2 ) v -SO 2 CH 3 , and -CO-(CH 2 ) v -O-COCH 3 where v is an integer from 0 to 12; R 4 -NH-, -N(-C 1-6 Alkyl), -N(-C 1-6 Alkyl)(-SO 2 CH 3 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -OH), -N(-C 1-6 Alkyl)(CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-(CH 2 ) v -O-CH 2 -NH-CO-CH 2 -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -COOH), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -NH 2 ), -N(-C 1-6 Alkyl)(-CO-CH(NH 2 )-(CH 2 ) v -phenyl), or

[0305] [ka] where v is an integer from 0 to 12; R 5 -H, -OH, -OCH 3 ,or

[0306] [ka] and R 6 is hydrogen, -C 1-6 Alkyl, -(CH 2 ) v -OH, -(CH 2 ) v -NH 2 , -(CH 2 ) v -phenyl, -(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CH 2 -phenyl, -(CH 2 ) v -NMe-CH 2 -phenyl-OMe, -(CH 2 ) v -NH-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-(CH 2 ) v -CCH, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -phenyl, -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH,

[0307] [ka] and v is independently an integer from 0 to 12; R 7 -H, -OH, -OCH 3 ,or

[0308] [ka] and the method is a) contacting a BA containing at least one glutamine residue with a compound L1-B' in the presence of a transglutaminase; b) contacting the product of step a) with one or more equivalents of a compound B″-L2-P, wherein group B″ is capable of being covalently linked to group B′, and one of groups B′ and B″ is —N 3 and

[0309] [ka] and the other of groups B' and B'' is selected from

[0310] [ka] and Z is C or N; and c) isolating the compound of formula (A) produced.

[0311] In one embodiment, the group B' is azide (-N 3 ).

[0312] In one embodiment, the group B″ comprises an alkyne. In one embodiment, the group B″ comprises

[0313] [ka] and Z is C or N.

[0314] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA. In another embodiment, the glutamine residue Gln is introduced into the BA by modifying one or more amino acids. In one embodiment, the Gln is Q295 or N297Q.

[0315] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG).In one embodiment, the transglutaminase is a bacterial transglutaminase (BTG).

[0316] Therapeutic Formulations and Administration The present disclosure provides a pharmaceutical composition comprising a protein-drug conjugate of the present disclosure.

[0317] In one aspect, the present disclosure provides a composition comprising a population of protein-drug conjugates according to the present disclosure having a drug-to-antibody ratio (DAR) of about 0.5 to about 30.0.

[0318] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0319] In one embodiment, the composition has a DAR of about 2.

[0320] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0321] In one embodiment, the composition has a DAR of about 4.

[0322] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0323] In one embodiment, the composition has a DAR of about 8.

[0324] In one embodiment, the composition has a DAR of about 10 to about 14.

[0325] In one embodiment, the composition has a DAR of about 12.

[0326] In one embodiment, the composition has a DAR of about 14 to about 18.

[0327] In one embodiment, the composition has a DAR of about 16.

[0328] In one embodiment, the composition has a DAR of about 20 to about 24.5.

[0329] In one embodiment, the composition has a DAR of about 24.

[0330] The compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0331] The dose of the protein-drug conjugate administered to a patient may vary depending on the age and size of the patient, the target disease, the condition, the route of administration, and the like. The appropriate dose is typically calculated according to body weight or body surface area. When the protein-drug conjugate of the present disclosure is used for therapeutic purposes in an adult patient, it may be advantageous to administer the protein-drug conjugate of the present disclosure intravenously, typically at a single dose of about 0.01 to about 20 mg per kg of body weight, more preferably about 0.02 to about 7 mg, about 0.03 to about 5 mg, or about 0.05 to about 3 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering the protein-drug conjugate can be empirically determined. For example, the progress of the patient can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0332] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, including, for example, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J.Biol.Chem.262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0333] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily applied in the delivery of the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen delivery device does not have a replaceable cartridge. Rather, a disposable pen delivery device is pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0334] Many reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN, to name just a few. STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices applicable in subcutaneous delivery of the pharmaceutical composition of the present disclosure include, but are not limited to, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0335] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0336] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by dissolving, suspending, or emulsifying, for example, the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. The aqueous medium for injection includes, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and these can be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, and the like can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations thus prepared are preferably filled into suitable ampoules.

[0337] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in a dosage form of unit dose suitable for the dosage of the active ingredient. Examples of such dosage forms of unit dose include tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody is usually about 5 to about 500 mg per dosage form in a unit dose. Particularly in the form of injection, the antibody is preferably contained in an amount of about 5 to about 100 mg and about 10 to about 250 mg for other dosage forms.

[0338] Therapeutic uses of protein-drug conjugates, linker-payloads, and payloads In another aspect, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for, inter alia, the treatment, prevention, and / or amelioration of a disease, disorder, or condition in need of such treatment.

[0339] In one embodiment, the invention provides a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to the present disclosure (e.g., an antibody-drug conjugate, a linker-payload, and / or a payload), or a composition comprising any compound according to the present disclosure.

[0340] In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating HER2+ breast cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating prostate cancer.

[0341] In one aspect, the present disclosure provides a method for selectively delivering a compound to a cell. In one embodiment, the method for selectively delivering a compound to a cell comprises linking the compound to a targeting antibody. In one embodiment, the compound is the payload described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of breast cancer cell, ovarian cancer cell, prostate cancer cell, lung cancer cell, liver cancer cell, or brain cancer cell.

[0342] In certain embodiments, the present disclosure provides a method for the preparation of a pharmaceutical composition comprising:

[0343] [Table 15-1]

[0344] [Table 15-2] The present invention provides a method for selectively delivering into a cell a compound having a structure selected from the group consisting of:

[0345] The present disclosure provides a method for selectively targeting an antigen on the surface of a cell with a compound. In one embodiment, the method for selectively targeting an antigen on the surface of a cell with a compound comprises linking the compound to a targeted antibody. In one embodiment, the compound is the payload described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of breast cancer cell, ovarian cancer cell, prostate cancer cell, lung cancer cell, liver cancer cell, or brain cancer cell.

[0346] In certain embodiments, the present disclosure provides a method for detecting an antigen on the surface of a cell by:

[0347] [Table 16-1]

[0348] [Table 16-2] The present invention provides a method of selectively targeting a compound having a structure selected from the group consisting of:

[0349] In certain embodiments, the present disclosure provides a method of treating a tumor and / or cancer, comprising administering to the tumor and / or cancer a therapeutic agent comprising:

[0350] [Table 17-1]

[0351] [Table 17-2] or a pharma- ceutically acceptable salt thereof.

[0352] Anti-Her2 antibody-drug conjugates In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for the treatment, prevention, and / or amelioration of, inter alia, any disease or disorder associated with or mediated by HER2 expression or activity, or treatable by binding to HER2 without competing for modified LDL, and / or by promoting HER2 receptor internalization and / or reducing the number of cell surface receptors.

[0353] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting immune responses is beneficial. In particular, the anti-HER2 protein-drug conjugates, including both monospecific anti-HER2 antibodies and bispecific anti-HER2 / HER2 antibodies of the present disclosure, can be used for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by HER2 expression or activity, or the proliferation of HER2+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved includes killing cells expressing HER2 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing HER2 that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0354] In one embodiment, the protein-drug conjugates of the present disclosure (and therapeutic compositions and dosage forms comprising same) comprise: a first antigen-binding domain (D1), and Second antigen-binding domain (D2) and a bispecific antigen-binding molecule comprising D1 specifically binds to the first epitope of human HER2; D2 specifically binds to a second epitope on human HER2.

[0355] In one embodiment of the above, D1 and D2 do not compete with each other for binding to human HER2.

[0356] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. According to certain embodiments of the present disclosure, anti-HER2 antibodies or anti-HER2 / HER2 bispecific antibodies are useful for treating patients suffering from breast cancer cells that are IHC2+ or higher. According to other related embodiments of the present disclosure, a method is provided that includes administering an anti-HER2 antibody or anti-HER2 / HER2 antibody disclosed herein to a patient suffering from breast cancer cells that are IHC2+ or higher. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a tumor that is castration-resistant.

[0357] In certain embodiments, the present disclosure further includes a method for treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0358] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting immune responses is beneficial. In particular, the protein-drug conjugates comprising the anti-HER2 antibody or anti-HER2 / HER2 antibody of the present disclosure can be used for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by HER2 expression or activity, or the proliferation of HER2+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved includes killing cells expressing HER2 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing HER2 that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0359] According to certain aspects, the disclosure provides a method for treating a disease or disorder associated with HER2 expression (e.g., breast cancer), comprising administering to a subject one or more of the anti-HER2 protein-drug conjugates or anti-HER2 / HER2 bispecific protein-drug conjugates described elsewhere herein after the subject has been determined to have breast cancer (e.g., and IHC2+ breast cancer). For example, the disclosure includes a method for treating breast cancer, comprising administering to a patient a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding molecule or an anti-HER2 / HER2 bispecific antibody or antigen-binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., antiandrogen therapy).

[0360] In certain embodiments, the present disclosure further includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by HER2-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment, or an anti-HER2 / HER2 bispecific antibody or antigen-binding fragment, disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0361] Combination Therapies and Formulations The present disclosure provides methods that include administering a pharmaceutical composition that includes any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined or administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of the present disclosure include, for example, HER2 antagonists (e.g., anti-HER2 antibodies [e.g., trastuzumab] or small molecule inhibitors of HER2 or anti-HER2 antibody-drug conjugates, or anti-HER2 / HER2 bispecific antibodies, or anti-HER2 / HER2 bispecific antibody-drug conjugates), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), HER2 / ErbB2, ErbB3, or other therapeutic agents. or an antagonist of another EGFR family member, such as ErbB4 (e.g., an anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds to EGFRvIII), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody), a VEGF antagonist (e.g., VEGF-Trap, see, e.g., US 7,087,See US 2009 / 0142354, US 2011 / 0027286, e.g., H1H685P, FOL2 antibodies, anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, e.g., H1H685P), FOL2 antibodies, anti-VEGF fusion proteins (e.g., anti-VEGF fusion proteins), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, e.g., H1H685P, FOL2 antibodies, anti-VEGF fusion proteins (e.g., anti-VEGF fusion proteins), ... Examples of the antagonists include H1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), and uroplakin antagonists (e.g., anti-uroplakin antibodies).

[0362] Other agents that may be beneficially administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of the present disclosure include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors. Pharmaceutical compositions of the disclosure (e.g., pharmaceutical compositions comprising an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 protein-drug conjugate (e.g., an antibody-drug conjugate disclosed herein)) can further comprise any of the following drugs: "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), "DHAP": dexamethasone (e.g., Deca dron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ), and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0363] The disclosure further includes therapeutic combinations comprising any of the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads referred to herein and one or more inhibitors of HER2, VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the foregoing cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen-binding molecules of the present disclosure may further be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of the present disclosure may further be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0364] The additional therapeutically active ingredient may be administered immediately prior to, simultaneously with, or immediately following administration of the antigen-binding molecule of the present disclosure (for purposes of the present disclosure, such administration regimens will be considered administration of the antigen-binding molecule "in combination" with the additional therapeutically active ingredient).

[0365] The present disclosure includes pharmaceutical compositions in which a protein-drug conjugate (e.g., an antibody-drug conjugate), linker-payload, and / or payload of the present disclosure are co-formulated with one or more additional therapeutically active ingredients, as described elsewhere herein.

[0366] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload can be administered to a subject over a defined time course. Methods according to this aspect of the present disclosure include sequentially administering to a subject multiple doses of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload of the present disclosure. As used herein, "sequentially administered" means that each dose of protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The disclosure includes methods comprising sequentially administering to a patient a single initial dose of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload, followed by one or more secondary doses of the protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload, optionally followed by one or more tertiary doses of the protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload.

[0367] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of the protein-drug conjugate of the present disclosure (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload. Thus, a "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"). A "secondary dose" is a dose administered after the initial dose. A "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of protein-drug conjugate (e.g., anti-HER2 or anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload, but generally may differ from each other with respect to frequency of administration. However, in certain embodiments, the amounts of protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload included in the initial dose, secondary dose, and / or tertiary dose are varied relative to one another (e.g., adjusted up or down as needed) over the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0368] In one exemplary embodiment of the present disclosure, the secondary and / or tertiary doses each comprise 1 to 26 of the immediately preceding dose (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, The vaccine is administered 13, 13 and 1 / 2, 14, 14 and 1 / 2, 15, 15 and 1 / 2, 16, 16 and 1 / 2, 17, 17 and 1 / 2, 18, 18 and 1 / 2, 19, 19 and 1 / 2, 20, 20 and 1 / 2, 21, 21 and 1 / 2, 22, 22 and 1 / 2, 23, 23 and 1 / 2, 24, 24 and 1 / 2, 25, 25 and 1 / 2, 26, 26 and 1 / 2, or more) weeks later. As used herein, the phrase "immediately preceding dose" refers to a dose of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload in a multiple administration sequence, without any intervening doses, that is administered to a patient prior to administration of the next dose in the sequence.

[0369] The method according to this aspect of the disclosure may include administering any number of secondary and / or tertiary doses of protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0370] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may further be adjusted during the course of treatment by the physician depending on the needs of the individual patient after clinical testing. EXAMPLES

[0371] The following examples illustrate certain aspects of the present invention. The examples should not be construed as limiting, but merely provide a more specific understanding and practice of the embodiments and various aspects thereof.

[0372] [Table 18-1]

[0373] [Table 18-2]

[0374] [Table 18-3]

[0375] Exatecan mesylate (P1), belotecan HCl salt (P8), 10-hydroxycamptothecin (P14), SN38 (P16), and irinotecan (P19) were commercially available from MCE or Bide Pharm. Compound 7A-1 was commercially available from TCI, and compound 7-2 was commercially available from Accela. Linker 5-1 was synthesized as described in WO2018089373 and WO2020146541, and linker 6-1 was synthesized as described in WO2020146541.

[0376] Example 1: Synthesis of Proexatecan (ProEXT) (Scheme 1)

[0377] The payloads and their characteristics according to the present disclosure are listed in Table 1 below.

[0378] [Table 19-1]

[0379] [Table 19-2]

[0380] [Table 19-3]

[0381] [Table 19-4]

[0382] [Table 19-5]

[0383] [ka]

[0384] Example 1A: General Procedure A for the Synthesis of Protected ProEXT (1-1)

[0385] [ka]

[0386] To a solution of protected amino acid (1.0 equiv.) in DMF (50 mM), exatecan mesylate (1.0 equiv.), HATU (1.5 equiv.), and DIPEA (2.0 equiv.) were added successively and the reaction mixture was stirred at room temperature overnight, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 1-1 as a pale yellow solid.

[0387] Example 1A-1: tert-butyl N-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)carbamate (Boc-GlyEXT,1-1a)

[0388] [ka]

[0389] Following the general procedure A starting from Boc-Gly-OH (18 mg, 0.10 mmol), Boc-GlyEXT (1-1a) (45 mg, 75% yield) was obtained as a pale yellow solid. ESI m / z: 593.3 (M+H). + .

[0390] Example 1A-2: tert-Butyl(4S)-4-{[(tert-butoxy)carbonyl]amino}-4-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 ,13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}butanoate (Boc-Glu(O t Bu)EXT,1-1b)

[0391] [ka]

[0392] N-Boc-Glu(O t According to the general procedure A starting from Boc-Glu(O Bu)-OH (CAS: 13726-84-6, 50 mg, 0.16 mmol), t Bu)EXT(1-1b) (91 mg, 77% yield) was obtained as a yellow solid. ESI m / z: 721.3 (M+H) + .

