HK40007827A - Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof - Google Patents

Description

Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof

Cross Reference to Related Applications

This application claims priority to U.S. provisional application serial No. 62/373,740, filed 2016, 8, 11, which is incorporated herein in its entirety.

Technical Field

The field of the disclosure generally relates to pyrrolobenzodiazepines with disulfide triggers, cyclic dione triggers, or arylboronic acid or arylboronic ester triggersProdrugs, and antibody conjugates thereof.

Background

Pyrrolobenzodiazepines are known(PBD) and its dimers interact with DNA and are potent cancer chemotherapeutic agents. The problem is the side effect(s) associated with some PBDsSuch as cardiotoxicity and acute tissue necrosis) limits use, dose, and effectiveness.

Conjugates comprising selective carrier-linker-PBD structures (e.g., antibody-PBD conjugates (ADCs)) are attractive selective chemotherapeutic molecules because they combine desirable properties of selectivity for target cells and cytotoxic drugs. By directing an effective cytotoxic drug to the target cells, the desired therapeutic effect in the target cells can be improved while minimizing the impact on non-target cells. Examples of such improvements include dose reduction, targeted delivery, and improved blood flow stability required to achieve a therapeutic effect. Nevertheless, PBD ADCs can still have undesirable side effects that limit use and/or dose.

Accordingly, there is a need for PBD compounds and formulations that provide reduced toxicity and improved bioefficacy.

Disclosure of Invention

In some embodiments, there is provided a PBD prodrug dimer-antibody conjugate composition of formula (I) comprising a first PBD prodrug monomer M1 and a second PBD-antibody monomer M2:

m1 is a PBD monomer. R²Is selected from-H, ═ CH₂-CN, -R, ═ CH-R, aryl, heteroaryl, bicyclic, and heterobicyclic. R³Is H. R⁶、R⁷And R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH, and SR. X is selected from S, O and NH. R¹⁰Is a prodrug moiety comprising (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester. R¹¹Selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, when X is S, wherein z is 2 or 3 and U is monovalent pharmaceutically acceptable cationIons. R is selected from the group consisting of lower alkyl groups having 1 to 10 carbon atoms and arylalkyl groups of up to 12 carbon atoms, (i) arylalkyl groups wherein the alkyl groups optionally contain one or more carbon-carbon double or triple bonds, or up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms. M1 contains an optional double bond, as indicated by the dashed line, between two of: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R²。

M2 is a PBD monomer. R^2’、R^3’、R^6’、R^7’、R^9’、R^11’And X' respectively correspond to R²、R³、R⁶、R⁷、R⁹、R¹¹And X, and are defined in the same manner as them. L is a self-immolative (self-immolative) linker comprising a disulfide moiety, a peptide moiety, or a peptidomimetic moiety. M2 contains an optional double bond, as indicated by the dashed line, between two of: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’。

M1 and M2 are bonded at the C8 position by a moiety-Q-T-Q '-wherein Q and Q' are independently selected from O, NH and S, and wherein T is optionally substituted C_1-12An alkylene group which is further optionally interrupted by one or more heteroatoms and/or aromatic rings. Ab is an antibody and p is an integer having a value of 1,2, 3, 4,5, 6,7, or 8 and represents the number of PBD prodrug dimers that can be conjugated or bonded to the antibody. Each asterisk independently represents a racemic or undefined stereochemical chiral center.

In some other embodiments, a PBD monomer prodrug composition of formula (II) is provided.

R²Is selected from-H, ═ CH₂-CN, -R, ═ CH-R, aryl, heteroaryl, bicyclic, and heterobicyclic. R³Is H. R⁶、R⁷、R⁸And R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH and SR, OR R⁷And R⁸Together with the carbon atom to which they are bonded form a group-O- (CH)₂)_n-O-, wherein n is 1 or 2. X is selected from S, O and NH. R¹⁰Is a prodrug moiety comprising (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester. R¹¹Selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S. R is selected from the group consisting of lower alkyl groups having 1 to 10 carbon atoms and arylalkyl groups of up to 12 carbon atoms, (i) aryl groups wherein the alkyl groups optionally contain one or more carbon-carbon double or triple bonds, or up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms. The PBD monomeric prodrug contains an optional double bond, as indicated by the dashed line, between two of: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R². Each asterisk independently represents a racemic or undefined stereochemical chiral center.

In some other embodiments, there is provided a PBD prodrug dimer compound of formula (VIII) comprising a first PBD prodrug monomer M1 and a second PBD monomer M2:

m1 is a PBD monomer. R²Is selected from-H, ═ CH₂、-CN、-R、-CH-R, aryl, heteroaryl, bicyclic and heterobicyclic. R³Is H. R⁶、R⁷And R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH, and SR. X is selected from S, O and NH. R¹⁰Is a prodrug moiety comprising (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester. R¹¹Selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S. R is selected from the group consisting of lower alkyl groups having 1 to 10 carbon atoms and arylalkyl groups of up to 12 carbon atoms, (i) aryl groups wherein the alkyl groups optionally contain one or more carbon-carbon double or triple bonds, or up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms. M1 contains an optional double bond, as indicated by the dashed line, between two of: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R²。

M2 is a PBD monomer. R^2’、R^3’、R^6’、R^7’、R^9’、R^11’And X' respectively correspond to R²、R³、R⁶、R⁷、R⁹、R¹¹And X. M2 contains an optional double bond, as indicated by the dashed line, between two of: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’。R¹²The bond between N10 'and C11' is absent when it is a double bond, or is selected from the group consisting of-C (O) O-L and-C (O) O-R¹⁰。

L is a self-immolative linker comprising at least one of a disulfide moiety, a peptide moiety, and a peptidomimetic moiety. M1 and M2 are bonded at the C8 position by a moiety-Q-T-Q '-wherein Q and Q' are independently selected from O, NH and S, and wherein T is optionally substitutedC_1-12An alkylene group which is further optionally interrupted by one or more heteroatoms and/or aromatic rings.

Each asterisk independently represents a racemic or undefined stereochemical chiral center.

In some other embodiments, a pharmaceutical composition is provided comprising the PBD prodrug dimer-antibody conjugate compound previously described, and a pharmaceutically acceptable diluent, antibody, or excipient.

In some other embodiments, there is provided a method of treating cancer comprising administering to a patient a pharmaceutical composition as previously described.

In other embodiments, there is provided the use of an antibody-drug conjugate compound as described previously in the manufacture of a medicament for the treatment of cancer in a mammal.

In other embodiments, there is provided an antibody-drug conjugate compound as previously described for use in a method of treating cancer.

In other embodiments, an article of manufacture is provided that includes a pharmaceutical composition as previously described, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat cancer.

Drawings

FIG. 1 depicts PBD monomeric disulfide prodrug activity [% ] against KPL-4 cells]Graph against the logarithm of prodrug concentration in moles/liter, and also depicts PBD monomer IC for KPL-4 cells₅₀Table of efficacy.

FIG. 2 depicts PBD monomeric disulfide prodrug activity [% ] against WSU-DLCL2 cells]Graph relative to the logarithm of prodrug concentration in moles/liter, and also depicts PBD monomer IC against WSU-DLCL2 cells₅₀Table of efficacy.

FIG. 3 depicts a plot of SK-BR-3 cell viability (% of control) versus PBD monomeric disulfide prodrug concentration (μ g/mL).

Figure 4 depicts a graph of PBD monomeric disulfide prodrug stability in human and rat whole blood evaluated at intervals of 4 hours and 24 hours, with results presented as the percentage of parent compound remaining relative to time 0.

Figure 5 depicts a plot of SK-BR-3 cell viability (% of control) versus PBD monomeric disulfide prodrug concentration (micromolar) and PBD dimeric disulfide prodrug concentration (μ g/mL).

Figure 6 depicts a graph of KPL-4 cell viability (% of control) versus PBD monomeric disulfide prodrug concentration (micromolar) and PBD dimeric disulfide prodrug concentration (μ g/mL).

FIG. 7 depicts a graph of UACC-257 and IGROV-1 relative cell viability (% of control) versus PBD dimer non-prodrug control concentration (nM), and also depicts PBD dimer IC for UACC-257 and IGROV-1 cells₅₀Table of efficacy.

FIG. 8 depicts a plot of relative cell viability (% of control) for UACC-257 and IGROV-1 versus concentration (nM) of PBD dimer with disulfide prodrug at position N10 of one PBD monomer and no prodrug at the other PBD monomer, and also depicts PBD dimer IC for indicated GSH concentrations for UACC-257 and IGROV-1 cells₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios were determined relative to the data for the PBD dimer non-prodrug control depicted in figure 7.

FIG. 9 depicts plots of relative cell viability (% of control) for UACC-257 and IGROV-1 versus concentration (nM) of PBD dimer with disulfide prodrug at N10 position for both PBD monomers, and also depicts PBD dimer IC for the indicated GSH concentrations for UACC-257 and IGROV-1₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios were determined relative to the data for the PBD dimer non-prodrug control depicted in figure 7.

Fig. 10 depicts a plot of SK-BR3 relative cell viability (% of control) versus concentration (nM) of: (i) a PBD dimer non-prodrug, (ii) a PBD dimer with a disulfide prodrug at the N10 position of one PBD monomer and no prodrug at the other PBD monomer, and (iii) a PBD dimer with a disulfide prodrug at the N10 position of both PBD monomers.

FIG. 11 depicts a plot of SK-BR-3 cell viability (% of control) versus the concentration of: (i) a 7C2HCA140C peptide-linked disulfide cyclopentyl prodrug ADC PBD dimer, (ii) a 7C2LC K149C peptide-linked disulfide thio-phenol prodrug ADC PBD dimer, (iii) a 7C2HC a140C ADC PBD dimer without a prodrug moiety, and (iv) a CD22HC a140C ADC PBD dimer without a prodrug moiety.

Figure 12 depicts a graph of KPL-4 cell viability (% of control) versus concentration of: (i) a 7C2HCA140C peptide-linked disulfide cyclopentyl prodrug ADC PBD dimer, (ii) a 7C2LC K149C peptide-linked disulfide thio-phenol prodrug ADC PBD dimer, (iii) a 7C2HC a140C ADC PBD dimer without a prodrug moiety, and (iv) a CD22HC a140C ADC PBD dimer without a prodrug moiety.

FIG. 13 depicts a plot of SK-BR-3 cell viability (% of control) versus concentration of: (i) a 7C2LCK149C peptide-linked disulfide cyclobutyl prodrug ADC PBD dimer, (ii) a 7C2LC K149C peptide-linked disulfide cyclopentyl prodrug ADC PBD dimer, (iii) a 7C2LC K149C peptide-linked disulfide thio-phenol prodrug ADC PBD dimer, (iv) a 7C2LC K149C peptide-linked disulfide isopropyl prodrug ADC PBD dimer, (V) a 4D5HC a118C ADC PBD dimer without a prodrug moiety, and (vi) a 4D5LC V205C ADC PBD dimer without a prodrug moiety.

Figure 14 depicts a graph of KPL-4 cell viability (% of control) versus concentration of: (i) a 7C2LCK149C peptide-linked disulfide cyclobutyl prodrug ADC PBD dimer, (ii) a 7C2LC K149C peptide-linked disulfide cyclopentyl prodrug ADC PBD dimer, (iii) a 7C2LC K149C peptide-linked disulfide thio-phenol prodrug ADC PBD dimer, (iv) a 7C2LC K149C peptide-linked disulfide isopropyl prodrug ADC PBD dimer, (V) a 4D5HC a118C ADC PBD dimer without a prodrug moiety, and (vi) a 4D5LC V205C ADC PBD dimer without a prodrug moiety.

Figure 15A depicts a graph of the normalized percentage of WSU-DLCL viable cells after 3 days compared to the number of cells at time 0 versus the concentration of: (i) having benzyl formate (C) at position N10 of one PBD monomer₆H₅-CH₂CD22 antibody ADC PBD dimer of the-O-C (O) -) moiety (negative control), (ii) CD22 antibody ADC PBD dimer aryl boronic acid prodrug ((OH)₂B-C₆H₄-CH₂-O-c (O), (iii) CD22 antibody ADC PBD dimer without prodrug moiety (positive control).

Figure 15B depicts a graph of WSU-DLCL cell killing versus drug concentration (μ g/mL) 3 days after exposure to: (i) CD22 antibody ADC PBD dimer with boronic acid prodrug (PBD dimer ADC boronic acid prodrug 1A); (ii) ly6E antibody ADC PBD dimer with boronic acid prodrug (PBD dimer ADC boronic acid prodrug 1B); (iii) CD22 antibody ADC PBD dimer without prodrug moiety (PBD dimer ADC boronic acid control 2) (positive control) and (iv) blank control.

FIG. 15C depicts a graph of MDA-MB-453 cell killing versus drug concentration (μ M) 3 days after exposure to: (i) PBD monomer control; (ii) a PBD monomer control having a benzyl formate moiety at the N10PBD position; and (iii) a PBD monomeric boronic acid prodrug.

FIG. 15D depicts a graph of MDA-MB-453 cell killing versus drug concentration (μ M) 3 days after exposure to: (i) silvestrol; (ii) PBD monomer control; (iii) a PBD monomer control having a benzyl formate moiety at the N10PBD position; (iv) PBD monomeric boronic acid prodrugs; (v) PBD monomer control and silvestrol; (vi) a PBD monomer control having a benzyl formate moiety at the N10PBD position and silvestrol; and (vii) PBD monomeric boronic acid prodrugs and silvestrol.7

FIG. 16 depictsA graph of normalized percentage of BJAB viable cells after 3 days compared to the number of cells at time 0 versus the concentration of: (i) having benzyl formate (C) at position N10 of one PBD monomer₆H₅-CH₂CD22 antibody ADC PBD dimer of the-O-C (O) -) moiety (negative control), (ii) CD22 antibody ADC PBD dimer aryl boronic acid prodrug ((OH)₂B-C₆H₄-CH₂-O-c (O), (iii) CD22 antibody ADC PBD dimer without prodrug moiety (positive control).

FIG. 17 depicts the activity [% ] against KPL-4 cells]Graph of concentration (M) relative to: (i) a PBD dimer with a quinone prodrug at position N10 of one PBD monomer and no prodrug or linker at position N10 of the other PBD monomer, and (ii) a PBD dimer without a prodrug or linker. FIG. 17 also depicts PBD dimer diaphorase prodrugs and control IC against KPL-4 cells₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios are based on prodrug IC versus PBD dimer control₅₀The value is obtained.

FIG. 18 depicts the activity [% ] against WSU cells]Graph of concentration (M) relative to: (i) a PBD dimer with a quinone prodrug at position N10 of one PBD monomer and no prodrug or linker at position N10 of the other PBD monomer, and (ii) a PBD dimer without a prodrug or linker. FIG. 18 also depicts PBD dimer diaphorase prodrugs and control IC against KPL-4 cells₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios are based on prodrug IC versus PBD dimer control₅₀The value is obtained.

FIG. 19 depicts the activity [% ] against KPL-4 cells]Graph of concentration (M) relative to: (i) a PBD monomer with a quinone prodrug at N10, and (ii) a PBD monomer without a prodrug or linker. FIG. 19 also depicts PBD dimer diaphorase prodrugs and control IC against KPL-4 cells₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios are based on prodrug IC versus PBD dimer control₅₀The value is obtained.

FIG. 20 depicts the activity [% ] against WSU cells]Graph of concentration (M) relative to: (i) a PBD monomer with a quinone prodrug at N10, and (ii) a PBD monomer without a prodrug or linker. FIG. 20 also depicts PBD dimer diaphorase prodrugs and control IC against WSU cells₅₀Potency and IC₅₀A table of ratios. IC (integrated circuit)₅₀Ratios are based on prodrug IC versus PBD dimer control₅₀The value is obtained.

FIG. 21 depicts a plot of SK-BR-3 cell viability (% of control) versus concentration (μ g/mL) of: (i)7C2LC K149C VC-PBD DT diaphorase quinone prodrug ADC PBD dimer, (ii) Ly6E LC K149C VC-PBDDT diaphorase quinone prodrug ADC PBD dimer, and (iii) 4D5HC a118C ADC PBD dimer without a prodrug moiety.

FIG. 22 depicts a graph of KPL-4 cell viability (% of control) versus concentration of: (i)7C2LC K149C VC-PBD DT diaphorase quinone prodrug ADC PBD dimer, (ii) Ly6E LC K149C VC-PBD DT diaphorase quinone prodrug ADC PBD dimer, and (iii) 4D5HC a118C ADC PBD dimer without a prodrug moiety.

FIG. 23 depicts tumor volume (mm)³) Graphs relative to days post-treatment with BJAB-luc human burkitt lymphoma in SCID mice: (i) histidine buffer vehicle; (ii) non-prodrug anti-CD 22HC-a118C PBD dimer ADC; (iii) anti-CD 22LC-K149C PDB dimer boronic acid prodrug ADC; (iv) anti-Ly 6E LC-K149C PDB dimer boronic acid prodrug ADC; (v) non-prodrug anti-CD 22LC-K149C PDB dimer ADC; and (vi) non-prodrug anti-Her 2HC-a118CPBD dimer ADC.

FIG. 24 depicts a graph of% weight change versus days following treatment with BJAB-luc human Burkitt's lymphoma in SCID mice: (i) histidine buffer vehicle; (ii) non-prodrug anti-CD 22HC-a118C PBD dimer ADC; (iii) anti-CD 22LC-K149C PDB dimer boronic acid prodrug ADC; (iv) anti-Ly 6E LC-K149C PDB dimer boronic acid prodrug ADC; (v) non-prodrug anti-CD 22LC-K149C PDB dimer ADC; and (vi) non-prodrug anti-Her 2HC-a118CPBD dimer ADC.

FIG. 25 depicts tumor volume (mm)³) Graphs relative days post-treatment with SCID-beige mice bearing KPL-4 human breast tumors: (i) a vehicle; (ii) non-prodrug anti-Her 2HC-a140C PBD dimer ADC; and (iii) anti-Her 2HC-a140CPBD thio-phenol prodrug ADC.

FIG. 26 depicts a graph of% weight change versus days following treatment of SCID-beige mice with KPL-4 human breast tumors with: (i) a vehicle; (ii) non-prodrug anti-Her 2HC-a140C PBD dimer ADC; and (iii) anti-Her 2HC-a140C PBD thio-phenol prodrug ADC.

Detailed Description

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

In some embodiments, the present disclosure relates generally to PBD monomer prodrug compounds of formula (II):

wherein R is²、R³、R⁶、R⁷、R⁸、R⁹、X、R¹⁰、R¹¹And defined in more detail elsewhere herein.

In some embodiments, the disclosure relates to PBD dimer prodrug compounds comprising a compound having R at the N10 position¹⁰The first PBD monomer of (a). The dimer further comprises a second PBD monomer having at position N10: (1) no substitution; (2) r¹⁰(ii) a Or (3) a linker. PBD dimers are typically one of two structures:

wherein R is²、R^2’、R³、R^3’、R⁶、R^6’、R⁷、R^7’、R⁹、R^9’、X、R¹⁰、R¹¹、R^11’Q, Q', T, # and linkers are defined in more detail elsewhere herein.

In some embodiments, the present disclosure relates to a PBD dimer prodrug compound comprising a first PBD monomer having at position N10: (i) a protecting group comprising a GSH-activated disulfide trigger, (ii) a protecting group comprising a DTD-activated quinone trigger, or (iii) a protecting group comprising a ROS-activated arylboronic acid or arylboronic ester trigger. The dimer further comprises a second PBD monomer having a linker conjugated to the thiol moiety of the antibody at position N10. PBD dimers are generally as follows:

wherein R is²、R^2’、R³、R^3’、R⁶、R^6’、R⁷、R^7’、R⁹、R^9’、X、R¹⁰、R¹¹、R^11’T, linker, antibody and p are defined in more detail elsewhere herein.

I. Definition of

Unless defined otherwise, scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs and are consistent with the following documents: singleton et al (1994) Dictionary of microbiology and Molecular Biology, 2 nd edition, J.Wiley & Sons, New York, NY; and Janeway, c., Travers, p., Walport, m., shmchik (2001) immunology, 5 th edition, garland publishing, New York.

A "prodrug" as defined herein is a PBD substituted at position N10 with a protecting group comprising a trigger, wherein the protecting group masks drug toxicity. The protecting group is activated (cleaved) enzymatically or chemically to generate the active drug by applying a stimulus to the trigger, such as an enzyme (e.g., DTD), ROS, or GSH. In some embodiments, the trigger is a disulfide, a cyclic diketone (e.g., quinone), or an arylboronic acid or arylboronic ester.

"protecting group" as defined herein refers to a moiety introduced into a drug molecule by chemical modification of a functional group that blocks or protects a particular functionality.

"DTD" refers to DT-diaphorase; "ROS" refers to reactive oxygen species; and "GSH" refers to glutathione.

A "linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an antibody-linker-drug conjugate having the general formula:

antibody- [ L-D]_p

Wherein p can be 1,2, 3, 4,5, 6,7, or 8. The linker typically comprises a linker to an antibody (Ab), an optional antibody spacer unit, an optional trigger unit for providing dissociation (mobilization), an optional drug (D) spacer unit, and a linker to a drug, and has the following general structure:

ab- [ Ab connecting part]- [ Ab spacer group]_{Optionally, the}- [ trigger)]_{Optionally, the}- [ D spacer group]_{Optionally, the}- [ D connecting part]-D。

In some embodiments, antibody-D conjugates can be prepared using linkers having reactive functionality for covalent attachment to drugs and antibodies. For example, in some embodiments, the cysteine thiol of a cysteine engineered antibody (Ab) may form a bond with a reactive functional group of a linker or drug-linker intermediate to make an ADC. In one embodiment, the linker has a functionality capable of reacting with free cysteines present on the antibody to form covalent disulfide bonds (see, e.g., Klussman et al (2004), Bioconjugate Chemistry 15(4): page 766-773, and the examples herein, which are incorporated herein by reference in their entirety). In some embodiments, the linker may comprise a cleavable self-immolative moiety, such as a peptide, peptidomimetic, or disulfide trigger. The linker may optionally comprise one or more "spacer" units between the self-immolative moiety and the drug moiety, such as a p-amino-benzyl ("PAB"), and/or between the self-immolative moiety and the antibody, such as a moiety derived from hexanoic acid. Non-limiting examples of spacers include valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), and aminobenzyloxycarbonyl ("PABC"). In some embodiments, the spacer may be self-immolative.

"self-immolative" and "self-immolative" refer to moieties, such as linkers, spacers, and/or prodrug triggers, that can be cleaved in vivo and/or in vitro, such as by enzymes (e.g., proteases or DTDs), GSH, ROS, and/or pH changes. Examples of self-immolative moieties include disulfides, peptides and peptidomimetics.

"peptide" refers to a short chain of two or more amino acid monomers linked by amide (peptide) bonds. The amino acid monomers can be naturally occurring and/or non-naturally occurring amino acid analogs.

"peptidomimetic" refers to a group or moiety that has a structure that is different from the general chemical structure of an amino acid or peptide, but functions in a similar manner to a naturally occurring amino acid or peptide.

"hindered linker" refers to a linker having a carbon atom with a sulfur capable of forming a disulfide bond, wherein the carbon atom is substituted with at least one substituent other than H, and more specifically is partially substituted with a hydrocarbyl or substituted hydrocarbyl group, as described in further detail below.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

By "cell targeting moiety" is meant an antibody that has binding affinity for the target expressing the antigen.

By "consisting essentially of" is meant at least 50%, at least 75%, at least 90%, at least 95%, or at least 99% of the referenced component on the recited basis, such as, for example and without limitation, on a weight/weight, volume/volume, weight/volume%, or equivalent% basis. "consisting essentially of" generally limits the features, compounds, compositions or methods to the enumerated elements and/or steps, but does not exclude the possibility of additional elements and/or steps not materially affecting the function, compound, composition and/or characteristics of the enumerated features, compounds, compositions or methods.

The terms "antibody" and "Ab" are used herein in the broadest sense and specifically encompass monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) journal. of Immunology 170: 4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. (Janeway, C., Travers, P., Walport, M., Sholomchik (2001) Immuno Biology, 5 th edition, Garland Publishing, New York). The target antigen typically has a number of binding sites, also referred to as epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, such targets including, but not limited to, one or more cancer cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be of any class (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species. However, in one embodiment, the immunoglobulin is of human, murine or rabbit origin.

An "antibody fragment" includes a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; minibodies (Olafsen et al (2004) Protein Eng. design & Sel.17(4): 315-; fragments produced from a Fab expression library; anti-idiotype (anti-Id) antibodies; CDRs (complementarity determining regions); and an epitope-binding fragment of any of the described herein that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor amounts of mutations that may naturally occur. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as it is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the present disclosure can be prepared by the hybridoma method initially described by Kohler et al (1975) Nature 256:495, or can be prepared by recombinant DNA methods (see, e.g., US 4816567; US 5807715). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the antibody library described in Clackson et al (1991) Nature,352: 624-; marks et al (1991) J.mol.biol.,222: 581-597.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of the above antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA,81: 6851-. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.) and human constant region sequences.

An "intact antibody" herein is an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH 3. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc constant region (either the native sequence Fc region or the amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors such as B cell receptors and BCR.

Intact immunoglobulin antibodies can be assigned different "classes" according to the amino acid sequence of their heavy chain constant domains, there are five major classes of IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. the heavy chain constant domains corresponding to different classes of antibodies are referred to as α, δ, ε, γ and μ, respectively.

A "cysteine engineered antibody" or "cysteine engineered antibody variant" is an antibody in which one or more residues of the antibody are substituted with cysteine residues. In accordance with the present disclosure, one or more thiol groups of cysteine engineered antibodies may be conjugated to prodrugs of the present disclosure to form THIOMAB^TMADC (i.e., THIOMAB)^TMDrug Conjugates (TDC)). In particular embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody, and can be used to conjugate the antibody to a drug moiety to produce an immunoconjugate as further described herein. For example, Thiomab^TMThe antibody may be an antibody having a single mutation from a non-cysteine natural residue to cysteine in the light chain (e.g., G64C, K149C, or R142C according to Kabat numbering) or in the heavy chain (e.g., D101C or V184C or T205C according to Kabat numbering). In a specific example, Thiomab^TMAntibodies have a single cysteine mutation in either the heavy or light chain such that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two engineered cysteine residues. US2012/0121615A 1 (incorporated herein by reference in its entirety) discloses cysteine engineered antibodies and methods of making.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is often characterized by uncontrolled cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., hodgkin's and non-hodgkin's lymphomas), blastoma, sarcoma, and leukemia. More specific examples of such cancers include Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), Chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia, Acute Promyelocytic Leukemia (APL), chronic myeloproliferative disorders, platelet leukemia (thrombocytic leukemia), precursor B cell acute lymphoblastic leukemia (pre-B-ALL), precursor T cell acute lymphoblastic leukemia (pre-ALL), Multiple Myeloma (MM), mastocytosis, mast cell leukemia, mast cell sarcoma, myelosarcoma, lymphocytic leukemia, and undifferentiated leukemia. In some embodiments, the cancer is myeloid leukemia. In some embodiments, the cancer is Acute Myeloid Leukemia (AML).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) is an amount effective to achieve the desired therapeutic or prophylactic result at the dosages and for periods of time necessary.

The term "epitope" refers to a specific site on an antigenic molecule to which an antibody binds. In some embodiments, the specific site on the antigen molecule to which the antibody binds is determined by hydroxyl radical footprinting (hydroxyradical printing).

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of immunological interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full-length antibody," "intact antibody," and "full antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "human consensus framework" is a framework representing the amino acid residues most frequently occurring in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subset of Sequences is a subset as described in Kabat et al, Sequences of Proteins of immunological interest, 5 th edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup k I, as described in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III, as described in Kabat et al, supra.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally can include at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) typically have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., Kindt et al KubyImmunology, 6 th edition, w.h.freeman and co., page 91 (2007)). In addition, antibodies that bind a particular antigen can be isolated using VH or VL domains from antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al, J.Immunol.150: 880-; clarkson et al, Nature 352: 624-.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the sequence of an antibody variable domain which are hypervariable and/or form structurally defined loops ("hypervariable loops"). Generally, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101 (H3). (Chothia and Lesk, J.mol.biol.196:901-917 (1987)) exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at the following amino acid residues: 24-34 of L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1 and 95-102 of H3. (Kabat et al, Sequences of Proteins of immunological Interest, Public Health Service 5 th edition, National Institutes of Health, Bethesda, Md. (1991)) in addition to the CDR1 in the VH, the CDRs typically contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDR is contained within a region of the CDR called the abbreviation-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at the following amino acid residues: 31-34 for L1, 50-55 for L2, 89-96 for L3, 50-58 for 31-35B, H2 for H1, and 95-102 for H3. (see Almagro and Fransson, front. biosci.13:1619-1633(2008)), unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated antibody" is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromanogr.b 848:79-87 (2007).

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, which consists of two identical light chains and two identical heavy chains bound by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy or variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings regarding the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence in order to achieve the maximum percent sequence identity, after aligning the sequences and, if necessary, introducing gaps, and not considering any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the% amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been submitted with the user document to the U.S. copyright office of Washington D.C. (20559), with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were unchanged.