[0393] Example 1A-3: tert-Butyl N[(1S)-5-{[(tert-butoxy)carbonyl]amino}-1-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}pentyl]carbamate (Boc-Lys(Boc)EXT,1-1c)

[0394] [ka]

[0395] Following the general procedure A starting from Boc-Lys(Boc)-OH (CAS: 2483-46-7, 0.12 g, 0.35 mmol), Boc-Lys(Boc)EXT(1-1c) (0.17 g, 64% yield) was obtained as a grey solid. ESI m / z: 764.2 (M+H). + , 664.3 (M-Boc+H) + .

[0396] Example 1A-4: (9H-fluoren-9-yl)methyl N-[(1S)-1-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}-2-phenylethyl]carbamate (Fmoc-PheEXT,1-1d)

[0397] [ka]

[0398] Following the general procedure A starting from N-Fmoc-Phe-OH (CAS: 35661-40-6, 50 mg, 0.13 mmol), N-Fmoc-PheEXT (1-1d) (73 mg, 70% yield) was obtained as a yellow solid. ESI m / z: 805.2 (M+H). + .

[0399] Example 1A-5: 2-amino-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 ,11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]acetamide (GlyEXT,P2)

[0400] [ka]

[0401] To a solution of Boc-GlyEXT(1-1a) (45 mg, 75 μmol) in DCM (3 mL) was added TFA (1 mL) and the mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.3%)) to give GlyEXT(P2) (35 mg, 90% yield) as a white solid. ESI m / z: 493.2 (M+H) + .

[0402] Example 1A-6: (4S)-4-amino-4-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}butanoic acid (GluEXT,P3)

[0403] [ka]

[0404] Following a similar procedure to P2, but starting from 1-1b (60 mg, 83 μmol) instead of 1-1a, P3 (34 mg, 60% yield, TFA salt) was obtained as a yellow solid. ESI m / z: 565.2 (M+H). + . 1 H NMR (400MHz, DMSO d6 ) δ 9.00(d,J=8.0Hz,1H),8.34-8.13(m,2H),7.86-7.83(m,1H),7.33(s,1H),6.55(s,1H),5.62-5.57(m,1H),5.43-5.38(m,3H),5.07- 5.02(m,1H),3.78(t,J=6.0Hz,1H),3.22-3.12(m,2H),2.43(s,3H),2.33-2.15(m,4H),1.98-1.79(m,4H),0.88(t,J=7.2Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ-73.5,-110.9ppm.

[0405] Example 1A-7: (2S)-2,6-diamino-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]hexanamide (LysEXT, P4)

[0406] [ka]

[0407] Following a similar procedure to P2, but starting from 1-1c (50 mg, 65 μmol) instead of 1-1a, P4 (34 mg, 66% yield, TFA salt) was obtained as a yellow solid. ESI m / z: 564.2 (M+H).+ . 1 H NMR (400MHz, DMSO d6 ) δ 9.09-9.06(m,1H),8.27(s,3H),7.85-7.83(m,1H),7.67(s,2H),7.35(s,1H),6.57(s, 1H),5.58-5.52(m,1H),5.48-5.43(m,2H),5.38-5.33(m,1H),5.13-5.08(m,1H),3.78( s,1H),3.24-3.15(m,2H),2.76(s,2H),2.42(s,3H),2.30-2.14(m,2H),1.90-1.84(m,2 H),1.80-1.70(m,2H),1.55-1.46(m,2H),1.39-1.28(m,2H),0.89(t,J=7.2Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -73.6,-110.9ppm.

[0408] Example 1A-8: (2S)-2-amino-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]-3-phenylpropanamide (PheEXT, P5)

[0409] [ka]

[0410] To a solution of compound 1-1d (50 mg, 62 μmol) in DMF (1 mL) was added diethylamine (0.1 mL) and the reaction mixture was stirred at room temperature for 30 min, which was monitored by LCMS. The resulting mixture was purified twice by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give P5 (14 mg, 39% yield) as a pale yellow solid. ESI m / z: 583.3 (M+H). + . 1 H NMR (400MHz, DMSO d6 ) δ 8.85(d,J=8.4Hz,1H),8.31(s,3H),7.86-7.83(m,1H),7.32(s,1H),7.02(s,1H),7.00(s ,1H),6.91(t,J=7.6Hz,2H),6.85(t,J=7.2Hz,1H),6.56(s,1H),5.59-5.52(m,1H),5.47 -5.41(m,2H),5.25-5.20(m,1H),4.62-4.58(m,1H),4.05(s,1H),3.27-3.10(m,2H),3.0 5-2.95(m,2H),2.43(s,3H),2.33-2.10(m,2H),1.98-1.85(m,2H),0.93-0.89(m,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -73.5,-111.0ppm.

[0411] Example 1B: Alternative synthesis of LysEXT(P4)

[0412] Example 1B-1: (9H-fluoren-9-yl)methyl N-[(1S)-5-azido-1-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]Tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}pentyl]carbamate (Fmoc-Lys(azide)EXT,1-2)

[0413] [ka]

[0414] Following the general procedure A starting from Fmoc-L-azido idine (CAS: 159610-89-6, 41 mg, 0.10 mmol), Fmoc-Lys(azido)EXT(1-2) (40 mg, 92% yield) was obtained as a pale yellow solid. 1 H NMR (400MHz, DMSO d6 ) δ 8.56(d,J=8.4Hz,1H),7.89-7.86(m,2H),7.80(d,J=11.0Hz,1H),7.75-7.54(m,3H),7.44-7.35(m,2H),7.35-7.20(m,3H),6.52(br s,1H),5.55-5.51(m,1H),5.39(d,J=16.2Hz,1H),5.33-5.22(m,2H),5.09( d,J=19.0Hz,1H),4.32-4.13(m,3H),4.10-3.95(m,1H),3.34-3.24(m,2H),3 .16-3.12(m,2H),2.40(s,3H),2.23-2.09(m,2H),1.89-1.77(m,2H),1.68-1 .62(m,2H),1.49-1.42(m,2H),1.40-1.22(m,2H),0.87(t,J=7.3Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -73.9,-111.2ppm.

[0415] Example 1B-2: (9H-fluoren-9-yl)methyl N-[(1S)-5-amino-1-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .04 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}pentyl]carbamate (1-3)

[0416] [ka]

[0417] To a yellow solution of compound 1-2 (80 mg, 86 μmol) in methanol (15 mL) and THF (5 mL) was added palladium on carbon (50 mg, 10% Pd) under nitrogen protection. The mixture was stirred at room temperature under hydrogen atmosphere for 5 h, which was monitored by LCMS. The resulting mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 1-3 (70 mg, crude) as a pale yellow solid. ESI m / z 393.5 (M / 2+H) + . 19 F NMR (376MHz, DMSO d6 ) δ -73.5,-111.1ppm.

[0418] Example 1B-3: (2S)-2,6-diamino-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]hexanamide (LysEXT, P4)

[0419] [ka]

[0420] To a solution of compound 1-3 (10 mg, crude, obtained above) in DMF (1 mL) was added diethylamine (0.2 mL) and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting solution was directly separated by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give P4 (5 mg, 41% yield from 1-2, TFA salt) as a pale yellow solid. ESI m / z: 564.2 (M+H) + .

[0421] Example 1C: N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]methanesulfonamide (MsEXT, P6)

[0422] [ka]

[0423] To a solution of exatecan mesylate (30 mg, 56 μmol) in dry DMA (1 mL), triethylamine (17 mg, 0.17 mmol) and methanesulfonyl chloride (7.1 mg, 62 μmol) were added successively at 0 °C, and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was directly separated by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.1%)) to give P6 (4.0 mg, 11% yield) as a yellow solid. ESI m / z 514.0 (M+H) + . 1H NMR (400MHz, DMSO d6 ) δ 7.81(d,J=1.6Hz,1H),7.79(d,J=4.8Hz,1H),7.32(s,1H),6.52(s,1H),5.43-5.42(m,3H),5.13-5.18(m,1H),3. 18(br,4H),2.39(s,3H),2.30-2.26(m,2H),1.93-1.83(m,3H),1.16(t,J=7.2Hz,1H),0.88(t,J=7.2Hz,3H)ppm. 19 F NMR (377MHz, DMSO d6 ) δ -73.44,-111.30ppm.

[0424] Example 1D: (10S,23S)-10-Ethyl-18-fluoro-10-hydroxy-23-[(2-methanesulfonylethyl)amino]-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaene-5,9-dione (MsEthEXT, P7)

[0425] [ka]

[0426] To a solution of exatecan mesylate (10 mg, 19 μmol) in methanol (2 mL), (methylsulfonyl)ethane (5.0 mg, 46 μmol) and triethylamine (10 mg, 92 μmol) were added and the reaction mixture was stirred at 65 °C for 4 h, which was monitored by LCMS. The resulting mixture was directly separated by reverse-phase flash chromatography (5-95% acetonitrile in aqueous TFA (0.1%)) to give P7 (5.0 mg, 40% yield, TFA salt) as a white solid. ESI m / z 542.5 (M+H) + .1 H NMR (400MHz, DMSO d6 ) δ 7.84(d,J=11.2Hz,1H),7.34(s,1H),6.60(s,1H),5.54-5.36(m,5H),5.00(s,1H),3.55-3.40(m, 5H),3.20-3.10(m,5H),2.65-2.60(m,1H),2.40(s,3H),1.89-1.85(m,2H),0.88-0.84(m,3H)ppm.

[0427] Example 2: Synthesis of payloads P9-P11 and P31-P34 (Scheme 2)

[0428] [ka]

[0429] Example 2A: N-{2-[(19S)-19-ethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]ethyl}-N-(propan-2-yl)methanesulfonamide (MsBLT, P9)

[0430] [ka]

[0431] To a solution of belotecan (30 mg, 64 μmol) in dry DMA (1 mL), triethylamine (19 mg, 0.19 mmol) and methanesulfonyl chloride (8.0 mg, 70 μmol) were added successively at 0 °C, and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was directly separated by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.1%)) to give P9 (6.0 mg, 18% yield) as a yellow solid. ESI m / z 512.3 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.32(d,J=8.4Hz,1H),8.20(d,J=8.4Hz,1H),7.89(t,J=7.6Hz,1H),7.79(t,J=7.6Hz,1H),7.35(s,1H),6.54(s,1H),5.48-5.40(m,4H) ),4.02-3.95(m,1H),3.55-3.46(m,2H),3.40(s,2H),3.00(s,3H),1.93-1.83(m,2H),1.15(d,J=4.0Hz,6H),0.88(t,J=7.6Hz,3H)ppm.

[0432] Example 2B: (19S)-19-Ethyl-19-hydroxy-10-{2-[(2-hydroxyethyl)(propan-2-yl)amino]ethyl}-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (HOEthBLT, P10)

[0433] [ka]

[0434] To a solution of belotecan (60 mg, 0.12 mmol) in methanol was added O-TBS-hydroxyacetaldehyde (24 mg, 0.14 mmol) and acetic acid (0.01 mL, cat.) at room temperature. The mixture was stirred at room temperature for 30 min, followed by the addition of sodium cyanoborohydride (16 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 18 h, which was monitored by LCMS. The resulting mixture was directly separated by reversed-phase flash chromatography (5–35% acetonitrile in aqueous TFA (0.01%)) to give compound 2-1 (43 mg, 51% yield, ESI m / z 592.4 (M+H)). + ) as a yellow solid.

[0435] Compound 2-1 (30 g, 42 μmol) obtained above was dissolved in a solution of hydrochloride salt (4N, 2 mL) in dioxane at 0° C., and the mixture was stirred at 0° C. for 1 h, which was monitored by LCMS. The resulting mixture was neutralized to pH 6-7 with saturated aqueous sodium bicarbonate at 0° C. and concentrated in vacuo to remove dioxane. The remaining mixture was separated by reverse-phase flash chromatography (0-20% acetonitrile in aqueous TFA (0.1%)) to give P10 (20 mg, 67% yield) as a yellow solid. ESI m / z 478.3 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 9.64(s,1H),8.42(d,J=8.7Hz,1H),8.23(d,J=8.5Hz,1H),7.91(t,J=8.3Hz,1H),7.81(t,J=8.0Hz,1H),7.37(s,1H),6.57(s,1H),6.58(s,1H) ,5.54-5.39(m,4H),3.90-3.79(m,4H),3.77-3.71(m,2H),2.48-2.41( m,2H),1.93-1.84(m,2H),1.34-1.24(m,8H),0.88(t,J=7.3Hz,3H)ppm.

[0436] Example 2C: 2-amino-N-{[2-({2-[(19S)-19-ethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]ethyl}(propan-2-yl)amino)ethoxy]methyl}acetamide (ProBLT, P11)

[0437] [ka]

[0438] To a solution of P10 (83 mg, 0.14 mmol) in DCM (20 mL) was added compound 2-2 (CAS: 1599440-06-8, 57 mg, 0.14 mmol) and pyridinium p-toluenesulfonate (PPTS) (7.0 mg, 28 μmol) in a sealed tube at 50 °C, and the reaction mixture was stirred at 50 °C for 72 h. After cooling, the volatiles were removed in vacuo, and the residue was separated by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give a pale yellow solid (50 mg, ESI m / z 393.6 (M / 2+H). + ) was dissolved in methanol (2 mL). Diethylamine (0.2 mL) was added to the solution and the reaction mixture was stirred at room temperature for 3 h until the Fmoc was completely removed by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give P11 (26 mg, 27% yield from P10) as a pale yellow solid. ESI m / z 564.3 (M+H) + . 1 H NMR (400MHz, DMSO d6) δ 9.99(s,1H),9.46(s,1H),8.95-8.81(m,1H),8.37(d,J=8.4Hz,1H),8.23(d ,J=8.2Hz,1H),8.12(d,J=7.4Hz,1H),7.90(dd,J=18.3,8.1Hz,2H),7.82(t ,J=7.5Hz,1H),7.60(t,J=9.3Hz,3H),7.51(t,J=6.0Hz,1H),7.37(s,1H),7 .25(d,J=8.1Hz,2H),6.57(s,1H),5.99(t,J=5.3Hz,1H),5.55-5.35(m,6H), 4.91(s,2H),4.71(s,2H),4.44-4.33(m,1H),4.30-4.19(m,2H),3.90-3.72 (m,6H),3.65-3.56(m,5H),3.53-3.30(m,16H),3.26-3.22(m,2H),3.03-2. 93(m,2H),2.36(d,J=7.2Hz,1H),2.29-2.01(m,4H),2.00-1.80(m,6H),1.8 0-1.53(m,7H),1.46-1.34(m,3H),1.32-1.24(m,6H),0.90-0.82(m,9H)ppm.

[0439] Example 3: Synthesis of payloads P14-P17, P20-P28, and P41-P44 (Scheme 3)

[0440] [ka]

[0441] Example 3A: (19S)-19-Ethyl-19-hydroxy-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (P15)

[0442] [ka]

[0443] A mixture of 10-hydroxycamptothecin P14 (0.73 g, 2.0 mmol) and potassium carbonate (0.55 g, 4.0 mmol) was suspended in DMF (5 mL) and heated to 85° C. until the brown reaction mixture became clear. The solution was then cooled to room temperature and methyl iodide (0.73 g, 2.0 mmol) was added to the solution in one portion. The solution was stirred at 85° C. under a nitrogen balloon for an additional 2.5 h until the reaction was complete, which was monitored by LCMS. A pale yellow solid precipitated. After cooling to room temperature, the reaction mixture was diluted with water (100 mL) and filtered to collect the pale yellow precipitate, which was dried in vacuum to give compound P15 (0.50 g, 67% yield) as a pale yellow solid. ESI m / z: 379.1 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.52(s, 1H), 8.04(d, J=10 Hz, 1H), 7.49-7.46(m, 2H), 7.26(s, 1H), 6.54(s, 1H), 5.41(s, 2H), 5.23(s, 2H), 3.93(s, 3H), 1.90-1.82(m, 2H), 0.88(t, J=7.6 Hz, 3H) ppm. Reference CN1587265, confirming the identity of P15, is incorporated by reference in its entirety.

[0444] Example 3B: (19S)-10,19-diethyl-19-hydroxy-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (P17)

[0445] [ka]

[0446] Following a similar procedure as compound P15, except using 7-ethyl-10-hydroxycamptothecin (SN-38, Example 2) instead of 10-hydroxycamptothecin, compound P17 (0.52 g, 84% yield, free base) was obtained as a pale yellow solid, which contained the lactone ring-opened product. 20 mg of the free base was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.3%)) to give Example 5 (15 mg, TFA salt) as a pale yellow solid. ESI m / z: 407.1 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.08(d,J=8.8Hz,1H), 7.53-7.49(m,2H), 7.27(s,1H), 6.52(s,1H), 5.43(s,2H), 5.30(s,2H), 3.99(s,3H), 3.20(q,J=7.2 Hz 2H), 1.93-1.81(m,2H), 1.33(t,J=7.6Hz,3H), 0.88(t,J=7.2Hz,3H)ppm. (No TFA protons observed). 19 F NMR (376MHz, DMSO d6 ) δ -73 ppm. Reference WO2005044821 confirming the identity of P17 is incorporated by reference in its entirety.

[0447] Example 3C: (19S)-19-Ethyl-19-hydroxy-10-(hydroxymethyl)-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (P20)

[0448] To a cooled (0° C. ice-water bath) suspension of compound P15 (0.30 g, 0.79 mmol) in a co-solvent of methanol (10 mL) and water (10 mL), 96% sulfuric acid (5.3 mL, 95 mmol) was added dropwise, and green vitriol (0.27 g, 0.95 mmol) was added. The suspension became clear and was cooled to −10° C. To the resulting solution, 30% hydrogen peroxide (1 mL, 9.5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 4 days, which was monitored by LCMS. The mixture was diluted with water, and a pale yellow solid precipitated. After collection by filtration and drying, compound P20 (0.29 g, 90% yield) was obtained as a pale yellow solid, which was pure enough without further purification. ESI m / z: 409.1 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.07(d,J=9.2Hz,1H),7.50(dd,J=9.2, 2.0Hz,1H),7.38(s,1H),7.27(s,1H),5.43(s,2H),5.38(s,2H),5.37(s,2H),5.23(br s,2H),3.96(s,3H),1.89-1.83(m,2H),0.89(t,J=7.2Hz,3H)ppm.

[0449] Example 3D: (19S)-10-(chloromethyl)-19-ethyl-19-hydroxy-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (int A)

[0450] [ka]

[0451] To a stirred suspension of compound P20 (0.12 g, 0.29 mmol) in dry pyridine (3 mL) was added methanesulfonyl chloride (67 mg, 0.59 mmol) at room temperature. The suspension was then stirred at room temperature for 2 h until P20 was completely consumed, which was monitored by LCMS. The reaction mixture was directly purified by reverse-phase flash chromatography (0-50% acetonitrile in aqueous TFA (0.01%)) to give the chloride (52 mg, crude) as a yellow solid, which was used in the next step. ESI m / z: 427.1 (M+H). + .

[0452] Example 3E: (19S)-10-(azidomethyl)-19-ethyl-19-hydroxy-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (P21)

[0453] [ka]

[0454] To a stirred yellow solution of int A (52 mg, crude, obtained above) in DMF (1.3 mL) was added sodium azide (30 mg, 0.47 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h until the reaction became clear. LCMS showed that the chloride was then consumed. The reaction mixture was directly purified by reverse-phase flash chromatography (0-100% methanol in aqueous TFA (0.01%)) to give compound P21 (25 mg, 26% yield from P20) as a pale yellow solid. ESI m / z: 434.1 (M+H) + . 1 H NMR (400MHz, DMSO d6) δ 8.13(d,J=9.2Hz,1H),7.58(d,J=2.4Hz,1H),7.57(dd,J=9.2, 2.8Hz,1H),7.29(s,1H),6.54(s,1H),5.42(s,2H),5.40(s,2H),5.27(s,2H),4.00(s,3H),1.91-1.83(m,2H),0.88(t,J=7.6Hz,3H)ppm.

[0455] Example 3F: (19S)-10-(aminomethyl)-19-ethyl-19-hydroxy-7-methoxy-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (P22)

[0456] [ka]

[0457] To a solution of compound P21 (20 mg, 46 μmol) in a co-solvent of THF (4 mL) and water (1 mL) was added triphenylphosphine (36 mg, 0.14 mmol). The clear solution was stirred overnight at room temperature under a nitrogen balloon until the reduction was complete according to LCMS. The reaction solution was concentrated in vacuo and the residue was purified by reverse phase flash chromatography (0-100% methanol in aqueous TFA (0.01%)) to give compound P22 (7 mg, 37% yield, TFA salt) as a pale yellow solid. ESI m / z: 408.2 (M+H) + . 1 H NMR (400MHz, DMSO d6) δ 8.44(s,3H),8.16(d,J=8.8Hz,1H),7.68(d,J=2.4Hz,1H),7.61(dd,J=9.2,2.4Hz,1H),7.31(s,1H),6.56 (s,1H),5.53(s,2H),5.45(s,2H),4.73(s,2H),4.05(s,3H),1.92-1.84(m,2H),0.88(t,J=7.6Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -73.62ppm.

[0458] Example 3G: General Procedure B for the Synthesis of Payload P23-26

[0459] To a solution of int A (1 equiv.) in dry DMF (10 mM) was added the corresponding amine (2.0 equiv.) and the reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was directly separated by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give the payloads P23-P26 as white solids.

[0460] Example 3G-1: Synthesis of (S)-11-(((3-azidopropyl)amino)methyl)-4-ethyl-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (P23)

[0461] [ka]

[0462] Following general procedure B, payload P23 (4.0 mg, 17% yield) was obtained as a white solid. ESI m / z: 491.2 (M+H) + . 1 H NMR (400MHz, DMSO d6) δ 8.07(d,J=8.8Hz,1H),7.54(s,1H),7.52-7.48(m,2H),5.50(d,J=16.4Hz,1H),5.38(s,2H),5.30(d,J=16.4Hz,1H),3 .98(s,3H),3.43(t,J=6.0Hz,2H),2.96-2.91(m,2H),1.94-1.84(m,5H),1.21-1.18(m,4H),0.91(t,J=7.2Hz,3H)ppm.

[0463] Example 3G-2: Synthesis of (S)-4-ethyl-4-hydroxy-9-methoxy-11-((prop-2-yn-1-ylamino)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (P24)

[0464] [ka]

[0465] Following general procedure B, payload P24 (1.3 mg, 6% yield) was obtained as a white solid. ESI m / z: 446.3 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.13-8.08(m,1H),7.67(d,J=2.8Hz,1H),7.54-7.51(m,1H),7.28(s,1H),6.50(s,1H),5.42-5.40(m,4H),4.36(br s,1H),3.97(s,3H),2.00-1.97(m,1H),1.90-1.83(m,2H),1.29-1.18(m,4H),0.88(t,J=7.2Hz,3H)ppm.

[0466] Example 3G-3: Synthesis of (S)-4-ethyl-4-hydroxy-9-methoxy-11-(((4-methoxybenzyl)(methyl)amino)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (P25)

[0467] [ka]

[0468] Following general procedure B, payload P25 (8.0 mg, 31% yield) was obtained as a white solid. ESI m / z: 542.2 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 7.96(d,J=9.2Hz,1H),7.48(s,1H),7.41-7.33(m,3H),7.10(br s,1H),6.92-6.89(m,2H),5.48(d,J=16.0Hz,1H),5.28(d,J=16.0Hz,1H),5.22(br,4H),4.01-3.97(m,1H) ),3.77-3.71(m,6H),2.59-2.43(m,3H),1.89-1.83(m,2H),1.21-1.18(m,1H),0.90(t,J=7.2Hz,3H)ppm.

[0469] Example 3G-4: Synthesis of (S)-11-((benzylamino)methyl)-4-ethyl-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (P26)

[0470] [ka]

[0471] Following general procedure B, payload P26 (2.0 mg, 8.5% yield) was obtained as a white solid. ESI m / z: 498.2 (M+H). + .

[0472] Example 3G-5: Synthesis of 2-((1-(((S)-4-ethyl-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazol-4-yl)oxy)acetic acid (P27)

[0473] [ka]

[0474] To a solution of P21 (15 mg, 35 μmol) in methanol (3.0 mL), COT (13 mg, 0.071 μmol) was added and the reaction mixture was stirred for 20 h at room temperature. The resulting mixture was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give P27 (5.5 mg, 26% yield) as a white solid. ESI m / z: 616.3 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.43(br s, 3H),8.13(d,J=9.2Hz,1H),7.56-7.51(m,2H),7.29(s,1H),6.58(br s,1H),6.26(s,2H),5.41(s,2H),5.16-4.98(m,3H),3.89(s,3H),3.73-3.6 2(m,4H),2.94-2.71(m,3H),1.89-1.80(m,4H),1.45-1.23(m,6H),1.00(br s,1H),0.87(t,J=7.2Hz,3H)ppm.

[0475] Example 3G-6: Synthesis of (S)-11-((4-benzyl-1H-1,2,3-triazol-1-yl)methyl)-4-ethyl-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (P28)

[0476] [ka]

[0477] To a solution of P21 (10 mg, 23 μmol) in THF (3 mL) was added benzylethene (2.7 mg, 23 μmol), sodium ascorbate (9.1 mg, 46 μmol), and aqueous copper(II) sulfate (2 mg in 1 mL of water), and the reaction mixture was stirred for 20 h at room temperature in the dark. The resulting mixture was directly separated by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give P28 (4.3 mg, 34% yield) as a white solid. ESI m / z: 550.1 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.51(br s,1H),8.07(d,J=9.2Hz,1H),7.84(s,1H),7.60(s,1H),7.48-7.42(m,2H),7.23-7.14(m,5H),6.22(s,2H),5.57(d,J=16.0 Hz,1H),5.37(d,J=16.0Hz,1H),5.36(s,2H),3.85(s,3H),1.97-1.94(m,2H),1.30-1.26(m,2H),0.99(t,J=7.2Hz,3H)ppm.

[0478] Example 4: Synthesis of payloads P13 and P18 (Scheme 4)

[0479] [ka]

[0480] Example 4A: Synthesis of tert-butyl N-{2-[(2S)-2-[12-ethyl-8-(hydroxymethyl)-2-methoxy-9-oxo-9H,11H-indolizino[1,2-b]quinolin-7-yl]-2-hydroxybutanamido]ethyl}carbamate (4-1a)

[0481] [ka]

[0482] A suspension of compound P17 (0.12 g, 0.30 mmol) and 1-Boc-ethylenediamine (0.20 g, 1.2 mmol) in dry acetonitrile (10 mL) was stirred at 50 °C for 3 days until the suspension became yellow and clear. The solution was directly purified by reverse-phase flash chromatography (0-80% acetonitrile in water) to give compound 4-1a (0.12 g, 71% yield) as a pale yellow solid. ESI m / z: 549.3 (MH 2 O+H) + .

[0483] Example 4B: Synthesis of {7-[(1S)-1-[(2-{[(tert-butoxy)carbonyl]amino}ethyl)carbamoyl]-1-hydroxypropyl]-12-ethyl-2-methoxy-9-oxo-9H,11H-indolizino[1,2-b]quinolin-8-yl}methyl acetate (4-2a)

[0484] [ka]

[0485] To a yellow solution of compound 4-1a (0.12 g, 0.21 mmol) in dry pyridine (2 mL) was added acetic anhydride (0.43 g, 4.2 mmol) and the reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The reaction mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 4-2a (55 mg, 42% yield) as a pale yellow solid. ESI m / z 549.3 (M-OAc). + .

[0486] Example 4C: Synthesis of {7-[(1S)-1-[(2-aminoethyl)carbamoyl]-1-hydroxypropyl]-12-ethyl-2-methoxy-9-oxo-9H,11H-indolizino[1,2-b]quinolin-8-yl}methyl acetate (P18)

[0487] [ka]

[0488] To a stirred yellow solution of compound 4-2a (6.1 mg, 10 μmol) in DCM (1.8 mL), TFA (0.2 mL) was added and the reaction mixture was stirred at room temperature for 2 h until the Boc was completely removed according to LCMS. The resulting solution was then directly purified by reverse-phase flash chromatography (0-80% acetonitrile in aqueous TFA (0.01%)) to give compound P18 (3.3 mg, 65% yield, TFA salt) as a yellow solid. ESI m / z: 449.2 (M-OAc) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.31(t,J=5.6Hz,1H),8.09(d,J=9.2Hz,1H),7.82(br s, 3H),7.53(d,J=2.8Hz,1H),7.51(s,1H),7.43(s,1H),6.31(s,1 H),5.37(d,J=10.8Hz,1H),5.29(d,J=10.8Hz,1H),5.29(s,2H),3.99(s,3H),3.43-3.39(m,1H),3.28-3.17(m,3H),2.85(br s,2H),2.22-2.11(m,2H),1.99(s,3H),1.33(t,J=7.2Hz,3H),0.88(t,J=7.2Hz,3H)ppm.

[0489] Example 4D: tert-Butyl N-{2-[(2S)-2-[(19S)-14-fluoro-19-(2-hydroxyacetamido)-6-(hydroxymethyl)-15-methyl-5-oxo-4,11-diazapentacyclo[10.7.1.0 2 , 10 .04 , 9 .0 16 , 20 Synthesis of ]icosa-1,6,8,10,12,14,16(20)-heptaen-7-yl]-2-hydroxybutanamido]ethyl}carbamate (4-1b)

[0490] [ka]

[0491] Following the same procedure as for compound 4-1a, except that compound DXd (P12) was used instead of compound P17, compound 4-1b (0.16 g, 84% yield) was obtained as a pale yellow solid. ESI m / z: 636.3 (M-OH). + . 1 H NMR (400MHz, DMSO d6 ) δ 8.45(d,J=9.2Hz,1H),8.14(t,J=5.6Hz,1H),7.81(d,J=11.2Hz,1H),7.42(s,1H),6.80(t,J=5.6H z,1H),6.48(s,1H),5.64-5.58(m.1H),5.20(d,J=18.8Hz,1H),5.12(d,J=18.8Hz,1H),4.82(d,J= 12.0 Hz, 1H), 4.66 (d, J=11.6 Hz, 1H), 3.97 (s, 2H), 3.20-3.10 (m, 4H), 3.03-2.98 (m, 2H), 2.39 (s, 3H), 2.21-2.12 (m, 4H), 1.43-1.38 (m, 9H), 0.87 (t, J=7.2 Hz, 3H) ppm. Reference Chemical & Pharmaceutical Bulletin, 1994, 2518-2525 confirming the identity of 4-1b is incorporated by reference in its entirety.