In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity (or may alternatively be expressed as a% amino acid sequence identity) of a given amino acid sequence a with, and or with, a given amino acid sequence B is calculated as follows: 100 times the score X/Y, where X is the number of amino acid residues that are scored as identical pairs by the sequence alignment program when aligning a and B of the ALIGN-2 program; and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless otherwise specifically stated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a formulation in a form such that the biological activity of the active ingredient contained therein is effective and does not contain additional components having unacceptable toxicity to the subject to which the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The terms "treatment" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean prolonging survival compared to the survival expected in the absence of treatment. Subjects in need of treatment include subjects already having the condition or disorder, as well as subjects susceptible to or to prophylaxis of the condition or disorder.

The term "therapeutically effective amount" refers to an amount of a drug that is effective for treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with cancer. To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For the treatment of cancer, efficacy can be determined, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).

The term "leaving group" as used herein refers to a moiety that leaves during a chemical reaction involving a group as described herein.

The term "hydrocarbyl" as used herein describes an organic compound or radical consisting only of the elements carbon and hydrogen. These moieties include, but are not limited to, alkyl, alkenyl, alkynyl, and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl and aryl moieties substituted with other aliphatic or cyclic hydrocarbyl groups, such as alkaryl, alkenaryl and alkynylaryl groups. Unless otherwise indicated, these moieties preferably include 1 to 20 carbon atoms, 1 to 10 carbon atoms, or1 to 6 carbon atoms.

The term "alkyl" as used herein by itself or as part of another substituent means a straight or branched chain hydrocarbon group (i.e., C) having the indicated number of carbon atoms_1-8Meaning 1 to 8 carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unless otherwise indicated, the alkyl groups described herein are preferably lower alkyl groups containing 1 to 10 or1 to 8 carbon atoms in the main chain. They may be linear or branched or cyclic, including but not limited toIn methyl, ethyl, propyl, isopropyl, allyl, benzyl, hexyl, and the like. The alkyl moiety may optionally contain one or more heteroatoms selected from O, S and N and is referred to as "heteroalkyl".

The terms "carbocycle", "carbocyclyl", "carbocycle", and "cycloalkyl" refer to a monocyclic ring (C) having 3 to 12 carbon atoms_3-12) Or a monovalent non-aromatic, saturated or partially unsaturated ring having 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms may be arranged, for example, as a bicyclo [4,5 ]]、[5,5]、[5,6]Or [6,6 ]]Systems, and bicyclic carbocycles having 9or 10 ring atoms may be arranged as a bicyclo [5,6]Or [6,6 ]]Systems, or bridge systems, such as bicyclo [2.2.1]]Heptane, bicyclo [2.2.2]Octane and bicyclo [3.2.2]Nonane. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like. The carbocyclic and cycloalkyl moieties may optionally contain one or more heteroatoms selected from O, S and N.

The term "alkoxy" refers to those alkyl groups that are attached to the rest of the molecule through an oxygen atom. The alkoxy moiety may optionally contain one or more heteroatoms selected from O, S and N and is referred to as "heteroalkoxy".

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, such as-CH₂CH₂CH₂CH₂CH₂-。

Unless otherwise indicated, alkynyl groups described herein are preferably lower alkynyl groups containing from 2 to 8 carbon atoms in the backbone and up to 20 carbon atoms. They may be straight or branched chain and include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

The term "aryl" as used herein alone or as part of another group denotes an optionally substituted homocyclic aromatic group, preferably a monocyclic or bicyclic group containing 5 to 20 carbons, 5 to 10 carbons, or 5 to 6 carbons in the ring portion, including but not limited to phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl. The aryl moiety may optionally contain one or more heteroatoms selected from O, S and N and is referred to as "heteroaryl" or "heterobicyclic". Such heteroaromatic compounds may contain 1 or 2 nitrogen atoms, 1 or 2 sulfur atoms, 1 or 2 oxygen atoms, and combinations thereof in the ring, with each heteroatom bonded to the remainder of the molecule through carbon. Non-limiting exemplary groups include pyridine, pyrazine, pyrimidine, pyrazole, pyrrole, imidazole, thiophene, thiopyrylium, p-thiazine, indole, purine, benzimidazole, quinolone, phenothiazine. Non-limiting exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amido, acetal, carbamoyl, carbocyclic and cyano, ester, ether, halogen, heterocyclic and hydroxy, ketone, ketal, phosphate, nitro, and thio groups.

The term "arylalkyl" as used herein refers to an aryl moiety substituted with at least one alkyl group, and optionally further substituted. An example of arylalkyl is phenylmethyl, also known as benzyl (C)₆H₅CH₃) Or benzylidene (-C)₆H₄CH₂-)。

As used herein, a "substituted" moiety is a moiety such as hydrocarbyl, alkyl, heteroaryl, bicyclic, and heterobicyclic substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a heteroatom such as nitrogen, oxygen, silicon, phosphorus, boron, sulfur, or a halogen atom. These substituents include, but are not limited to, halogen, heterocyclyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy, hydroxy, keto, acyl, acyloxy, nitro, tertiary amino, amido, nitro, cyano, thio, sulfinate, sulfamoyl, ketal, acetal, ester, and ether.

The terms "halogen" and "halo" as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.

The term "cyclic diketone" refers to cyclic and heterocyclic compounds having an even number of-c (o) -groups. In some embodiments, the cyclic compound is an aryl (quinone). In some embodiments, the heterocyclic compound is a heteroaryl. A non-exclusive list of cyclic diketones includes naphthoquinones and indole diketones.

The term "pharmaceutically acceptable cation", denoted U, refers to a monovalent cation. Examples of pharmaceutically acceptable monovalent cations are discussed in Berge et al, J pharm. sci.,66,1-19(1977), which is incorporated herein by reference. In some aspects, the pharmaceutically acceptable cation is inorganic, including but not limited to alkali metal ions (e.g., sodium or potassium ions) and ammonia. For example, in some aspects, part of the SO_zU may be SO₃Na、SO₃K or SO₃NH₄。

Certain compounds of the present disclosure may have asymmetric carbon atoms (optical centers) or double bonds. Such compounds have the same molecular formula but differ in their atomic bonding properties or order or their spatial arrangement of atoms, and are referred to as "isomers". Isomers that differ in their arrangement in atomic space are referred to as "stereoisomers". Diastereomers are stereoisomers with opposite configuration at one or more chiral centers, which are not enantiomers. Stereoisomers with one or more asymmetric centers that are non-superimposable mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, one pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of one or more of their asymmetric centers and described by the R-and S-sequence rules of Cahn, Ingold and Prelog, or in such a way that the molecule rotates the plane of polarized light and is designated as dextrorotatory or levorotatory (i.e., the (+) or (-) -isomers, respectively). The chiral compounds may be present as individual enantiomers or as mixtures thereof. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures". In certain embodiments, the compounds are enriched in at least about 90% by weight of a single diastereomer or enantiomer. In other embodiments, the compounds are enriched in at least about 95%, 98%, or 99% by weight of a single diastereomer or enantiomer. The compounds of the present disclosure encompass racemates, diastereomers, geometric isomers, positional isomers, and individual isomers thereof (e.g., individual enantiomers), and all such are intended to be encompassed within the scope of the present disclosure.

Prodrug monomers, dimers and conjugates

In some embodiments, the drug is a PBD monomer or a PBD dimer. In some embodiments, the PDB dimer recognizes and binds to a specific DNA sequence. The natural product ampamycin, a PBD, was originally reported in 1965 (Leimgruber et al, (1965) J.Am.chem.Soc.,87: 5793-5795; Leimgruber et al, (1965) J.Am.chem.Soc.,87: 5791-5793). Since then, a number of PBDs have been reported as naturally occurring and analogous (Thurston et al, (1994) chem. Rev.1994, 433-465), including dimers of tricyclic PBD scaffolds (US 6884799; US 7049311; US 7067511; US 7265105; US 7511032; US 7528126; US 7557099), although not intending to be bound by any particular theory, it is believed that the dimer structure confers an appropriate three-dimensional shape for homohelicity with the minor groove of type B DNA (isohelicity) leading to a slip fit (snug fit) at the binding site (Kohn, in antibodies III Springer-Chelag, New York, page 3-11 (1975); Hurley and New ham-VanAntiner, (1986) Acc.Chem.Res.,19: 230. Chestn.83. C2 aryl substituents as toxic compounds (Biocide J2923, 2010. 76, 2010) and K19H 92. J. 9 H. 76 H. 9 H. J. 9, and H. 9. J. H. 9, No. 23, No. 11, No. 9, No. 23,2, No. 2, 9, No. 2, No. 2, 9, No. 2, No. 2, No. 2 Each of the references cited in (a) is incorporated herein by reference in its entirety.

PBD monomers and PBD dimers within the scope of the present disclosure are known. See, for example, US 2010/0203007, WO 2009/016516, US 2009/304710, US 2010/047257, US 2009/036431, US 2011/0256157, WO 2011/130598), WO 00/12507, WO 2005/085250, and WO 2005/023814, each of which is incorporated herein by reference in its entirety. PBD dimers within the scope of the present disclosure are formed from two PBD monomers connected at the C8 carbon atom of each PBD monomer.

A. Conjugates

In some embodiments, the PBD prodrug dimer-antibody conjugate has formula (I) comprising a first PBD prodrug monomer M1 and a second PBD-antibody monomer M2:

m1 is a PBD monomer, wherein the dashed line represents an optional double bond between two of: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R². In some embodiments, C₁And C₂A single bond between, C₂And C₃Is a single bond, and C2 and R²The bond between is a double bond.

R²Is selected from-H, ═ CH₂-CN, -R, ═ CHR, aryl, heteroaryl, bicyclic, and heterobicyclic. In some embodiments, R²Is ═ CH₂、

R³Is hydrogen; x is selected from S, O and NH; and R is¹¹Selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S.

R⁶、R⁷And R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH, and SR. In some embodiments, R⁶And R⁹Is H. In some embodiments, R⁷Is OCH₃。

R¹⁰Are prodrug moieties, described in more detail elsewhere herein, which include (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester.

R is selected from the group consisting of lower alkyl groups having 1 to 10 carbon atoms and arylalkyl groups of up to 12 carbon atoms, (i) aryl groups wherein the alkyl groups optionally contain one or more carbon-carbon double or triple bonds, or up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms.

M2 is a PBD monomer, wherein the dashed line represents an optional double bond between two of: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’. In some embodiments, C_1’And C_2’A single bond between, C_2’And C_3’Is a single bond, and C_2’And R^2’The bond between is a double bond.

R^2’、R^3’、R^6’、R^7’、R^9’、R^11’And X' respectively correspond to R²、R³、R⁶、R⁷、R⁹、R¹¹And X, and are defined in the same manner as them.

L is a self-immolative linker comprising at least one of a disulfide moiety, a peptide moiety, and a peptidomimetic moiety. In some embodiments, the linker comprises a disulfide moiety or a peptide moiety.

Each asterisk independently represents a racemic or undefined stereochemical chiral center.

M1 and M2 are bonded at the C8 position by a moiety-Q-T-Q '-wherein Q and Q' are independently selected from O, NH and S, and wherein T is optionally substituted C_1-12An alkylene group which is further optionally interrupted by one or more heteroatoms and/or aromatic rings. In some embodiments, Q and Q' are O, and T is C₃Alkylene or C₅An alkylene group.

The Ab is an antibody as defined elsewhere herein, in some embodiments, the antibody comprises at least one cysteine thiol moiety, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of (1) BMPR1 (bone morphogenetic protein receptor-IB type), E (LAT, SLC 7A), 3) STEAP (transmembrane epithelial antigen of prostate), 4) MUC (07125), 5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin), 6) Napi2 (NAPI-3B, TIIb, SLC34A, solute carrier family 34 (sodium phosphate) 2, II-dependent phosphate transporter 3B), 7) Sema5B (FLJ10372, KIAA1445, 15, SEMA5, SEMA, CEP, CEPS 5, CEPS, SEMA, SEMEN, SEMA, SEMEK, SEMA, SEMEN, SEMA, SEPTS, SEMEN, SEMEK, SEMA, SEMEK, SEPTS, SEMA, SES, SEMA, SEPTS, SE.

The integer p is 1,2, 3, 4,5, 6,7 or 8. In some embodiments, p is 1,2, 3, or 4. In some embodiments, a composition is provided comprising a mixture of PBD prodrug dimer-antibody conjugates, wherein the average drug loading per antibody in the mixture of conjugate compounds is about 2 to about 5.

In some embodiments, R⁷And R^7’is-OCH₃；R³、R^3’、R⁶、R^6’、R⁹And R^9’Are all H; and R is²And R^2’Is ═ CH₂、

In some embodiments, C of M1₁And C₂The bond between (A) and (B) is a single bond; c of M1₂And C₃The bond between (A) and (B) is a single bond; c of M2_1’And C_2’The bond between (A) and (B) is a single bond; c of M2_2’And C_3’The bond between (A) and (B) is a single bond; c of M1₃Is divided into two R³Is substituted by the radicals R³Each of the groups is H; c of M2_3’Is divided into two R^3’Is substituted by the radicals R^3’Each of the groups is H; c of M1₂And R²The bond between is a double bond; and C of M2_2’And R^2’The bond between is a double bond.

In some embodiments, the PBD prodrug dimer-antibody conjugate compound has formula (Ia):

wherein R is¹⁰L, p and Ab are as defined elsewhere herein.

B. Monomer

In some embodiments, the PBD monomer compound has formula (II):

wherein R is²、R³、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹The bonding schemes in, X,. and pyrrolidine ring a are as described elsewhere herein in connection with the PBD dimer.

In some embodiments, the PBD monomeric compound has formula (IIa):

wherein R is¹⁰As defined elsewhere herein.

C. Dimer

In some embodiments, the PBD prodrug dimer compound has formula (VIII) comprising a first PBD prodrug monomer M1 and a second PBD monomer M2:

wherein R is²、R^2’、R³、R^3’、R⁶、R^6’、R⁷、R^7’、R⁹、R^9’、R¹⁰、R¹¹、R^11’The bonding schemes in X, X ', Q, Q', T,. and pyrrolidine rings a and B are as described elsewhere herein in connection with the PBD dimers.

In some embodiments, R¹²Is absent and N10' and C_11’The bond between is a double bond. In some embodiments, R¹²Selected from the group consisting of-C (O) O-L and-C (O) O-R¹⁰Wherein R is¹⁰Is a prodrug moiety as described elsewhere herein. L is as defined elsewhere herein.

In some embodiments, the PBD prodrug dimer compound has formula (VIIIa):

wherein R is¹⁰As defined elsewhere herein.

In some embodiments, the PBD prodrug dimer compound has formula (VIIb):

wherein R is¹⁰And L is as defined elsewhere herein.

Prodrug protecting group-triggers

Prodrug protecting groups containing triggers within the scope of the present disclosure include disulfides, cyclic diketones, arylboronic acids, and arylboronic acid esters. The prodrug protecting group comprising the trigger is conjugated to the PBD at the N10 position through a carbamate moiety. The protecting group is cleaved enzymatically or chemically by application of a stimulus, such as an enzyme (e.g., DTD), ROS, or GSH, to generate the active drug.

A. Disulfide protecting group-triggers

Disulfide protecting group-triggers of the present disclosure¹⁰Moieties having the general formula (V):

where the wavy line indicates the point of connection to the PBD N10 location (i.e.,)。

R⁵⁰selected from optionally substituted C_1-8Alkyl or C_2-6Alkyl, optionally substituted C_1-8Or C_2-6Heteroalkyl groups, optionally substituted cycloalkyl groups containing from 2 to 6 carbon atoms, and optionally substituted heterocycloalkyl groups containing from 2 to 6 carbon atoms. In some particular embodiments, R⁵⁰Is selected from-CH₂-CH₃、-CH(CH₃)₂、-C(CH₃)₃、-CH₂-CH₂OH、-CH₂-CH₂-C(O)OH、-CH₂-CH₂-O-CH₃And 3 to 6 membered cycloalkyl or heterocycloalkyl. In some embodiments, cycloalkyl and heterocycloalkyl R⁵⁰Moieties are selected from:

in some embodiments, R⁵⁰Is selected from CH₃CH₂-、(CH₃)₂CH-and (CH)₃)₃C-。

R⁵¹Is optionally substituted C₂Alkylene or optionally substituted benzylidene. In some such embodiments, R⁵¹Having the formula:

in some embodiments, R⁶¹And R⁶²Independently selected from H and optionally substituted C_1-6Alkyl, and optionally substituted C_1-6A heteroalkyl group. In some particular embodiments, R⁶¹And R⁶²Independently selected from H and optionally substituted C_1-4Alkyl, and optionally substituted C_1-4Ethers or tertiary amines. In some other specific embodiments, R⁶¹And R⁶²Is H. In other particular embodiments, R⁶¹And R⁶²Is H and R⁶¹And R⁶²is-CH₃、C_1-4Ether or C tertiary amine. In some other specific embodiments, R⁶¹And R⁶²Each is H, or R⁶¹And R⁶²Each is CH₃。

In some embodiments, R⁶¹And R⁶²Together with the carbon atoms to which they are bonded form an optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl moiety, each ring substitution containing from 2 to 6 carbon atoms.

In some embodiments, R⁶³And R⁶⁴Independently selected from H and CH₃. In some other specific embodiments, R⁶³And R⁶⁴Is H.

Exemplary R⁵¹A non-limiting list of parts is as follows:

in some alternative embodiments, R⁵¹Is an arylalkyl group. In one such embodiment, R⁵¹Comprises the following steps:

a non-limiting list of exemplary disulfide protecting group-trigger moieties is as follows:

in accordance with the present disclosure and based on experimental evidence in vitro to date, it has been found that the disulfide protecting group-triggers of the present disclosure are cleaved intracellularly in proliferating cells (such as cancer cells) to express elevated GSH, and are generally stable in non-proliferating cells expressing normal GSH levels, as well as in whole blood/plasma. More specifically, it is known that blood concentrations of GSH are very low, such as in the micromolar range, while intracellular GSH concentrations are typically up to three orders of magnitude greater, such as in the millimolar range. It is also believed that GSH concentrations in cancer cells are even higher due to increased activity of the reductase.

It has also been found that the difference between the intracellular reduction potentials (expressed in mV) between proliferating cells and non-proliferating cells can be used to achieve disulfide trigger activation and drug release in proliferating cells while providing prodrug stability in non-proliferating cells, whole blood and plasma. More specifically, the ratio of reduced GSH to oxidized GSH (also referred to as GSH disulfide or "GSSG") in a GSH/GSSG redox pair is believed to be related to the reduction potential (typically expressed in mV). It is also believed that certain GSH/GSSG ratios are characteristic of proliferating cells. The negativity of the reduction potential increases with increasing GSH/GSSG ratio (i.e., with increasing relative concentration of GSH). Typical GSH/GSSG reduction potentials are presented in the following table:

	cytoplasm is alsoOriginal potential (mV)
		Blood/plasma	-140
Proliferating cells	-260 to-230
		Growth arrested cells	-220 to-190
Apoptotic cells	-170 to-150

The reduction potential of the cysteine (Cys) and dithiocysteine (CySS) redox pair also allows intracellular release of the drug from the disulfide prodrug, with the blood/plasma Cys/CySS reduction potential typically ranging from-80 mV to 0mV and the Cys/CySS reduction potential in the cytoplasm typically being about-160 mV.

B. Cyclic diketone protecting group-trigger

In some embodiments, R¹⁰The cyclic diketone protecting group-trigger is a1, 4-or 1, 2-quinone having the general formula:

A. d, E, G and J are independently selected from C and N, where N is a secondary amine, tertiary amine or imine (═ N-). Each m is independently selected from 0 and 1. The dotted line represents an optional double bond between E-D or D-A. When present, R^A、R^D、R^E、R^GAnd R^JIndependently selected from H, OH, optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen, with the proviso that R^A、R^D、R^E、R^GAnd R^JIs C covalently bonded to the oxygen atom of the carbamate moiety at the PBD N10 position_1-4An optionally substituted alkyl or heteroalkyl linker. In some embodiments, R^A、R^DAnd R^EOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker. In some other embodiments, A- (R) is^A)_m、D-(R^D)_mAnd E- (R)^E)_mOne of them isIn some other embodiments, A- (R) is^A)_mIs composed ofD is C and R^DIs C₁Linker, E- (R)^E)_mIs composed ofWherein the bond between D and E is a double bond. In other embodiments, G and J are C (carbon). In other embodiments, G and J are C, and R^GAnd R^JOne of them is-O-CH₃. In some embodiments, the cyclic diketone is a1, 4-cyclic diketone.

In some embodiments, the cyclic dione prodrug moiety is a quinone selected from:

wherein the hydroxyl moiety provides a point of attachment to the PBD.

In some particular embodiments, the cyclic diketone is an indole diketone having one of the following formulae:

where the wavy line indicates the point of attachment to the oxygen atom at the position of PBD N10 (i.e.,)。

in some embodiments, R¹⁰The cyclic diketone protecting group-trigger is a1, 4-or 1, 2-cyclic diketone having the formula:

A. d, E, F, G and J are independently selected from C and N, wherein at least one of A, D, E and F is C and at least one of G and J is C, and wherein N is a secondary amine, tertiary amine or imine (═ N-). Each n is independently selected from 0 and 1. The dotted line represents an optional double bond. When present, R^A、R^D、R^E、R^F、R^GAnd R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen, with the proviso that R^A、R^D、R^E、R^F、R^GAnd R^JIs present and is C covalently bonded to the oxygen atom of the carbamate moiety at position PBDN10_1-4An optionally substituted alkyl or heteroalkyl linker. In some embodiments, R^A、R^D、R^EAnd R^FOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker. In some embodiments, D-R^DOr E-R^ETo have a bond to C_1-4An optionally substituted alkyl or heteroalkyl linker, and A, F and at least one of the other of D and E is N. In some embodiments, R^A、R^EAnd R^FIs independently selected from H, optionally substituted C_1-4Alkyl or heteroalkyl and optionally substituted C_1-4Alkoxy or heteroalkoxy, and wherein D is C and R^DIs C₁And (4) a joint. In some other embodiments, G- (R)^G)_nAnd J- (R)^J)_nAt least one of which isIn some other embodiments, G-R^GAnd J-R^JOne of them is C-O-CH₃，G-R^GAnd J-R^JIs CH, wherein the bond between G and J is a double bond. In some other embodiments, the ring formed by A, D, E and F is unsaturated or partially saturated. In other embodiments, the ring formed by A, B, D and E is unsaturated or partially saturated.

Prodrugs with cyclic diketone protecting group-triggers can be activated with DTD two-electron reductases, which are believed to be overexpressed in endothelial cells of many human tumors and blood vessels. DTD is NAD (P) H quinone oxidoreductase type I (NQO1) enzyme (EC1.6.99.2) that catalyzes the direct two-electron transfer of quinones using NADH or NADPH as a cofactor (see, e.g., Mendoza et al, "Human NAD (P) H: quinone oxido reductase type I (HNQO1) activation of quinone propionic acid trigger groups", Biochemistry, 10.9.2012; 51(40):8014-8026, incorporated herein by reference). DTD is believed to be a prodrug activator under both aerobic and anoxic conditions.

Human breast and lung cancers are known to express high levels of DTD. For example, NQO1 expression in nRPKM (where nRPKM refers to a normalized reading per Kb transcript length per million plotted reads) is about 30 to 2000, compared to hematologic and lymphatic cancers that typically have nRPKM values in the range of about 0.5 to about 20. As disclosed in the table below, DTD is overexpressed in many cancers relative to normal tissue (see s. danson et al, "DT-diaphorase: atarget for new anticancer drugs", Cancer Treatment Reviews (2004)30,437-449), where "NS" means insignificant:

cells	DTD ratio to Normal tissue
		Primary of human colon cancer	2.5 to 3.9
Metastatic property of human colon cancer	47
		Human breast cancer	NS to 9.5
Human NSCLC	8.2 to 19.2
		Human liver cancer	3.8 to 50

Without being bound by any particular theory, it is believed that the intracellular prodrug release mechanism generally proceeds according to the following mechanism, as represented by a quinone species, where prodrug quinone trigger activation and drug release are mediated by DTD two-electron reduction:

C. arylboronic acids and arylboronic acid ester protecting group-triggers

Arylboronic acids and arylboronic acid ester protecting group-triggers R of the present disclosure¹⁰Moieties have the general formula (IVa):

wherein the wavy line indicates the point of attachment to the oxygen atom at the PBD N10 position (i.e., -O-C (O) -N)₁₀<PBD)。R²⁰And R²¹Independently selected from H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, and optionally substituted aryl or heteroaryl. Alternatively, R²⁰And R²¹Together are an optionally substituted moiety- (CH)₂)_n-, wherein n is 2 or 3, said moieties together with the O atom to which they are attached together with the B atom forming a heterocycloalkyl ring. The heterocycloalkyl ring can optionally include a fused heteroalkyl ring, a fused aryl ring, or a fused heteroaryl ring. The wavy line indicates the point of connection to the position of PBD N10.

In some embodiments, the arylboronic acid and arylboronic ester triggers have the formula:

R³⁰、R³¹、R³²、R³³、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴and R⁴⁵Independently selected from H, halogen, -CN, -OH, -NH₂、-COOH、-CONH₂、-NO₂、-SH、-SO₂Cl、-SO₃H、-SO₄H、-SO₂NH₂、-NHNH₂、-ONH₂、-NHC＝(O)NHNH₂Optionally substituted C_1-8An alkyl or heteroalkyl group, an optionally substituted cycloalkyl or heterocycloalkyl group containing from 2 to 7 carbon atoms, an optionally substituted aryl or heteroaryl group. In some embodiments, (i) R³⁰Or R³¹And R³²Or R³³(iii) R⁴⁰Or R⁴¹And R⁴²Or R⁴³And/or (iii) R⁴²Or R⁴³And R⁴⁴Or R⁴⁵Form an optionally substituted fused ring having 2 to 7 carbon atomsAn alkyl ring, a fused heterocycloalkyl ring, a fused aryl ring, or a fused heteroaryl ring. The wavy line indicates the point of connection to the position of PBD N10.

A non-limiting list of exemplary arylboronic acid and arylboronic ester protecting group-triggers is as follows:

in one embodiment, the protecting group-trigger is an arylboronic acid, i.e., wherein R is²⁰And R²¹Is H.

Prodrugs with arylboronic acid or arylboronic acid ester protecting group-triggers can be treated with ROS (such as H)₂O₂) And (4) activating. (see, e.g., Kuang, Y. et al, "Hydrogen Peroxide indicator DNA Cross-Linking Agents: Targeted Anticancer reagents", J.Am. chem. Soc. (2011),133(48), 19278-19281; Peng, X. et al, "ROS-activated antagonist reagents: a new strand for molecular dam", the r Deliv. (2012),3(7), 823-833; and Chen, W. et al, "Reactive Oxygen Species (ROS) indicator DNA Cross-Linking Agents and the ideal effect on Cancer Cells and the Normal Cells", J.4557, 20148, each of which is incorporated herein by reference in its entirety)

Cancer cells are thought to exhibit increased oxidative stress as compared to normal, non-cancerous cells, and are thought to have increased ROS (such as H)₂O₂) Cell concentration of wherein H₂O₂The concentration can be increased 10-fold in cancer cells, such as up to 0.5nmol/10⁴One cell/h. (see, e.g., Peng; Chen; Zieba, M. et al, "company of Hydrogen generation and the content of lipid oxidation products in Lung Cancer and plasma Research", Respiratory Medicine (2000),94, 800. 805; Szatowski, T. et al, "Production of Large animals of Hydrogen Peroxide in human turbine cell", Cancer Research (1991),51, 794. 798, whereEach of which is incorporated herein by reference. ) It is believed that the high ROS concentration in cancer cells and the concomitant ROS signaling become major factors in tumor formation, development, proliferation and survival through DNA mutation, metastasis, angiogenesis and reduced sensitivity to therapeutic agents (see, e.g., Peng).

Without being bound by any particular theory, it is believed that the ROS-activated intracellular prodrug release mechanism proceeds according to the following mechanism:

IV. joint

The linkers of the present disclosure are bifunctional chemical moieties capable of covalently linking an antibody and a drug ("D") together as a triple molecule. Linkers within the scope of the present disclosure are not strictly limited and have the general structure:

ab- [ Ab connecting part]- [ Ab spacer group]_{Optionally, the}- [ trigger)]_{Optionally, the}- [ D spacer group]_{Optionally, the}- [ D connecting part]And comprises an Ab linker, an optional Ab spacer unit, an optional self-immolative (trigger) unit, and an optional drug spacer, and a drug linker.

In some embodiments, the linker comprises a self-immolative moiety (trigger). Non-limiting examples of self-immolative moieties within the scope of the present disclosure include peptides, peptidomimetics, and disulfides.

In some embodiments, the linker includes a self-immolative peptide unit that allows enzymatic cleavage of the linker, such as by a protease, to facilitate release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al (2003) nat. Biotechnol.21:778- > 784). Exemplary peptide units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), valine-alanine (va or val-ala), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly). The peptide units may comprise naturally occurring amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogues, such as citrulline. The peptide units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsin B, C and D, or plasmin protease.

In some embodiments, the linker comprises a self-immolative peptidomimetic unit that allows cleavage of the linker. Exemplary peptidomimetic units include, but are not limited to, triazoles, cyclobutane-1-1-dicarboxaldehyde-citrulline, olefins, halogenated olefins, and isoxazoles. Some examples of peptidomimetic units include the following, where the wavy line to the left of the peptidomimetic unit is the point of attachment to a spacer or an antibody linking moiety and the wavy line to the right of the peptidomimetic unit is the point of attachment to a spacer or a drug linking moiety:

some examples of Ab- [ peptidomimetic linker units ] -drug groups within the scope of the present disclosure are as follows, wherein "AA" refers to amino acids, wherein AA1 and AA2 may be the same, or different, naturally occurring or non-naturally occurring amino acids:

in some embodiments, the linker comprises a self-cleaving disulfide unit that allows the linker to be cleaved. The disulfide linker typically has the formula:

wherein: s_CIs an antibody cysteine sulfur atom; r⁷⁰And R⁷¹Independently selected from H and C_1-3Alkyl radical, wherein R⁷⁰And R⁷¹Only one of which may be H, or R⁷⁰And R⁷¹Together with the carbon atom to which they are bonded form a four-to six-membered ring optionally containing oxygen heteroatoms; and, the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety at the position of PBD N10. In some other embodiments, R⁷⁰And R⁷¹Independently selected from H, -CH₃and-CH₂CH₃Wherein R is⁷⁰And R⁷¹Only one of which may be H, or R⁷⁰And R⁷¹Together with the carbon atom to which they are bonded, form a ring selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuran and tetrahydropyran.

In some embodiments, the linker may comprise a spacer unit. In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In some such embodiments, a p-aminobenzyl alcohol spacer unit is linked to an amino acid unit through an amide bond, and a carbamate, methylcarbamate, or carbonate linkage is formed between the benzyl alcohol and the drug (Hamann et al (2005) Expert opin. ther. patents (2005)15: 1087-. In other embodiments, the linker-antibody moiety is attached to the oxygen atom of the carbamate moiety at position PBD N10 as follows:

in some other embodiments, spacers include, but are not limited to, aromatic compounds that are electronically similar to PAB groups, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al (1999) bioorg.Med.chem.Lett.9:2237) and ortho-or para-aminobenzyl acetals.

In some embodiments, spacers which undergo cyclization upon hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutanoic acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo [2.2.1] and bicyclo [2.2.2] ring systems (Storm et al (1972) J.Amer.chem.Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al (1990) J.org.chem.55: 5867). the linkage of the drug to the α -carbon of the glycine residue is another example of a spacer which may be useful (Kingsbury et al (1984) J.Med.chem.27: 1447). in some aspects, the spacer unit is self-immolative.

The linker comprises a reactive group suitable for covalent conjugation to an antibody. In some embodiments, the antibody comprises at least one reactive thiol moiety and the linker comprises a reactive sulfur atom, maleimide, bromoacetamide, and iodoacetamide, or an alkene, wherein the antibody is conjugated to the linker by a covalent bond formed by reaction of the antibody reactive thiol with the linker reactive sulfur atom, maleimide, bromoacetamide, iodoacetamide, or alkene, according to methods known to those skilled in the art.

Non-limiting examples of protocols for conjugating an antibody having a reactive thiol moiety to a drug-linker moiety are shown below:

examples of some specific drug- [ L ] conjugates are as follows, wherein x is 1 to 8 and wherein [ conjugated ] refers to a reactive group as described elsewhere herein:

in embodiments comprising an autodisintegrable disulfide, conjugation to Ab may be accomplished according to the methods described in application No. PCT/CN2015/092084, which is incorporated herein by reference in its entirety. In general, an activated leaving group-disulfide-drug compound having the formula:

contacting an antibody having at least one sulfhydryl moiety with a leaving group (X)_L) Is replaced and the sulfur atom is covalently bonded to a mercapto sulfur atom to form a disulfide. In the above formula: x_LIs a thiol leaving group; the leaving group and the linker are bonded by a disulfide bond; r⁷⁰And R⁷¹As defined elsewhere herein; and, Sp is an optional spacer as described elsewhere herein, wherein n is 0 or 1. The joint may be considered a hindered joint because R⁷⁰And R⁷¹Only one of which may be H.

In some embodiments, the leaving group may suitably be selected from the following:

in such embodiments, the wavy line indicates the point of attachment of the leaving group to the S atom of the hindered linker, thereby forming a disulfide bond. X¹、X²、X³、X⁴And X⁵Independently C, N, S or O, provided that X¹To X⁵Is N, the dotted line represents an optional double bond, and a represents a six-membered ring. Y is¹、Y²、Y³And Y⁴Independently C, N, S or O, provided that Y¹To Y⁴Is N, the dotted line represents an optional double bond, and B represents a five-membered ring. Z¹、Z²、Z³、Z⁴、Z⁵And Z⁶Independently C, N, S or O, with the proviso that Z¹And Z²Is N, the dotted line represents an optional double bond, C represents a six-membered ring, and D represents a fused five-membered ring. Each R³Independently selectself-NO₂、-NH₂、-C(O)OH、R⁵S(O)(O)-、-C(O)N(R⁵)(R⁵) -Cl, -F, -CN and-Br. Each R⁵Independently selected from H, optionally substituted C_1-6Hydrocarbyl, optionally substituted C_5-6Carbocycle, and optionally substituted C_5-6And q is 1,2 or 3. Each carbon atom in the ring structure of leaving group 1, leaving group 2, leaving group 3 and/or leaving group 4 is replaced by R⁵Optionally substituted. Each nitrogen atom in the ring structure of leaving group 1, leaving group 2, leaving group 3 and/or leaving group 4 is replaced by R⁵Optionally substituted to form a tertiary or quaternary amine.

In some particular such embodiments, X¹、X²、X³、X⁴And X⁵Independently is C or N, X¹To X⁵No more than two of which are N, and ring a is unsaturated. In other particular embodiments, Y¹、Y²Y³And Y⁴Independently is C or N, and the B ring is unsaturated. In other particular embodiments, Z¹Is N, Z²Selected from N, S and O, Z³To Z⁶Selected from C and N, Z³To Z⁶No more than two of which are N, and ring C is unsaturated. In other particular embodiments, each R is³Independently selected from-NO₂、-NH₂、-C(O)OH、H₃CS (O) and-C (O) N (CH)₃)₂。

In some embodiments, the leaving group isWherein the wavy line indicates the point of attachment of the leaving group to the S atom of the hindered linker, thereby forming a disulfide bond. In some particular embodiments, C_1-4The alkyl group is a methyl group.

Some examples of leaving groups of the present disclosure are exemplified below:

examples of hindered disulfide linkers are as follows, wherein the wavy line at the sulfur atom refers to the point of attachment to a leaving group as defined elsewhere herein, and wherein the wavy line at the carbonyl moiety refers to the point of attachment to the PBD N10 atom:

v. antibody

The antibodies of the present disclosure are any cell-targeted biological compound that binds to one or more tumor-associated antigens or cell surface receptors, the antibody comprising at least one reactive cysteine thiol moiety suitable for conjugation to a linker.

Certain types of cells, such as cancer cells, express surface molecules (antigens) that are unique compared to the surrounding tissue. Cell targeting moieties bound to these surface molecules are capable of specifically targeted delivery of the drugs described elsewhere herein to target cells. For example and without limitation, the cell targeting moiety can bind to and be internalized by a lung, breast, brain, prostate, spleen, pancreas, cervix, ovary, head and neck, esophagus, liver, skin, kidney, leukemia, bone, testis, colon, or bladder cell.

A. Tumor associated antigens

In some particular embodiments of the present disclosure, the target cell is a cancer cell that expresses a Tumor Associated Antigen (TAA) or comprises a cell surface receptor. Tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art. In order to find effective cellular targets for cancer diagnosis and treatment, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal, non-cancerous cells. Typically, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cell than on the surface of the non-cancerous cell. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target cancer cells for destruction by antibody-based therapies.

Examples of tumor associated antigens TAAs include, but are not limited to, TAAs (1) - (53) listed herein. For convenience, information relating to these antigens (all of which are known in the art) is set forth herein and includes names, alias names, Genbank accession numbers, and one or more primary references, which follow the nucleic acid and protein sequence identification convention of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to TAAs (1) - (53) are available in public databases such as GenBank. Tumor associated antigens targeted by antibodies include all amino acid sequence variants and isoforms having at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to sequences identified in the cited references, or exhibiting substantially the same biological properties or characteristics as TAAs having the sequences found in the cited references. For example, TAAs having variant sequences are generally capable of specifically binding to antibodies that specifically bind to TAAs having the corresponding sequences listed. The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession No. NM-001203) ten Dijke, P. et al Science 264(5155): 101-; WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1 (pages 38-39); WO2002102235 (claim 13; page 296); WO2003055443 (pages 91-92); WO200299122 (example 2; page 528 and 530); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; page 183); WO200254940 (page 100-101); WO200259377 (page 349 and 350); WO200230268 (claim 27; page 376); WO200148204 (example; fig. 4) NP _001194 bone morphogenic protein receptor, type IB/pid — NP _ 001194.1-cross-reference: MIM: 603248, respectively; NP-001194.1; AY 065994.

(2) E16(LAT1, SLC7A5, Genbank accession No. NM-003486) biochem Biophys Res Commun.255(2), 283-; WO2004048938 (example 2); WO2004032842 (example IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (example 2); WO200299074 (claim 19; page 127 and 129); WO200286443 (claim 27; page 222, 393); WO2003003906 (claim 10; page 293); WO200264798 (claim 33; pages 93-95); WO200014228 (claim 5; page 133 and 136); US2003224454 (fig. 3); WO2003025138 (claim 12; page 150); NP _003477 solute carrier family 7 (cationic amino acid transporter, y + system), member 5/pid NP _ 003477.3-Homo sapiens (Homo sapiens) cross-reference: MIM: 600182, respectively; NP-003477.3; NM-015923; NM _003486_ 1.

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession No. NM-012449) cancer Res.61(15),5857-5860(2001), Hubert, R.S. et al (1999) Proc. Natl.Acad.Sci.U.S.A.96(25): 14523-14528); WO2004065577 (claim 6); WO2004027049 (fig. 1L); EP1394274 (example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (example 5); US2003185830 (example 5); US2003064397 (fig. 2); WO200289747 (example 5; page 618 and 619); WO2003022995 (example 9; FIG. 13A, example 53; page 173, example 2; FIG. 2A); NP _036581 six transmembrane epithelial antigens of the prostate were cross-referenced: MIM: 604415, respectively; NP-036581.1; NM _012449_ 1.

(4)0772P (CA125, MUC16, Genbank accession No. AF361486) J.biol.chem.276(29): 27371-; WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; page 116-121); US2003124140 (example 16); US 798959. Cross-referencing: GI: 34501467, respectively; AAK 74120.3; AF361486_ 1.

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin, Genbank accession No. NM-005823) Yamaguchi, N. et al biol. chem.269(2),805-808(1994), Proc. Natl. Acad. Sci. U.S.A.96(20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A.93(1):136-140(1996), J.biol. chem.270(37):21984-21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; pp 287-288); WO2002101075 (claim 4; pp 308-309); WO200271928 (pp 320-321); WO9410312 (pp 52-57); cross references MIM: 601051; NP-005814.2; NM-005823-1.

(6) Napi3B (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3B, Genbank accession No. NM-006424) J.biol.chem.277(22): 19665-; WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17 to 19); WO200157188 (claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; page 139-140); cross-referencing: MIM: 604217, respectively; NP-006415.1; NM _006424_ 1.

(7) Sema5B (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5B Hlog, Sema domain, 7 thrombospondin repeats (type 1 and type 1), transmembrane domain (TM) and brachlocytic domain (semaphorin)5B, Genbank accession AB040878) Nagase T. et al (2000) DNA Res.7(2): 143-; WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; page 50); WO200188133 (claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); accession number: Q9P 283; EMBL; AB 040878; BAA 95969.1. Genew; HGNC: 10737.

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession No. AY 358628); ross et al (2002) Cancer Res.62: 2546-; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (example 5); WO2003105758 (claim 12); US2003206918 (example 5); EP1347046 (claim 1); WO2003025148 (claim 20); cross-referencing: GI: 37182378, respectively; AAQ 88991.1; AY358628_ 1.

(9) ETBR (endothelin type B receptor, Genbank accession No. AY 275463); nakamuta m. et al biochem. biophysis. res. commun.177,34-39,1991; ogawa y, et al biochem, biophysis, res, commun.178,248-255,1991; arai H. et al Jpn.Circ.J.56,1303-1307,1992; arai h. et al j.biol.chem.268,3463-3470,1993; sakamoto a., Yanagisawa m., et al biochem, biophysis, res, commun.178,656-663,1991; elshourbagy n.a. et al j.biol.chem.268,3873-3879,1993; haendler B.et al J.Cardiovasc.Pharmacol.20, S1-S4,1992; tsutsumi M. et al Gene 228,43-49,1999; straussberg r.l. et al proc.natl.acad.sci.u.s.a.99,16899-16903,2002; bourgeois c, et al j.clin.endocrinol.meta.82, 3116-3123,1997; okamoto Y, et al, biol.chem.272,21589-21596,1997; verheij j j.b. et al am.j.med.genet.108,223-225,2002; hofstra r.m.w. et al eur.j.hum.genet.5,180-185,1997; puffenberger E.G. et al Cell 79,1257-1266, 1994; attie t. et al, hum.mol.genet.4,2407-2409,1995; auricchio A. et al hum. mol. Genet.5: 351-; amiel J, et al hum.mol.Genet.5,355-357,1996; hofstra r.m.w. et al nat. genet.12,445-447,1996; svensson p.j. et al hum.genet.103,145-148,1998; FuchsS. et al mol.Med.7,115-124,2001; pingault V. et al (2002) hum. Genet.111, 198-206; WO2004045516 (claim 1); WO2004048938 (example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (fig. 1); WO2003016494 (fig. 6); WO2003025138 (claim 12; page 144); WO200198351 (claim 1; page 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; page 297-299); US 2003109676; US6518404 (fig. 3); US5773223 (claim 1 a; columns 31-34); WO 2004001004.

(10) MSG783(RNF124, putative protein FLJ20315, Genbank accession No. NM — 017763); WO2003104275 (claim 1); WO2004046342 (example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (example 6); cross-referencing: locus ID: 54894; NP-060233.2; NM _017763_ 1.

(11) STEAP2(HGNC _8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession AF455138) Lab. invest.82(11):1573-1582 (2002)); WO 2003087306; US2003064397 (claim 1; FIG. 1); WO200272596 (claim 13; pages 54 to 55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; fig. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; figure 10); cross-referencing: GI: 22655488, respectively; AAN 04080.1; AF455138_ 1.

(12) TrpM4(BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channels, subfamily M member 4, Genbank accession No. NM-017636) Xu, X.Z. et al Proc. Natl. Acad. Sci. U.S.A.98(19): 10692-; US2003143557 (claim 4); WO200040614 (claim 14; page 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268 (claim 27; page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIGS. 1A-D); cross-referencing: MIM: 606936, respectively; NP-060106.2; NM _017636_ 1.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factors, Genbank accession NP-003203 or NM-003212) Ciccodiocola, A. et al EMBO J.8(7):1987-1991(1989), am. J.hum. Genet.49(3):555-565 (1991)); US2003224411 (claim 1); WO2003083041 (example 1); WO2003034984 (claim 12); WO200288170 (claim 2; pages 52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; pages 94-95, 105); WO200222808 (claim 2; FIG. 1); US5854399 (example 2; columns 17-18); US5792616 (fig. 2); cross-referencing: MIM: 187395, respectively; NP-003203.1; NM _003212_ 1.

(14) CD21(CR2 (complement receptor 2) or C3DR (C3d/EB virus receptor) or Hs.73792, Genbank accession number M26004) Fujisaku et al (1989) J.biol.chem.264(4): 2118. 2125); weis J.J. et al J.exp.Med.167,1047-1066,1988; moore M. et al Proc.Natl.Acad.Sci.U.S.A.84,9194-9198,1987; barelm, et al mol.Immunol.35,1025-1031,1998; weis j.j. et al proc.natl.acad.sci.u.s.a.83,5639-5643,1986; sinha s.k. et al (1993) j.immunol.150, 5311-5320; WO2004045520 (example 4); US2004005538 (example 1); WO2003062401 (claim 9); WO2004045520 (example 4); WO9102536 (fig. 9.1-9.9); WO2004020595 (claim 1); accession number: p20023; q13866; q14212; EMBL; m26004; AAA 35786.1.

(15) CD79 56567 (CD79B, CD79 β, IGb (immunoglobulin related β), B29, Genbank accession No. NM _000626 or 11038674) Proc. Natl. Acad. Sci. U.S.A. (2003)100(7) 4126-4131, Blood (2002)100(9) 3068-3076, Muller et al (1992) Eur.J.Immunol.22(6):1621-1625), WO2004016225 (claim 2, FIG. 140), WO2003087768, US 2001874104 (claim 1, page 102), WO2003062401 (claim 9), WO200278524 (example 2), US2002150573 (claim 5, page 15), US5644033, WO 20030482482 (claim 1, pages 306 and 309), WO 20027855, US 65351, NM 3448313, NM-4611-NP 35; WO 200055-11435; WO 20030424, WO 34387).

(16) FcRH2(IFGP4, IRTA4, SPAP1A (phosphatase-anchored protein 1a containing SH2 domain), SPAP1B, SPAP1C, Genbank accession No. NM-030764, AY358130) Genome Res.13(10): 2265-; WO2004016225 (claim 2); WO 2003077836; WO200138490 (claim 5; FIGS. 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25); cross-referencing: MIM: 606509, respectively; NP-110391.2; NM _030764_ 1.

(17) HER2(ErbB2, Genbank accession number M11730) Coissens L. et al Science (1985)230(4730): 1132-1139); yamamoto T. et al Nature 319,230-234, 1986; semba k, et al proc.natl.acad.sci.u.s.a.82,6497-6501,1985; swiercz J.M. et al J.cell biol.165,869-880,2004; kuhns j.j. et al j.biol.chem.274,36422-36427,1999; cho H. -S. et al Nature 421,756-760, 2003; ehsani A. et al (1993) Genomics 15, 426-429; WO2004048938 (example 2); WO2004027049 (fig. 1I); WO 2004009622; WO 2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US 2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIGS. 1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (example 13; pages 95 to 107); WO200212341 (claim 68; FIG. 7); WO200213847 (pages 71-74); WO200214503 (page 114-; WO200153463 (claim 2; pages 41-46); WO200141787 (page 15); WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); US5869445 (claim 3; column 31-38); WO9630514 (claim 2; pages 56 to 61); EP1439393 (claim 7); WO2004043361 (claim 7); WO 2004022709; WO200100244 (example 3; FIG. 4); accession number: p04626; EMBL; m11767; aaa35808.1. embl; m11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession number M18728); barnett T. et al Genomics 3,59-66,1988; tawaragi Y, et al biochem. Biophys. Res. Commun.150,89-96,1988; strausberg R.L. et al Proc.Natl.Acad.Sci.U.S.A.99: 16899-169903, 2002; WO 2004063709; EP1439393 (claim 7); WO2004044178 (example 4); WO 2004031238; WO2003042661 (claim 12); WO200278524 (example 2); WO200286443 (claim 27; page 427); WO200260317 (claim 2); accession number: p40199; q14920; EMBL; m29541; aaa59915.1. embl; and M18728.

(19) MDP (DPEP1, Genbank accession BC017023) Proc. Natl. Acad. Sci. U.S.A.99(26): 16899-169903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 (fig. 6-8); WO9946284 (fig. 9); cross-referencing: MIM: 179780, respectively; AAH 17023.1; BC017023_ 1.

(20) IL20R α (IL20Ra, ZCYTOR7, Genbank accession AF184971), Clark H.F. et al, GenomeRes.13,2265-2270,2003, Mungall A.J. et al, Nature 425,805-811,2003, Blumberg H. et al, Cell 104,9-19,2001, Dumoutier L. et al, J.munol.167, 19,2001-3549,2001, Parrish-NovakJ. et al, J.biol.Chem.277,47517-47523,2002, Pletnev S. et al (2003) Biochemistry 42:12617-12624, Sheikh F. et al (2004) J.munol.172, 2006-2010, EP1394274 (example 11), US 2005320 (example 5), WO 20030262 (page 74-302193), WO 2009259-WO 20040059, WO 2003759, WO 2003757-3759, WO 20040055, WO 20014659-3759, WO 2003757-3659, WO 20040055, WO 2003757-3659, WO 2003759, WO 2003757-3659, WO 3, WO 20040055, WO 3-3757, WO 3-3759, WO 3, WO.

(21) Short proteoglycans (BCAN, BEHAB, Genbank accession No. AF229053) Gary S.C. et al Gene256,139-147,2000; clark H.F. et al Genome Res.13,2265-2270,2003; straussberg r.l. et al proc.natl.acad.sci.u.s.a.99,16899-16903,2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1).

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession NM-004442) Chan, J. and Watt, V.M., Oncogene 6(6), 1057-; WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (page 128-132); WO200053216 (claim 1; page 42); cross-referencing: MIM: 600997, respectively; NP-004433.2; NM _004442_ 1.

(23) ASLG659(B7h, Genbank accession number AX092328) US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (fig. 3); US2003165504 (claim 1); US2003124140 (example 2); US2003065143 (fig. 60); WO2002102235 (claim 13; page 299); US2003091580 (example 2); WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7 b); WO200202624 (claim 13; FIGS. 1A-1B); US2002034749 (claim 54; pages 45 to 46); WO200206317 (example 2; page 320-321, claim 34; page 321-322); WO200271928 (page 468 and 469); WO200202587 (example 1; FIG. 1); WO200140269 (example 3; page 190-192); WO200036107 (example 2; page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-; WO 0116318.

(24) PSCA (prostate stem cell antigen precursor, Genbank accession No. AJ297436) Reiter r.e. et al proc.natl.acad.sci.u.s.a.95,1735-1740,1998; gu Z, et al Oncogene 19,1288-1296, 2000; biochem, biophysis, res, commun, (2000)275(3) 783-788; WO 2004022709; EP1394274 (example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO2003003906 (claim 10; page 288); WO200140309 (example 1; FIG. 17); US2001055751 (example 1; FIG. 1 b); WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; FIG. 1B); accession number: o43653; EMBL; AF 043498; AAC 39607.1.

(25) GEDA (Genbank accession number AY 260763); AAP14954 lipoma HMGIC fusion partner-proteid/pid ═ AAP 14954.1-homo sapiens species: homo sapiens WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (example 3, claim 20); US2003194704 (claim 45); cross-referencing: GI: 30102449, respectively; AAP 14954.1; AY260763_ 1.

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF 116456); BAFF receptor/pid NP _ 443177.1-Thompson homo, j.s. et al Science 293(5537),2108-2111 (2001); WO 2004058309; WO 2004011611; WO2003045422 (examples; pages 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; page 615 and 616); WO200294852 (column 136 and 137); WO200238766 (claim 3; page 133); WO200224909 (example 3; FIG. 3); cross-referencing: MIM: 606269, respectively; NP-443177.1; NM _052945_ 1; AF 132600.

(27) CD22(B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK 026467); wilson et al (1991) J.Exp.Med.173: 137-146; WO2003072036 (claim 1; FIG. 1); cross-referencing: MIM: 107266, respectively; NP-001762.1; NM _001771_ 1.

(28) CD79a (CD79A, CD79 α, immunoglobulin-related α), a B cell-specific protein which interacts covalently with Ig β (CD79B) and forms complexes with Ig M molecules on the surface, transducing signals involved in B cell differentiation, pI: 4.84, MW: 25028, TM: 2[ P ] gene chromosome 19q13.2, Genbank accession No. NP-001774.10), WO2003088808, US20030228319, WO2003062401 (claim 9), US2002150573 (claim 4, pages 13-14), WO9958658 (claim 13, FIG. 16), WO9207574 (FIG. 1), US 565633, Ha et al (1992) J.Immunol.148(5): 1526-1531; Mueller et al (1992) Eur.J.Biochem.22:1 1625; Hashito et al (19835) J.35; Psuiman 3462. 19851-57) 19835, 19851-52, 19835, 19851, 19835, 19851, 153, and 3435, 19835, 19851, 19835, and 3435, 19835, immunol, 19835.

(29) CXCR5 (burkitt lymphoma receptor 1, a G protein-coupled receptor, activated by CXCL13 chemokines, plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and may play a role in the development of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI: 8.54, MW: 41959, TM: 7[ P ] Gene chromosome: 11q23.3, Genbank accession No. NP _001707.1) WO 2004040000; WO 2004015426; US2003105292 (example 2); US6555339 (example 2); WO200261087 (fig. 1); WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (example 1, page 152-153, example 2, page 254-256); WO9928468 (claim 1, page 38); US5440021 (example 2, columns 49-52); WO9428931 (pages 56 to 58); WO9217497 (claim 7, fig. 5); dobner et al (1992) Eur.J.Immunol.22: 2795-2799; barella et al (1995) biochem.J.309: 773-779.

(30) HLA-DOB (β subunit of MHC class II molecules (Ia antigen) which binds peptides and presents them to CD4+ T lymphocytes), 273aa, pI: 6.56, MW: 30820, TM: 1[ P ] gene chromosome 6P21.3, Genbank accession No. NP 002111.1) Tonnelle et al (1985) EMBO J.4(11) 2839. multidot. 2847; Jonsson et al (1989) Immunogenetics 29(6) 411. multidot. 413; Beck et al (1992) J.mol.228: 433. multidot. 441; Strausberg et al (2002) Proc. Natl.Acad.Sci. USA99: 16899. multidot. 169903; 1987) J.biol.chem.262: 8759; Beausberg et al (2002) J.141255. multidot. 35. multidot. 19835; 19835) WO 35. multidot. 19835; 19835: 19835; 19835: 19835; 1989) Biogene # WO 35: 1989).

(31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and defects may contribute to pathophysiological conditions of idiopathic detrusor instability); 422aa), pI: 7.63, MW: 47206, TM: 1[ P ] Gene chromosome: 17p13.3, Genbank accession NP-002552.2) Le et al (1997) FEBS Lett.418(1-2): 195-; WO 2004047749; WO2003072035 (claim 10); touchman et al (2000) Genome Res.10: 165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82).

(32) CD72(B cell differentiation antigens CD72, Lyb-2) prion sequence full length maease. 8.66, MW: 40225, TM: 1[ P ] Gene chromosome: 9p13.3, Genbank accession No. NP _001773.1) WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (page 105-106); von Hoegen et al (1990) J.Immunol.144(12): 4870-4877; strausberg et al (2002) Proc. Natl. Acad. Sci USA99: 16899-.

(33) LY64 (lymphocyte antigen 64(RP105), type I membrane protein of the Leucine Rich Repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147, TM: 1[ P ] Gene chromosome: 5q12, Genbank accession No. NP _005573.1) US 2002193567; WO9707198 (claim 11, pages 39-42); miura et al (1996) Genomics 38(3) 299-304; miura et al (1998) Blood 92: 2815-2822; WO 2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24 to 26).

(34) FcRH1(Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing the C2-type Ig-like and ITAM domains, likely to play a role in B lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925, TM: 1[ P ] Gene chromosome: 1q21-1q22, Genbank accession No. NP _443170.1) WO 2003077836; WO200138490 (claim 6, fig. 18E-1-18-E-2); davis et al (2001) Proc. Natl. Acad. Sci USA 98(17) 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7).

(35) IRTA2 (putative immunoreceptors with potential role in B cell development and lymphomata, with receptor translocation related 2 of the immunoglobulin superfamily; gene dysregulation due to translocation occurs in some B cell malignancies); 977aa, pI: 6.88, MW: 106468, TM: 1[ P ] Gene chromosome: 1q21, Genbank accession No.: AF343662, AF343663, AF343664, AF343665, AF369794, AF 39453, AK090423, AK090475, AL834187, AY 358085; mice: AK089756, AY158090, AY 506558; NP _ 112571.1. WO2003024392 (claim 2, fig. 97); nakayama et al (2000) biochem. Biophys. Res. Commun.277(1): 124-127; WO 2003077836; WO200138490 (claim 3, fig. 18B-1-18B-2).

(36) TENB2(TMEFF2, tomorgulin, TPEF, HPP1, TR, putative transmembrane proteoglycans associated with growth factors of the EGF/heregulin family and follistatin); 374aa, NCBI accession number: AAD55776, AAF91397, AAG49451, NCBI RefSeq NP-057276; NCBI gene: 23671, respectively; OMIM: 605734, respectively; SwissProt Q9UIK 5; genbank accession No. AF 179274; AY358907, CAF85723, CQ782436 WO2004074320(SEQ ID NO 810); JP2004113151(SEQ ID NO2, 4, 8); WO2003042661(SEQ ID NO 580); WO2003009814(SEQ id no 411); EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304(SEQ ID NO 2706); US 2004249130; US 2004022727; WO 2004063355; US 2004197325; US 2003232350; US 2004005563; US 2003124579; horie et al (2000) Genomics 67: 146-; uchida et al (1999) biochem. Biophys. Res. Commun.266: 593-602; liang et al (2000) Cancer Res.60: 4907-12; Glynne-Jones et al (2001) Int J cancer.10 month 15; 94(2):178-84.

(37) PMEL17 (silver homolog; SILV; D12S 53E; PMEL 17; SI; SIL); ME 20; gp100) BC 001414; BT 007202; m32295; m77348; NM-006928; McGlinchey, r.p. et al (2009) proc.natl.acad.sci.u.s.a.106(33), 13731-; kummer, M.P. et al (2009) J.biol.chem.284(4), 2296-.

(38) TMEF 1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1); h7365; c9orf 2; c9ORF 2; u19878; x83961; NM-080655; NM-003692; harms, P.W, (2003) GenesDev.17(21), 2624-2629; gery, S. et al (2003) Oncogene 22(18): 2723-2727.