[0492] Example 4E: {[(19S)-6-[(acetyloxy)methyl]-7-[(1S)-1-[(2-{[(tert-butoxy)carbonyl]amino}ethyl)carbamoyl]-1-hydroxypropyl]-14-fluoro-15-methyl-5-oxo-4,11-diazapentacyclo[10.7.1.0 2, 10 .0 4 , 9 .0 16 , 20 Synthesis of ]icosa-1,6,8,10,12,14,16(20)-heptaen-19-yl]carbamoyl}methyl acetate (4-2b)

[0493] [ka]

[0494] Following the same procedure as for compound 4-2a, except using compound 4-1b instead of compound 4-1a, compound 4-2b (50 mg, 73% yield, TFA salt) was obtained as a pale yellow solid. ESI m / z: 678.3 (M-OAc). + . 1 H NMR (400MHz, DMSO d6 ) δ 8.68(d,J=8.8Hz,1H),8.05-8.03(m,1H),7.83(d,J=10.8Hz,1H),7.45(s,1H),6.78(d,J=4.4Hz,1H),6.21(br s,1H),5.59-5.58(m,1H),5.37(d,J=11.2Hz,1H),5.30(d,J=10.8Hz,1H),5.25-5.13(m,2H),4.52(d,J=2.0Hz,2H),3.20-3.17(m ,3H),3.06-2.99(m,3H),2.41(s,3H),2.22-2.10(m,4H),2.09(s,3H),1.98(s,3H),1.38-1.35(m,9H),0.87(t,J=7.2Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -74(TFA),-111(Ar-F)ppm.

[0495] Example 4F: {[(19S)-6-[(acetyloxy)methyl]-7-[(1S)-1-[(2-aminoethyl)carbamoyl]-1-hydroxypropyl]-14-fluoro-15-methyl-5-oxo-4,11-diazapentacyclo[10.7.1.0 2 , 10 .04 , 9 .0 16 , 20 Synthesis of ]icosa-1,6,8,10,12,14,16(20)-heptaen-19-yl]carbamoyl}methyl acetate (P14)

[0496] [ka]

[0497] Following the same procedure as for compound P18, except using compound 4-2b instead of compound 4-2a, compound P13 (26 mg, 60% yield, TFA salt) was obtained as a white solid. ESI m / z: 638.3 (M+H). + . 1 H NMR (400MHz, DMSO d6 ) δ 8.69(d,J=8.4Hz,1H),8.32(t,J=6.0Hz,1H),7.83(d,J=10.8Hz,1H),7.72(br s, 3H),7.48(s,1H),6.34(s,1H),5.61-5.58(m.1H),5.37-5.14(m,4H),4.55(d,J=14.8Hz,1H),4 .50(d,J=14.8Hz,1H),3.41-3.35(m,1H),3.26-3.23(m,1H),3.22-3.18(m,2H),2.84-2.83(br s,2H),2.42(s,3H),2.21-2.12(m,4H),2.08(s,3H),1.99(s,3H),0.88(t,J=7.2Hz,3H)ppm.

[0498] Table 2 below provides a list of exemplary linker-payloads (LPs) of the present disclosure. Table 3 below provides chemical properties of exemplary LPs. Table 4 below shows HCT-15 data for exemplary payloads and linker-payloads.

[0499] [Table 20-1]

[0500] [Table 20-2]

[0501] [Table 20-3]

[0502] [Table 20-4]

[0503] [Table 21-1]

[0504] [Table 21-2]

[0505] [Table 22-1]

[0506] [Table 22-2]

[0507] Example 5: Synthesis of vcPAB linker-payload (Schemes 5A and 5B)

[0508] [ka]

[0509] [Table 23]

[0510] Example 5A: General Procedure C

[0511] To a solution of amine-containing or protected payload (1.0 equiv.) in dry DMF (2 mM), compound 5-1 (1 equiv.) and DIPEA (3 equiv.) were added, and the reaction mixture was stirred at room temperature for 16 h, which was monitored by LCMS. The resulting solution was directly purified by preparative HPLC (0-70% acetonitrile in aqueous TFA (0.01%)) to give the corresponding linker-payloads (23-81% yields, TFA salts) as solids.

[0512] Example 5A-1: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (LP1)

[0513] [ka]

[0514] Following general procedure C starting from exatecan with 5-1a, linker-payload LP1 (36 mg, 81% yield, TFA salt) was obtained as a white solid after purification by preparative HPLC (0-100% acetonitrile in aqueous ammonium bicarbonate (10 mM)), treated with aqueous TFA (0.1 M) to pH 4.0, concentrated in vacuo, and lyophilized. ESI m / z: 626.8 (M / 2+H). + . 1 H NMR (400MHz, DMSOd6 ) δ 10.00(s,1H),8.13(d,J=6.8Hz,1H),8.07(d,J=9.2Hz,1H),7.88(d,J=8.4Hz,1H),7.78 (d,J=10.8Hz,1H),7.61(d,J=8.4Hz,3H),7.36(d,J=8.4Hz,2H),7.32(s,1H),6.54(s,1H) ),6.00(s,1H),5.45(s,3H),5.29(s,3H),5.08(s,2H),4.41-4.35(m,1H),4.29-4.21(m, 2H),3.87(d,J=14.4Hz,1H),3.75(d,J=14.4Hz,1H),3.62-3.57(m,2H),3.50-3.48(m,14 H), 3.27-3.23 (m, 3H), 3.15-2.90 (m, 4H), 2.38-2.33 (m, 4H), 2.24-2.07 (m, 5H), 1.96-1.67 (m, 9H), 1.59-1.53 ​​(m, 3H), 1.46-1.33 (m, 3H), 0.89-0.82 (m, 9H) ppm. (No TFA protons were observed). 19 F NMR (376MHz, DMSO d6 ) δ -74 (TFA),-111 (Ar-F)ppm.

[0515] Example 5A-2: {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-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl carbamate (LP2)

[0516] [ka]

[0517] Following general procedure C starting from exatecan with 5-1b, linker-payload LP2 (with lactone ring-opened product, 10 mg, 26% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% methanol in aqueous ammonium bicarbonate (10 mM)).

[0518] Lactone: HPLC purity: 67%, retention time: 8.16 minutes, ESI m / z: 459.4 (M / 3+H) + , 688.3(M / 2+H) + ,

[0519] Ring-opening product: HPLC purity: 33%, retention time: 6.98 minutes, ESI m / z: 465.2 (M / 3+H) + , 697.3(M / 2+H) + .

[0520] 1 H NMR (400MHz, DMSO d6) δ 9.99(s,1H),8.18-8.11(m,1H),8.06(d,J=8.8Hz,1H),7.94-7.86(m.1H),7.79-7.75(m.2H) ,7.68(d,J=1.6Hz,1H),7.67-7.56(m.3H),7.51-7.42(m,3H),7.38-7.28(m,6H),6.53(s,0.6 H),6.05-6.00(m,0.4H),5.99-5.96(m,0.6H),5.45-5.41(m,3H),5.29-5.28(m,2H),5.26-5 .21(m,1H),5.08(s,2H),5.02(d,J=14.0Hz,1H),4.75-4.70(m,0.4H),4.41-4.36(m,1H),4.2 5-4.21(m,1H),3.61-3.57(m,3H),3.47-3.42(m,13H),3.24-3.22(m,2H),3.21-3.19(m,1H) ,3.14-3.05(m,3H),3.03-3.00(m,1H),2.97-2.91(m,1H),2.61-2.55(m,1H),2.48-2.44(m,1 H),2.41-2.35(m,4H),2.27-2.09(m,4H),2.03-1.95(m,2H),1.90-1.83(m,1H),1.80-1.72( m,2H),1.65-1.54(m,1H),1.47-1.23(m,2H),1.04(t,J=6.8Hz,0.4H),0.90-0.81(m,9H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -111ppm.

[0521] Example 5A-3: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .06 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)carbamate (LP5)

[0522] [ka]

[0523] Following general procedure C starting from P2 with 5-1a, linker-payload LP5 (22 mg, 32% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 655.5 (M / 2+H) + , 1309.6(M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 9.99(s,1H),8.50(d,J=8.5Hz,1H),8.13(d,J=7.5Hz,1H),7.88(d,J=8.5Hz,1H),7.81(d,J=10.9Hz,1H),7.59-7.57(m,3H),7.34-7. 25(m,3H),6.53(s,1H),5.99-5.95(m,1H),5.60-5.54(m,1H),5.42(s,4H),5.31-5.17(m,2H),4.94(s,2H),4.41-4.34(m,1H),4.27- 4.21(m,2H),3.88-3.73(m,2H),3.68-3.58(m,4H),3.56-3.44(m,14H),3.43-3.40(m,2H),3.25-3.23(m,2H),3.19-3.15(m,1H),3.0 3-2.92(m,2H),2.40-2.38(m,4H),2.23-2.04(m,5H),1.96-1.67(m,9H),1.62-1.52(m,3H),1.45-1.34(m,3H),0.87-0.81(m,9H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -111ppm.

[0524] Example 5A-4: (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP6)

[0525] [ka]

[0526] Following general procedure C starting from P2 with 2-1c, linker-payload methylate-LP6 (15 mg, 45% yield, ESI m / z: 727.0 (M / 2+H) + ) was obtained as a white solid after purification by reverse-phase flash chromatography (5-70% acetonitrile in aqueous TFA (0.01%)). To a solution of the resulting methylated-LP6 (10 mg, 6.9 μmol) in THF (2 mL) was added aqueous lithium hydroxide (0.5 mg, 20 mM, 1 mL) and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was directly purified by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)) to give LP6 (3.0 mg, 30% yield) as a white solid. ESI m / z: 720.0 (M / 2+H) + . 1H NMR (400MHz, DMSO d6 ) δ 10.03(s,1H),8.51(d,J=8.8Hz,1H),8.18(d,J=7.6Hz,1H),8.08(d,J=8.0Hz,1H),7.81(d,J= 11.2Hz,1H),7.74(d,J=7.6Hz,1H),7.63-7.56(m,3H),7.44(t,J=6.0Hz,1H),7.31(s,1H),7.2 7(d,J=8.8Hz,2H),6.54(s,1H),6.01-5.96(m,1H),5.60-5.55(m,1H),5.43(m,4H),5.32-5.1 6(m,2H),4.93(s,2H),4.39-4.31(m,2H),4.29-4.25(m,1H),4.22-4.17(m,1H),3.90-3.84(m, 1H),3.78-3.73(m,1H),3.67-3.62(m,1H),3.60-3.55(m,2H),3.51-3.46(m,13H),3.44-3.40 (m,3H),3.27-3.23(m,3H),3.06-3.00(m,2H),2.97-2.90(m,2H),2.43-2.39(m,4H),2.36-2.3 0(m,2H), 2.26-2.20(m,3H), 2.18-2.15(m,1H), 2.08(s,1H), 1.95-1.84(m,5H), 1.76-1.69(m,3H), 1.60-1.55(m,2H), 1.43-1.35(m,3H), 0.87-0.80(m,11H) ppm. (COOH protons were not revealed.)

[0527] Example 5A-5: (4S)-4-{[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]amino}-4-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .06 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}butanoic acid (LP9)

[0528] [ka]

[0529] Following general procedure C starting from P3 with 5-1a, linker-payload LP9 (22 mg, 38% yield) was obtained as a pale yellow solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 691.3 (M / 2+H). + . 1 H NMR (400MHz, DMSO d6 ) δ 10.00(s,1H),8.55(d,J=8.5Hz,1H),8.13(d,J=7.1Hz,1H),7.88(d,J=8.8Hz, 1H),7.82(d,J=10.9Hz,1H),7.69-7.53(m,3H),7.44(d,J=7.6Hz,1H),7.31(s, 1H),7.23(d,J=8.5Hz,2H),5.99(s,1H),5.52(s,1H),5.42(s,2H),5.33(d,J=1 8.7Hz,1H),5.08(d,J=18.7Hz,1H),4.97-4.87(m,2H),4.43-4.35(m,1H),4.30 -4.19(m,4H),4.02-3.95(m,1H),3.87(d,J=14.7Hz,1H),3.75(d,J=14.8Hz,1 H),3.61-3.57(m,2H),3.54-3.48(m,14H),3.26-3.21(m,2H),3.17(s,2H),3.0 5-2.94(m,2H),2.46(d,J=7.0Hz,1H),2.41-2.36(m,4H),2.29-2.02(m,7H),2. 01-1.81(m,11H),1.64-1.52(m,3H),1.51-1.30(m,3H),0.97-0.68(m,9H)ppm.

[0530] Example 5A-6: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-1-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}-2-phenylethyl)carbamate (LP10)

[0531] [ka]

[0532] Following general procedure C starting from P5 with 5-1a, linker-payload LP10 (55 mg, 51% yield) was obtained as a yellow solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 700.3 (M / 2+H). + . 1 H NMR (400MHz, DMSO d6) δ 9.99(s,1H),8.65(d,J=8.5Hz,1H),8.13(d,J=7.5Hz,1H),7.88(d,J=8.6H z,1H),7.82(d,J=11.0Hz,1H),7.68-7.49(m,4H),7.31(s,1H),7.26-7.13 (m,4H),7.06(t,J=7.4Hz,2H),7.00-6.94(m,1H),6.00(s,1H),5.58-5.51 (m,1H),5.43(s,2H),5.23(d,J=19.0Hz,1H),4.89(t,J=9.2Hz,2H),4.42-4 .21(m,4H),3.85(d,J=14.8Hz,1H),3.75(d,J=14.8Hz,1H),3.67-3.58(m, 14H),3.43-3.40(m,2H),3.27-3.17(m,4H),3.09-2.89(m,4H),2.85-2.78 (m,1H),2.46(d,J=7.0Hz,1H),2.43-2.36(m,4H),2.26-2.03(m,6H),1.99 -1.81(m,6H),1.80-1.66(m,4H),1.66-1.30(m,7H),0.93-0.78(m,9H)ppm.

[0533] Example 5A-7: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(5S)-5-amino-5-{[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}pentyl]carbamate (LP11)

[0534] [ka]

[0535] Following general procedure C starting from 1-3 with 5-1a, linker-payload Fmoc-LP11 (38 mg (>95% purity) and 60 mg (>75% purity), ESI m / z: 802.2 (M+H) + ) was obtained as a pale yellow solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). Diethylamine (0.1 mL) was added to a solution of Fmoc-LP11 (38 mg, >95% purity, 21 μmol) in dry DMF (1 mL) and the reaction mixture was stirred at room temperature for 2 h until complete removal of Fmoc, which was monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous formic acid (0.1%)) to give LP11 (14 mg, 11% yield from 1-3) as a white solid. ESI m / z: 691.2 (M / 2+H) + . 1 H NMR (400MHz, DMSO d6) δ 10.00(s,1H),8.46(s,1H),8.29(s,1H),8.14(d,J=7.3Hz,1H),7.89(d, J=8.8Hz,1H),7.81(d,J=10.9Hz,1H),7.64-7.54(m,3H),7.31(s,1H),7 .25(d,J=8.5Hz,2H),7.17(s,1H),6.54(s,1H),6.01(s,1H),5.53(s,1H) ),5.42(m,4H),5.28-5.16(m,2H),4.89(s,2H),4.37(m,1H),4.24(m,2H) ,3.81(m,3H),3.59(m,4H),3.53-3.47(m,12H),3.45-3.40(m,2H),3.24 (m,2H),3.17(m,2H),3.02-2.89(m,3H),2.43-2.32(m,5H),2.26-2.15( m,3H),2.13-2.02(m,3H),1.99-1.90(m,2H),1.88-1.79(m,3H),1.79-1 .66(m,3H),1.64-1.52(m,4H),1.48-1.33(m,7H),0.91-0.78(m,9H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -111.2ppm.