(39) GDNF-Ra1(GDNF family receptor α 1; GFRA 1; GDNFR; GDNFRA; RETL 1; TRNR 1; RET 1L; GDNFR- α 1; GFR- α -1), U95847; BC 014962; NM-145793 NM-005264; Kim, M.H. et al (2009) mol.cell.biol.29(8), 2264-2277; Treanor, J.et al (1996) Nature 382(6586): 80-83).

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); NP-002337.1; NM-002346.2; de Nooij-van Dalen, A.G. et al (2003) int.J. cancer 103(6), 768-; zammit, D.J. et al (2002) mol.cell.biol.22(3): 946-952.

(41) TMEM46(SHISA homolog 2 (African toads); SHISA 2); NP-001007539.1; NM-001007538.1; furushima, K. et al (2007) Dev.biol.306(2), 480-492; clark, H.F. et al (2003) Genome Res.13(10): 2265-.

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT 1); NP-067079.2; NM-021246.2; mallyya, M. et al (2002) Genomics 80(1) 113-; ribas, G, et al (1999) J.Immunol.163(1): 278-287.

(43) LGR5 (G protein-coupled receptor 5 containing leucine-rich repeats; GPR49, GPR 67); NP-003658.1; NM-003667.2; salanti, G, et al (2009) am.J.Epidemiol.170(5): 537-545; yamamoto, Y. et al (2003) Hepatology 37(3): 528-533.

(44) RET (RET proto-oncogene; MEN 2A; HSCR 1; MEN 2B; MTC 1; PTC; CDHF 12; Hs.168114; RET 51; RET-ELE 1); NP-066124.1; NM-020975.4; tsukamoto, H. et al (2009) Cancer Sci.100(10): 1895-; narita, N. et al (2009) Oncogene 28(34) 3058-3068.

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY 6K; HSJ 001348; FLJ 35226); NP-059997.3; NM-017527.3; ishikawa, N. et al (2007) Cancer Res.67(24): 11601-; deNooij-van Dalen, A.G. et al (2003) int.J.cancer 103(6) 768-774.

(46) GPR19(G protein-coupled receptor 19; Mm.4787); NP-006134.1; NM-006143.2; montpetit, A. and Sinnett, D. (1999) hum. Genet.105(1-2): 162-164; o' Down, B.F. et al (1996) FEBSLett.394(3): 325-.

(47) GPR54(KISS1 receptor; KISS 1R; GPR 54; HOT7T 175; AXOR 12); NP-115940.2; NM-032551.4; nanvenot, J.M. et al (2009) mol.Pharmacol.75(6): 1300-; hata, K. et al (2009) Anticancer Res.29(2): 617-623.

(48) ASPHD 1(1 containing aspartate β -hydroxylase domain; LOC253982), NP-859069.2; NM-181718.3; Gerhard, D.S. et al (2004) Genome Res.14(10B): 2121-2127.

(49) Tyrosinase (TYR; OCAIA; OCA 1A; tyrosinase; SHEP 3); NP-000363.1; NM-000372.4; bishop, D.T. et al (2009) nat. Genet.41(8): 920-; nan, H, et al (2009) int.J. cancer 125(4): 909-917.

(50) TMEM118 (Vickers, transmembrane 2; RNFT 2; FLJ 14627); NP-001103373.1; NM-001109903.1; clark, H.F. et al (2003) Genome Res.13(10): 2265-2270; scherer, S.E. et al (2006) Nature 440(7082) 346-.

(51) GPR172A (G protein-coupled receptor 172A; GPCR 41; FLJ 11856; D15Ertd747 e); NP-078807.1; NM-024531.3; ericsson, T.A. et al (2003) Proc. Natl. Acad. Sci. U.S.A.100(11): 6759-6764; takeda, S. et al (2002) FEBS Lett.520(1-3): 97-101.

(52) CD33, a member of the sialic acid binding immunoglobulin-like lectin family, is a 67-kDa glycosylated transmembrane protein. In addition to committed myeloid monocytes and erythroid progenitors, CD33 is expressed on most myeloid and monocytic leukemia cells. It was not seen on the earliest pluripotent stem cells, mature granulocytes, lymphoid cells or non-hematopoietic cells (Sabbath et al (1985) J. Clin. invest.75: 756-56; Andrews et al (1986) Blood 68: 1030-5). CD33 contains two tyrosine residues on its cytoplasmic tail, each followed by a hydrophobic residue, similar to the Immunoreceptor Tyrosine Inhibitory Motif (ITIM) seen in many inhibitory receptors.

(53) CLL-1(CLEC12A, MICL and DCAL2) encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have a wide variety of functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune responses. The proteins encoded by such genes are negative regulators of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is tightly linked to other CTL/CTLD superfamily members in the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) curr. Opin. struct. biol.9(5): 585-90; van Rhenen A et al, (2007) Blood 110(7): 2659-66; Chen CH et al (2006) Blood 107(4): 1459-67; Marshall AS et al (2006) Eur. J. Immunol.36(8): 2159-69; Bakker AB et al (2005) Cancer Res.64(22): 8443-50; Marshall AS et al (2004) J.biol.m.279 (15): 14792-802). CLL-1 has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is predicted to not bind calcium or sugars), a stem region, a transmembrane domain, and a short cytoplasmic tail containing the ITIM motif.

In any antibody embodiment of the disclosure, the antibody is humanized. In one embodiment, the antibody comprises an HVR as in any embodiment of the disclosure, and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is a human VL kappa I consensus (VLKI) framework and/or a VH framework VH 1. In certain embodiments, the human acceptor framework is a human VL kappa I consensus (VLKI) framework and/or a VH framework VH1, comprising any one of the following mutations.

In another embodiment, the antibody comprises a VH as in any of the embodiments provided herein, and a VL as in any of the embodiments provided herein.

In another embodiment of the present disclosure, the antibody according to any embodiment herein is a monoclonal antibody, including a human antibody. In one embodiment, the antibody is an antibody fragment, such as an Fv, Fab ', scFv, diabody, or F (ab')2 fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgG1 antibody, an IgG2a antibody, or other antibody classes or isotypes as defined herein. In some particular embodiments, the antibody is selected from anti-HER 2, anti-CD 22, anti-CD 33, anti-Napi 2b, and anti-CLL-1.

In another embodiment, an antibody according to any embodiment herein may bind any feature, alone or in combination, as described herein.

B. Affinity of antibody

In certain embodiments, an antibody provided herein has ≤ 1 μ M, ≦ 100nM, ≦ 50nM, ≦ 10nM, ≦ 5nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM or ≦ 0.001nM, and optionally ≦ 10nM^-13Dissociation constant (Kd) of M. (e.g., 10)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) which is performed using a Fab version of the antibody of interest and its antigen, as described by the following assay. The solution binding affinity of Fab to antigen was measured by: with minimum concentration of (in the presence of a titration series of unlabelled antigen¹²⁵I) The labeled antigen equilibrates the Fab, and the bound antigen is then captured with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.Mol.biol.293:865-881 (1999)). To establish the assay conditions, theMulti-well plates (Thermo Scientific) were coated overnight with 5. mu.g/ml capture anti-Fab antibodies (Cappellabs) in 50mM sodium carbonate (pH 9.6) and subsequently with 2% (weight/volume) fetal bovine serum albumin in PBSThe white was blocked at room temperature (about 23 ℃) for 2 to 5 hours. In a non-absorbent board (Nunc #269620), 100pM or 26pM of [ alpha ], [ beta ]¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (e.g.in accordance with the evaluation of anti-VEGF antibodies, Fab-12, in Presta et al, Cancer Res.57:4593-4599 (1997)). Then incubating the target Fab overnight; however, incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution was then removed and the plate was plated with 0.1% polysorbate 20 in PBSAnd washing eight times. When the plate had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and place the plate in TOPCOUNT^TMCount on a gamma counter (Packard) for ten minutes. The concentration of each Fab that produced a maximum binding of less than or equal to 20% was selected for competitive binding assays.

According to another embodiment, Kd is measured using a surface plasmon resonance assay using an immobilized antigen CM5 chip at about 10 Response Units (RU) at 25 ℃Or(BIAcore, Inc., Piscataway, NJ). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate (pH 4.8) and/or HBS-P (0.01M Hepes pH7.4, 0.15M NaCl, 0.005% surfactant P20) before injection at a flow rate of 5. mu.l/min and/or 30. mu.l/min to achieve approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, approximately 25. mu.l at 25 ℃ are usedTwo-fold serial dilutions of Fab (0.78nM to 500nM) were injected at minute flow rate into the injection well with 0.05% polysorbate 20 (TWEEN-20)^TM) Surfactant in pbs (pbst). Using a simple one-to-one Langmuir (Langmuir) binding model (Evaluation Software version 3.2) calculation of association rates (k) by simultaneous fitting of association and dissociation sensorgrams_on) And dissociation rate (k)_off). The equilibrium dissociation constant (Kd) is calculated as the ratio k_off/k_on. See, for example, Chen et al, J.mol.biol.293: 865-. If the association rate measured by the surface plasmon resonance assay described herein exceeds 10⁶M^-1s^-1Then the association rate can be determined by: such as in spectrometers such as the spectrophotometer equipped with stop-flow (Aviv Instruments) or 8000-series SLM-AMINCO with stirred cell^TMMeasured in a spectrophotometer (thermospectonic) using a fluorescence quenching technique (excitation 295 nM; emission 340nM, 16nM band pass) that measures the increase or decrease in fluorescence emission intensity of 20nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increasing concentrations of antigen at 25 ℃.

C. Antibody fragments

In certain embodiments, The Antibodies provided herein are antibody fragments, including but not limited to Fab, Fab ' -SH, F (ab ')2, Fv, and scFv fragments, as well as other fragments described herein for a review of specific antibody fragments, see Hudson et al Nat. Med.9:129-134(2003) a review of scFv fragments, see, e.g., Pluckth ü n, in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore editors, (Springer-Verlag, New York), p.269-315 (1994), see also WO 93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458 for a discussion of Fab and F (ab ')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetraantibodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., e.

D. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody, wherein the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, wherein HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc. nat' lAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describing "surface reconstruction"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer,83:252-260(2000) (describing the "guided selection" method for FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" approach (see, e.g., Sims et al J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of human antibodies having particular subsets of light or heavy chain variable regions (see, e.g., Carter et al Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al J.Immunol.,151:2623 (1993)); human mature (somatomerism) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived from FR library screening (see, e.g., Baca et al, J.biol. chem.272:10678-10684(1997) and Rosok et al, J.biol. chem.271:22611-22618 (1996)).

E. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, curr. opin. pharmacol.5:368-74(2001), and Lonberg, curr. opin. immunol.20: 450-.

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which is present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For an overview of the methods for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, XENOMOUSE^TMU.S. Pat. nos. 6,075,181 and 6,150,584 to technology; describeU.S. patent numbers 5,770,429 for technology; describes K-MU.S. Pat. No. 7,041,870 to Art and describesU.S. patent application publication No. US2007/0061900 of the art. The human variable regions from whole antibodies generated by such animals may be further modified, for example, by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York,1987), and Boerner et al, J.Immunol.,147:86 (1991)), human antibodies produced by human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268(2006) (describing human-human hybridomas). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histology and Histopathology,20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Described herein are techniques for selecting human antibodies from antibody libraries.

F. Antibodies derived from libraries

Antibodies of the present disclosure can be isolated by screening combinatorial libraries of antibodies with a desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such Methods are reviewed, for example, in Hoogenboom et al, in Methods in Molecular Biology178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ,2001) and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in molecular biology 248:161-175(Lo editor, Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5): 1073-; fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In some phage display methods, pools of VH and VL genes are separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries, which can then be screened against antigen-binding phage, as described in Winter et al, Ann. Rev. immunol.,12:433-455 (1994). Phage typically display antibody fragments as single chain fv (scfv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., by humans) to provide a single source of antibody to multiple non-self antigens as well as self antigens without the need for any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, natural libraries can also be prepared synthetically by: unrearranged V-gene segments were cloned from stem cells and PCR primers containing random sequences were used to encode the hypervariable CDR3 regions and to effect rearrangement in vitro as described in Hoogenboom and Winter, J.Mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. patent publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Herein, an antibody or antibody fragment isolated from a human antibody library is considered a human antibody or human antibody fragment.

G. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies comprising antigen binding domains with polyepitopic specificity (i.e., capable of specifically binding to two or more different epitopes on one biomolecule or capable of specifically binding to epitopes on two or more different biomolecules). In some embodiments, the multispecific antibody is a monoclonal antibody having binding specificity for at least two different sites. In some embodiments, the antigen binding domain of a multispecific antibody (such as a bispecific antibody) comprises two VH/VL units, wherein a first VH/VL unit specifically binds a first epitope and a second VH/VL unit specifically binds a second epitope, wherein each VH/VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies; an antibody having two or more VL and VH domains; antibody fragments such as Fab, Fv, dsFv, scFv; a diabody; bispecific diabodies and triabodies; antibody fragments that have been covalently or non-covalently linked. A VH/VL unit that also comprises at least a portion of a heavy chain variable region and/or at least a portion of a light chain variable region may also be referred to as an "arm" or "hemi-polymer" or "half antibody". In some embodiments, the semimer comprises a heavy chain variable region portion sufficient to allow intramolecular disulfide bond formation with the second semimer. In some embodiments, the hemimer comprises a knob mutation (knob mutation) or a hole mutation (hole mutation), for example to allow heterodimerization with a second hemimer or hemiantibody comprising a complementary hole mutation or knob mutation. Knob mutations and hole mutations are discussed further herein.

In certain embodiments, the multispecific antibodies provided herein can be bispecific antibodies. The term "bispecific antibody" is used in the broadest sense and encompasses multispecific antibodies comprising antigen-binding domains capable of specifically binding to two different epitopes on one biomolecule or capable of specifically binding to epitopes on two different biomolecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or being "dual specific". Bispecific antibodies can be prepared as full length antibodies or antibody fragments. The term "biparatopic antibody" as used herein refers to a bispecific antibody wherein a first antigen-binding domain and a second antigen-binding domain bind to two different epitopes on the same antigenic molecule, or it may bind to epitopes on two different antigenic molecules.

In some embodiments, the first antigen-binding domain and the second antigen-binding domain of a biparatopic antibody can bind to two epitopes within the same antigen molecule (intramolecular binding). For example, the first antigen-binding domain and the second antigen-binding domain of a biparatopic antibody may bind to two different epitopes on the same antibody molecule. In certain embodiments, the two different epitopes bound by a biparatopic antibody are epitopes that are not normally bound simultaneously by a monospecific antibody (such as a conventional antibody or an immunoglobulin single variable domain).

In some embodiments, the first antigen-binding domain and the second antigen-binding domain of a biparatopic antibody can bind to an epitope located within two different antigen molecules.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537(1983)), WO 93/08829, and trauecker et al, EMBO j.10:3655(1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168), WO2009/089004, US2009/0182127, US2011/0287009, Marvin and Zhu, Acta pharmacol.sin. (2005)26(6): 649-. The term "knob-into-hole" or "KnH" technology as used herein refers to a technology that directs the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (pore) into the other polypeptide at an interacting interface. For example, KnH has been introduced into the Fc: Fc binding interface, CL: CH1 interface or VH/VL interface of an antibody (see, e.g., US2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al 1997, Protein Science 6: 781-. In some embodiments, KnH drives two different heavy chains to pair together during the manufacture of a multispecific antibody. For example, a multispecific antibody having KnH in its Fc region may also comprise a single variable domain linked to each Fc region, or further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technique can also be used to pair together two different receptor extracellular domains or to pair any other polypeptide sequences (e.g., including affibodies, peptibodies, and other Fc fusions) that comprise different target recognition sequences.

The term "knob mutation" as used herein refers to a mutation that introduces a protrusion (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a pore mutation.

"protuberance" refers to at least one amino acid side chain that extends from an interface of a first polypeptide and thus can be located in a complementary cavity of an adjacent interface (i.e., an interface of a second polypeptide) in order to stabilize the heteromultimer and thereby facilitate, for example, heteromultimer formation rather than homomultimer formation. The protrusions may be present at the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the first polypeptide is altered to encode a protuberance. To achieve this, a nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced by a nucleic acid encoding at least one "import" amino acid residue having a larger side chain volume than the original amino acid residue. It should be understood that there may be more than one original and corresponding input residue. The side chain volumes of the various amino residues are shown, for example, in table 1 of US 2011/0287009. Mutations that introduce "processes" may be referred to as "knob mutations".

In some embodiments, the import residue for forming the protuberance is a naturally occurring amino acid residue selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the import residue is tryptophan or tyrosine. In some embodiments, the original residue used to form the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

By "cavity" is meant at least one amino acid side chain that is recessed from the interface of a second polypeptide and thus accommodates a corresponding protrusion on the adjacent interface of a first polypeptide. The cavity may be present at the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced by DNA encoding at least one "import" amino acid residue having a smaller side chain volume than the original amino acid residue. It should be understood that there may be more than one original and corresponding input residue. In some embodiments, the import residue for cavity formation is a naturally occurring amino acid residue selected from the group consisting of alanine (a), serine (S), threonine (T), and valine (V). In some embodiments, the import residue is serine, alanine, or threonine. In some embodiments, the original residue used to form the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Mutations introduced into "cavities" may be referred to as "pore mutations".

The protuberance can be "located in" the cavity, which means the spatial location of the protuberance and cavity, respectively, at the interface of the first polypeptide and the second polypeptide, and the protuberance and cavity are sized such that the protuberance can be located in the cavity without significantly interfering with the normal association of the first and second polypeptides at the interface. Since protrusions such as Tyr, Phe, and Trp do not generally extend perpendicularly from the axis of the interface and have a preferred conformation, alignment of a protrusion with a corresponding cavity may in some cases rely on modeling the protrusion/cavity pair based on three-dimensional structure, such as that obtained by X-ray crystallography or Nuclear Magnetic Resonance (NMR). This can be accomplished using art-recognized techniques.

In some embodiments, the knob in the IgG1 constant region is mutated to T366W. In some embodiments, the pore mutations in the IgG1 constant region comprise one or more mutations selected from T366S, L368A, and Y407V. In some embodiments, the pore mutations in the IgG1 constant region include T366S, L368A, and Y407V.

In some embodiments, the knob in the IgG4 constant region is mutated to T366W. In some embodiments, the pore mutations in the IgG4 constant region comprise one or more mutations selected from T366S, L368A, and Y407V. In some embodiments, the pore mutations in the IgG4 constant region include T366S, L368A, and Y407V.

Multispecific antibodies can also be prepared by: engineering electrostatic steering to make antibody Fc-heterodimer molecules (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., US4676980, and Brennan et al, Science,229:81 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., Kostelny et al, j. immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to prepare bispecific antibody fragments (Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sFv) dimers (Gruber et al, J.Immunol.,152:5368 (1994)); and making trispecific antibodies as described, for example, in Tutt et al j.immunol.147:60 (1991).

Also included herein are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVDs) (US 2006/0025576a1, and Wu et al (2007) Nature Biotechnology).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, the disclosure encompasses antibody variants with some, but not all, effector functions, which make the antibody a desirable candidate for applications in which the in vivo half-life of the antibody is critical, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/loss of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding capacity (and thus may lack ADCC activity), but retains FcRn binding capacity. Primary cells for mediating ADCC NK cells express only Fc (RIII, whereas monocytes express Fc (RI, Fc (RII and Fc (RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu. Rev. Immunol.9: 457-A492 (1991); non-limiting examples of in vitro assays to assess ADCC activity of a target molecule are described in US 5500362 (see, e.g., Hellstrom, I. et al Proc. nat 'l Acad. Sci. USA 83:7059-7063(1986)) and Hellstrom, I et al, Proc. nat' l Acad. Sci. USA 82: 1499-A (1985); 5,821,337 (see Bruggen, M. et al J. Exp. Med.166:1351 (1987); 1361) (see Brugmann, M. et al, J. Exp. 166. Med.166: 1351)) In (c) (ii). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or in addition, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al Proc. nat' l Acad. Sci. USA 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg, M.S. et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 are substituted (US 6,737,056, which is incorporated by reference in its entirety). Such Fc mutants include Fc mutants substituted at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (US 7332581, which is incorporated by reference in its entirety).

In certain embodiments, Pro329 of the wild-type human Fc region is replaced with glycine or arginine or an amino acid residue sufficiently large to disrupt the proline sandwich within the Fc/Fc γ receptor interface formed between proline 329 of Fc and the tryptophan residues Trp 87 and Trp 110 of FcgRII (Sondermann et al: Nature 406,267-273 (20.7.2000)). In another embodiment, the at least one additional amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in another embodiment, the at least one additional amino acid substitution is L234A and L235A of the human IgG1Fc region or S228P and L235E of the human IgG4Fc region (US 8969526, which is incorporated by reference in its entirety).

In certain embodiments, the polypeptide comprises an Fc variant of a wild-type human IgG Fc region, wherein Pro329 of the human IgGFc region of said polypeptide is substituted with glycine and wherein the Fc variant comprises at least two additional amino acid substitutions at L234A and L235A of the human IgG1Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EU index of Kabat (U.S. patent No. 8,969,526, which is incorporated by reference in its entirety). In certain embodiments, polypeptides comprising P329G, L234A, and L235A substitutions exhibit reduced affinity for human fcyriiia and fcyriia for down-regulating ADCC to at least 20% of the ADCC induced by a polypeptide comprising a wild-type human IgG Fc region, and/or for down-regulation of ADCP (U.S. patent No. 8,969,526, which is incorporated by reference in its entirety).

In a specific embodiment, a polypeptide comprising an Fc variant of a wild-type human Fc polypeptide comprises the triple mutation: amino acid substitution at position Pro329, L234A and L235A mutations (P329/LALA) (U.S. Pat. No. 8,969,526, which is incorporated by reference in its entirety). In particular embodiments, the polypeptide comprises the following amino acid substitutions: P329G, L234A and L235A.

Certain antibody variants with improved or impaired binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9(2):6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues)).

In some embodiments, alterations that result in altered (i.e., improved or attenuated) C1q binding and/or Complement Dependent Cytotoxicity (CDC) are made in the Fc region, for example, as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249(1994)) are described in US2005/0014934A1(Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826).

For additional examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

H. Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, e.g., "THIOMAB^TMAn antibody ", wherein one or more residues of the antibody are substituted with a cysteine residue. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody, and can be used to conjugate the antibody to a drug moiety to produce an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); k149(Kabat numbering) of the light chain; a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No. 7,521,541.

At one endIn some embodiments, Thiomab^TMThe antibody comprises one of the heavy or light chain cysteine substitutions listed in table a below.

TABLE A

In other embodiments, Thiomab^TMThe antibody comprises one of the heavy chain cysteine substitutions listed in table B.

TABLE B

In some other embodiments, Thiomab^TMThe antibody comprises one of the light chain cysteine substitutions listed in table C.

Watch C

In some other embodiments, Thiomab^TMThe antibody comprises one of the heavy or light chain cysteine substitutions listed in table D.

Table D

Cysteine engineered antibodies that may be used in the antibody-drug conjugates (ADCs) of the present disclosure for cancer therapy include, but are not limited to, antibodies directed against cell surface receptors and Tumor Associated Antigens (TAAs). Tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art. In order to find effective cellular targets for cancer diagnosis and treatment, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal, non-cancerous cells. Typically, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cell than on the surface of the non-cancerous cell. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target cancer cells for destruction by antibody-based therapies.

Examples of tumor associated antigens TAAs include, but are not limited to, TAAs (1) - (53) listed herein. For convenience, information relating to these antigens (all of which are known in the art) is set forth herein and includes names, alias names, Genbank accession numbers, and one or more primary references, all following the nucleic acid and protein sequence identification conventions of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to TAAs (1) - (53) are available in public databases such as GenBank. Tumor associated antigens targeted by the antibodies include all amino acid sequence variants and isoforms having at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to sequences identified in the cited references, or exhibiting substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references. For example, TAAs having variant sequences are generally capable of specifically binding to antibodies that specifically bind to TAAs having the corresponding sequences listed. The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

Prodrug preparation

Methods for preparing PBD monomer prodrugs and PBD dimer prodrugs within the scope of the present disclosure are described elsewhere herein.

Linker-drug conjugation

Conjugation of the linker to the PBD amine may suitably be carried out according to the methods of WO 2013/055987, WO 2015/023355 and WO 2015/095227, each of which is incorporated herein in its entirety by reference.

In some such embodiments, an activating linker as described elsewhere herein is combined with a solution of PBD monomers or dimers to form a linker-PBD conjugate. In general, any solvent that is capable of providing a solution comprising about 0.05 to about 1 mole per liter of PBD is suitable. In some embodiments, the solvent is DCM. In some other linker-PBD conjugation embodiments, a solution of PBD, a stoichiometric excess of triphosgene (or diphosgene or phosgene), and a base (e.g., 4-dimethylaminopyridine) is formed in a solvent (e.g., anhydrous DCM). The linker intermediate having an alcohol moiety (as described elsewhere herein) is compounded with the PBD solution to form a reaction mixture, which is stirred until the reaction is complete to form a product mixture comprising the linker-PBD conjugate. The reaction mixture may suitably comprise from about 0.005 moles/liter to about 0.5 moles/liter of PBD, from about 2 to about 10 equivalents of linker intermediate per equivalent of PBD, and from about 0.02 to about 0.5 equivalents of base. After the reaction is complete, the linker-PBD conjugate can be isolated, such as by solvent evaporation, and purified by methods known in the art, such as one or more of extraction, reverse phase high pressure liquid chromatography, ion exchange chromatography, or flash chromatography.

Preparation of disulfide conjugate Compounds

In some embodiments, disulfide conjugated compounds of the present disclosure having the formula:

can be prepared by forming a reaction mixture comprising (1) a solvent system comprising water, (2) a source of an antibody comprising at least one cysteine having a thiol moiety, and (3) a stoichiometric excess of a source of a linker-PBD conjugate having the formula:

antibody, X_L、R¹、R²Sp, n and D are as described elsewhere herein. Reacting the reaction mixture to form a mixture comprising a disulfide conjugate compound of formula (II), wherein p is 1,2, 3, 4,5, 6,7, or 8. Although individual disulfide conjugate compounds in a mixture may have a p-value of 1 to 8, and some antibody molecules in such a mixture may be unconjugated (p ═ 0), the ratio of PBD to antibody, expressed as the ratio of PBD equivalents to antibody equivalents, of the plurality of disulfide conjugate compounds formed in the product mixture is about 1 to about 5, about 1.5 to about 3, about 1.5 to about 2.5, about 1.7 to about 2.3, about 1.8 to about 2.2, or about 2.

In any of the various embodiments of such embodiments, the source of the antibody can be provided in a solution in an aqueous buffer. In some such embodiments, the buffer may suitably be N- (2-acetylamino) -aminoethanesulfonic acid ("ACES"); acetate salt; n- (2-acetylamino) -iminodiacetic acid ("ADA"); 2-aminoethanesulfonic acid, taurine ("AES"); 2-amino-2-methyl-1-propanol ("AMP"); 2-amino-2-methyl-1, 3-propanediol, aminobutanediol (Ammediol) ("AMPD"); n- (1, 1-dimethyl-2-hydroxyethyl) -3-amino-2-hydroxypropanesulfonic acid ("AMPSO"); n, N-bis- (2-hydroxyethyl) -2-aminoethanesulfonic acid ("BES"); n, N' -bis (2-hydroxyethyl) -glycine ("Bicine"); [ BIS- (2-hydroxyethyl) -imino ] -Tris- (hydroxymethyl methane) ("BIS-Tris"); 1, 3-BIS [ Tris (hydroxymethyl) -methylamino ] propane ("BIS-Tris-propane"); dimethylarsinic acid ("cacylate"); 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid ("CAPSO"); cyclohexylaminoethanesulfonic acid ("CHES"); a citrate salt; 3- [ N-bis (hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid ("DIPSO"); n- (2-hydroxyethyl) -piperazine-N' -ethanesulfonic acid ("HEPES"); n- (2-hydroxyethyl) -piperazine-N' -3-propanesulfonic acid ("HEPPS, EPPS"); n- (2-hydroxyethyl) -piperazine-N' -2-hydroxypropanesulfonic acid ("HEPPSO"); 2- (N-morpholino) -ethanesulfonic acid ("MES"); 3- (N-morpholino) -propanesulfonic acid ("MOPS"); 3- (N-morpholino) -2-hydroxypropanesulfonic acid ("MOPSO"); piperazine-N, N' -bis (2-ethanesulfonic acid) ("PIPES"); piperazine-N, N' -bis (2-hydroxypropanesulfonic acid) ("POPSO"); a salt of succinic acid; 3- { [ tris (hydroxymethyl) -methyl ] -amino } -propanesulfonic acid ("TAPS"); 3- [ N-tris (hydroxymethyl) -methylamino ] -2-hydroxypropanesulfonic acid ("TAPSO"); 2-aminoethanesulfonic acid, AES ("taurine"); triethanolamine ("TEA"); 2- [ tris (hydroxymethyl) -methylamino ] -ethanesulfonic acid ("TES"); n- [ tris (hydroxymethyl) -methyl ] -glycine ("Tricine"); TRIS (hydroxymethyl) -aminomethane ("TRIS"). In some such embodiments, the buffer may be succinate or tris. The buffer concentration is suitably about 5mM, about 10mM, about 15mM, about 20mM, about 50mM, about 100mM, or about 150nM, such as about 5mM to about 150mM, about 5mM to about 30mM, about 5mM to about 20mM, about 5mM to about 10mM, or about 50mM to about 100 nM. The concentration of antibody in the buffer can be about 1mg/mL, about 5mg/mL, about 10mg/mL, or higher. The pH of the antibody solution is suitably from about 4 to about 8, from about 4 to about 6, or about 5.

In any of the various embodiments of such embodiments, the source of the linker-PBD conjugate compound ("activated linker-PBD conjugate") can generally be formed by dissolving the activated hindered linker-PBD conjugate in a solvent comprising at least one polar aprotic solvent selected from the group consisting of acetonitrile, tetrahydrofuran, ethyl acetate, acetone, N-dimethylformamide, dimethylacetamide, dimethylsulfoxide, propylene glycol, ethylene glycol, and dichloromethane. In some embodiments, the solvent comprises, consists essentially of, or consists essentially of: n, N-dimethylformamide and/or dimethylacetamide.

The source of antibody and the source of activated linker-PBD conjugate may be suitably mixed to form a reaction mixture. The concentration of antibody in the reaction mixture is suitably about 1mg/mL, about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, or about 25mg/mL, and ranges thereof, such as about 1mg/mL to about 25mg/mL, about 1mg/mL to about 20mg/mL, about 1mg/mL to about 15mg/mL, about 1mg/mL to about 10mg/mL, about 5mg/mL to about 25mg/mL, about 5mg/mL to about 20mg/mL, or about 5mg/mL to about 15 mg/mL. The equivalent ratio of activated linker-PBD conjugate to antibody is suitably about 2:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and ranges thereof, such as about 2:1 to about 20:1, about 3:1 to about 15:1, about 3:1 to about 10:1, or about 5:1 to about 10: 1. In some embodiments of the present disclosure, the reaction mixture solvent system comprises primarily water. In some other embodiments, the reaction mixture solvent system may generally comprise at least 75 vol/vol% of a buffer as described elsewhere herein, and about 5 vol/vol%, about 10 vol/vol%, about 15 vol/vol%, about 20 vol/vol%, about 25 vol/vol%, or about 30 vol/vol%, and ranges thereof, such as from about 5 vol/vol% to about 30 vol/vol%, from about 5 vol/vol% to about 20 vol/vol%, from about 5 vol/vol% to about 15 vol/vol%, or about 10 vol/vol% of a polar aprotic solvent as described elsewhere herein. In some other embodiments, the reaction mixture solvent system may typically comprise at least 50 vol/vol% of a buffer as described elsewhere herein, and between about 10 vol/vol% and about 50 vol/vol%, such as about 10 vol/vol%, about 20 vol/vol%, about 30 vol/vol%, about 40 vol/vol%, or more than 50 vol/vol% propylene glycol or ethylene glycol. Alternatively, the reaction mixture solvent system can comprise from about 50 vol/vol%, about 60 vol/vol%, about 70 vol/vol%, 75 vol/vol%, about 80 vol/vol%, about 85 vol/vol%, about 90 vol/vol%, or about 95 vol/vol% water to about 95 vol/vol%, and ranges thereof, such as from about 50 vol/vol% to about 95 vol/vol% water, from about 75 vol/vol% to about 95 vol/vol% water, from about 80 vol/vol% to about 95 vol/vol% water, or from about 85 vol/vol% to about 95 vol% water. The pH of the reaction mixture is suitably about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0, and ranges thereof, such as about 5.0 to about 9.0, about 6.0 to about 9.0, about 6.5 to about 9.0, or about 7.0 to about 9.0, or about 7.5 to about 8.5.

Incubating the reaction mixture at about 10 ℃, about 15 ℃, about 20 ℃, about 25 ℃, about 30 ℃, about 35 ℃, about 40 ℃, about 45 ℃ or about 50 ℃, and ranges thereof, such as about 10 ℃ to about 50 ℃, about 15 ℃ to about 45 ℃, about 15 ℃ to about 40 ℃, about 20 ℃ to about 35 ℃, or about 20 ℃ to about 30 ℃ for about 0.5 hour, about 1 hour, about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, or about 48 hours, and ranges thereof, such as about 0.5 hours to about 48 hours, or about 1 hour to about 24 hours, to form a product mixture comprising the disulfide-conjugated compound of formula (II).

The product mixture may comprise at least 60A%, at least 65 area%, at least 70 area%, at least 75 area%, at least 80 area%, at least 85 area%, or at least 90 area% of the antibody-linker-PBD conjugate as determined by MS/LC. The product mixture may further comprise at least one leaving group by-product species. In some embodiments, the area percentage of the total leaving group byproduct species as compared to the area percentage of the disulfide conjugate compound formed is less than 10 area%, less than 5 area%, less than 4 area%, less than 3 area%, less than 2 area%, less than 1 area%, less than 0.5 area%, as measured by MS/LC. In the case of antibodies, and based on experimental evidence to date, leaving group by-product species may include:

wherein X_LAs defined elsewhere herein, and wherein Q refers to X having no sulfur linking atom_LAnd (4) partial. Exemplary X and corresponding Q are illustrated as follows:

the reaction mixture may further comprise an unconjugated antibody compound including (i) an unconjugated antibody monomeric species comprising at least one disulfide bond formed by reaction of two cysteine thiol moieties, (ii) an unconjugated antibody dimeric species comprising at least one disulfide bond formed by reaction of two cysteine thiol moieties, and (iii) a combination of the unconjugated antibody monomeric species and the unconjugated antibody dimeric species. In some embodiments, the total concentration of unconjugated antibody compound is less than 10 area%, less than 5 area%, less than 4 area%, less than 3 area%, less than 2 area%, less than 1 area%, less than 0.5 area%, less than 0.3 area%, less than 0.1 area%, or is undetectable compared to the area percentage of disulfide-conjugated compound formed, as measured by MS/LC.

PBD prodrug treatment methods

It is contemplated that the PBD prodrug conjugate compounds of the present disclosure may be useful in the treatment of various diseases or disorders characterized, for example, by the overexpression of tumor antigens. Exemplary conditions or hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemias and lymphoid malignancies. Others include neuronal, glial, astrocyte, hypothalamic, glandular, macrophage, epithelial, interstitial, blastocoel, inflammatory, angiogenic, and immunological (including autoimmune) disorders.

In one embodiment, the PBD prodrug conjugate compounds provided herein are used in a method of inhibiting the proliferation of a cancer cell, the method comprising exposing the cell to an antibody-prodrug conjugate under conditions that allow the binding of the antibody or antibody-prodrug conjugate to a tumor-associated antigen on the surface of the cell, thereby inhibiting the proliferation of the cell. In certain embodiments, the method is an in vitro or in vivo method. In additional embodiments, the cell is a lymphocyte, lymphoblast, monocyte, or myelomonocytic cell.

In vitroCell proliferation inhibition can be achieved by using CellTiter-Glo available from Promega (Madison, Wis.)^TMLuminescent Cell Viability Assay. The assay determines the number of viable cells in culture based on the quantification of ATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J.Immunol.meth.160:81-88, U.S. Pat. No. 6602677. The assay can be performed in 96-well or 384-well format, making it amenable to automated High Throughput Screening (HTS). See Cree et al (1995) AntiCancer Drugs6: 398-404. The assay procedure involves the addition of a single reagent directly to the cultured cells: (Reagent). This results in cell lysis and the generation of a luminescent signal by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is proportional to the number of viable cells present in the culture. Data may be recorded by a photometer or a CCD camera imaging device. Luminous output is expressed as Relative Light Units (RLU).

In another embodiment, a PBD prodrug conjugate compound for use as a medicament is provided. In additional embodiments, a PBD prodrug conjugate compound for use in a method of treatment is provided. In certain embodiments, a PBD prodrug conjugate compound is provided for use in the treatment of cancer. In certain embodiments, the present disclosure provides a PBD prodrug conjugate compound for use in a method of treating a subject, the method comprising administering to the subject an effective amount of the PBD prodrug conjugate compound.

The PBD prodrug conjugate compounds of the present disclosure may be used alone or in combination with other agents in therapy. For example, an antibody or immunoconjugate of the present disclosure may be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anthracycline. In some embodiments, the anthracycline is daunorubicin or idarubicin. In some embodiments, the additional therapeutic agent is cytarabine. In some embodiments, the additional therapeutic agent is cladribine. In some embodiments, the additional therapeutic agent is fludarabine or topotecan. In some embodiments, the additional therapeutic agent is 5-azacytidine or decitabine.

Such combination therapies noted herein encompass both combined administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case administration of the compounds of the disclosure may occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants. The compounds of the present disclosure may also be used in combination with radiation therapy.

The compounds of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration for local treatment, as necessary. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over various time points, bolus administration, and pulsed infusion.

The compounds of the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of agent delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The compounds of the present disclosure are formulated without, but optionally with, one or more agents currently used for preventing or treating such disorders. The effective amount of the other agent will depend on the amount of the compound of the present disclosure in the formulation, the type of condition being treated, and other factors discussed herein. These agents are typically used at the same doses as described herein and with the administration routes as described herein, or at about 1% to 99% of the doses described herein, or at any dose and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of a compound of the disclosure (either alone or in combination with one or more other additional therapeutic agents) will depend upon the type of disease being treated, the type of compound, the severity and course of the disease, whether the compound is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attendant physician. The compounds are suitable for administration to a patient at one time or via a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of a compound of the present disclosure may be administered to a patient as a first candidate dose, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned herein. For repeated administrations over several days or longer, depending on the condition, the treatment should generally be continued until the desired suppression of disease symptoms occurs. An exemplary dose of the compound will range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about two to about twenty doses, or, for example, about six doses of antibody). A higher loading dose may be administered initially, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

Intracellular release of drug from the PBD prodrug conjugate compound in target cells is believed to be caused by a combination of linker self-cleavage and (i) GSH activation of a disulfide trigger, (ii) DTD activation of a quinone trigger, or (iii) ROS activation of an arylboronic acid or arylboronic ester trigger.

With regard to linkers comprising a disulfide moiety, GSH-mediated release provides advantages over certain linkers known in the prior art (such as acid-labile hydrazine linkers). More specifically, it is known that blood concentrations of GSH are very low, such as in the micromolar range, while intracellular GSH concentrations are typically up to three orders of magnitude greater, such as in the millimolar range. It is also believed that GSH concentrations in cancer cells are even higher due to increased activity of the reductase. It is also believed that steric hindrance at the linker carbon atom with the sulfur atom provides improved blood flow stability and improved intracellular release. Thus, it is believed that the disulfide conjugated compounds of the present disclosure provide improved stability in the bloodstream and improved intracellular release rates.

With respect to PBD prodrugs, GSH, DTD, or ROS activation of the PBD N10 protecting group masks toxicity in blood stream and plasma, and provides selective toxicity advantages over PBD drugs that do not include a protective prodrug moiety. More specifically, it is known that GSH, DTD, and ROS are low in blood concentration as compared to cancer cells. Thus, it is believed that the PBD prodrug conjugate compounds of the present disclosure provide reduced blood flow toxicity and targeted intracellular activation of PBD.

X. product

In another embodiment of the present disclosure, an article of manufacture is provided that contains materials useful in the treatment, prevention, and/or diagnosis of the conditions described herein. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective in treating, preventing and/or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a PBD prodrug conjugate compound of the present disclosure. The label or package insert indicates that the composition is used to treat the selected condition. Further, an article of manufacture can comprise (a) a first container having a composition contained therein, wherein the composition comprises a PBD prodrug conjugate compound of the present disclosure; and (b) a second container having a composition therein, wherein the composition comprises another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may also include a package insert indicating that the composition is useful for treating a particular condition. Alternatively or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Examples

The following are examples of the methods and compounds of the present disclosure. It is to be understood that various other embodiments may be practiced in view of the general description provided herein.

The structures of the PBD monomer and dimer compounds disclosed in the examples and the corresponding reference labels are depicted below, where the asterisk refers to the chiral center of racemic or undefined stereochemistry.

Example 1: general procedure for ADC preparation

In some aspects of the disclosure, cysteine engineered antibodies (such as those listed in tables A-D) are made reactive for conjugation to linker-drug intermediates by treatment with a reducing agent such as DTT (Cleland reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride; see Getz et al (1999) anal. biochem. Vol. 273:73-80 (incorporated herein by reference); Soltec vents, Beverly, MA), followed by reformation of interchain disulfide bonds (reoxidation) using a mild oxidizing agent such as dehydroascorbic acid. Full-length cysteine engineered monoclonal antibodies (Thiomabt) expressed in CHO cells (see Gomez et al (2010) Biotechnology and dbioeng.105(4):748-^M) Reduction overnight, which removes cysteine and GSH adducts and reduces chains in antibodiesAn inter-disulfide bond. Adduct removal was monitored by reverse phase liquid chromatography/mass spectrometry ("LC/MS") using a PLRP-S column. The reduced Thiomab was diluted and acidified by addition of at least 4 volumes of 10mM sodium succinate (pH5 buffer)^TM. Alternatively, the antibody is diluted and acidified by adding at least 4 volumes of 10mM succinate (pH5) and titrating with 10% acetic acid until the pH is approximately 5. Followed by reduction of pH and dilution of Thiomab^TMThe sample was loaded onto a HiTrap S cation exchange column, washed with several column volumes of 10mM sodium acetate (pH5) and eluted with 50mM Tris (pH 8.0), 150mM sodium chloride.

By performing reoxidation, disulfide bonds are reestablished between cysteine residues present in the parent Mab. Eluting the above reduced Thiomab^TMTreatment with 15X dehydroascorbic acid (DHAA) for about 3 hours, or alternatively, 200nM to 2mM aqueous copper sulfate (CuSO4) overnight at room temperature. Other oxidizing agents (i.e., oxidizing agents) and oxidizing conditions known in the art may be used. Ambient air oxidation may also be effective. This mild partial reoxidation step effectively forms intrachain disulfides with high fidelity. Reoxidation was monitored by reverse phase LC/MS using a PLRP-S column. Thiomab to be reoxidized^TMDiluted with succinate buffer as described above to reach a pH of about 5 and purified on an S-column as described above except that elution was performed using a gradient of 10mM succinate (pH5) (300 mM sodium chloride in 10mM succinate (pH5, buffer a) (buffer B)). Addition of EDTA to eluted Thiomab^TMTo a final concentration of 2mM and, if desired, concentrated to achieve a final concentration of greater than 5 mg/mL.

The resulting Thiomab suitable for conjugation^TMStored in aliquots at-20 ℃. LC/MS analysis was performed on a 6200 series TOF or QTOF Agilent LC/MS. Heating the sample to 80 deg.CChromatography was performed on a 1000A, microwell column (50mmx2.1mm, Polymer Laboratories, Shropshire, UK). A linear gradient of 30-40% B (solvent A: 0.05% TFA in water, solvent B) was used: 0.04% TFA in acetonitrile) and the eluent was directly ionized using an electrospray source. Data were collected and deconvoluted by MassHunter software. Prior to LC/MS analysis, the antibody or drug conjugate (50. mu.g) was treated with peptide N-glucosidase F ("PNGase F") (2 units/ml; PROzyme, San Leandro, Calif.) at 37 ℃ for 2 hours to remove the N-linked carbohydrate. Alternatively, the antibody or drug conjugate was partially digested with LysC (0.25 μ g/50 μ g antibody or conjugate) at 37C for 15 minutes to give Fab and Fc fragments for analysis by LC/MS. Peaks in the deconvoluted LC/MS spectra were assigned and quantified. The drug-to-antibody ratio (DAR) is calculated by calculating the ratio of the intensity of one or more peaks corresponding to the drug-conjugated antibody relative to the intensity of all peaks observed.

Thiomab for conjugation in 10mM succinate (pH5), 150mM NaCl, 2mM EDTA with 1M Tris^TMAdjusting the pH value to 7.5-8.5. An excess of about 3 to 20 molar equivalents of the linker-drug intermediate of the present disclosure having a thiol-reactive pyridyl disulfide group is dissolved in DMF or DMA and added to the reduced, reoxidized, and pH adjusted antibody. The reaction was incubated at room temperature or 37 ℃ and monitored until complete (1 to about 24 hours) as determined by LC/MS analysis of the reaction mixture. When the reaction is complete, the conjugate can be purified by one or any combination of several methods to remove remaining unreacted linker-drug intermediate and aggregated protein (if present at significant levels). For example, the conjugate can be diluted with 10mM histidine-acetate at pH 5.5 until the final pH is about 5.5 and purified by S cation exchange chromatography using a HiTrap S column connected to an Akta purification system (GE Healthcare) or using a S maxi spin column (Pierce). Alternatively, the conjugate can be purified by gel filtration chromatography using an S200 column connected to an Akta purification system or using a Zeba spin column. Alternatively, dialysis may be used. Thiomab was filtered using gel filtration or dialysis with 240mM sucrose^TMThe drug conjugate was formulated as 20mM His/acetate (pH 5). The purified conjugate was concentrated by centrifugal ultrafiltration, filtered through a 0.2- μm filter under sterile conditions and frozen for storage. The antibody-drug conjugates were characterized by: BCA assay to determine protein concentration; analytical SEC (size exclusion chromatography) for aggregation analysis; and LC/MS after treatment with lysine C endopeptidase (LysC) to calculate DAR.

The conjugate was subjected to size exclusion chromatography using a Shodex KW802.5 column in 0.2M potassium phosphate (pH 6.2) with 0.25mM potassium chloride and 15% IPA at a flow rate of 0.75 ml/min. The aggregation state of the conjugate was determined by integration of the absorbance of the elution peak area at 280 nm.

The conjugates can be analyzed by LC/MS using an Agilent QTOF 6520ESI instrument. For example, the antibody-drug conjugate was treated with 1:500 w/w endoproteinase Lys C (Promega) in Tris (pH 7.5) for 30min at 37 ℃. The resulting cut fragments were loaded to a temperature of 80 deg.C(Angstrom), 8 μm (micron) PLRP-S (highly crosslinked polystyrene) column, and elution was performed using a gradient of 30% B to 40% B over 5 minutes. Mobile phase a was H with 0.05% TFA₂O, and mobile phase B was acetonitrile with 0.04% TFA. The flow rate was 0.5 ml/min. Protein elution was monitored by uv absorbance detection at 280nm prior to electrospray ionization and MS analysis. Chromatographic resolution (chromatographically resolution) of unconjugated Fc fragment, residual unconjugated Fab and pharmacologically Fab is generally achieved. Using Mass Hunter^TMThe software (Agilent Technologies) deconvolutes the obtained m/z spectra to calculate the mass of the antibody fragments.

Example 2: herceptin a118C antibody-probe conjugates

Various probe compounds containing a linker and a thiol leaving group were conjugated to herceptin a 118C. In each conjugation, 5mg/mL of antibody in a solvent system was contacted with a probe-linker-leaving group compound in an equivalent ratio of probe compound to antibody of 10:1, wherein the probe-linker was conjugated to the antibody via a disulfide bond. The solvent system contained 75mM Tris (pH8.5) and 10 v/v% DMF. The conjugation reaction was carried out at room temperature for 24 hours. The reaction product mixture was analyzed by LC/MS to determine the ratio of drug to antibody (DAR), the area percentage of the leaving group by-product compared to the antibody-probe conjugate, the area percentage of unconjugated dimer compared to the antibody-probe conjugate, and the area percentage of unconjugated monomer compared to the antibody-probe conjugate.

The probe has the following formula, wherein the wavy line indicates the point of attachment of the joint:

the probe-linker-leaving group compounds evaluated included:

the results are reported in table 1 below, where "DAR" refers to the drug-antibody ratio, "% LG" refers to the percentage of leaving group by-product, "% unconjugated dimer" refers to the percentage of unconjugated antibody dimer and "% unconjugated monomer" refers to the percentage of unconjugated antibody dimer.

TABLE 1

Linker-leaving group	DAR	％LG	% unconjugated dimer	% unconjugated monomer
					dimethyl/PDS	0.6	64	3	6
Dimethyl/5-nitro PDS	0.7	35	22	1
					Dimethyl/3-nitro PDS	0.2	69	18	2
dimethyl/Ellman	0.2	89	2	1
					Cyclopropyl/5-nitro PDS	2	1	0	0
Ethyl/5-Nitro PDS	2	1	0	0
					methyl/PDS	1.1	39	0	3
Methyl/5-nitro PDS	1.4	27	1	1
					Methyl/3-nitro PDS	2	2	0	0
methyl/Ellman	1.7	17	1	0

Example 3: herceptin K149C antibody-probe conjugates

The probe compounds of example 2 were evaluated for conjugation to herceptin K149C by disulfide bonding under the reaction conditions of example 2. The reaction product mixture was analyzed by LC/MS to determine the drug to antibody ratio (DAR) and the percentage area of leaving group by-product compared to the antibody-probe conjugate. The results are reported in table 2 below.

TABLE 2

Linker-leaving group	DAR	％LG
			dimethyl/PDS	0.3	84
Dimethyl/5-nitro PDS	0.6	68
			Dimethyl/3-nitro PDS	0.2	92
dimethyl/Ellman	0.2	91
			Cyclopropyl/5-nitro PDS	2	0.4
Ethyl/5-nitro PDS	2	0.3
			methyl/PDS	0.7	62
Methyl/5-nitro PDS	1.6	20
			Methyl/3-nitro PDS	1.8	9
methyl/Ellman	1.8	9

Example 4: herceptin A118C ADC

Various drug compounds containing a linker and a 5-nitro PDS thiol leaving group were conjugated to herceptin 24D5 HC a118C antibody via a disulfide bond. In each conjugation, 5mg/mL of antibody in the solvent system was contacted with the drug-linker-leaving group compound, wherein the equivalent ratio of drug-linker-leaving group compound to antibody was 3: 1. The solvent system included 75mM Tris (pH 8.5). The conjugation reaction was carried out at room temperature for 3 hours. The reaction product mixture was analyzed by LC/MS to determine the drug to antibody ratio (DAR) and the percentage area of leaving group by-product compared to the antibody-probe conjugate.

The drug has the formula, wherein the wavy line indicates the point of attachment of the linker:

drug-linker-leaving group compounds evaluated included the following, where "D" represents a drug:

the results are reported in table 3 below.

TABLE 3

Example 5: xCD22K149C ADC

In a first evaluation, a linker-drug compound comprising a mesylate leaving group (MTS) (linker 1) was conjugated to the xCD22K149C antibody via a disulfide bond. In a second evaluation, a linker-drug compound comprising an MTS leaving group (linker 2) is conjugated to an antibody in a second evaluation. In each conjugation, 5mg/mL of antibody in the solvent system was contacted with the drug-linker-leaving group compound, wherein the equivalent ratio of probe compound to antibody was 3: 1. The solvent system included 75mM Tris (pH8.5). The conjugation reaction was carried out at room temperature for 3 hours. Linkers 1 and 2 are exemplified as follows:

the reaction product mixture was analyzed by LC/MS to determine the drug to antibody ratio (DAR) and the percentage area of leaving group by-product compared to the antibody-probe conjugate. For linker 1, the drug to antibody ratio was 2.0, the area percent of antibody-drug conjugate was about 91%, the area percent of unconjugated antibody was less than 0.1%, and the area percent of MTS byproduct was about 0.5%. For linker 2, the drug to antibody ratio was 1.5, the area percent of antibody-drug conjugate was about 91%, the area percent of unconjugated antibody was less than 0.1%, and the area percent of MTS byproduct was about 0.5%.

Example 6: toxicity of PBD monomeric disulfide prodrugs on KPL-4 and WSU-DLCL2 cell lines

Toxicity of PBD monomeric disulfide prodrug and diaphorase prodrug on KPL-4, WSU-DLCL2, HCT-116, HCC1395 and Jurkat cell cultures was evaluated. KPL-4 (breast cancer cell line) expresses Her2 and exhibits high GSH levels of about 12 to about 24 mM. KPL-4 cells also have high DTD expression of about 839nRPKM (where nRPKM refers to normalized reads per Kb of transcript length per million plotted reads). WSU-DLCL2 (non-Hodgkin lymphoma cell line) showed low GSH levels of about 1.4mM and low DTD expression of about 1.4 nRPKM.

The efficacy of disulfide and DT-diaphorase prodrugs was determined by cell proliferation using the following protocol (CELLTITER GLO)^TMLuminescent Cell visual Assay, Promega Corp.). First, 40ul aliquots of cell cultures containing about 8000 cells of WSU-DLCL2 cells (low NQO1 gene and GSH levels), 4000 KPL-4 cells (high NQO1 gene and GSH levels), HCT-116 cells, HCC1395 cells, or Jurkat cells in RPMI-1640 medium (supplemented with 10% fetal bovine serum, 2mM glutamine, 50uM cystine, and 0.015 g/LL-methionine) were deposited into each well of 384 well flat bottom clear white polystyrene tissue culture treated microplates (Corning, NY). Second, control wells containing media with and without cells were prepared. Third, compounds with and without disulfide or DT-diaphorase prodrug (n ═ 3) were added in triplicate using ECHO sonic pipetting technology (labcytec inc, Sunnyvale, CA) using a 1:3x series of dilutionsInto experimental wells to generate a 10-point dose-response curve. Fourth, cells were cultured in a humidified incubator set at 37 ℃ and maintained at 5% CO₂Of the atmosphere (c). Fifth, the plates were equilibrated to room temperature for approximately 30 minutes. Sixth, add a volume of CELLTITER GLO^TMA reagent equal to the volume of cell culture medium present in each well. Seventh, the contents were mixed on an orbital shaker for 10 minutes in the dark to induce cell lysis. Eighth, the plate was incubated at room temperature for 30 minutes to stabilize the luminescence signal. Ninth, luminescence was recorded and reported as% activity in the figure, where RLU (relative luminescence units) was normalized to control (no compound control minus no cell control). Tenth, data for each replicate (n-3) for each antibody is plotted as individual dots as the mean of luminescence for each replicate with standard deviation error bars.

IC₅₀The efficacy results are presented in Table 4 below, in nM, where the data in parentheses represent the IC repeatedly evaluated₅₀As a result:

TABLE 4

The results for PBD monomer control 1 and PBD monomer disulfide prodrugs 1-4 for KPL-4 are depicted in figure 1 and the results for PBD monomer control 1 and PBD monomer disulfide prodrugs 1-4 for WSU-DLCL2 are depicted in figure 2, where each figure is activity [% ]]Graph against the logarithm of prodrug concentration in moles per liter. Activity [% ]]Expressed as negative values, this refers to a decrease in cell viability. E.g., -50 activity [% ]]Means that the cell viability is reduced by 50%. In addition to prodrug 4, for KPL-4, for prodrugs 1 to 3, the parent compound has the greatest IC₅₀The ratio of prodrug of (prodrug 2) is about 1.5. In addition to prodrug 4, for WSU-DLDL2, for prodrugs 1 through 3, the parent compound has the greatest IC₅₀The ratio of prodrug of (prodrug 2) is about 9.6. Difference in disulfide prodrugs was observed in both high GSH cell lines (KPL-4) and low GSH cell lines (WSU-DLCL2)And (4) activating. Less difference was observed for the KPL-4 cell line compared to the WSU-DLCL2 cell line.

In addition to efficacy assessments, the release of PBD dimer diaphorase prodrug 1 was also assessed. The release was measured as 12% after 90min incubation according to the following method. First, a master mix reaction containing 0.5uM of the PBD prodrug, 50uM of PBD, 200mM NADPH was prepared in a 96-well polypropylene plate, quenched and diluted with 40uL of 0.1% formic acid. Next, the reaction mixture was incubated at room temperature for 5, 30, 90 min. Third, standards in DMSO were prepared in triplicate in 96-well polypropylene plates using the ECHO sonic pipetting technique (Labcyte Inc, Sunnyvale, CA) in 0.1% formic acid using a 1:2.5x series of dilutions to generate a 10-point dose-response curve. Fourth, 10uL of sample and standard were injected into AB SCIEX combined with Waters liquid chromatography6500 mass spectrometer. The LC gradient used was Phenomenex Kinetex C18, 1.7 μm,100X2.1 mm column, mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) 0-0.5min 5% B, 1-1.10min 70-95% B, 2.49-2.5min 95% B, 2.5-3.0min 95-5% B, flow rate of 0.8mL/min (column temperature 30 ℃). The retention time was 1.06 min. Multiple Reaction Monitoring (MRM) in MS was switched to (585/257.0) and (585/504.2). The compound-dependent MS parameters of MRM (585/257.0) were CE, CXP, DP and EP for 51, 20, 206 and 10, respectively, and the parameters of MRM (585/504.2) were CE, CXP, DP and EP for 27, 12, 206 and 10, respectively. Finally, the data were analyzed using multisant analysis software and the criteria were calculated using GraphPad Prism 6 linear regression fitting.

Example 7: toxicity of PBD monomeric disulfide prodrugs to SK-BR-3 cell line

Toxicity of PBD monomer disulfide prodrugs 1 to 4, PBD dimer disulfide prodrugs 1 and 4, PBD monomer control 1, and PBD dimer control 1 on SK-BR-3 cell cultures was evaluated. The SK-BR-3 cell line is a breast cancer cell that overexpresses HER2 and exhibits GSH levels of about 3 to about 10 mM. After verification by sequence analysis, a HER2 positive breast cancer cell line SK-BR-3 was obtained internally to Genentech. Cells were grown in DMEM + 10% FBS supplemented with 2mM L-glutamine at 37 degrees celsius for the duration of the assay. On the first Day of the experiment, designated "Day-1 (Day-1)", SK-BR-3 cells were harvested from tissue culture flasks, counted, and then seeded into 96-well plates at a concentration of 7,500 cells per well. The next day, designated "day 0", the cell culture medium was removed from the cells and replaced with 100uL of fresh medium containing serial dilutions (1:3) of the compounds in table 5 below. The plate was then returned to the 37 ℃ incubator and allowed to incubate for 3 days. On the "day 3" of the assay, the plate was removed from the incubator and allowed to equilibrate to room temperature, then 100uL of room temperature CellTiter-Glo reagent (Promega) was added. The plate was then stirred at 300rpm for 10 minutes. The luminescence signal generated by this reaction was recorded on an Envision microplate reader (Perkin-Elmer). Data were arranged in excel (microsoft) and plotted in kaleidagraph (synergy software).