[0536] Example 5A-8: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[({[2-({2-[(19S)-19-ethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]ethyl}(propan-2-yl)amino)ethoxy]methyl}carbamoyl)methyl]carbamate (LP12)

[0537] [ka]

[0538] Following general procedure C starting from P11 with 5-1a, linker-payload LP12 (10 mg, 40% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 690.8 (M / 2+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 9.99(s,1H),9.46(s,1H),8.95-8.81(m,1H),8.37(d,J=8.4Hz,1H),8.23(d,J=8.2Hz,1H),8.12(d,J=7.4Hz, 1H),8.00-7.85(m,2H),7.82(t,J=7.5Hz,1H),7.70-7.48(m,4H),7.37(s,1H),7.25(d,J=8.1Hz,2H),6.57(br s,1H),5.99(br t,J=5.3Hz,1H),5.55-5.35(m,6H),4.91(br s,2H),4.71(br s,2H),4.44-4.33(m,1H),4.30-4.19(m,2H),3.90-3.72(m,6H),3.65 -3.56(m,5H),3.53-3.45(m,16H),3.26-3.22(m,2H),3.03-2.93(m,2H) ),2.40-2.32(m,1H),2.29-2.01(m,4H),2.00-1.80(m,6H),1.80-1.53(m,7H),1.46-1.34(m,3H),1.32-1.24(m,6H),0.90-0.82(m,9H)ppm.

[0539] Example 5A-9: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-{[(19S)-19-ethyl-19-hydroxy-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]methyl}carbamate (LP20)

[0540] [ka]

[0541] Following general procedure C starting from P21 with 5-1a, linker-payload LP20 (16 mg, 57% yield, TFA salt) was obtained as a pale yellow solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 613.0 (M / 2+H) + . 1 H NMR (400MHz, DMSO d6) δ 9.97(s,1H),8.17(t,J=6.4Hz,1H),8.10(d,J=9.2Hz,2H),7.87(d,J=8.4Hz,1H), 7.66(s,1H),7.61-7.56(m,3H),7.53(dd,J=8.8,2.4Hz,1H),7.29(s,2H),7.27(br s,1H),6.51(s,1H),5.97(t,J=6.4Hz,1H),5.48(s,2H),5.44(s,2H),5.41(s,2H),4.97(s,2H),4.79(d,J=6.0Hz, 2H),4.41-4.34(m,1H),4.28-4.20(m,2H),3.96(s,3H),3.87(d,J=14.8Hz,1H),3.75(d,J=14.8Hz,1H),3.62-3.58 (m, 2H), 3.50-3.48(m, 12H), 3.46-3.42(m, 2H), 3.26-3.20(m, 2H), 3.03-2.91(m, 2H), 2.47-2.33(m, 1H), 2.21-2.03(m, 4H), 1.98-1.68(m, 8H), 1.61-1.46(m, 3H), 1.42-1.31(m, 3H), 0.91-0.82(m, 9H) ppm. (No TFA protons were observed). 19 F NMR (376MHz, DMSO d6 ) δ -74ppm.

[0542] Example 5A-10: {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-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-{[(19S)-19-ethyl-19-hydroxy-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]methyl}carbamate (LP21)

[0543] [ka]

[0544] Following general procedure C starting from P21 with 5-1b, linker-payload LP21 (10 mg, 38% yield) was obtained as a pale yellow solid after purification by reverse-phase flash chromatography (0-100% methanol in aqueous ammonium bicarbonate (10 mM)).

[0545] Lactone: HPLC purity: 77%, retention time: 7.67 minutes, ESI m / z: 449.9 (M / 3+H) + , 674.3(M / 2+H) + ,

[0546] Ring-opening product: HPLC purity: 23%, retention time: 6.57 minutes, ESI m / z: 455.9 (M / 3+H) + , 683.4(M / 2+H) + .

[0547] 1 H NMR (400MHz, DMSO d6) δ 9.98(s,1H),8.18(t,J=6.0Hz,1H),8.13-8.09(m,2H),7.87(d,J=8.4Hz,1H),7.77(d,J=5.6Hz,1H),7.69-7.65(m.2H),7.63-7.6 0(m,1H),7.57(d,J=8.4Hz,2H),7.54-7.44(m,4H),7.39-7.34(m,2H),7.32-7.27(m,4H),6.51(s,1H),5.99-5.96(m,1H),5.48(br s,2H),5.44(s,2H),5.41(s,2H),5.02(d,J=13.6Hz,1H),4.97(s,2H),4.79(d,J=6.0Hz,2H),4.40-4.35(m,1H),4 .25-4.21(m,1H),3.96(s,3H),3.62-3.57(m,3H),3.48-3.42(m,12H),3.30-3.28(m,2H),3.09-3.07(m,2H),3.03 -2.99(m,1H),2.97-2.91(m,1H),2.60-2.55(m,1H),2.46-2.44(m,1H),2.41-2.37(m,1H),2.28-2.19(m,1H),2.0 3-1.93(m,2H),1.89-1.83(m,2H),1.80-1.65(m,2H),1.61-1.53(m,1H),1.48-1.31(m,2H),0.90-0.81(m,9H)ppm.

[0548]

change

[0549]

Table 24

[0550] Example 5B: {7-[(1S)-1-[(2-{[({4-[(2S)-2-[(2S)-2-[1-(4-{2-アザトリシクロ[10.4.0.0 4 , 9Synthesis of ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl]amino}ethyl)carbamoyl]-1-hydroxypropyl]-12-ethyl-2-methoxy-9-oxo-9H,11H-indolizino[1,2-b]quinolin-8-yl}methyl acetate (LP19)

[0551] [ka]

[0552] Following general procedure C starting from P18 with 5-1b, linker-payload LP19 (19 mg, 82% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-80% acetonitrile in water). ESI m / z: 695.0 ((M-OAc) / 2+H). + . 1 H NMR (400MHz, DMSO d6) δ 9.98(s,1H),8.12-8.09(m,2H),8.06-8.03(m,1H),7.87(d,J=8.4Hz,1H),7.77(t,J=6.0Hz,1H),7.69(dd,J=8.4 and 2.0Hz,1H),7.62(d,J=7.2Hz,1H),7.57(d,J=8.4Hz,2H),7.53-7.44(m,5H),7.41 -7.39(m,1H),7.38-7.28(m,3H),7.24(d,J=8.8Hz,2H),7.20-7.18(m,1H),6.18( s,1H),5.98(t,J=6.0Hz,1H),5.42(s,2H),5.37(d,J=10.8Hz,1H),5.30(d,J=10. 8Hz,1H),5.29(s,2H),5.03(d,J=14.0Hz,1H),4.90(s,2H),4.41-4.35(m,1H),4.2 3(q,J=6.8Hz,1H),3.99(s,3H),3.62-3.57(m,3H),3.48-3.42(m,12H),3.30-3.2 8(m,1H),3.22-3.17(m,3H),3.10-3.07(m,5H),3.04-2.93(m,2H),2.68-2.55(m,1 H),2.46-2.33(m,2H),2.26-2.11(m,4H),2.10-1.96(m,5H),1.78-1.63(m,2H),1 .60-1.53(m,1H),1.47-1.35(m,2H),1.32(t,J=7.2Hz,3H),0.88-0.81(m,9H)ppm.

[0553] Example 6: Synthesis of sugar-PAB リンカー-ペイロード LP3, LP4, and およびLP7 (スキーム6)

[0554]

change

[0555] Example 6A: LP3

[0556] Example 6A-1: Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenoxy]oxane-2-carboxylate (6-2a)

[0557] [ka]

[0558] To a solution of compound 6-1a (26 mg, 30 μmol) in DMF (1.0 mL) was added exatecan mesylate (16 mg, 30 μmol), HOBt (4.0 mg, 30 μmol), and DIPEA (7.7 mg, 60 μmol). The reaction mixture was stirred at room temperature for 16 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 6-2a (18 mg, 52% yield) as a yellow solid. ESI m / z: 1152.5 (M+H) + .

[0559] Example 6A-2: (2S,3S,4S,5R,6S)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (LP3)

[0560] [ka]

[0561] To a mixture of compound 6-2a (22 mg, 19 μmol) in methanol (2 mL) was added lithium hydroxide monohydrate (7.2 mg, 0.17 mmol) and water (2 mL). The reaction mixture was stirred for 1 h at 25° C. and was monitored by LCMS. The resulting mixture was acidified to pH 3-4 with aqueous hydrochloric acid (1N) and then purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)) to give LP3 (6.0 mg, 28% yield, TFA salt) as a white solid. ESI m / z: 1012.5 (M+H) + . 1 H NMR (400MHz, DMSO d6) δ 8.56(s,1H),8.09(d,J=9.2Hz,1H),7.98-7.92(m,1H),7.79-7.76(m,2H),7.54-7.52(m,1H),7.34(d,J=8.4Hz,1H),7.30(s,1H),6.52(br s,1H),5.66(br s,1H), 5.44(s,2H), 5.28(s,3H), 5.15-5.06(m,3H), 4.89(s,1H), 4.25(s,1H), 3.88-3.71(m,2H), 3.25-3.05(m,9H), 2.38-2.33(m,4H), 2.23-2.01(m,6H), 1.98-1.79(m,4H), 1.74-1.65(m,2H), 1.65-1.46(m,2H), 1.28(m,1H), 0.88(t,J=7.2Hz,3H)ppm. (acid protons not revealed.)

[0562] Example 6B: LP4

[0563] Example 6B-1: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl]-4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenoxy]oxan-2-yl]methyl acetate (6-2b)

[0564] [ka]

[0565] To a solution of compound 6-1b (55 mg, 63 μmol) in DMF (3.0 mL), exatecan mesylate (34 mg, 63 μmol), HOBt (8.5 mg, 63 μmol), and DIPEA (16 mg, 0.13 mmol) were added, and the reaction mixture was stirred at room temperature for 16 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.1%)) to give compound 6-2b (34 mg, 46% yield) as a yellow solid. ESI m / z: 1166.5 (M+H) + .

[0566] Example 6B-2: [3-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}phenyl]methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (LP4)

[0567] [ka]

[0568] To a mixture of compound 6-2b (15 mg, 13 μmol) in methanol (2 mL) was added lithium hydroxide monohydrate (4.9 mg, 0.12 mmol) and water (2 mL). The reaction mixture was stirred for 1 h at 15° C. and was monitored by LCMS. The resulting mixture was acidified to pH 3-4 with aqueous hydrochloric acid (1N) and then purified by preparative HPLC (5-95% acetonitrile in aqueous formic acid (0.1%)) to give LP4 (4.0 mg, 31% yield) as a yellow solid. ESI m / z: 998.2 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.49-8.43(m,4H),8.10(d,J=8.8Hz,1H),7.83-7.72(m,3H),7.54(d,J=8.4Hz,1H),7.37(d,J=8.8Hz,1H),7.31(s,1H),6.54(s,1H),5.78(br s,1H),5.44(s,2H),5.33-5.28(m,4H),5.16-5.08(m,3H),4.91(d,J=8.4Hz,1H),4.68(br s,1H),4.28-4.23(m,1H),3.88-3.72(m,4H),3.20-2.94(m,3H),2.38-2.33(m,4H), 2.17-1.97(m,8H),1.96-1.68(m,6H),1.56-1.38(m,3H),0.88(t,J=7.6Hz,3H)ppm.

[0569] Example 3C: LP7

[0570] Example 3C-1: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .020 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenoxy]oxan-2-yl]methyl acetate (4-2c)

[0571] [ka]

[0572] To a solution of compound 6-1b (31 mg, 36 μmol) in DMF (1.0 mL), P2 (17 mg, 36 μmol), HOBt (5.0 mg, 36 μmol), and DIPEA (9.2 mg, 71 mmol) were added, and the reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 6-2c (43 mg, 88% yield) as a yellow solid. ESI m / z: 1223.5 (M+H) + , 1245.5(M+Na) + .

[0573] Example 3C-2: [3-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}phenyl]methyl N-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)carbamate (LP7)

[0574] [ka]

[0575] To a mixture of compound 6-2c (43 mg, 35 μmol) in methanol (2 mL), aqueous lithium hydroxide (0.16 mM, 2 mL) was added and the reaction mixture was stirred for 2 h at 15° C., which was monitored by LCMS. The resulting mixture was acidified to pH 3-4 with aqueous hydrochloric acid (1N) and then purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)) to give LP7 (9.8 mg, 57% yield) as a white solid. ESI m / z: 1055.5 (M+H). + .

[0576] Example 7: Synthesis of Peptide Linker-Payload LP8 (Schemes 7A and 7B)

[0577] [ka]

[0578] Example 7A: Synthesis of 2-[2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)acetamido]acetic acid (7A-3)

[0579] [ka]

[0580] To a solution of compound 7-2 (0.10 g, 0.37 mmol) in DMSO (2 mL), peptide 7A-1 (H-Gly-Gly-Gly-Gly-OH, 90 mg, 0.37 mmol) and DIPEA (94 mg, 0.73 mmol) were added and the reaction mixture was stirred at room temperature overnight. The resulting mixture was directly purified by reverse-phase flash chromatography (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 7A-3 (23 mg, 15% yield) as a white solid. ESI m / z: 411.2 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.23-8.07(m,3H), 7.83(t,J=5.7Hz,1H), 4.33(d,J=6.4Hz,1H), 3.93(d,J=14.9Hz,1H), 3.77(m,9H), 2.25-2.04(m,3H), 1.98-1.84(m,2H), 1.82-1.71(m,2H), 1.59(s,2H), 1.40(m,1H) ppm. (No TFA protons observed).

[0581] Example 7B: 2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)-N-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methyl)acetamide (LP8)

[0582] [ka]

[0583] To a solution of compound 7A-3 (12 mg, 29 μmol) in dry DMF (3 mL), HATU (17 mg, 44 μmol) and DIPEA (12 mg, 88 μmol) were added, and the mixture was stirred at room temperature for 10 min, followed by the addition of exatecan (13 mg, 29 μmol). The reaction mixture was stirred at room temperature for 4 h until exatecan was completely consumed by LCMS. The resulting mixture was directly purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to give linker-payload LP8 (11 mg, 39% yield, TFA salt) as a white solid. ESI m / z: 828.5 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 8.40(d,J=8.5Hz,1H),8.18-8.09(m,3H),7.84-7.77(m,2H),7.32(s,1H),5.62- 5.52(m,1H),5.43(s,2H),5.23(d,J=3.7Hz,2H),4.31(t,J=5.6Hz,1H),3.92(d,J =14.7Hz,1H),3.82-3.63(m,9H),3.18-3.16(m,2H),2.41(s,3H),2.23-2.09(m,5 H),1.93-1.75(m,6H),1.60-1.53(m,2H),1.24(s,2H),0.87(t,J=7.3Hz,3H)ppm.

[0584] [ka]

[0585] Example 7C: 2-amino-N-{[(19S)-19-ethyl-19-hydroxy-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]methyl}acetamide (7B-1)

[0586] [ka]

[0587] To a solution of Fmoc-Gly-OH (37 mg, 0.13 mmol) in DMF (5.0 mL), DIPEA (0.04 mL, 0.25 mmol), HATU (56 mg, 0.15 mmol) were added, and the reaction mixture was stirred at room temperature for 15 min, followed by the addition of payload P17 (50 mg, 0.12 mmol). The mixture was then stirred at room temperature overnight until P17 was completely consumed, which was monitored by LCMS. Piperidine (0.3 mL) was then added to the mixture, and the resulting mixture was stirred at room temperature for 30 min until Fmoc was completely removed by LCMS. The mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give 7B-1 (35 mg, 61% yield, TFA salt) as a yellow solid. ESI m / z: 465.2 (M+H) + .

[0588] Example 7D: (2S)-2-[2-(2-aminoacetamido)acetamido]-N-[({[(19S)-19-ethyl-19-hydroxy-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]methyl}carbamoyl)methyl]-3-phenylpropanamide (7B-2)

[0589] [ka]

[0590] To a solution of peptide Fmoc-Gly-Gly-Phe-OH (32 mg, 65 μmol) in DMF (6 mL) was added DIPEA (0.02 mL, 0.13 mmol) and HATU (29 mg, 77 μmol), the reaction mixture was stirred at room temperature for 15 min, followed by the addition of compound 7B-1 (30 mg, 65 μmol). The mixture was stirred overnight at room temperature, which was monitored by LCMS. Then piperidine (0.3 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 min until the Fmoc was completely removed by LCMS. The reaction mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give crude 7B-2 (35 mg, TFA salt, crude (68% pure by LCMS)) as a yellow solid, which was used in the next step without further purification. ESI m / z: 363.8 (M / 2+H) + .

[0591] Example 7E: (2S)-2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)-N-[({[(19S)-19-ethyl-19-hydroxy-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]methyl}carbamoyl)methyl]-3-phenylpropanamide (LP22)

[0592] [ka]

[0593] To a solution of compound 7B-2 (crude, 35 mg) in DMF (3 mL) obtained above, DIPEA (0.03 mL, 0.20 mmol) and compound 7-2 (14 mg, 49 μmol) were added and the reaction mixture was stirred at room temperature for 1.5 h, which was monitored by LCMS. The resulting mixture was then directly purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to give linker-payload LP22 (2.0 mg, 5% yield) as an off-white solid. ESI m / z: 890.3 (M+H) + .

[0594] Example 8: Synthesis of vcPAB-Carbonate Linker-Payload LP18 (Scheme 8)

[0595] [ka]

[0596] Example 8A: {4-[(2S)-2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl(19S)-10,19-diethyl-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaen-19-yl Carbonate (8-1)

[0597] [ka]

[0598] A suspension of compound P16 (0.12 g, 0.30 mmol) and DMAP (0.11 g, 0.90 mmol) in dry DCM (18 mL) was cooled in ice water. To the suspension, a solution of triphosgene (45 mg, 0.15 mmol) in dry DCM (2 mL) was added dropwise over 10 min. Upon addition, the yellow suspension became clear. After stirring the reaction solution for 20 min at room temperature, a chloroformate intermediate was formed, which was monitored by TLC (an aliquot of the reaction solution was quenched with dry methanol and then compared for mobility with starting P16 by TLC elenting in MeOH / DCM, v / v=1 / 15). The methyl carbonate of the chloroformate intermediate was reacted with R f 0.75, and P16 is R f 0.5). Then, Boc-vcPAB (0.14 g, 0.30 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 20 min, which was monitored by LCMS. The resulting solution was concentrated in vacuo below 30° C., and the residue was purified by reverse-phase flash chromatography (0-65% acetonitrile in aqueous TFA (0.01%)) to give compound 8-1 (0.15 g, 56% yield) as a white solid. ESI m / z: 812 (M-Boc+H) + ,406.7(Fragment,M P16 +H) + .

[0599] Example 8B: {4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl(19S)-10,19-diethyl-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-heptaen-19-yl carbonate (8-2)

[0600] [ka]

[0601] To a cooled (<10 °C) mixture of 8-1 (90 mg, 98 μmol) in DCM (9 mL) was added TFA (1 mL) and the resulting clear yellow solution was stirred at <10 °C for 1 h until the Boc was removed by LCMS. The reaction solution was then quenched with saturated aqueous sodium bicarbonate to pH 6.0, after which the volatiles were removed in vacuo. The remaining aqueous mixture was purified by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.01%)) to give compound 8-2 (58 mg, 73% yield) as a yellow solid. ESI m / z: 812.3 (M+H) + .

[0602] Example 8C: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl(19S)-10,19-diethyl-7-methoxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20 ]Synthesis of Henicosa-1(21),2,4,6,8,10,15(20)-heptaen-19-yl carbonate (LP18)

[0603] [ka]

[0604] To a yellow solution of compound 8-2 (20 mg, 25 μmol) in dry DMF (2 mL), DIPEA (9.5 mL, 74 μmol) and 8-3 (13 mg, 25 μmol) were added, and the reaction solution was stirred at room temperature for 6 h until the starting material 8-2 was nearly consumed, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.01%)) to give linker-payload LP18 (18 mg, 60% yield) as a pale yellow solid. ESI m / z: 612.5 (M / 2+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 10.03(s,1H),8.14(d,J=7.2Hz,1H),8.11(d,J=9.2Hz,1H),7.89(d,J=8.8Hz,1H),7 .60(d,J=8.4Hz,3H),7.56-7.51(m,2H),7.30(d,J=8.4Hz,2H),6.97(s,1H),6.00(br s,1H),5.52(s,2H),5.34(d,J=3.2Hz,2H),5.09(q,J=12Hz,2H),4.40-4.35(m,1H),4.29-4.22(m,2H) ,4.00(s,3H),3.87(d,J=14.8Hz,1H),3.75(d,J=14.8Hz,1H),3.62-3.57(m,2H),3.50-3.48(m,16H), 3.27-3.19(m,4H), 3.04-2.91(m,2H), 2.46-2.33(m,2H), 2.25-2.06(m,5H), 2.03-1.66(m,6H), 1.61-1.56(m,3H), 1.45-1.36(m,3H), 1.32(t,J=7.6Hz,3H), 0.92-0.81(m,9H)ppm.(No TFA protons were observed). 19 F NMR (376MHz, DMSO d6 ) δ -73ppm.

[0605] Example 9: Synthesis of Linker-Payload LP13, LP14, and LP15 (Scheme 9)

[0606] [ka]

[0607] Example 9A-1: Synthesis of 2-({[(4-azidophenyl)methoxy]carbonyl}amino)acetic acid (9-1)

[0608] [ka]

[0609] To a solution of 4-aminobenzyl alcohol (10 g, 81 mmol) and azido(trimethyl)silane (11 g, 97 mmol, 13 mL) in acetonitrile (600 mL) was added tert-butyl nitrite (13 g, 0.12 mol, 15 mL) dropwise at 0° C. After the mixture was stirred at room temperature for 2 h, a solution of bis(4-nitrophenyl)carbonate (32 g, 0.11 mol) and DIPEA (21 g, 0.16 mol, 28 mL) in THF (300 mL) was added and the mixture was stirred at room temperature for 12 h, which was monitored by TLC (25% ethyl acetate in petroleum ether). The volatiles were removed in vacuo and the residue was dissolved in acetonitrile (500 mL). To the solution, glycine (15 g, 0.20 mol) and aqueous sodium bicarbonate (0.8 M, 250 mL, 0.2 mol) were added dropwise, and the mixture was stirred at room temperature for 16 h, which was monitored by TLC (25% ethyl acetate in petroleum ether). The resulting mixture was washed with ethyl acetate (150 mL x 6). The aqueous solution was acidified to pH 2-3 with concentrated HCl, and then extracted with ethyl acetate (150 mL x 3). The combined organic solution was dried over anhydrous sodium sulfate and concentrated in vacuo to give 9-1 (13 g, 61% yield) as a brown solid, which was used in the next step without further purification. 1 H NMR (400MHz, MeOD d4 ) δ 7.46-7.34(m,2H),7.12-6.97(m,2H),5.08(s,2H),3.83(s,2H)ppm.

[0610] Example 9A-2: ({[(4-azidophenyl)methoxy]carbonyl}amino)methyl acetate (9-2)

[0611] [ka]

[0612] To a solution of compound 9-1 (13 g, 51 mmol) in THF (150 mL), lead acetate (45 g, 0.10 mol) and copper diacetate (0.93 g, 5.1 mmol) were added, and the reaction mixture was stirred at 40 °C for 1 h, which was monitored by TLC (25% ethyl acetate in petroleum ether). The reaction was quenched with water (200 mL) and extracted with ethyl acetate (200 mL x 2). The combined organic solution was washed with brine (150 mL x 2), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-25% ethyl acetate in petroleum ether) to give compound 9-2 (7.8 g, 55% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3) δ 7.36(br d,J=8.2Hz,2H),7.03(d,J=8.4Hz,2H),5.93(br s,1H),5.21(br d,J=7.5Hz,2H),5.11(s,2H),2.07(s,3H)ppm.

[0613] Example 9A-3: Synthesis of 2-[({[(4-azidophenyl)methoxy]carbonyl}amino)methoxy]acetic acid (9-3b')

[0614] [ka]

[0615] To a solution of 9-2 (0.20 g, 0.76 mmol) in DCM (3 mL) was added PPTS (38 mg, 0.15 mmol) and hydroxyacetic acid (0.17 g, 2.3 mmol) and the reaction mixture was stirred at 50 °C for 16 h in a sealed tube, which was monitored by LCMS. The resulting mixture was cooled and the volatiles were removed in vacuo. The residue was purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 9-3b' (0.10 g, 47% yield) as a yellow solid. ESI m / z: 303 (M+Na) + .

[0616] Example 9A-4: (4-azidophenyl)methyl N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamate (9-3b)

[0617] [ka]

[0618] To a yellow solution of compound 9-3b' (50 mg, 0.13 mmol) and DIPEA (49 mg, 0.38 mmol) in dry DMF (1.5 mL), HATU (59 mg, 0.15 mmol) was added, and the mixture was stirred at room temperature for 30 min, followed by the addition of exatecan (60 mg, 0.11 mmol). The reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 9-3b (58 mg, 65% yield) as a pale yellow solid. ESI m / z: 698 (M+H) + .

[0619] Example 9A-5: {4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]propanamido]phenyl}methyl N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamate (9-4c)

[0620] [ka]

[0621] To a 25 mL vial charged with a stir bar and THF (3 mL) was added 9-3b (42 mg, 52 μmol), 4A molecular sieves (0.50 g), followed by trimethylphosphine (0.16 mL, 0.16 mmol). The reaction mixture was stirred for 5 min, and then Fmoc-Val-Ala-OPFP (33 mg, 57 μmol) was added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 min, which was monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give Fmoc-9-4c (53 mg, TFA salt) as a pale yellow solid, which was dissolved in dry DMF (1 mL). To the solution was added diethylamine (0.1 mL). The reaction mixture was stirred at room temperature for 1 h until the Fmoc was completely removed by LCMS. The resulting solution was directly separated by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 9-4c (28 mg, 57% yield, TFA salt) as a pale yellow solid. ESI m / z: 422 (M / 2+H). + .

[0622] NMR of Fmoc-9-4c: 1 H NMR (400MHz, DMSO d6 ) δ 10.03(s,1H),8.48-8.29(m,1H),8.26-8.11(m,1H),7.89(d,J=7.5Hz,2H),7.79-7.72(m,3H),7.70-7.52(m,2H),7. 44-7.41(m,3H),7.34-7.25(m,3H),7.20-7.17(m,2H),6.54(d,J=9.0Hz,1H),5.59(s,2H),5.42(s,2H),5.21(s,1H) ,4.87(s,1H),4.54(d,J=6.5Hz,2H),4.41-4.32(m,1H),4.35-4.17(m,3H),4.00(s,1H),3.98-3.83(m,1H),3.15(s, 2H),2.38(s,3H),2.18-2.11(m,3H),2.09-1.95(m,2H),1.93-1.71(m,2H),1.30-1.14(m,6H),0.89-0.77(m,9H)ppm.19 F NMR (376MHz, DMSO d6 ) δ -73.45,-111.33ppm.

[0623] NMR of 9-4c: 1 H NMR (400MHz, DMSO d6 ) δ 10.20(br,1H),8.84-8.67(m,1H),8.46(br s,1H),8.21(br s,2H),8.06(s,2H),7.80(d,J=10.5Hz,1H),7.65-7.50(m,2H),7.31(s,1H),7.23(t,J=8.2Hz,1H),6.53(s, 1H),5.60(s,1H),5.42(s,2H),5.22(s,1H),4.88(d,J=5.8Hz,2H),4.60-4.45(m,3H),4.00(s,1H),3.61(br s,1H),3.17(br s,1H),2.93-2.90(m,2H),2.39(s,3H),2.33-2.06(m,2H),1.93-1.76(m,2H),1.34( t,J=7.4Hz,3H),1.16(t,J=7.3Hz,3H),0.97-0.93(m,6H),0.85(t,J=7.4Hz,3H)ppm. 19 F NMR (376MHz, DMSO d6 ) δ -73.44,-111.33ppm.

[0624] Example 9A-6: {4-[(2S)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]propanamido]phenyl}methyl N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamate (LP15)

[0625] [ka]

[0626] To a yellow solution of compound 8-3 (14 mg, 27 μmol) in dry DMF (1.5 mL), DIPEA (14 mg, 0.11 mmol) and compound 9-4c (23 mg, 24 μmol) were added and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous formic acid (0.1%)) to give linker-payload LP15 (9.0 mg, 26% yield) as a white solid. ESI m / z: 1253 (M+H) + . 1 H NMR (400MHz, DMSO d6 ) δ 9.96(s,1H),8.45(m,1H),8.21(m,1H),7.89(d,J=8.6Hz,1H),7.79(d,J=10.9Hz,1H),7.66-7.52(m,3H) ),7.30(s,1H),7.25-7.19(m,2H),6.53(s,1H),5.59(s,1H),5.42(s,2H),5.22(s,2H),4.86(s,2H),4.5 4(d,J=6.7Hz,2H),4.45-4.30(m,1H),4.29-4.08(m,2H),4.00(s,2H),3.87(d,J=14.8Hz,1H),3.75(d,J =14.7Hz,1H),3.58(d,J=6.2Hz,2H),3.51-3.45(m,12H),3.44-3.40(m,2H),3.29-3.21(m,2H),3.16(br ppm. 19 F NMR (376MHz, DMSO d6) δ -111.33ppm.

[0627] Example 9B-1: {4-[(2S)-2-amino-5-(carbamoylamino)pentanamido]phenyl}methyl N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamate (9-4b)

[0628] [ka]

[0629] Following the same procedure as for 9-4c, except using Fmoc-Cit-OPFP instead of Fmoc-Val-Ala-OPFP, compound 9-4b (41 mg, 50% yield) was obtained as a pale yellow solid. ESI m / z: 829 (M+H).