IC₅₀The efficacy results are presented in table 5 below:

TABLE 5

PBD agents	SK-BR-3IC50(nM)
		PBD monomer control 1	124
PBD monomeric disulfide prodrug 1	331
		PBD monomeric disulfide prodrug 3	616
PBD monomeric disulfide prodrug 2	1757
		PBD monomeric disulfide prodrug 4	>10,000
PBD dimer control 1	0.144
		PBD dimer disulfide prodrug 1	2.39
PBD dimer disulfide prodrug 4	3.09

The results for SK-BR-3 are depicted in fig. 3 and 10, where the graphs are a plot of cell viability (% of control) versus PBD compound concentration (nM). Based on the results, it is believed that the more bulky disulfide prodrug moiety has reduced potency. Furthermore, based on experimental results to date, it is believed that potency correlates with disulfide stability.

Example 8: whole blood stability of PBD monomer and dimer prodrugs

The whole blood stability of various PBD monomer and PBD dimer prodrugs of the present invention was evaluated.

Whole blood stable samples were prepared as follows. Stability samples were generated in mouse (CB17SCID), rat (Sprague-Dawley), cynomolgus Monkey (Cynomologus Monkey) and human whole blood plasma as well as buffer (0 and 24 hours). Blood was collected by bioremodelling and then cold transported overnight and samples were generated immediately upon arrival of whole blood. To generate stability samples, initial dilutions of the source material were prepared in buffer (1 × PBS, 0.5% BSA, 15PPM Proclin) so that all molecules were at a concentration of 1 mg/mL. A1: 10x dilution (36uL of 1mg/mL initial dilution +324uL of blood or buffer) was then performed to generate a final concentration of 100ug/mL of stability sample. Once mixed, 150 μ Ι _ of whole blood/buffer stable sample was aliquoted into two separate sets of tubes for two different time points. The 0 hour time point was then placed in a-80 ℃ freezer, while the 24 hour time point was placed on a shaker in a 37 ℃ incubator. When the 24 hour samples reached the given time point, they were also placed in a-80 ℃ freezer.

Whole blood stability samples were evaluated by affinity capture LC-MS assay. First, streptavidin-coated magnetic beads (Life Technologies Corporation, Grand Island, NY) were washed 2X with HBS-EP buffer (GEHealthcare, Sunnyvale, Calif.), then mixed with biotinylated CD22 anti-idiotypic antibody using KingFisher Flex (Thermo Fisher scientific, Waltham, Mass.) and incubated for 2 hours at room temperature with gentle stirring. After 2 hours, the SA-bead/biotin-xId Ab complex was washed 2x with HBS-EP buffer, mixed with diluted whole blood stability samples, and then incubated for 2 hours at room temperature with gentle stirring. After 2 hours, the SA-bead/biotin-xidb/sample complex was washed 2x with HBS-EP buffer, mixed with deglycosylase PNGase F (New England BioLabs, Ipswich, MA), and incubated overnight at 37 ℃ with gentle stirring. After overnight incubation, deglycosylated SA-bead/biotin-xId Ab/sample complex was washed 2X with HBS-EP buffer, then with water (Optima H)₂O, FisherScientific, Pittsburgh, PA) 2x and finally 1x with 10% acetonitrile. The beads were then eluted in 30% acetonitrile/0.1% formic acid where they were incubated at room temperature with gentle agitation for 30min before collecting the beads. The eluted sample was injected and loaded onto a Thermo Scientific PepSsoft RP monolith (500 μm. times.5 cm) maintained at 65 ℃. The samples were separated on the column using a Waters Acquity UPLC system at a flow rate of 20 μ L/min using the following gradient: 20% B (95% acetonitrile + 0.1% formic acid), 0-2 min; 35% B, 2.5 min; 65% B,5 min; 95% B, 5.5 min; 5% B, 6 min. This column was combined with a Waters Synapt G2-S Q-ToF mass spectrometer operating at positive ESI with a mass number range of 500 to 5000Th (m/z) collected for on-line detection.

The in vitro whole blood stability of PBD monomeric disulfide prodrugs 2 through 6, 8, 10 and 13 and PBD monomeric control 1 were evaluated at 4 hour and 24 hour intervals in human and rat. The results are presented in figure 4 as the percentage of parent compound remaining relative to time 0.

The in vitro whole blood stability of PBD dimer ADC boronic acid prodrug 1, PBD dimer ADC diaphorase prodrug 1B, and PBD dimer ADC disulfide prodrug 1, 2A, 2B, and 3 to 5 was evaluated in mice, rats, cynomolgus monkeys, and humans and the results are presented in table 6 below, where "DAR" refers to the ratio of drug to antibody, "Δ% DAR 2" refers to Δ DAR2 at 24 hours relative to buffer at 0 hours, and "prodrug loss" refers to prodrug loss at 24 hours relative to buffer at 0 hours.

Table 6:

example 9: toxicity of PBD monomeric disulfide prodrugs and PBD dimeric disulfide prodrugs to various cell lines

Toxicity of various PBD monomeric disulfide prodrugs and PBD dimeric disulfide prodrugs were evaluated on UACC-257, Igrov-1 and A2058 cell lines. Seeding the cells in 384-well plates andand treated with the drug 24 hours later. After 4 days of drug incubation, cell viability was determined using Promega CellTiter-Glo luminescence reagent, which measures ATP levels (an indirect measure of cell number). The luminescence intensity was measured on a PerkinElmer Envision reader. Relative cell viability was calculated by normalization to non-drug treated controls and plotted using the KleidaGraph software package. IC (integrated circuit)₅₀Values were determined as the concentration that achieved 50% of the maximum cell killing.

IC₅₀The results are presented in table 7A below:

TABLE 7A

PBD agents	UACC-257	Igrov-1	A2058
				PBD monomer control 1	181.4	68.5	56.9
PBD monomer control 2	>20,000	>20,000	>20,000
				PBD monomeric disulfide prodrug 1	275.7	122.8	82.9
PBD monomeric disulfide prodrug 2	1858.8	1871.8	635.2
				PBD monomeric disulfide prodrug 3	356.0	253.9	75.2
PBD monomeric disulfide prodrug 4	19164.1	12640.1	8109.9
				PBD monomeric disulfide prodrug 5	892.1	765.1	395.3
PBD monomeric disulfide prodrug 6	497.4	3262.3	566.6
				PBD monomeric disulfide prodrug 7	>30,000	>30,000	10956
PBD monomeric disulfide prodrug 8	194.1	171.8	47.2
				PBD monomeric disulfide prodrug 9	2109.1	1046.9	291.8
PBD monomeric disulfide prodrug 10	4732.3	4807.6	1827.9
				PBD monomeric disulfide prodrug 11	331.1	206.7	108.3
PBD monomeric disulfide prodrug 12	155.8	83.6	31.1
				PBD monomeric disulfide prodrug 13	>10,000	>10,000	>10,000
PBD monomeric disulfide prodrug 15	1500.2	928.2	304.2
				PBD monomeric disulfide prodrug 16	19460.1	18509.1	5593.4
PBD monomeric disulfide prodrug 19	147.3	77.4	37.7
				PBD monomeric disulfide prodrug 22	80.6	39.8	23.7
PBD monomeric disulfide prodrug 23	866.2	687.2	206.3
				PBD monomeric disulfide prodrug 24	2694.2	1479.9	596.1
PBD monomeric disulfide prodrug 25	241.6	129.4	47.7
				PBD monomeric disulfide prodrug 26	328.3	245.8	84.5
PBD monomeric disulfide prodrug 27	939.1	957.9	261.2
				PBD monomeric disulfide prodrug 28	2102.2	2304.1	920.1
PBD monomeric disulfide prodrug 29	142.1	57.8	29.1
				PBD monomeric disulfide prodrug 30	307.6	234.8	107.6
PBD dimer control 1 (in GSH cell group)	0.95	0.061	----
				PBD dimer disulfide prodrug 1	2.2	0.21	----
PBD dimer disulfide prodrug 4	7.8	2.5	----

It should be noted that the IC of each prodrug compared to the corresponding control can be calculated₅₀A ratio. For example, IC for cell line UACC-257 and IGROV-1, PBD dimer disulfide prodrug 1₅₀The ratios were 2.3 and 3, respectively.4, the calculation is as follows: (PBD dimer disulfide prodrug 1UACC-257IC₅₀2.2)/(PBD dimer control 1UACC-257IC₅₀0.95) ratio 2.3); and (PBD dimer disulfide prodrug 1IGROV-1 IC)₅₀0.21)/(PBD dimer control 1IGROV-1IC₅₀0.061) ═ ratio 3.4).

PBD dimer control 1 and PBD dimer control 2 were further evaluated against a variety of cell lines in a standard cell group. IC50 results are reported in table 7B below.

Table 7B:

cell lines	PBD dimer control 1	PBD dimer control 2
			MES-SA	0.028	10.0
MES-SA/Dx5	0.56	>100
			BJAB	0.015	4.9
BJAB/Pgp	----	53.7
			KPL-4	0.053	67.4
HCC1569X2	0.097	----
			T-47D	0.032	----
HCC1937	0.15	----
			NCI-H1781	0.011	----
SW 900	0.078	----
			MDA-MB-231	----	73.2
HCT 116	----	15.0
			A2058	----	5.3
DLD-1	----	>100
			HL-60	----	2.4

The results for PBD monomer disulfide prodrug 2,3,5, 6, PBD dimer disulfide prodrug 1 to 3, PBD monomer control 1 and PBD dimer control 1 are depicted in fig. 5 and the results for KPL-4 are depicted in fig. 6, where each graph is a plot of cell viability (% of control) versus PBD compound concentration (μ g/mL). Differential disulfide prodrug activation was observed in both the high GSH cell line (KPL-4) and the low GSH cell line (WSU-DLCL 2). Less difference was observed for the KPL-4 cell line compared to the WSU-DLCL2 cell line.

The results for PBD dimer control 1 against UACC-257 and IGROV-1 cell lines are depicted in FIG. 7. The results for PBD dimer disulfide prodrug compound 1 against UACC-257 and IGROV-1 cell lines are depicted in FIG. 8. The results for PBD dimer disulfide prodrug compound 4 against UACC-257 and IGROV-1 cell lines are depicted in FIG. 9.

Example 10: toxicity of PBD dimer disulfide prodrug-antibody conjugates to SK-BR-3 and KPL-4 cell lines

Various PBD dimer ADC disulfide prodrugs, PBD dimer ADC diaphorase prodrugs, and non-prodrug PBD dimer ADC controls were evaluated for toxicity to KPL-4 and SK-BR-3 cell cultures. Cells were plated in black-walled 96-well plates (4000 per well for SK-BR-3; 1200 cells per well for KPL-4) and allowed to stand at 37 ℃ in 5% CO₂Was attached overnight in a humid atmosphere. The medium was then removed and replaced with fresh medium containing different concentrations of ADC. After 5 days, CellTiter-Glo reagent was added to the wells for 10min at room temperature and luminescence signal was measured using PerkinElmer EnVision.

IC₅₀The results are presented in Table 8 below：

TABLE 8

The results for PBD dimer ADC disulfide prodrug 2A, PBD dimer ADC disulfide prodrug 1B, non-prodrug PBD dimer ADC control 1, and non-prodrug PBD dimer ADC control 2 for SK-BR-3 are depicted in fig. 11 and the results for KPL-4 are depicted in fig. 12, where each graph is a plot of cell viability after 5 days (% of control) versus PBD compound concentration (μ g/mL). Differential activation of disulfide prodrugs by HER2 conjugates was observed in HER2 cell line.

Example 11: toxicity of PBD dimer disulfide prodrug-antibody conjugates to SK-BR-3 and KPL-4 cell lines

PBD dimer ADC disulfide prodrugs 1, 2B, 3 and 4 and non-prodrug PBD dimer ADC controls 1 and 2 were evaluated for toxicity to SK-BR-3 and KPL-4 cell lines for cell viability after 5 days. Cells were plated in black-walled 96-well plates (4000 per well for SK-BR-3; 1200 cells per well for KPL-4) and allowed to stand at 37 ℃ in 5% CO₂Was attached overnight in a humid atmosphere. The medium was then removed and replaced with fresh medium containing different concentrations of ADC. After 5 days, CellTiter-Glo reagent was added to the wells for 10min at room temperature and luminescence signal was measured using PerkinElmer EnVision.

The results are reported in fig. 13 and 14.

Example 12: whole blood stability of PBD dimer-antibody conjugate disulfide prodrugs

Stability of PBD dimer ADC disulfide prodrug 1 and 2B was evaluated in buffer ("buffer"), cynomolgus whole blood ("cynomolgus WB"), human whole blood ("human WB"), mouse whole blood ("mouse WB"), and rat whole blood ("rat WB"). The experimental protocol is described in example 8. The results are reported in table 9 below, where the percent loss refers to the total loss of prodrug compared to the prodrug concentration at time 0. The loss of prodrug includes loss of imine form (i.e., the hydroxyl moiety at C11 adjacent to the substitution point of the N10 prodrug).

TABLE 9

Example 13: toxicity of ROS-activated PBD dimer-antibody conjugate aryl boronic acid prodrugs

Toxicity of various PBD monomer and dimer boronic acid prodrug compounds were evaluated for cytotoxicity. In an in vitro tumor cell killing assay, cells were added to each well of a 96-well microtiter plate at 8,000 cells per well and at 37 ℃ in 5% CO₂Was incubated overnight in a humid atmosphere. Cells were exposed to various concentrations of the indicated prodrug, positive control, and negative control compounds. In the case of the silvestrol evaluation, 1:3 serial dilutions were used. After 3 days of incubation, Cell titer-Glo reagent (Promega, Madison WI) was added to wells at 100 μ L per well, followed by incubation at room temperature for 10 minutes and measurement of luminescence signal using a Packard/Perkin-Elmer TopCount.

The toxicity of PBD dimer ADC boronic acid prodrugs 1A and 1B on WSU-DLCL and BJAB tumor cell lines was evaluated for cell viability. PBD dimer ADC boronic acid control 1A was used as a negative control and PBD dimer ADC boronic acid control 2 was used as a positive control. Results are reported in fig. 15A, 15B and 16 as normalized percentage of viable cells after 3 days compared to the number of cells at time 0 relative to dose (μ g/mL). PBD dimer ADC boronic acid prodrug 1A provided an EC of 1.701nM₅₀And non-prodrug PBD dimer ADC control 2 provided an EC of 0.0236nM₅₀. PBD dimer ADC boronic acid prodrug 1A provided an EC of 0.0509nM₅₀PBD dimer ADC boronic acid prodrug 1B provided an EC of 15.86nM₅₀And PBD dimer ADC boronic acid control 2 provided an EC of 0.0274₅₀。

The toxicity of PBD monomeric boronic acid prodrug 1 on MDA-MB-453 tumor cell line was evaluated for cell viability. PBD monomer control 1 was used as a positive control and PBD monomer control 2 was used as a negative control. The toxicity study was repeated, with the PBD prodrug and control administered in combination with silvestrol. FIG. 15C depicts a graph of MDA-MB-453 cell killing versus drug concentration (μ M) 3 days after exposure to: (i) PBD monomer control; (ii) a PBD monomer control having a benzyl formate moiety at the N10PBD position; and (iii) a PBD monomeric boronic acid prodrug. FIG. 15D depicts a graph of MDA-MB-453 cell killing versus drug concentration (μ M) 3 days after exposure to: (i) silvestrol; (ii) PBD monomer control; (iii) a PBD monomer control having a benzyl formate moiety at the N10PBD position; (iv) PBD monomeric boronic acid prodrugs; (v) PBD monomer control and silvestrol; (vi) a PBD monomer control having a benzyl formate moiety at the N10PBD position and silvestrol; and (vii) PBD monomeric boronic acid prodrug and silvestrol. PBD monomer control 1 provided an EC of 0.1635nM₅₀PBD monomer boronic acid prodrug 1 provided an EC of 1.846nM₅₀And PBD monomer prodrug 2 provided an EC of 267,356nM₅₀。

The results indicate that ROS arylboronic acid prodrug provides increased cell killing relative to the negative control.

Example 14: efficacy of anti-CD 22 and anti-Her 2 antibody drug conjugates

The efficacy of anti-CD 22 antibody-drug conjugates (ADCs) was studied in a mouse xenograft model of BJAB-luc human burkitt lymphoma. The BJAB-luc cell line was obtained from the Genentech cell line bank. This cell line was identified by Short Tandem Repeat (STR) profiling using Promega PowerPlex16 system and compared to the external STR profile of the cell line to determine cell line progenitors. The BJAB-luc cell line expresses CD22 as determined by FACS and IHC. To establish the xenograft model, female C.B-17SCID mice (Charles river laboratories) were each inoculated subcutaneously in the flank region with BJAB-luc cells (2000 ten thousand cells suspended in 0.2mL Hank's balanced salt solution (Invitrogen)).

The efficacy of anti-Her 2 antibody-drug conjugates (ADCs) was studied in a mouse xenograft model of KPL4 human breast cancer. The KPL4 cell line was obtained from dr.j. kurebayashi laboratory (japan), and this cell line expresses HER2 as determined by FACS and IHC. To establish the xenograft model, female c.b-17SCID-beige mice (Charles river laboratories) were each inoculated with KPL4 cells (300 ten thousand cells suspended in a 1:1 mixture of 0.2mL of Hank's balanced salt solution (Invitrogen) and Matrigel (Matrigel) (BD Biosciences) in the region of the thoracic breast fat pad. KPL4 xenografts are a model for inducing cachexia, in which animals lose about 5% of their initial body weight in response to the tumor itself. Administration of anti-Her 2ADC attenuated this tumor-associated weight loss and was well tolerated in animals.

When the tumor reaches 100-300mm³At the mean tumor volume, animals were randomly divided into groups of 5-10 mice each and received a single intravenous injection of ADC (referred to as day 0). In a first assessment as depicted in fig. 23 and 24, mice were treated with vehicle (histidine buffer #8, 100 μ Ι _ IV once), non-prodrug PBD dimer ADC control 2(0.1mg/kg and 0.2mg/kg IV once), PBD dimer ADC boronic acid prodrug 1A (0.2, 0.5, 1,2, and 5mg/kg IV once), PBD dimer ADC boronic acid control 1A (1mg/kg IV once), PBD dimer ADC boronic acid prodrug 1B (1mg/kg IV once), and non-prodrug PBD dimer ADC control 3(0.2 and 1mg/kg IV once). In a second assessment depicted in fig. 25 and 26, mice were treated with vehicle (histidine buffer #8, 100 μ Ι _ IV once), non-prodrug PBD dimer ADC control 1(0.3, 1, and 3mg/kg IV once), and PBD dimer ADC disulfide prodrug 1B (1, 3, 6, and 10mg/kg IV once). Throughout the study, mice were measured for tumors and body weights 1-2 times a week. Mice were immediately euthanized when weight loss was > 20% of their initial body weight. When the tumor reaches 3000mm³Or show thatAll animals were euthanized before signs of ulceration occurred. Tumor volume was measured in two dimensions (length and width) using calipers and calculated using the formula: tumor size (mm3) is 0.5x (length x width).

The results are presented in fig. 23 to 26.

FIG. 23 shows the efficacy of anti-CD 22ADC in C.B-17SCID mice with BJAB-luc human Burkitt's lymphoma. The highest dose evaluated (0.2mg/kg) of non-prodrug PBD dimer ADC control 2 resulted in only a moderate delay in tumor growth. In contrast, PBD dimer ADC boronic acid prodrug 1A was very effective and pushed tumor regression at doses as low as 0.2 mg/kg. PBD dimer ADC boronic acid control 1 did not show any effect on tumor growth. PBD dimer ADC boronic acid prodrug 1A and non-prodrug PBD dimer ADC control 3 conjugate have some antitumor activity; however, the activity of the anti-CD 22 conjugate was more excellent at matching dose levels.

Figure 24 shows the effect of anti-CD 22ADC (1) non-prodrug PBD dimer ADC control 2, (2) PBD dimer ADC boronic acid prodrug 1A, and (3) PBD dimer ADC boronic acid control 1 on body weight of c.b-17SCID mice with BJAB-luc human burkitt lymphoma. Administration of anti-CD 22ADC was well tolerated in animals in which no weight loss was observed.

FIG. 25 shows the efficacy of anti-Her 2ADC in C.B-17SCID-beige mice bearing KPL-4 human breast tumors. Non-prodrug PBD dimer ADC control 1 exhibited dose-dependent inhibition of tumor growth and tumor regression at 1mg/kg or higher. Similarly, PBD dimer ADC disulfide prodrug 1 also showed dose-dependent efficacy and tumor regression at 6mg/kg or higher.

FIG. 26 shows the effect of non-prodrug PBD dimer ADC control 1 and PBD dimer ADC disulfide prodrug 1 (anti-Her 2ADC) on body weight of C.B-17SCID-beige with KPL4 human breast tumor. KPL4 xenografts are a model for inducing cachexia, in which animals lose about 5% of their initial body weight in response to the tumor itself. Administration of anti-Her 2ADC attenuated this tumor-associated weight loss and was well tolerated in animals.

Example 15: toxicity of DTD-activated PBD monomer and dimer quinone prodrugs

Toxicity of PBD dimer diaphorase prodrug 1, PBD monomer diaphorase prodrug 2, PBD dimer control 1 and PBD monomer control 1 on KPL-4 and WSU cell lines was evaluated for cell viability. The KPL-4 cell line is a high DTD cell line characterized by a NQO1nRPKM of 839, and the WSU cell line is a low DTD cell line characterized by a NQO1nRPKM of 1.36. IC (integrated circuit)₅₀Ratios are based on prodrug IC versus PBD dimer control₅₀The value is obtained.

The results are reported below in table 10 and fig. 17 to 20.

Watch 10

Example 16: toxicity of DTD-activated PBD monomer and dimer quinone prodrugs

The toxicity of PBD dimer ADC diaphorase prodrugs 1A and 1B and non-prodrug PBD dimer ADC control 5 on KPL-4 and SK-BR-3 cell lines was evaluated for cell viability. The KPL-4 cell line is a high DTD cell line characterized by NQO1nRPKM of 839, and the SK-BR03 cell line is a low DTD cell line characterized by NQO1nRPKM of 171. The results are reported below in fig. 21 and 22.

Example 17: disulfide cleavage and DNA oligonucleotide binding of PBD analogs

Cleavage of various prodrug disulfide compounds of the present disclosure after 24 hours of exposure to cysteine and Glutathione (GSH) was evaluated, and DNA binding of PBD analogs was evaluated.

For disulfide cleavage determination, compounds were incubated at 15 μ M with 0.2mM cysteine or 4mM GSH in 100mM Tris buffer (pH 7.0) containing 5% methanol at 37 ℃. Aliquots were taken at the indicated time points and the samples were analyzed by LC/MS on a Hypersil Gold C18 column (100X2.1, 1.9. mu.M, Thermo Scientific) on a Sciex TripleTOF 5600. The column was eluted at 0.4mL/min by a gradient of buffer A (0.1% formic acid in 10mM ammonium acetate) to buffer B (0.1% formic acid in 10mM ammonium acetate in 90% acetonitrile), 5% B0-0.5 min, 5-25% B0.5-8 min, 25-75% B8-13 min, and 75-95% B13-13.5 min, 95% B13.5-14.5 min, 95-5% B14.5-15 min. All products were isolated and characterized by LC/MS/MS in positive ESI ion mode. All analytes had the protonated molecule MH + as the major species, and few source fragments. The full scan accurate mass peak area is used to estimate the relative abundance of each component.

For DNA binding determination, compounds were incubated at 100. mu.M for 1 hour at 37 ℃ in 10mM bis-Tris (pH 7.1) with 100. mu.M double stranded DNA oligonucleotides 1 and 2. Single-stranded DNA oligonucleotides (5'-TATAGAATCTATA-3' and 3 '-ATATCTTAGATAT-5') were synthesized in Genentech. Samples were analyzed by LC/MS/UV (210-. The column was eluted at 0.4mL/min by a gradient of buffer A (50mM hexafluoroisopropanol and 15mM diisopropylethylamine) to buffer B (50% A and 50% 1:1 methanol: acetonitrile), 5% 0-0.5min, 5-25% B0.5-25 min, 25-95% B25-40min, and 95% B40-42 min. The remaining% is the average of the starting DNA oligonucleotides remaining in the incubation (n-2). The product was characterized by LC/MS in negative ESI ion mode.

The results are reported in table 11 below.

Table 11:

example 18: formation of GSH adducts and quinone stability

The stability of quinones and PBD monomers and dimeric diaphorase prodrugs within the scope of the present disclosure upon exposure to GSH and the formation of related GSH adducts were evaluated.

For degradation analysis, compounds were incubated at 25 μ M with 15mM GSH in 200mM Tris buffer (pH 7.0) containing 5% methanol at 37 ℃ for 3 hours. Control incubations were performed without GSH. Samples were analyzed by LC/MS on a Hypersil gold C18 column (100X2.1, 1.9. mu.M, Thermo Scientific) on a Sciex TripleTOF 5600. The column was eluted at 0.4mL/min by a gradient of buffer A (0.1% formic acid in 10mM ammonium acetate) to buffer B (0.1% formic acid in 10mM ammonium acetate in 90% acetonitrile), 5% B0-0.5 min, 5-25% B0.5-8 min, 25-75% B8-13 min, and 75-95% B13-13.5 min, 95% B13.5-14.5 min, 95-5% B14.5-15 min. All products were isolated and characterized by LC/MS/MS in positive ESI ion mode. The full scan accurate mass peak area is used to estimate the relative abundance of each component.

In vitro DT diaphorase activated drug release assay was used to measure NADPH loss. DT diaphorase-activated norfloxacin and Payload (PBD) release was measured by in vitro depletion of NADPH. The assay method was improved by absorbance measurements of NADPH at A340 (Osman et al, Chemico-Biological Interactions 147(2004)99-108) due to compound interference. The loss of NADPH was measured by monitoring the decrease in fluorescence intensity of NADPH at 480nM after excitation at 340 nM. Materials and reagents were as follows: (1) bovine Serum Albumin (BSA): sigma catalog number A7030-50G (≧ 98%(agarose gel electrophoresis), lyophilized powder, substantially free of fatty acids, substantially free of globulin, (2) assay buffer 50mM Tris-HCl/0.007% BSA buffer (pH 7.4), (3) DT diaphorase preparation by dissolving lyophilized human DT diaphorase (Sigma Cat. No. D1315, lot SLBJ9723V, MW 32,253U/mg, 1.6mg protein/vial, cytochrome C reduced by 1 micromole per minute in one unit in the presence of menadione substrate at 37 ℃ in 8.1mL H2O as 50U/0.2mg/mL (6.25uM protein), storing aliquots at-20 ℃ prior to assay, diluting the stock solution 2.5 fold in assay buffer as 5X working solution (20U/mL, 2.5uM, 0.08; ug/mL) nicotinamide (4) β -adenine dinucleotide phosphate (NADPH, sodium salt, 655-phosphate dihydrate, Sigma G-655,>94% (HPLC): a48 mM stock of NADPH was prepared in assay buffer and stored at-20C. Prior to assay, stock was diluted to 1mM as a 5X working solution; (5) DT diaphorase specific inhibitors: a 40mM stock of dicoumarin (Sigma, cat # M1390-5G, MW 336.29) was prepared in 0.13N NaOH and stored at 4 ℃. Diluting the stock solution to 250uM to be used as a 5X working solution; (6) a compound: norfloxacin and PBD conjugate compounds were diluted to 250uM in assay buffer as 5X working solution; and (7) 384 well black plates with transparent bottom. For the assay procedure: (1) the reaction mixture containing 0.5uM DT-D, 50uM compound, 200mM NADPH was placed in 384 well plates at 50 ul/well. For the control with DT diaphorase inhibitor dicoumarin was added to the reaction mixture, with a final 50uM of each compound. Reaction mixtures containing only NADPH and compound were also added as baseline controls. DT diaphorase was added in the last step; and (2) the reaction mixture was incubated at room temperature for 5, 30, 90 min. The fluorescence intensity (RFU) of DNAPH was recorded on a M1000 plate reader (Tecan) with excitation at 340nM and emission at 480 nM. For data analysis, data was analyzed and plotted using prism graphpad 6. The NADPH loss at the reaction time point of 90min was calculated by the following formula, where: % NADPH loss ═ RFU_{Non-inhibitor}-(RFU_{Has an inhibitor}/RFU_{Non-inhibitor})]*100。

The results are reported in table 12 below, wherein: "remaining%" means the percentage remaining after 3 hours; "degradation (Degr)" means degradation; "payload release (pay. rel)" refers to the release of payload (MRM quinat) based on 2 μ M starting material (% recovery at 90-min versus 0 hours); and "NADPH depletion (NADPH Dep.)" refers to NADPH depletion at 90 minutes.