[0630] Example 9B-2: Synthesis of (4S)-4-{[(2S)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-methyl-1-oxobutan-2-yl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoic acid (9-5)

[0631] [ka]

[0632] H-Glu(O) in DMF (200 mL) tTo a solution of (Bu)-Val-OH (29 g, 56 mmol) fmoc-PEG in DCM (200 mL) 4 A solution of -OSu (31 g, 53 mmol) and DIPEA (7.2 g, 56 mmol, 9.7 mL) was added and the reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. Then, the volatiles were removed in vacuo. The residue was diluted with water (100 mL), washed with MTBE (80 mL x 3), and acidified to pH 5 with citric acid. The mixture was extracted with ethyl acetate (120 mL x 2), and the combined organic solution was washed with brine (60 mL x 2), dried over anhydrous sodium sulfate, and concentrated in vacuo to give Fmoc-PEG 100 as a yellow oil. 4 -Glu(O t Bu)-Val-OH (33 g) was obtained. ESI m / z: 772 (M+H) + .

[0633] To a solution of HOSu (7.9 g, 68 mmol) in DMF (150 mL) and DCM (150 mL) was added DIC (6.5 g, 51 mmol) and the Fmoc-PEG obtained above. 4 -Glu(O t Bu)-Val-OH (33 g) was added. The reaction mixture was stirred at room temperature for 12 h, which was monitored by TLC and LCMS. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The residue was diluted with water (200 mL) and extracted with ethyl acetate (150 mL x 3). The combined organic solution was washed with brine (150 mL x 3), dried over anhydrous sodium sulfate, and concentrated in vacuo to give Fmoc-PEG 2H2O. 4 -Glu(O t Bu)-Val-OSu (14 g) was obtained as a yellow oil. ESI m / z 870 (M+H) + . 1H NMR(400MHz,CDCl3) δ 7.76(d,J=7.5Hz,2H),7.64-7.58(m,2H),7.42-7.37(m,2H),7.34-7.28(m,2H),4.86-4.76( m,1H),4.56-4.34(m,3H),4.28-4.18(m,1H),3.76-3.73(m,3H),3.66-3.56(m,13H),3.39(br d,J=4.9Hz,1H),2.81(br s, 6H),2.50-2.46(m,2H),2.40-2.32(m,2H),2.14-2.04(m 1H),1.97-1.81(m,4H),1.44(s,9H),1.05(s,3H),1.04(s,3H)ppm.

[0634] Fmoc-PEG in DCM (0.5 mL) 4 -Glu(O t To a solution of Bu)-Val-OSu (obtained above, 0.14 g, 0.16 mmol) was added TFA (0.72 g, 0.47 mL, 6.3 mmol) and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was triturated in MTBE (10 mL x 2). The off-white precipitate was collected by centrifugation to give compound 9-5 (Fmoc-PEG 4 -Glu-Ala-OSu) (0.13 g, H-Glu(O t 22% yield from Bu)-Val-OH as an off-white solid. ESI m / z: 813 (M+H) + .

[0635] Example 9B-3: (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecane-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-({4-[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11.0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (9-6b)

[0636] [ka]

[0637] Following a similar procedure as LP15, except using compound 9-5 instead of intermediate 8-3 and 9-4b instead of 9-4c, Fmoc-9-6b (26 mg) was obtained as a pale yellow solid, which was dissolved in DMF (2 mL). Diethylamine (0.2 mL) was added to the solution, and the reaction mixture was stirred at room temperature for 1 h until the Fmoc was completely removed by LCMS. The mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give 9-6b (13 mg, 20% yield) as a white solid. ESI m / z: 653 (M+H) + .

[0638] Example 9B-4: (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-({4-[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24Synthesis of ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}oxy}methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP14)

[0639] [ka]

[0640] To a yellow solution of compound 7-3 (3.3 mg, 12 μmol) in dry DMF (1 mL), DIPEA (2.6 mg, 20 μmol) and compound 9-6b (13 mg, 10 μmol) were added and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5-95% acetonitrile in aqueous formic acid (0.1%)) to give linker-payload LP14 (4.5 mg, 31% yield) as a white solid. ESI m / z: 735 (M / 2+H) + .

[0641] Example 9C-1: (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[2-({2-[(19S)-19-ethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2 , 11 .0 4 , 9 .0 15 , 20Synthesis of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]ethyl}(propan-2-yl)amino)ethoxy]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP13)

[0642] [ka]

[0643] Following a similar procedure to LP14, except using P10 instead of hydroxyacetic acid, linker-payload LP13 (8 mg from P10, 1% yield over 4 steps) was obtained as an off-white solid. ESI m / z: 727.3 (M / 2+H). + .

[0644] Example 10: Synthesis of branched sugar-PAB linker-payload (Scheme 10)

[0645] [ka]

[0646] Example 10A: Synthesis of [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-{2-[(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethyl)carbamoyl]-4-(hydroxymethyl)phenoxy}oxan-2-yl]methyl acetate (10-2)

[0647] [ka]

[0648] To a solution of compound 10-1 (0.10 g, 0.2 mmol) in DMF (5 mL), N-Fmoc-PEG 2 -amine (74 mg, 0.2 mmol) and DIPEA (52 mg, 0.4 mmol) were added and the reaction mixture was stirred at room temperature for 10 min, after which HATU was added to the reaction. The mixture was stirred at room temperature for 4 h, which was monitored by LCMS. The resulting mixture was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 10-2 (0.14 g, 81% yield) as a white solid. ESI m / z: 851.3 (M+H) + .

[0649] Example 10B: Synthesis of [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-{2-[(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethyl)carbamoyl]-4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenoxy}oxan-2-yl]methyl acetate (10-3)

[0650] [ka]

[0651] To a solution of 10-2 (0.14 g, 0.16 mmol) and bis(4-nitrophenyl)carbonate (0.25 g, 0.82 mmol) in DMF (5 mL), DIPEA (0.11 g, 0.82 mmol) and DMAP (20 mg, 0.16 mmol) were added and the reaction mixture was stirred at room temperature for 4 h, which was monitored by LCMS. The resulting mixture was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound 10-3 (0.13 g, 75% yield) as a white solid. ESI m / z: 1017.2 (M+H) + .

[0652] Example 10C: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamoyl)-4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenoxy]oxan-2-yl]methyl acetate (10-4)

[0653] [ka]

[0654] To a solution of 10-3 (0.10 g, 98 μmol) in DMF (5 mL), exatecan (43 mg, 98 μmol), HOAt (7 mg, 49 μmol), and DIPEA (26 mg, 0.20 mmol) were added and the reaction mixture was stirred at room temperature for 4 h, which was monitored by LCMS. The resulting mixture was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)) to give compound Fmoc-10-4 (0.11 g, 86% yield, ESI m / z: 1318.3 (M+H). + ) was obtained as a white solid, which was dissolved in DMF (5 mL). To the solution was added DIPEA (13 mg, 0.17 mmol) and the reaction mixture was stirred for 1 h at room temperature. The resulting mixture was isolated by reverse phase flash chromatography (85 mg, 78% yield) as a white solid. ESI m / z: 1091.3 (M+H) + .

[0655] Example 10D: {3-[(2-{2-[2-(3-{2-[3-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethoxy}ethoxy)propanamide]-3-{2-[(2-{2-[2-({5-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]-2-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}phenyl}formamido)ethoxy]ethoxy}ethyl)carbamoyl]ethoxy}propoxy}propanamide )ethoxy]ethoxy}ethyl)carbamoyl]-4-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl carbamate (LP16)

[0656] [ka]

[0657] To a stirred mixture of 10-4 (85 mg, 78 μmol) and 10-5 (35 mg, 39 μmol) in DMF (5 mL) was added DIPEA (20 mg, 0.16 mmol) and the mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The resulting mixture was purified by reversed-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give a white solid, which was dissolved in methanol (5 mL). To the solution was added aqueous lithium hydroxide (36 mM, 5 mL). The reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was directly separated by reversed-phase flash chromatography (5-100% acetonitrile in aqueous TFA (0.01%)) to give LP16 (30 mg, 33% yield) as a white solid. ESI m / z: 789.2 (M / 3+H) + .

[0658] Example 11. Synthesis of branched vcPAB linker-payload (Scheme 11)

[0659] [ka]

[0660] Example 11A: (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-({4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-(3-{3-[2-({2-[2-(2-{[(1S)-1-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-({4-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Synthesis of tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2-methylpropyl]carbamoyl}-3-carboxypropyl]carbamoyl}ethoxy)ethoxy]ethyl}carbamoyl)ethoxy]-2-[3-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethoxy}ethoxy)propanamido]propoxy}propanamido)ethoxy]ethoxy}propanamido)butanoic acid (LP17)

[0661] [ka]

[0662] To a yellow solution of 11-1 (50 mg, 23 μmol) in DMF (1.5 mL) was added exatecan (12 mg, 23 μmol), HOBt (3.1 mg, 23 μmol), and DIPEA (8.9 mg, 69 μmol) and the reaction mixture was stirred at room temperature for 6 h. The resulting mixture was purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to give LP17 (14 mg, 22% yield) as a white solid. ESI m / z: 928 (M / 3+H) + .1 H NMR (400MHz, DMSO d6 ) δ 10.06(s,2H),8.28(br s,2H),8.14(d,J=7.2Hz,2H),8.06(d,J=8.4Hz,2H),7.95(t,J=5.2Hz,2H),7.84-7.74(m,5H ),7.64-7.57(m,5H),7.35(d,J=8.4Hz,2H),7.30(s,2H),6.52(s,2H),6.22-6.09(m,2H),5.5 6-5.47(m,4H),5.44(s,4H),5.33-5.21(m,6H),5.07(s,4H),4.42-4.23(m,6H),4.22-4.15( m,2H),3.96-3.90(m,1H),3.88-3.84(m,1H),3.80-3.75(m,1H),3.62-3.53(m,11H),3.47(br s,14H),3.41-3.38(m,6H),3.26-3.15(m,10H),3.13-3.07(m,2H),3.03-2.91(m,4H),2.40(br s, 6H),2.35-2.29(m,9H),2.23-2.10(m,10H),2.03-1.95(m,2H),1.93-1.80(m,8H), 1.76-1.55(m,11H),1.48-1.33(m,6H),1.07-0.99(m,1H),0.90-0.80(m,18H)ppm.

[0663] Example 12. General procedure for generating site-specific conjugates

[0664] This example generally demonstrates a method for site-specific conjugation of a payload to an antibody or antigen-binding fragment thereof according to an embodiment of the present disclosure. The method includes a two-step process as shown in Figure 1. The first step is the addition of an amine linker azide, e.g., azide-PEG, to the antibody. 3 -amine (AL, amino-PEG 4 The first step was microbial transglutaminase (MTG)-mediated conjugation of the N-azide (N-azide) and an extra amine reagent (AL) was used to avoid potential cross-linking of antibody chains. The second step was the conjugation of the alkyne-conjugated payload linker payload (LP) via strain-promoted azide-alkyne cycloaddition (SPAAC).3 A conjugate tagged with Ab-AL 4 ) were conjugated to the AL. The number of LP molecules added to the antibody depends on the number of conjugation sites and the number of azide functional groups in the AL (n=1). For antibodies with WT Fc domains that have been enzymatically deglycosylated or have an N297D Fc mutation and have azides functionalized with an AL linker, the expected DAR=2×n×m, where n is the number of azide functional groups on the AL and m is the number of LP payloads. For antibodies with an N297Q Fc mutation and azide functionalized with an AL linker, the expected DAR=4. For all parent antibodies (Ab), azide-functionalized antibodies (Ab-(N 3 )n), the final ADCs and corresponding linker-payloads (LPs) produced as specific examples, as well as their ES-MS results and DAR values ​​of the ADCs are summarized in Tables 4 and 5.

[0665] [Table 25]

[0666] [Table 26-1]

[0667] [Table 26-2]

[0668] Nonglycosylated human antibody IgG (IgG1, IgG4, etc.) containing N297Q or N297D mutations were used for ADC conjugation. Two approaches (I and II) were performed by a two-step process (Figure 1), and the conjugation results with MS-DAR values ​​were summarized in the ADC list (Table 7).

[0669] [Table 27-1]

[0670] [Table 27-2]

[0671] Step 1: Site-specific conjugation of Handle-functionalized amines to antibodies generated drug conjugates containing 2, 4, or 8 handles per antibody.

[0672] Nonglycosylated human antibody IgG containing the N297Q or N297D mutation in BupH buffer (pH 7.4) was mixed with ≥100 molar equivalents of unbranched handle-amine (AL) or branched handle-amine (BL). The resulting solution was mixed with transglutaminase (25 U / mL, 1 U mTG / mg antibody, Zedira, Darmstadt, Germany, or 10 U / mL, 5.5 U MTG / mg antibody, Modernist Pantry-ACTIVA TI containing maltodextrin, Ajinomoto, Japan) to obtain a final concentration of 0.5–20 mg / mL of antibody. The reaction mixture was incubated for 24 h at 25–37 °C with gentle shaking while being monitored by ESI-MS. Upon completion, excess amine and mTG were removed by size-exclusion chromatography (SEC) or protein A column chromatography. The conjugates were characterized by UV-Vis, SEC, and ESI-MS.

[0673] Step 2: Click reaction between the handle-functionalized antibody and the linker-payload of Table 2 to generate site-specific ADCs.

[0674] Handle-functionalized antibody (Ab-(AL)) in PBS (pH 7.4) n or Ab-(BL) n, 1-20 mg / mL) was incubated with ≥2-10 molar equivalents of linker-payload (LP) dissolved in an organic solvent such as DMSO or DMA (10 mg / mL) to obtain a reaction mixture containing 5-15% organic solvent (v / v) for 1-48 h at 25-37 °C with gentle shaking. The reaction was monitored by ESI-MS. Upon completion, excess amounts of LP and organic solvent were removed by a desalting column with BupH (pH 7.4) and protein aggregates (if present) were removed by size exclusion chromatography (SEC). The purified conjugate, Ab-(AL-LP) n ADC or Ab-(BL-LP) 4 The ADC was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. The conjugate monomer purity was >95% by SEC.

[0675] T-DXd was conjugated using our in-house trastuzumab. Both T-DXd and isotype Ab-DXd ADCs were conjugated with Daiichi's maleimide-tetrapeptide GGFG-linker-DXd, and antibody interchain cysteine ​​conjugation with the maleimide linker payload was achieved using conventional procedures.

[0676] All ADCs were purified by SEC using a Cytiva AKTA instrument using a 16 / 600 Superdex® 200 column and eluting with DPBS at pH 7.4 at a flow rate of 1.5 mL / min. The DAR values ​​of the ADCs were measured by ESI-MS. Ab-[AL] 4 A 4× increase in mass of LP from Ab-[BL] was observed and correlated with the 4DAR ADC. 4 A further mass increase of 7–8×LP from was observed, indicating a 7–8 DAR ADC.

[0677] Example 13. Detailed conjugation procedure A representative 4DAR ADC from Approach I is illustrated below (Figure 1). A non-glycosylated anti-Her2 human IgG antibody containing the N297Q mutation was mixed with >200 molar equivalents of azido-dPEG3-amine (Handle, MW 708.41 g / mol). The resulting solution was mixed with microbial transglutaminase (10 U / mL, 5,5 U mTG / mg antibody, Modernist Pantry-ACTIVA TI contains maltodextrin from Ajinomoto, Japan) to give a final concentration of 5 mg / mL of antibody. The reaction mixture was incubated at 37°C for 24 hours with gentle shaking while being monitored by ESI-MS. Upon completion, excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The azide linker attached to the antibody results in a mass increase of 808 Da compared to the mAb, and the four handles are represented as an antibody with four azide handles (Ab-(handle) 4 The site-specific antibody-azide conjugate (2.1 mg / mL) in PBS (pH 7.4) was mixed with 7 molar equivalents of linker-payload in 2 mM DMSO to give a reaction mixture containing 5% organic solvent (v / v), and the solution was set at 32° C. for 36 h with gentle shaking. The reaction was monitored by ESI-MS. Upon completion, excess amounts of linker-payload and protein aggregates were removed by size-exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. The conjugate monomer purity was 99.8% by SEC. The drug-bound antibody results in a mass increase corresponding to the DAR4 conjugate. The conjugate monomer purity was >99% by SEC.