Table 12:

example 19: preparation of PBD monomeric disulfide prodrugs

Example 19 general scheme 1

The overall reaction scheme of general scheme 1 is as follows:

example 19 general scheme 1, step 1:

to Compound 1(1.13kg, 4.59mol, 1.00 mu m) in two portions at 0 deg.CAmount) to a solution in THF (10L) LiBH was added₄(99.90g, 4.59mol, 1.00 eq) (in LiBH₄With little temperature change during the addition). The suspension was stirred at 0 ℃ for 1h and then at 10-20 ℃ for 18 h. The mixture was cooled to 0 ℃ and NH was added₄Aqueous Cl (5L). The layers were separated and the aqueous layer was extracted with EA (5Lx 3). The combined organics were washed with brine. Subjecting the organic layer to Na₂SO₄Dried, filtered and concentrated to give compound 2 as a clear oil (1600g, 7.36mol, 80.22% yield).

Example 19 general scheme 1, step 2:

A50-L flask was charged with Compound 2(1.60kg, 7.36mol, 1.00 eq), DCM (20L), and then TEA (1.12kg, 11.05mol, 1.50 eq) and acetyl chloride (635.54g, 8.10mol, 1.10 eq) were added dropwise with stirring at 0 ℃. After addition, the resulting solution was stirred at 15-25 ℃ for 18h, quenched by the addition of 5L water and extracted with 3x2L of DCM. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo to afford compound 3 as a colorless oil (2.46kg, 9.49mol, 128.90% yield).

Example 19 general scheme 1, step 3:

A20L 3-neck round bottom flask was charged with Compound 3(1.23kg, 4.75mol, 1.00 eq.) in DCM (12L) and PCC (1.54kg, 7.13mol) was added in portions at 15 ℃. The resulting solution was stirred at 15-25 ℃ for 18 h. The solid was filtered off and the filtrate was concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate-petroleum ether (1:5) to give compound 4(1.13kg, 4.38mol, 46.15% yield) as a pale yellow liquid.

Example 19 general scheme 1, step 4:

A10-L3-necked round-bottomed flask was charged with methyl (triphenyl) phosphonium bromide (958.03g, 2.68mol), THF (2.5L), and then t-BuOH (300.94g, 2.68mol) was added portionwise over 2h at 0 ℃. To this was added dropwise a solution of compound 4(460.00g, 1.79mol) in THF (2.5L) with stirring at 0 ℃. The resulting solution was stirred at-5-0 ℃ for 20min, quenched by addition of 500mL water and extracted with 3 × 500mL ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate-petroleum ether (1:20) to give compound 5(275.00g, 1.08mol, 30.09%) as a pale yellow liquid.

Example 19 general scheme 1, step 5:

a mixture of compound 5(330.00g, 1.29mol) in HCl (gas)/EtOAc (3L, 4M/L) was stirred at 0 ℃ for 20 min. The mixture was then stirred at 10-30 ℃ for 1 h. The mixture was concentrated in vacuo to give compound 6(250.00g, 1.30mol, 101.12%) as a yellow solid, which was used in the next step without purification.

Example 19 general scheme 1, step 6:

A3000-mL 3-necked round-bottomed flask purged with and maintained under an inert atmosphere of nitrogen was charged with a solution of Compound 7(354.42g, 1.56mol, 1.30 equiv.) in THF (1.5L), then SOCl was added dropwise with stirring₂(1.71kg, 14.33mol, 11.94 eq.). The resulting solution was stirred at 20-30 ℃ for 4h and then concentrated in vacuo. Another 3000-mL 3-necked round bottom flask, purged and maintained with a nitrogen inert atmosphere, was charged with a solution of Compound 6(230.00g, 1.20mol, 1.00 eq.) in DCM (2.5L). Et was added dropwise thereto at-40 ℃ with stirring₃N (485.75g, 4.80mol, 4.00 eq) was then added to the solution in the first flask at-40 ℃. The temperature was allowed to warm to 0 ℃ naturally, quenched by the addition of 3000mL of water/ice, and extracted with 3 × 1000mL dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with EtOAc: PE (1:3) to give compound 8(210.00g) as a light brown oil, which was used in the next step without purification.

Example 19 general scheme 1, step 7:

to a mixture of compound 8(90.00g, 247.02mmol, 1.00 equiv.) in THF (400mL), MeOH (100mL), H2O (400mL) was added NaOH (29.64g, 741.05mmol, 3.00 equiv) all at once at 0 ℃. The mixture was stirred at 20-30 ℃ for 18 h. The aqueous phase was extracted with EtOAc (300mLx 3). The combined organic phases were washed with saturated brine (100mL) and anhydrous Na₂SO₄Dried, filtered and concentrated in vacuo to afford compound 9(90.26g, crude product) as a yellow solid, which was used in the next step without further purification.

Example 19 general scheme 1, step 8:

in a 2000mL three-necked round bottom flask equipped with a temperature probe, magnetic stirrer and nitrogen inlet was added TBDMSCl (126.62g, 840.12mmol), imidazole in DMF (1L) (57.20g, 840.12mmol, 3.00 equiv). A solution of compound 9(90.26g, 280.04mmol, 1.00 eq.) in DMF (1L) was then added to the mixture at 0 ℃. The resulting reaction mixture was stirred at 25-30 ℃ for 2 h. The reaction mixture was poured into ice water (1L) and then extracted with DCM (200mLx 3). The combined organic phases were washed with brine (100mL) and Na₂SO₄Dried and concentrated in vacuo to give the residue to give compound 10(126.00g) as a yellow oil, which was used in the next step without further purification.

Example 19 general scheme 1, step 9:

to a mixture of compound 10(126.00g, 288.61mmol, 1.00 equiv.) in AcOH (1L) was added Zn (188.72g, 2.89mol) in portions by maintaining a temperature below 30 ℃. The mixture was stirred at 20-30 ℃ for 30 min. The residue was poured into EtOAc (500mL) and filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (PE/EtOAc ═ 10/1, 1/1) to give 11as a yellow oil (58.00g, 142.64mmol, 49% yield). 1H NMR (400MHz, chloroform-d) d ppm 6.71(s,1H)6.22(s,1H)4.85-4.97(m,2H)4.52(br.s.,1H)4.14-4.23(m,1H)3.99-4.13(m,1H)3.82(s,3H)3.77(s,3H)3.59(d, J ═ 5.73Hz,1H)2.63-2.72(m,2H)2.01-2.04(m,1H)1.23(t, J ═ 7.06Hz,1H)0.85(s,9H) -0.06-0.06(m, 5H).

Example 19 general scheme 2 below is a general scheme for preparing PBD disulfide prodrugs of the present disclosure:

asterisks in structure C and elsewhere depicted in example 19 represent chiral centers. In some aspects, R as recited in the schemes above¹Corresponds to R as described herein⁶¹R as cited in the above scheme²Corresponds to R as described herein⁶²And R as cited in the above schemes³Corresponds to R as described herein⁵⁰。

Example 19A: preparation of PBD monomeric disulfide prodrug 10

Example 19A general procedure IA-carbonate formation method a:

coupling (2-amino-4, 5-dimethoxy-phenyl) - [ (2S) -2- [ [ tert-butyl (dimethyl) silyl)]Oxymethyl radical]-4-methylene-pyrrolidin-1-yl]Methanone (222mg, 0.5460mmol) was dissolved in 3mL of DCM and Et was added by pipette₃N followed by the addition of triphosgene. After a total of 20min, 2- (tert-butyldisulfanyl) -2-methyl-propan-1-ol (B, 1.05 eq, 0.5733mmol, 100 mass%) in 2mL of DCM (including 0.5mL of the rinse) was added, followed by 10uL of dibutyltin diacetate (20 uL, 0.07487 mmol). After about 1.5h, another 35uL (28mg) of dithiol and 8uL of dibutyltin diacetate (2:57pm) were added and the reaction was stirred overnight. The reaction was diluted with EtOAc and then washed with 1n hcl solution. The organics were washed with saturated sodium bicarbonate. The organic layer was dried over sodium sulfate and then concentrated. The residue was purified by flash chromatography (25g silica gel, 20% -30% -50% EtOPc/Hept) to give it as freeDesired carbonate as a colored oil (227mg, 67% yield).

Example 19A general procedure II-removal of TBS group by HOAc:

[2- (tert-Butyldisulfanyl) -2-methyl-propyl ] N- [2- [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidine-1-carbonyl ] -4, 5-dimethoxy-phenyl ] carbamate (200mg, 0.319mmol) was dissolved in 3mL of THF, followed by addition of water (0.8mL) and acetic acid (3.4mL) at room temperature. After the reaction was complete, sodium carbonate was added to neutralize the acetic acid. The mixture was extracted three times with EtOAc. The combined EtOAc extracts were dried over sodium sulfate and concentrated to give the crude alcohol (233mg), which was used in the next step without purification.

Example 19A general procedure III-DMP oxidation and cyclization:

to 2- (isopropyldisulfanyl) -2-methyl-propyl in DCM (3.5mL) at room temperature]N- [2- [ (2S) -2- (hydroxymethyl) -4-methylene-pyrrolidine-1-carbonyl]-4, 5-dimethoxy-phenyl]Dess-Martin periodinane (86.1mg, 0.203mmol, 1.02 equiv.) was added to carbamate (99.2mg, 0.199mmol, 100 mass%). The reaction mixture was diluted with DCM and then with a mixture of saturated NaHCO₃Washed (about 3mL) with 1M sodium sulfite (about 3mL), dried over sodium sulfate, concentrated to give about 120mg of crude product (oil), which was purified by reverse phase HPLC to give the desired carbonate (28.9mg) and recovered alcohol starting material (10.3 mg). LCMS (5-95, AB,5min), RT 2.69min, M/z 497[ M +1]]+；1H NMR(400MHz,DMSO-d6)δ7.08(s,1H),6.82(s,1H),6.64(d,J＝6.0Hz,1H),5.49-5.33(m,1H),5.13(d,J＝7.3Hz,2H),4.21-4.05(m,2H),4.04-3.91(m,1H),3.81(s,4H),3.79-z3.72(m,1H),3.46(t,J＝9.3Hz,1H),3.31(s,3H),2.99-2.76(m,2H),2.63-2.51(m,1H),1.38-0.94(m,12H)。

Example 19B: preparation of PBD monomeric disulfide prodrug 4

The title compound was synthesized in the same manner as example 19A, except that the following general procedure IB was used for disulfide formation.

Example 19B general procedure Ib-carbonate formation method B:

(2-amino-4, 5-dimethoxy-phenyl) - [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidin-1-yl ] methanone (121mg, 0.2976mmol) and N, N-diisopropylamine (3 equiv., 0.8928mmol) were mixed in 3mL of THF, then (4-nitrophenyl) chloroformate (75mg, 0.3720mmol) was added at room temperature. The mixture was heated to 75 ℃. After about 1h 50min, 2- (isopropyldisulfanyl) -2-methyl-propan-1-ol (71mg, 0.3938mmol) in 1.5mL of THF was added. The mixture was stirred at 75 ℃ overnight, cooled to room temperature, diluted in EtOAc, washed with 1N HCl and then saturated sodium bicarbonate. The solution was concentrated and the resulting residue was purified by silica gel chromatography (20% followed by 30% followed by 50% EtOAc/Hept) to give the disulfide (34mg, 19% yield).

LCMS:(5-95,AB,5min),RT＝2.78min,m/z＝511[M+1]+；1H NMR(400MHz,DMSO-d6)δ7.08(s,1H),6.81(s,1H),6.63(d,J＝5.9Hz,1H),5.49-5.34(m,1H),5.13(d,J＝6.1Hz,2H),4.19-3.91(m,3H),3.81(s,3H),3.78-3.67(m,1H),3.46(t,J＝9.2Hz,1H),3.32(s,35H),2.96-2.82(m,1H),2.61-2.53(m,1H),1.18(s,9H),1.04(d,J＝3.7Hz,6H)。

Example 19C: preparation of PBD monomeric disulfide prodrug 15

PBD monomeric disulfide prodrug 15 was prepared according to reaction scheme 3 below:

triphosgene (116mg, 0.39mmol) was dissolved in 2mL of DCM, and then a solution of (2R) -2- [ (5-nitro-2-pyridyl) disulfanyl ] propan-1-ol (277mg, 1.125mmol) and pyridine (0.14mL, 1.761mmol) in 2mL of DCM was added. After 30min, this solution was added to a solution of (2-amino-4, 5-dimethoxy-phenyl) - [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidin-1-yl ] methanone (410mg, 0.978mmol, 97% by mass) in 3mL of DCM plus 0.5mL of rinse. After completion of the reaction, the mixture was diluted with EtOAc, washed with 1N aqueous HCl, followed by saturated sodium bicarbonate. The organics were dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (25g silica gel, 30% followed by 40% followed by 50% EtOAc/Hept) to give [ (2R) -2- [ (5-nitro-2-pyridinyl) disulfanyl ] propyl ] N- [2- [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidine-1-carbonyl ] -4, 5-dimethoxy-phenyl ] carbamate (480mg, 72% yield).

To [ (2R) -2- [ (5-nitro-2-pyridyl) disulfanyl ] propyl ] N- [2- [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidine-1-carbonyl ] -4, 5-dimethoxy-phenyl ] carbamate (45mg, 0.066mmol, 100 mass%) in DMF (0.5mL) was added propane-2-thiol (40mg, 0.53mmol) by syringe without using any solvent. The mixture was heated to 59 ℃ in a sealed vial. After completion of the reaction, the mixture was concentrated under reduced pressure and azeotroped (azotroped) once with toluene. The resulting residue was purified by silica gel chromatography (40g silica, 20% -35% -50% EtOAc/Hept) to give [ (2R) -2- (isopropyldisulfanyl) propyl ] N- [2- [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidine-1-carbonyl ] -4, 5-dimethoxy-phenyl ] carbamate as a yellow oil (134mg, 89% yield).

PBD monomeric disulfide prodrug 15 was then prepared according to general procedures II and III of example 19A.

LCMS:(5-95,AB,5min),RT＝2.58min,m/z＝483[M+1]+；1H NMR(400MHz,DMSO-d6)δ7.07(s,1H),6.82(s,1H),6.63(d,J＝6.1Hz,1H),5.38(dd,J＝9.7,6.1Hz,1H),5.20-5.07(m,2H),4.20(dd,J＝11.1,6.1Hz,1H),4.15-4.05(m,1H),3.97(d,J＝15.4Hz,2H),3.80(s,6H),3.45(t,J＝9.3Hz,1H),3.10-2.82(m,3H),2.58-2.53(m,1H),1.18(t,J＝6.2Hz,7H),1.04(d,J＝6.9Hz,3H)。

Example 19D: preparation of PBD monomeric disulfide prodrug 13

PBD monomeric disulfide prodrug 13 was prepared according to the method of example 19C. LCMS (5-95, AB,5min), RT ═ 2.70min, M/z ═ 497[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.07(s,1H),6.81(s,1H),6.65(d, J ═ 5.9Hz,1H),5.38(dd, J ═ 9.7,5.9Hz,1H),5.19-5.04(m,2H),4.20(dd, J ═ 11.2,5.9Hz,1H),4.10(d, J ═ 15.8Hz,1H),4.04-3.91(m,2H),3.80(s,6H),3.45(t, J ═ 9.3Hz,1H),3.04-2.82(m,2H),2.58-2.53(m,1H),1.23(s,9H),1.03(d, J ═ 6.9, 3H).

Example 19E: preparation of PBD monomeric disulfide prodrug 2

PBD monomeric disulfide prodrug 2 was prepared according to the following reaction scheme:

in some aspects, R³Corresponding to R as described elsewhere herein⁵⁰。

Triphosgene (268mg, 0.9046mmol, 100 mass%) was dissolved in 3mL DCM in a flask, then a solution of 2- [ (5-nitro-2-pyridinyl) disulfanyl ] ethanol (604mg, 2.601mmol) in 6mL DCM was added, then pure pyridine (1.8 equivalents, 4.071mmol, 100 mass%) was added. After 30min, this solution was added to a solution of (2-amino-4, 5-dimethoxy-phenyl) - [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidin-1-yl ] methanone (948mg, 2.262mmol) in 5mL of DCM plus 0.8mL of rinse. After about 20min, the mixture was diluted with EtOAc, washed with 1N HCl, followed by saturated sodium bicarbonate. The organics were dried over sodium sulfate, concentrated and column chromatographed (40g silica gel, 25% followed by 45% EtOAc/Hept) to give the carbamate as a yellow fluffy solid (1.07g, 71% yield).

2- [ (5-Nitro-2-pyridyl) disulfanyl ] ethyl N- [2- [ (2S) -2- [ [ tert-butyl (dimethyl) silyl ] oxymethyl ] -4-methylene-pyrrolidine-1-carbonyl ] -4, 5-dimethoxy-phenyl ] carbamate (1.067g, 1.61mmol) was dissolved in THF, then water (1.5mL) was added followed by acetic acid (8mL) at room temperature. The mixture was heated to 55 ℃ and stirred overnight. After the mixture was cooled to room temperature, sodium carbonate was added to neutralize the acetic acid. The mixture was extracted three times with EtOAc. The combined EtOAc extracts were dried over sodium sulfate and concentrated to give the crude alcohol (1.26g), which was used in the next step without purification.

Dess-Martin periodinane (715mg, 1.685mmol) was added to the above alcohol (884mg, 1.61mmol) in DCM (16mL) at room temperature. After about 70min, an additional 105mg of D-M periodinane was added. After 2.5h, 76mg of D-M periodinane were added. Once all starting material was consumed, the mixture was diluted with DCM and then with mixed saturated NaHCO₃Washed (about 6mL) and 1M sodium sulfite (about 6mL), dried over sodium sulfate and concentrated under reduced pressure. The resulting product was purified by silica gel column chromatography (40g silica gel, 50% followed by 80% followed by 100% EtOAc/Hept)The residue to give 2- [ (5-nitro-2-pyridyl) disulfanyl as a yellow solid]Ethyl (6S,6aR) -6,6 a-dihydroxy-2, 3-dimethoxy-8-methylene-11-oxo-7, 9-dihydro-6H-pyrrolo [2, 1-c)][1,4]Benzodiazepine-5-Carboxylic acid ester (765mg, 84% yield).

Injection of 2- [ (5-nitro-2-pyridyl) dithioalkyl in 0.2mL of DMSO]Ethyl (6aS) -6-hydroxy-2, 3-dimethoxy-8-methylene-11-oxo-6, 6a,7, 9-tetrahydropyrrolo [2,1-c][1,4]BenzodiazepineTo 5-Carboxylic acid ester (63mg, 0.115mmol) was added propane-2-thiol (0.5mL), followed by heating to 59 ℃. After completion of the reaction, the mixture was cooled to room temperature and co-evaporated twice with EtOAc in a rotary evaporator to remove as much thiol as possible. The resulting residue was purified by reverse phase HPLC to give 2-sulfanylethyl (6aS) -6-hydroxy-2, 3-dimethoxy-8-methylene-11-oxo-6, 6a,7, 9-tetrahydropyrrolo [2,1-c ]][1,4]Benzodiazepine-5-Carboxylic acid ester (38.6 mg).

LCMS:(5-95,AB,5min),RT＝2.42min,m/z＝469[M+1]+；1HNMR(400MHz,DMSO-d6)δ7.06(s,1H),6.80(s,1H),6.62(s,1H),5.37(dd,J＝9.6,5.4Hz,1H),5.13(d,J＝7.0Hz,2H),4.37(dt,J＝12.2,6.3Hz,1H),4.14-3.93(m,4H),3.80(s,6H),3.45(t,J＝9.3Hz,1H),3.01-2.83(m,3H),1.23-1.16(m,6H)。

Example 19F: preparation of PBD monomeric disulfide prodrug 16

PBD monomeric disulfide prodrug 16 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT 2.56min, M/z 483[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.79(s,1H),6.62(s,1H),5.37(dd, J ═ 9.7,5.9Hz,1H),5.16-5.09(m,2H),4.35(dt, J ═ 12.2,6.3Hz,1H),4.10(d, J ═ 16.0Hz,1H),3.98(d, J ═ 16.0Hz,2H),3.80(s,6H),3.44(t, J ═ 9.3Hz,1H),2.88(t, J ═ 12.6Hz,3H),2.54(s,1H),1.25(s,9H),0.08(s, 1H).

Example 19G: preparation of PBD monomeric disulfide prodrug 29

PBD monomeric disulfide prodrug 29 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 1.72min, M/z ═ 471[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.79(d, J ═ 4.9Hz,1H),6.62(s,1H),5.37(dd, J ═ 9.7,5.8Hz,1H),5.13(d, J ═ 7.0Hz,2H),4.88-4.80(m,1H),4.45-4.34(m,1H),4.14-4.05(m,1H),4.02-3.93(m,1H),3.80(s,6H),3.79-3.78(m,1H),3.60(dq, J ═ 14.2,6.5Hz,2H),3.49-3.40(m,1H),2.94-2.69(m,4H),2.54(s, 1H).

Example 19H: preparation of PBD monomeric disulfide prodrug 28

PBD monomeric disulfide prodrug 28 was prepared according to the method of example 19D. LCMS (5-95, AB,5min), RT ═ 2.68min, M/z ═ 495[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.80(s,1H),6.62(s,1H),5.37(dd, J ═ 9.7,5.8Hz,1H),5.13(d, J ═ 6.8Hz,2H),4.44-4.33(m,1H),4.15-4.05(m,1H),4.02-3.93(m,1H),3.81(s,6H),3.44(t, J ═ 9.1Hz,1H),2.88(d, J ═ 7.8Hz,3H),2.55(dt, J ═ 5.7,2.5Hz,2H),2.48-2.42(m,1H),1.89(d, J ═ 8.4, 2H),1.64(s, 4H), 1H (s,4H), 4H (s, 4H).

Example 19I: preparation of PBD monomeric disulfide prodrug 27

PBD monomeric disulfide prodrug 27 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 1.83min, M/z ═ 499[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.80(s,1H),5.37(d, J ═ 9.7Hz,1H),5.16-5.09(m,2H),4.45-4.34(m,1H),4.16-3.93(m,2H),3.80(s,6H),3.44(t, J ═ 9.2Hz,1H),3.05-2.79(m,5H),2.65-2.50(m,1H),2.46(s,1H),2.07(s, 3H).

Example 19J: preparation of PBD monomeric disulfide prodrug 7

PBD monomeric disulfide prodrug 7 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 2.62min, M/z ═ 495[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.07(s,1H),6.82(s,1H),6.66-6.60(m,1H),5.41-5.36(m,1H),5.16-5.09(m,2H),4.30(d, J ═ 11.7Hz,1H),4.15-4.06(m,1H),4.02-3.88(m,2H),3.81(s,6H),3.45(t, J ═ 9.3Hz,1H),2.91(td, J ═ 16.0,8.6Hz,2H),2.57-2.53(m,1H),1.14(dd, J ═ 11.4,6.6Hz,6H),0.96-0.81(m, 4H).

Example 19K: preparation of PBD monomeric disulfide prodrug 1

PBD monomeric disulfide prodrug 1 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 2.20min, M/z ═ 455[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.80(s,1H),6.62(s,1H),5.37(dd, J ═ 9.7,5.1Hz,1H),5.16-5.09(m,2H),4.39(dt, J ═ 12.2,6.3Hz,1H),4.15-3.93(m,3H),3.80(s,6H),3.45(t, J ═ 9.3Hz,1H),2.88(dd, J ═ 16.1,9.0Hz,3H),2.65(q, J ═ 12.6,6.8Hz,2H),2.54(d, J ═ 2.3Hz,1H),1.23-1.14(m, 3H).

Example 19L: preparation of PBD monomeric disulfide prodrug 26

PBD monomeric disulfide prodrug 26 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT 2.08min, M/z 497[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.08(s,1H),6.82(s,1H),6.65(s,1H),5.38(d, J ═ 9.7Hz,1H),5.13(d, J ═ 6.6Hz,2H),4.77(s,3H),4.45-4.34(m,1H),4.26-4.06(m,3H),4.02-3.93(m,1H),3.81(s,6H),3.45(t, J ═ 9.3Hz,1H),3.09(s,1H),2.94-2.83(m,1H),2.62-2.53(m,1H),1.04(d, J ═ 6.9Hz,2H), -0.03 — 0.13(m, 1H).

Example 19M: preparation of PBD monomeric disulfide prodrug 25

PBD monomeric disulfide prodrug 25 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 2.16min, M/z ═ 499[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.07(s,1H),6.82(s,1H),6.63(brs,1H),5.38(d, J ═ 9.7Hz,1H),5.14(s,2H),4.2-4.1(m,2H),3.99(s,1H),3.81(s,6H),3.46(s,4H),3.22(s,3H),2.79(s,2H),2.69-2.54(m,3H),1.05(s, 2H).

Example 19N: preparation of PBD monomeric disulfide prodrug 24

PBD monomeric disulfide prodrug 24 was prepared according to the method of example 19E.

The thiol was synthesized according to the following scheme:

to a solution of tetrahydro-2H-pyran-4-carbaldehyde 14a (5g, 43.8mmol) in MTBE (50mL) was added S₂Cl₂(2.96g, 21.9 mmol). The reaction mixture was stirred at 55 ℃ under nitrogen for 16h, then cooled to ambient temperature, the solvent removed in vacuo and purified by silica gel column chromatography (silica: 200 mesh, PE/EtOAc: 5/1) to give disulfide 14b as a yellow oil (5g, 58%). 1H NMR (300MHz, CDCl 3). delta.9.05 (s,1H),3.92-3.42(m,8H),2.22-1.58(m, 8H).

To a solution of 4,4' -dithioalkanediyl-bis (tetrahydro-2H-pyran-4-carbaldehyde) 7(5g, 12.82mmol) in THF (50mL) was added LiAlH in portions₄(0.97g, 25.64 mmol). After addition, the reaction mixture was stirred at ambient temperature for 2 hours, then acidified to PH 6 with HCl (3N), extracted with ethyl acetate (30mLx3), and filtered over Na₂SO₄Drying, removal of solvent and purification by silica gel column chromatography (silica: 200-300 mesh, PE/EA-10/1) gave thiol 14c as a yellow oil (2.02g, 34%). 1H NMR (300MHz, CDCl 3). delta.3.86-. 383(m,4H),3.53(s,2H),2.25(s,1H),1.86-1.43(m, 5H). GCMS (m/z) ES 148.

LCMS:(5-95,AB,5min),RT＝2.24min,m/z＝525[M+1]+；1H NMR(400MHz,DMSO-d6)δ7.07(s,1H),6.82(s,1H),6.61(s,1H),5.37(d,J＝9.6Hz,1H),5.13(d,J＝7.0Hz,2H),4.23(dd,J＝11.1,6.0Hz,1H),4.10(d,J＝15.7Hz,1H),4.02-3.83(m,4H),3.81(s,6H),3.45(t,J＝9.3Hz,1H),3.02(dt,J＝11.1,4.1Hz,1H),2.94-2.83(m,2H),2.59-2.52(m,2H),1.95-1.76(m,2H),1.55-1.37(m,2H),1.27-1.21(m,1H),1.05(d,J＝6.9Hz,3H)。

Example 19O: preparation of PBD monomeric disulfide prodrugs

The above PBD monomeric disulfide prodrug was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT 1.86min, M/z 485[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.06(s,1H),6.79(s,1H),6.62(s,1H),5.37(dd, J ═ 9.7,6.0Hz,1H),5.13(d, J ═ 7.0Hz,2H),4.88(t, J ═ 5.7Hz,1H),4.38(d, J ═ 7.5Hz,1H),4.16-3.95(m,2H),3.80(s,6H),3.46(d, J ═ 11.3Hz,2H),3.38-3.34(m,1H),2.86(s,4H),2.58-2.53(m,1H),1.19(dd, J ═ 12.6,6.6, 3H).

Example 19P: preparation of PBD monomeric disulfide prodrug 12

PBD monomeric disulfide prodrug 12 was prepared according to the method of example 19E. LCMS (5-95, AB,5min), RT ═ 2.40min, M/z ═ 469[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.07(s,1H),6.82(s,1H),6.63(s,1H),5.38(dd, J ═ 9.7,5.9Hz,1H),5.13(d, J ═ 6.7Hz,2H),4.21(dd, J ═ 11.1,6.2Hz,1H),4.10(d, J ═ 16.0Hz,1H),3.97(d, J ═ 16.1Hz,2H),3.81(d, J ═ 2.3Hz,6H),3.45(t, J ═ 9.3Hz,1H),3.05(s,1H),2.94-2.83(m,1H),2.70-2.53(m,3H),2.45(p, 1H), 1H), 1.02(m, 1H), 1H).

Example 19Q: preparation of PBD monomeric disulfide prodrug 3

PBD monomeric disulfide prodrug 3 was prepared according to the method of example 19E.

P-nitropyridine disulfide was synthesized according to the following scheme:

to a solution of cyclobutanecarboxaldehyde 17a (9.8g, 120mmol) in MTBE (80mL) was added S₂Cl₂(8.1g, 60 mmol). The reaction mixture was stirred under nitrogen at 55 ℃ for 16 hours. The reaction mixture was cooled to ambient temperature, the solvent removed in vacuo and purified by silica gel column chromatography (silica: 200-mesh 300, PE/EtOAc: 20/1) to give 1,1' -disulfanediylbicyclobutanecarboxaldehyde as a brown oil (11.2g, 83%). 1H NMR (300MHz, CDCl 3). delta.9.28 (s,2H),3.06-1.20(m, 12H).