[0678] [Table 28]

[0679] Example 6: In vitro cell killing activity in SKBR3 cell line

[0680] In vitro cytotoxicity assays were performed to test the ability of the disclosed anti-Her2 drug conjugates (ADCs) (see Table 5) to kill human cell lines. The in vitro cytotoxicity of the ADCs, isotype control ADCs, and reference free payloads was assessed using the CellTiter-Glo 2.0 assay kit (Promega, catalog number G9243), and the amount of ATP present was used to determine the number of viable cells in the culture.

[0681] For the assay, SK-BR-3 cells were seeded at 1000 cells / well in white 96-well BioCoat plates (Corning #356693) coated with poly-D-lysine in complete growth medium and incubated at 37 °C for 1 h at 5% CO 2 Cells were grown overnight at 37 °C under low-temperature conditions. Three-fold serial dilutions of anti-Her2 ADC or isotype control ADC were prepared in dilution medium (Optimem + 0.1% BSA) and added to the cells at final concentrations ranging from 100 nM to 0.015 nM (concentrations were corrected for DAR (drug-antibody ratio) and dosed based on effective payload concentration). Three-fold serial dilutions of free payload were prepared in 100% DMSO, transferred to fresh dilution medium, and then added to the cells at a final constant DMSO concentration of 0.2% and final payload concentrations ranging from 100 nM to 0.015 nM. The last well of each dilution series (untreated well) served as a blank control containing only medium (ADC) or medium + 0.2% DMSO (payload) and were plotted as a continuation of the three-fold serial dilutions. After 6 days, 100 μL of CellTiter Glo2.0 was added to each well, the plate was mixed on an orbital shaker for 2 minutes, and the plate was incubated at room temperature for 10 minutes. Relative light units (RLU) were measured with an Envision luminometer (PerkinElmer) and cell viability was expressed as a percentage of untreated (100% viable) cells. IC 50Values ​​were determined using a four-parameter logistic equation across a 10-point dose-response curve (GraphPad Prism). Maximum % killing was also determined for each test article as follows: 100-minimum percent survival. One experiment was performed and the results are shown in Figures 2 and 3, with the EC values ​​for each test article. 50 Values ​​and maximum % mortality are reported.

[0682] [Table 29] As shown in Table 9 above, anti-Her2 ADCs conjugated via glutamine killed high Her2-expressing SK-BR-3 cells and increased IC 50 The EC values ​​ranged from 0.17 nM to 4.1 nM, and the % maximum killing values ​​ranged from >60% to 95%. Trastuzumab deruxtecan (T-DXD via cysteine ​​conjugation), which killed SK-BR-3 cells, was used as a positive control in the assay and showed a EC 50 The EC value was 372 pM and the maximal % killing value was 90.9%. The unconjugated anti-Her2 antibody was weakly cytotoxic in all lines tested (results not shown). The free exatecan payload released from the ADC killed the cells and 50 The value was 516 pM, with maximum % killing in the range of approximately 95%.

[0683] Since various changes in the above subject matter are possible without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description or defined in the claims be construed as illustrative and exemplary of the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the appended claims.

[0684] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. Formula (A): BA-(L1-B-L2-P) ｎ (A) A compound having the structure: BA is an antibody or antigen-binding fragment thereof; L1 is a first linker, B is a triazole-containing moiety; L2 is a second linker, P is P-I to P-IV 【Chemistry 1】 is selected from the group consisting of R １ is hydrogen, C １－６ Alkyl, -(CH ２ ) ｖ -OH, -(CH ２ ) ｖ -NH ２ , -(CH ２ ) ｖ -C(O)OH, -(CH ２ ) ｖ -phenyl, and -(CH ２ ) ｖ -SO ２ CH ３ and v is an integer from 0 to 12; R ２ is hydrogen, C １－６ Alkyl, -(CH ２ ) ｖ -OH, -(CH ２ ) ｖ -NH ２ , -(CH ２ ) ｖ -C(O)OH, -(CH ２ ) ｖ -phenyl, and -(CH ２ ) ｖ -SO ２ CH ３ and v is an integer from 0 to 12; R ３ is hydrogen, -C １－６ Alkyl, -(CH ２ ) ｖ -OH, -(CH ２ ) ｖ -NH ２ , -(CH ２ ) ｖ -C(O)OH, -(CH ２ ) ｖ -phenyl, -(CH ２ ) ｖ -SO ２ CH ３ , and —CO—(CH ２ ) ｖ -O-COCH ３ and v is an integer from 0 to 12; R ４ is -NH-, -N(-C １－６ alkyl), -N(-C １－６ alkyl) (-SO ２ CH ３ ), -N(-C １－６ alkyl) (-(CH ２ ) ｖ -OH), -N(-C １－６ alkyl) (CO-CH ２ -NH ２ ), -N(-C １－６ alkyl) (-(CH ２ ) ｖ -O-CH ２ —NH—CO—CH ２ -NH ２ ), -N(-C １－６ alkyl) (—CO—CH(NH ２ )-(CH ２ ) ｖ -COOH), -N(-C １－６ alkyl) (—CO—CH(NH ２ )-(CH ２ ) ｖ -NH ２ ), -N(-C １－６ alkyl) (—CO—CH(NH ２ )-(CH ２ ) ｖ -phenyl), or 【Chemistry 2】 and v is an integer from 0 to 12; R ５ is H, -OH, -OCH ３ ,or 【Transformation 3】 and R ６ is hydrogen, -C １－６ Alkyl, -(CH ２ ) ｖ -OH, -(CH ２ ) ｖ -NH ２ , -(CH ２ ) ｖ -phenyl, -(CH ２ ) ｖ -N ３ , -(CH ２ ) ｖ -NH-CH ２ -phenyl, -(CH ２ ) ｖ -NMe-CH ２ -phenyl-OMe, -(CH ２ ) ｖ -NH-(CH ２ ) ｖ -N ３ , -(CH ２ ) ｖ -NH-(CH ２ ) ｖ -CCH, -(CH ２ ) ｖ -NH-CO-CH(NH ２ )-(CH ２ ) ｖ -phenyl, -(CH ２ ) ｖ -NH-CO-CH(NH ２ )-(CH ２ ) ｖ -N ３ , -(CH ２ ) ｖ -NH-CO-CH(NH ２ )-(CH ２ ) ｖ -NH ２ , -(CH ２ ) ｖ -NH-CO-CH(NH ２ )-(CH ２ ) ｖ -COOH, 【Chemistry 4】 and v is independently an integer from 0 to 12; R ７ is H, -OH, -OCH ３ ,or 【Transformation 5】 and A compound wherein n is an integer from 1 to 12.

2. The compound of claim 1 , wherein the first linker L1 is connected to the side chain of a glutamine residue of the BA.

3. The compound described in claim 1, wherein the BA contains one or more glutamine residues, which are either naturally present in the BA or are introduced into the BA by site-specific modification of one or more amino acids.

4. The compound of claim 1, wherein the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

5. The compound described in claim 1, wherein the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, and brain cancer.

6. The compound described in claim 2, wherein the glutamine residue is Q295 or N297Q.

7. The compound of claim 1, wherein L1 comprises C 1-6 alkyl, phenyl, aralkyl-NH-, -C(O)-, -(CH 2 ) u -NH-C(O)-, -(CH 2 ) u -C(O)-NH-, -(CH 2 -CH 2 -O) v -, -(CH 2 ) u -(O-CH 2 -CH 2 ) v -C(O)-NH-, a peptide unit comprising 2 to 4 amino acids, or a combination thereof, each of which may be optionally substituted with one or more of -S-, -S(O 2 )-, -C(O)-, -C(O 2 )-, and -CO 2 H, and the subscripts u and v are independently integers from 1 to 8.

8. L1 is Table 1-1 Table 1-2 Table 1-3 2. The compound of claim 1 selected from the group consisting of:

9. B is 【Transformation 6】 and Z is C or N.

10. L2 is a compound of formula (L2): -SP1-AA-SP2- (L2) wherein: SP1 is absent or is the first spacer unit; AA is absent or is a peptide unit containing 2-4 amino acids; SP2 is absent or is a second spacer unit covalently attached to P; 2. The compound of claim 1, wherein at least one of SP1, AA, and SP2 is not absent.

11. SP1 is absent or 【Transformation 7】 , C 1-6 alkyl, -(CH 2 -CH 2 -O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH 2 ) u -, -NH-(CH 2 ) u -C(O)-, -NH-(CH 2 -CH 2 -O) v -, -NH-(CH 2 -CH 2 -O) v -C(O)-, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -, -NH-(CH 2 -CH 2 -O) v -(CH 2 ) u -C(O)-, -(CH 2 ) u -NH-C(O)-, -NH-(CH 2 ) u 11. The compound of claim 10, selected from the group consisting of -NH-C(O)-, -NH-(CH 2 ) u -C(O)-NH-, and combinations thereof, wherein the subscripts u and v are independently integers from 1 to 8.

12. The compound described in claim 10, wherein AA is a peptide unit containing 2 to 4 amino acids selected from alanine, glycine, valine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

13. SP2 is absent, or 【Transformation 8】 and combinations thereof, wherein R c is independently, at each occurrence, absent, or 【Chemistry 9】 11. The compound of claim 10, wherein the group is selected from:

14. L2 is Table 2-1 Table 2-2 Table 2-3 2. The compound of claim 1 selected from the group consisting of:

15. The compound of claim 1, wherein n is selected from 2, 4, and 8.

16. The compound comprising: Table 3-1 Table 3-2 Table 3-3 10. The compound of claim 1, having a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

17. Formula (Alk-L2-P): Alk-SP1-AA-SP2-P (Alk-L2-P) or a pharmaceutically acceptable salt thereof, wherein Alk is an alkyne-containing moiety; SP1 is absent or is the first spacer unit; AA is absent or is a peptide unit containing 2-4 amino acids; SP2 is absent or is a second spacer unit; P is P-I to P-IV 【Chemistry 10】 10. A compound selected from the group consisting of:

18. The compound of formula (Alk-L2-P), Table 4-1 Table 4-2 Table 4-3 Table 4-4 18. The compound of claim 17, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

19. A composition comprising a population of compounds described in any one of claims 1 to 18, having a drug-to-antibody ratio (DAR) of about 0.5 to about 12.

0.

20. The composition described in claim 19, having a DAR selected from about 2, about 4, and about 8. 【Request Item 21】 【Table 5-1】 Table 5-2 1. A compound having a structure selected from the group consisting of:

22. A pharmaceutical composition comprising a compound according to any one of claims 1 to 18 and 21, and a diluent, carrier, and / or excipient.

23. Use of a compound according to claim 21 in the manufacture of a medicament for use in a method for treating a tumor and / or cancer, said method comprising contacting said tumor and / or cancer with said compound.

24. Use of a compound according to any one of claims 1 to 18 and 21 in the manufacture of a medicament for use in a method of treating a disease in a subject in need thereof, said method comprising administering a therapeutically effective amount of said compound to said subject, and said disease being cancer.

25. The use of claim 24, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

26. A pharmaceutical composition for use in a method for selectively delivering a compound to a cell, comprising a compound described in any one of claims 1 to 18 and 21.

27. ​​A pharmaceutical composition for use in a method for selectively targeting an antigen on the surface of a cell with a compound, the pharmaceutical composition comprising a compound described in any one of claims 1 to 18 and 21.

28. Formula (A): BA-(L1-B-L2-P) n (A) 1. A method for producing a compound having the structure: BA is an antibody or antigen-binding fragment thereof; L1 is a first linker that covalently attaches to the side chain of a glutamine residue of the BA; B is a triazole-containing moiety; L2 is a second linker covalently bonding to P; P is P-I to P-IV 【Chemistry 11】 an antitumor agent selected from the group consisting of R 1 is hydrogen, C 1-6 alkyl, —(CH 2 ) v —OH, —(CH 2 ) v —NH 2 , —(CH 2 ) v —C(O)OH, —(CH 2 ) v —phenyl, and —(CH 2 ) v —SO 2 CH 3 , where v is an integer from 0 to 12; R 2 is hydrogen, C 1-6 alkyl, —(CH 2 ) v —OH, —(CH 2 ) v —NH 2 , —(CH 2 ) v —C(O)OH, —(CH 2 ) v —phenyl, and —(CH 2 ) v —SO 2 CH 3 , where v is an integer from 0 to 12; R 3 is hydrogen, —C 1-6 alkyl, —(CH 2 ) v —OH, —(CH 2 ) v —NH 2 , —(CH 2 ) v —C(O)OH, —(CH 2 ) v —phenyl, —(CH 2 ) v —SO 2 CH 3 , and —CO—(CH 2 ) v —O—COCH 3 , where v is an integer from 0 to 12; R 4 is —NH—, —N(—C 1-6 alkyl), —N(—C 1-6 alkyl)(—SO 2 CH 3 ), —N(—C 1-6 alkyl)(—(CH 2 ) v —OH), —N(—C 1-6 alkyl)(CO—CH 2 —NH 2 ), —N(—C 1-6 alkyl)(—(CH 2 ) v —O—CH 2 —NH—CO—CH 2 —NH 2 ), —N(—C 1-6 alkyl)(—CO—CH(NH 2 )—(CH 2 ) v —COOH), —N(—C 1-6 alkyl)(—CO—CH(NH 2 )—(CH 2 ) v —NH 2 ), —N(—C 1-6 alkyl)(—CO—CH(NH 2 )—(CH 2 ) v -phenyl), or 【Chemistry 12】 and v is an integer from 0 to 12; R 5 is H, —OH, —OCH 3 , or 【Chemistry 13】 and R 6 is hydrogen, —C 1-6 alkyl, —(CH 2 ) v —OH, —(CH 2 ) v —NH 2 , —(CH 2 ) v -phenyl, —(CH 2 ) v —N 3 , —(CH 2 ) v —NH—CH 2 -phenyl, —(CH 2 ) v —NMe-CH 2 -phenyl-OMe, —(CH 2 ) v —NH—(CH 2 ) v —N 3 , —(CH 2 ) v —NH—(CH 2 ) v —CCH, —(CH 2 ) v —NH—CO—CH(NH 2 )—(CH 2 ) v -phenyl, —(CH 2 ) v —NH—CO—CH(NH 2 )—(CH 2 ) v -N 3 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -NH 2 , -(CH 2 ) v -NH-CO-CH(NH 2 )-(CH 2 ) v -COOH, 【Chemistry 14】 and v is independently an integer from 0 to 12; R 7 is H, —OH, —OCH 3 , or 【Chemistry 15】 and the method comprises: a) contacting BA containing at least one glutamine residue with compound L1-B' in the presence of transglutaminase; b) contacting the product of step a) with one or more equivalents of compound B''-L2-P, wherein group B'' can be covalently linked to group B', and one of said groups B' and B'' is -N 3 and 【Chemistry 16】 and the other of said groups B′ and B″ is selected from: 【Chemistry 17】 and Z is C or N; c) isolating the compound of formula (A) produced.

29. The compound of formula (A) Table 6-1 Table 6-2 Table 6-3 or a pharmaceutically acceptable salt thereof.

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