To a solution of 1,1' -disulfanediylbicyclobutane carboxaldehyde 5(11.2g, 49mmol) in THF (200mL) LiAlH was added portionwise₄(3.7g, 97 mmol). After addition, the reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was acidified with HCl (3N) to pH 6, extracted with ethyl acetate (200mLx3), and filtered over Na₂SO₄Drying, removal of solvent and purification by silica gel column chromatography (silica: 200-300 mesh, PE/EtOAc: 10/1) gave thiol 17c as a yellow oil (3.8g, 33%). 1H NMR (300MHz, CDCl3) δ 3.64(s,2H),2.35-2.09(m,5H),2.05-1.87(m,2H),1.81(s,1H). gcms (es) m/z ═ 118.

A mixture of 17c (4.88g, 41.35mmol) and 1, 2-bis (5-nitropyridin-2-yl) disulfane (12.82g, 41.35mmol) in MeOH (100mL) was stirred at ambient temperature under nitrogen for 16 h. The solution was concentrated in vacuo and the residue was purified by silica gel column chromatography (silica: 200-300 mesh, PE/EA ═ 10/1) to give the target compound 17d (2.01g, 18%) as a yellow solid. 1H NMR (400MHz, DMSO) δ 9.30(s,1H),8.34(dd, J ═ 8.8,2.6Hz,1H),7.60(d, J ═ 8.8Hz,1H),3.57(s,2H),3.41(s,1H),2.27-2.14(m,5H),2.08-1.93(m, 1H); lcms (es) M/z +273(M + 1).

LCMS:(5-95,AB,5min),RT＝2.61min,m/z＝495[M+1]+；1HNMR(400MHz,DMSO-d6)δ7.08(s,1H),6.82(s,1H),6.64(d,J＝6.0Hz,1H),5.45-5.36(m,1H),5.13(d,J＝7.2Hz,2H),4.30(d,J＝11.5Hz,1H),4.10(d,J＝15.8Hz,1H),3.97(d,J＝13.7Hz,2H),3.80(s,6H),3.46(td,J＝9.5,1.8Hz,1H),2.89(dd,J＝15.8,9.2Hz,1H),2.62-2.52(m,2H),1.91(s,6H),1.63(s,1H),1.13(t,J＝7.5Hz,3H)。

Example 19R: preparation of PBD monomeric disulfide prodrug 5

PBD monomeric disulfide prodrug 5 was prepared according to the method of example 19E.

P-nitropyridine disulfide was synthesized according to the following scheme:

to a solution of cyclopentanecarboxaldehyde 18a (9.0g, 92mmol) in MTBE (30mL) was added S₂Cl₂(7.4g, 55 mmol). The reaction mixture was stirred under nitrogen at 55 ℃ for 16 hours. The reaction mixture was cooled to ambient temperature, the solvent removed in vacuo and purified by silica gel column chromatography (silica: 200-mesh 300, PE/EtOAc: 80/1) to give 1,1' -disulfanediyldicyclopentanecarboxaldehyde 18b as a brown oil (5.5g, 46%). 1HNMR (300MHz, CDCl3) delta 9.23(s,2H),2.41-1.51(m, 16H).

To a solution of 1,1' -disulfanediyldicyclopentanecarboxaldehyde 18b (8.5g, 32.9mmol) in THF (60mL) was added LiAlH in portions₄(2.5g，65.8mmol)。After addition, the reaction mixture was stirred at ambient temperature for 2 hours, then the solution was acidified to pH 6 with HCl (3N), extracted with ethyl acetate (150mLx2), and taken over Na₂SO₄Dried, the solvent removed and purified by silica gel column chromatography (silica: 200 mesh, 300 mesh, PE/EtOAc ═ 20/1) to give 18c as a yellow oil (5.5g, 63%). 1H NMR (300MHz, CDCl 3). delta.3.51 (s,2H),2.04(s,1H),1.85-1.67(m,7H),1.64(s, 1H). Gcms (es) m/z + 132.

A mixture of 18c (3.5g, 26.5mmol) and 1, 2-bis (5-nitropyridin-2-yl) dithiolane 3(12.3g, 39.8mmol) in MeOH (50mL) was stirred at ambient temperature under nitrogen for 16 h. After the reaction was complete, the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography (silica: 200-300 mesh, PE/EtOAc ═ 10/1) to give the title compound 18d as a yellow solid (1.9g, 25%). 1H NMR (400MHz, DMSO): δ 9.24-9.20(m,1H),8.58(dd, J ═ 8.9,2.7Hz,1H),8.17(dd, J ═ 8.9,0.5Hz,1H),5.18(t, J ═ 5.5Hz,1H),3.40(d, J ═ 5.5Hz,2H),1.63-1.82(m, 8H); lcms (es) M/z +287(M + 1).

LCMS:(5-95,AB,5min),RT＝2.48min,m/z＝509[M+1]+；1HNMR(400MHz,DMSO-d6)δ7.08(s,1H),6.82(s,1H),6.73-6.60(m,1H),5.40(d,J＝7.7Hz,1H),5.13(d,J＝7.2Hz,2H),4.22(d,J＝11.0Hz,1H),4.10(d,J＝15.9Hz,1H),4.01-3.84(m,2H),3.81(s,6H),3.46(t,J＝9.2Hz,1H),2.88(dd,J＝15.9,9.3Hz,1H),2.55(dd,J＝4.1,2.3Hz,2H),1.81-1.39(m,10H),1.12(t,J＝7.3Hz,3H)。

Example 19S: preparation of PBD monomeric disulfide prodrugs 11 and 30

PBD monomeric disulfide prodrugs 11 and 30 correspond to the above structures and are diastereomers having different configurations at one or more chiral centers marked with an asterisk. A compound was prepared according to the procedure for example 19E.

Using the procedure described in example 19N, p-nitropyridine disulfide was synthesized according to the following scheme.

PBD monomeric disulfide prodrug 11LCMS (5-95, AB,5min), RT ═ 1.95min, M/z ═ 511[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.08(s,1H),6.83(s,1H),6.64(s,1H),5.39(d, J ═ 9.7Hz,1H),5.13(d, J ═ 7.1Hz,2H),4.34(d, J ═ 11.5Hz,1H),4.10(d, J ═ 16.0Hz,1H),3.98(t, J ═ 15.3Hz,2H),3.81(d, J ═ 2.4Hz,6H),3.78-3.72(m,1H),3.63(s,1H),3.54(s,2H),3.46(t, J ═ 9.2Hz,1H),2.89(dd, J ═ 15.7,9.4, 1H),2.62 (s,2H), 3.83H, 3.83 (d, 1H), 3.7, 3.2H, 3.83 (d, 3.3.2H).

PBD monomeric disulfide prodrug 30LCMS (5-95, AB,5min), RT ═ 1.99min, M/z ═ 511[ M +1] +; 1H NMR (400MHz, DMSO-d6) δ 7.08(s,1H),6.82(s,1H),6.66(s,1H),5.48-5.34(m,1H),5.21-5.07(m,2H),4.33(d, J ═ 11.5Hz,1H),4.18-3.91(m,3H),3.81(s,6H),3.79-3.70(m,0H),3.48(d, J ═ 17.5Hz,2H),2.97-2.82(m,1H),2.55(dd, J ═ 4.2,2.2Hz,2H),2.47-2.29(m,1H),1.85(d, J ═ 13.2Hz,2H),1.13(t, J ═ 7.4, 3H).

Example 20: preparation of PBD dimer disulfide prodrugs

Example 20A: preparation of PBD dimer disulfide prodrug 4

PBD dimer disulfide prodrug 4 was prepared according to the following reaction scheme:

each asterisk in the above structure and elsewhere depicted in example 20 represents a chiral center.

To a solution of triphosgene (83.2mg, 0.280mmol) in DCM (2.0mL) at 0 deg.C was added a solution of compound A2 and pyridine in DCM (3.0 mL). The mixture was stirred at 20 ℃ for 30min and concentrated in vacuo. It was dissolved in DCM (5.0mL) and added dropwise to a solution of pyridine (18.5mg, 0.234mmol) and Compound A1(124mg, 0.516mmol) at 20 ℃. After stirring the reaction mixture at 20 ℃ for 2h, it was concentrated in vacuo and purified by column chromatography (0-50% EtOAc in petroleum ether) to give compound a3(150mg, 54%) as a yellow solid. LCMS (5-95, AB,1.5min): RT ═ 1.419min, M/z ═ 1261.4[ M +1] +.

Compound A3(150mg, 0.119mmol) in THF (4.0mL), H₂The solution in O (4.0mL) and HOAc (6.0mL) was stirred at 10 ℃ for 8 h. The mixture was diluted with EtOAc (15mL) and washed with H₂O(10mL)、NaHCO₃Aqueous solution (10mL) and brine (10 mL). Subjecting the organic layer to Na₂SO₄Dried, filtered, concentrated and purified by preparative TLC (5% CH in DCM)₃OH) to give compound a4(75mg, 60.4%) as a colorless oil. LCMS (5-95, AB,1.5min): RT ═ 0.941min, M/z ═ 1033.3[ M + 1-]+。

A mixture of Compound A4(44mg, 0.04mmol) and DMP (54mg, 0.13mmol) in DCM (15mL) was stirred at 13 ℃ for 16 h. The reaction mixture was concentrated in vacuo and purified by preparative TLC (5.6% MeOH in DCM, Rf ═ 0.5) followed by preparative HPLC (10mM, NH)₄HCO₃-ACN) to give PBD dimer disulfide prodrug 4 as a white solid (15mg, 34%). LCMS (5-95, AB,1.5min): RT ═ 0.941min, M/z ═ 1051.2[ M + 23-]+；1HNMR(400MHz,CDCl3)δ7.19(s,2H),6.73(s,2H),5.59-5.57(m,2H),5.12(s,4H),4.43(d,J＝10.8Hz,2H),4.26-4.15(m,1H),4.11(s,1H),4.02-3.98(m,3H),3.96(s,4H),3.88(s,6H),3.80-3.65(m,2H),3.62(m,2H),2.90-2.80(m,2H),2.72-2.60(m,6H),2.18(br,2H),2.02-1.92(br,13H),1.70-1.63(m,3H),1.22-1.19(m,6H)。

Example 20B: preparation of PBD dimer disulfide prodrug 1

PBD dimer disulfide prodrug 1 was prepared according to the following reaction scheme:

to a solution of triphosgene (42.02mg, 0.140mmol) in DCM (4.0mL) was added dropwise a solution of compound A1(300.0mg, 0.310mmol) and triethylamine (63.68mg, 0.630mmol) in DCM (4 mL). After stirring the mixture at 0 ℃ for 30min, a solution of compound A2(112.16mg, 0.630mmol) and triethylamine (127mg, 1.26mmol) in DCM (2.0mL) was added and the mixture was stirred at 18 ℃ for 18 h. The mixture was partitioned between water (20.0mL) and DCM (40.0mL), and the organic layer was washed with water (20.0mL), brine (20.0mL), and concentrated. It was purified by column chromatography (EtOAc: petroleum ether 1:2) to give compound A3(240mg, 65%) as a yellow oil. LCMS (5-95, AB,1.5min): RT 1.296min, M/z 1157.4[ M +1] +.

To a solution of compound a3(240.0mg, 0.210mmol) in THF (1.5mL) was added dropwise a mixture of HOAc/H2O (4.0mL, 3/1). The mixture was stirred at 8 ℃ for 18 h. With NaHCO₃The solution was adjusted to pH8 and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with Na₂SO₄Dried, filtered and concentrated. It was purified by column chromatography (DCM: MeOH ═ 20:1) to give compound a4(130mg, 68%) as a yellow oil. LCMS (5-95, AB,1.5min): RT ═ 0.868min, M/z ═ 929.3[ M + 1-]+。

To a solution of compound A4(60.0mg, 0.0600mmol) in DCM (6.0mL) was added DMP (95.83mg, 0.230mmol), and the mixture was stirred at 18 ℃ for 1.0 h. The mixture was filtered and washed with Na₂SO₃Aqueous solution (20.0mL), brine (20.0mL), and water (20.0 mL). The organic layer was washed with Na₂SO₄Dry, concentrate, and purify by preparative TLC (7% MeOH in DCM) to give compound a5 as a white solid (30mg, 49%). LCMS (5-95, AB,1.5min): RT 0.804min, M/z 925.3[ M + 1%]+。

TFA (1.0mL) was added dropwise to compound a5(30.0mg, 0.030mmol) at 0 ℃. After stirring for 20min, the mixture was added dropwise to saturated NaHCO at 0 deg.C₃To solution (40.0mL) and extracted with DCM (3 × 15 mL). The combined organic layers were washed with Na₂SO₄Dried, concentrated, and purified by preparative TLC (18% MeOH in DCM, Rf ═ 0.6) to give PBD dimer disulfide prodrug 1as a white solid (5.8mg, 22%). LCMS (5-95, AB,1.5min): RT ═ 0.890min, M/z ═ 807.2[ M + 1-]+。

Example 20C: preparation of PBD dimer disulfide prodrug 2

PBD dimer disulfide prodrug 2 was prepared according to the following reaction scheme:

to triphosgene (89.43mg, 0.300mmol) andto a solution of molecular sieves (50mg) in DCM (5.0mL) was added a solution of compound A2(165.0mg, 0.710mmol) and pyridine (168.58mg, 2.13mmol) in DCM (5.0 mL). The mixture was stirred at 0 ℃ for 30 min. The resulting mixture was added dropwise to Compound A1(745mg, 0.780mmol), pyridine (169mg, 2.13mmol) andMS in DCM (5.0 mL). Stir at 0 ℃ for 30min and wash with water (5.0 mL). The organic phase was dried, concentrated and purified by flash column chromatography (5% MeOH in DCM) to give product a3(698mg, 81%) as a yellow oil. LCMS (5-95, AB,1.5min): RT 1.187min, M/z 606.5[ M/2+ 1[ ]]+。

To a solution of compound A3(698.0mg, 0.580mmol) in DCM (10.0mL) was added 2-propanethiol (439mg, 5.76 mmol). Will be provided withThe mixture was stirred at 20 ℃ for 1h, MnO was added₂(100mg) and stirred for 5min and filtered. The filtrate was concentrated and purified by preparative TLC (50% EtOAc in petroleum ether) to give compound a5(620mg, 95%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.221min, M/z 1131.4[ M +1]+。

To a solution of compound A5(620.0mg, 0.550mmol) in THF (6.0mL) and water (6.0mL) was added HOAc (3.29g, 54.8 mmol). The mixture was stirred at 40 ℃ for 16h and concentrated. Purification by column chromatography (10% MeOH in DCM) gave compound a6(208mg, 42%) as a yellow oil. LCMS (5-95, AB,1.5min): RT ═ 0.854min, M/z ═ 903.3[ M +1] +.

To a solution of Compound A6(208.0mg, 0.230mmol) in DCM (8.0mL) was addedMolecular sieves, DMP (224.7mg, 0.530 mmol). The mixture was stirred at 20 ℃ for 2h and saturated NaHCO₃And Na₂S2O₃The solution was quenched (2.0mL/2.0 mL). After stirring for 5min, DCM (5.0mL) was added and separated. The DCM phase was washed with water (2 × 5 mL). It was dried, concentrated and purified by preparative TLC (5% MeOH in DCM, Rf ═ 0.2) to give compound a7(121mg, 58%) as a light yellow foam. LCMS (5-95, AB,1.5min): RT ═ 0.783min, M/z ═ 781.3[ M-100+ 1-]+。

TFA (1.0mL, 13.5mmol) was added to Compound A7(121.0mg, 0.130mmol) at 0 ℃. After stirring the mixture for 10min, it was added to cold saturated NaHCO₃Solution (20mL) and extracted with DCM (3 × 10 mL). The combined organic layers were concentrated and purified by preparative TLC (10% MeOH in DCM, Rf ═ 0.2) followed by preparative HPLC (ACN, acetonitrile: 42-62%, 0.225% FA) to give PBD dimer disulfide prodrug 2(7.2mg, 7.0%). LCMS (5-95, AB,1.5min): RT ═ 0.868min, M/z ═ 781.3[ M + 1-]+。

Example 20D: preparation of PBD dimer disulfide prodrug 3

PBD dimer disulfide prodrug 3 was prepared according to the following reaction scheme:

at 0 ℃ and N₂To a solution of triphosgene (65.37mg, 0.220mmol) in DCM (2.0mL) was added a mixture of compound A1(420.0mg, 0.440mmol) and triethylamine (89.16mg, 0.880mmol) in DCM (3.0 mL). After the mixture was stirred at 21 ℃ for 30min, it was concentrated and DCM (18mL) was added. At 0 ℃ and N₂Next, a solution of A2(75.0mg, 0.390mmol) and triethylamine (78.91mg, 0.780mmol) in DCM (2.0mL) was added. After stirring the reaction mixture at 20 ℃ for 1h, it was concentrated in vacuo and purified by column chromatography (0-50% EtOAC in petroleum ether) to give compound a3(350mg, 74%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.379min, M/z 1171.5[ M + 1%]+。

To a solution of compound A3(415.2mg, 0.350mmol) in THF (30mL) and water (15mL) was added HOAc (2.02mL, 35.2 mmol). The reaction solution was stirred at 20 ℃ for 12 h. It was concentrated in vacuo and diluted with EtOAc (200mL) and H₂O (2X100mL) wash followed by NaHCO₃Aqueous (2 × 60mL) washes. The EtOAc layer was washed with Na₂SO₄Dried, filtered and concentrated. It was purified by column chromatography (0-10% MeOH in DCM) to give compound a4(320mg, 87.1%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.080min, M/z 943.5[ M + 1%]+。

To a solution of compound A4(170.0mg, 0.180mmol) in DCM (20mL) was added DMP (229.3mg, 0.540 mmol). After stirring the reaction mixture at 18 ℃ for 1H, it is washed with H₂O (20mL) dilution and Na addition₂SO₃Aqueous solution (20mL) and NaHCO₃Aqueous solution (20 mL). The mixture was extracted with EtOAc (3 × 60 mL). The combined organic layers were passed over Na₂SO₄Dried, filtered, concentrated, and purified by column chromatography (0-5% MeOH in DCM) to giveTo compound a5(150mg, 75%) as a white solid. LCMS (5-95, AB,1.5min): RT 0.814min, M/z 961.5[ M + 23%]+。

A solution of compound A5(75.0mg, 0.080mmol) in TFA (9.5mL) and water (0.50mL) was stirred at 14 ℃ for 1 h. The reaction mixture was poured into cold saturated NaHCO₃(100mL) and extracted with DCM (2 × 100 mL). The combined organic layers were dried, concentrated, and purified by preparative TLC (4% MeOH in DCM, Rf ═ 0.5) then by preparative HPLC (Waters Xbridge Prep OBD C18150 × 305 u, conditions: 0.225% FA-CAN) to give PBD dimer disulfide prodrug 3 as a white solid (9.5mg, 14%). LCMS (5-95, AB,1.5min): RT ═ 0.956min, M/z ═ 821.3[ M + 23-]+。

Example 20E: preparation of PBD dimer control 1

PBD dimer control 1 was prepared according to the following reaction scheme:

compound A1(1.80g, 1.71mmol) in HOAc/THF/H₂A solution in O (9.0mL/4.5mL/3.0mL) was stirred at room temperature for 48 h. The solution was diluted with EtOAc (150mL) and washed with H₂O(4x40mL)、NaHCO₃Aqueous solution (4X40mL) and H₂O (40mL) wash. The EtOAc layer was washed with Na₂SO₄Dry, filter and concentrate to give compound a2 as an oil (1.38g, 91%).

To a stirred solution of compound A2(800mg, 0.99mmol) in DCM (20mL) was added DMP (1.26g, 2.97mmol) at 0 ℃. The reaction mixture was stirred at room temperature for 3 h. It was diluted with EtOAc (100mL) and Na at 0 deg.C₂SO₃Aqueous solution (30mL) was quenched. Subjecting the organic layer to H₂O(3x30mL)、NaHCO₃Aqueous solution (30mL) and H₂O (30mL) wash. Subjecting it to Na₂SO₄Drying, filtering, concentrating, and dredgingPurification by preparative TLC (DCM/MeOH ═ 15:1) to give compound a3(400mg, 50.0%) as a colourless solid. LCMS (ESI,5-95AB/1.5min): RT ═ 0.767min, [ M + Na-]+＝843.4。

Compound A3(300mg, 0.36mmol) in 95% TFA/H₂The solution in O (4.0mL) was stirred at 0 ℃ for 2 h. The solution was then added dropwise to saturated NaHCO at 0 deg.C₃In solution (120 mL). The mixture was extracted with DCM (3 × 20 mL). The combined organic layers were passed over Na₂SO₄Dry, filter, dry, concentrate and purify by preparative HPLC to give PBD dimer control 1as a white solid (70mg, 33%). LCMS (ESI,5-95AB/1.5min): RT ═ 0.767min, [ M + Na-]+＝843.4 1H NMR(400MHz,CDCl3)δppm 7.69(d,J＝4.80Hz,2H),7.50(s,2H),6.81(s,2H),5.19(d,J＝10.80Hz,4H),4.29(s,5H),4.02-4.19(m,4H),3.94(s,6H),3.83-3.92(m,3H),3.09-3.16(m,2H),2.90-2.99(m,2H),1.98-1.94(m,4H),1.66-1.70(m,2H)。

Example 20F: preparation of PBD dimer control 2

PBD dimer control 2 was prepared according to the following reaction scheme:

compound A1(1.80g, 1.71mmol) in HOAc/THF/H₂A solution in O (9.0mL/4.5mL/3.0mL) was stirred at room temperature for 48 h. It was diluted with EtOAc (150mL) and washed with H₂O(4x40mL)、NaHCO₃Aqueous solution (2X40mL) and H₂O (40mL) wash. The EtOAc layer was washed with Na₂SO₄Dry, filter and concentrate to give compound 2 as an oil (1.38g, 91%).

To compound A2(800mg, 0.99mmol) at 0 deg.CTo a stirred solution in DCM (20mL) was added DMP (1.26g, 2.97mmol) and the reaction mixture was stirred at rt for 3 h. The mixture was then diluted with EtOAc (100mL) and Na at 0 deg.C₂SO₃Aqueous solution (30mL) was quenched. Subjecting the organic layer to H₂O (3X30mL), saturated NaHCO₃Solution (30mL) and H₂O (30mL) over Na₂SO₄Dried, filtered and concentrated. The residue was purified by preparative TLC (DCM/MeOH ═ 15:1) to give compound a3(400mg, 50.0%) as a colourless solid. LCMS (ESI,5-95AB/1.5min): RT ═ 0.867min, [ M + Na-]+＝843.4。1H NMR(400MHz,CDCl3)δ7.20(s,2H),6.61(s,2H),5.49(d,J＝9.2Hz,2H),5.14(d,J＝4.8Hz,4H),4.32-4.14(m,5H),4.07-3.99(m,5H),3.90(s,6H),3.62(t,J＝9.2Hz,2H),2.96-2.89(m,2H),2.73-2.69(m,2H),1.98-1.94(m,4H),1.69-1.67(m,2H),1.37(s,18H)。

A solution of compound A3(120mg, 0.147mmol) in 95% TFA/H2O (2.0mL) was stirred at 0 ℃ for 2H. The solution was then added dropwise to saturated NaHCO at 0 deg.C₃In solution (120 mL). The mixture was extracted with DCM (3 × 20 mL). The combined organic layers were passed over Na₂SO₄Dried and concentrated to give compound 4(86mg, 100%) as a crude product. LCMS (ESI,5-95AB/1.5min): RT ═ 0.764min, [ M + H ]]+＝585.3。

To a solution of Compound A4(86mg, 0.147mmol) in anhydrous DCM/MeOH (5.0mL/2.5mL) was added NaBH₃CN (92mg, 1.47 mmol). The reaction mixture was stirred at room temperature overnight. It was concentrated and the residue was taken up with saturated NaHCO₃Diluted (20mL) and extracted with DCM (3 × 20 mL). The combined DCM layers were washed with Na₂SO₄Dried, filtered and concentrated. The residue was purified by preparative hplc (fa) to give PBD dimer control 2 as a white solid (15.6mg, 18.1%). LCMS (ESI,5-95AB/1.5min): RT ═ 0.808min, [ M + H-]+＝589.3。1H NMR(400MHz,CDCl3)δ7.58(s,2H),6.05(s,2H),5.06(d,J＝11.6Hz,4H),4.42-4.27(m,6H),3.98(t,J＝6.8Hz,6H),3.84(s,6H),3.55(d,J＝12.0Hz,2H),3.35-3.30(dd,J＝12.4,9.6Hz,2H),2.93-2.87(m,2H),2.46-2.42(m,2H),1.94-1.89(m,4H),1.62-1.60(m,2H)。

Example 21: preparation of PBD dimer disulfide prodrugs comprising a linker for conjugation to an antibody

Example 21A: preparation of PBD dimer disulfide prodrug 4 comprising a linker

PBD dimer disulfide prodrug 4 comprising a linker was prepared according to the following reaction scheme:

each asterisk in the above structure and elsewhere depicted in example 21 represents a chiral center.

To a mixture of A6(400.0mg, 1.47mmol) in DCM (10mL) was added ethanethiol A7(2.74g, 44.1 mmol). The reaction mixture was stirred at 40 ℃ for 30 h. Subjecting the mixture to MnO₂(0.20g) for 5min and filtered. The filtrate was concentrated and the residue was purified by preparative TLC (100% DCM, Rf ═ 0.5) to give compound a2(110mg, 42%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ 3.74(s,2H),2.75-2.70(m,2H),2.13-1.87(m,6H),1.84(s,1H),1.30(t, J ═ 7.6Hz, 3H).

To a solution of triphosgene (82.4mg, 0.28mmol) in DCM (4.0mL) was added dropwise a solution of A2(110.0mg, 0.620mmol) and pyridine (146.4mg, 1.85mmol) in DCM (4.0 mL). The mixture was stirred at 15 ℃ for 30min and concentrated. It was dissolved in DCM (5.0mL) and added dropwise to a solution of A1(1.05g, 1.23mmol) and pyridine (145.87mg, 1.84mmol) in DCM (15.0mL) at 0 ℃. After stirring the mixture at 15 ℃ for 2h, it was concentrated and the residue was purified by column chromatography (0-50% EtOAc in petroleum ether) to give a3(310mg, 45.8%) as a yellow oil. LCMS (5-95, AB,1.5min): RT 1.151min, M/z 1057.4[ M +1] +.

To a solution of triphosgene (26.5mg, 0.090mmol) in DCM (5.0mL) at 0 deg.C was added a solution of A3(210mg, 0.200mmol) and triethylamine (60.28mg, 0.60mmol) in DCM (5.0 mL). The reaction mixture was stirred at 15 ℃ for 30 min. To the above mixture was added dropwise a solution of MC-VC-PAB (166.0mg, 0.290mmol) and triethylamine (59.0mg, 0.58mmol) in DMSO (3.0 mL). The reaction mixture was stirred at 40 ℃ for 2 h. The mixture was diluted with DCM (30mL) and washed with water (3 × 10 mL). The combined organic layers were dried, concentrated, and purified by column chromatography (0-10% MeOH in DCM) to give a4(160mg, 48.4%) as a yellow oil. LCMS (5-95, AB,1.5min): RT 1.271min, M/z 828.6[ M/2+1] +.

To a mixture of A4(160.0mg, 0.100mmol) in THF (3.0mL) and water (3.0mL) was added acetic acid (4.5 mL). The reaction mixture was stirred at 15 ℃ for 15 h. The mixture was diluted with EtOAc (30mL), and washed with water (2X10mL), saturated NaHCO₃(2 × 10mL) and brine (10 mL). Passing the organic phase over Na₂SO₄Dried and concentrated to give crude product a5(120mg, 82.7%) as a yellow oil, which was used in the next step without further purification. LCMS (5-95, AB,1.5min): RT 0.801min, M/z 714.6[ M/2+ 1[ ]]+。

To a mixture of A5(60.0mg, 0.040mmol) in DMSO (3.0mL) was added IBX (58.8mg, 0.21 mmol). The reaction mixture was stirred at 40 ℃ for 16 h. The mixture was purified by preparative HPLC (ACN 40-70%/0.225% aqueous FA) to give (15mg, 25.1%) as a white solid. LCMS (5-95, AB,1.5min): RT 0.758min, M/z 712.5[ M/2+1] +.

In some aspects of the disclosure, PBD dimer disulfide prodrug 4 comprising a linker can be conjugated to an antibody to form PBD dimer ADC disulfide prodrug 4.

Example 21B: preparation of PBD dimer disulfide prodrug 3 comprising linker

PBD dimer disulfide prodrug 3 comprising a linker was prepared according to the following reaction scheme:

to a solution of triphosgene (486.9mg, 1.64mmol) in DCM (10mL) was added a solution of compound A1(1.40g, 1.64mmol) and triethylamine (664mg, 6.56mmol) in DCM (10 mL). The mixture was stirred at 8 ℃ for 10 min. The mixture was concentrated to give the crude product as a yellow solid (1.48g, 99.6%). To a solution of the above crude product (1.41g, 1.56mmol) in DCM (15mL) was added a solution of compound A2(150.0mg, 0.780mmol) and triethylamine (158mg, 1.56mmol) in DCM (6.0 mL). After stirring the mixture at 8 ℃ for 1h, it was concentrated and purified by flash column chromatography (50% EtOAc in petroleum ether) to give the product compound a3(300mg, 30%) as a yellow solid. LCMS (5-95, AB,1.5min): RT ═ 1.193min, M/z ═ 1071.5[ M +1] +.

To a solution of triphosgene (41.6mg, 0.140mmol) in DCM (6.0mL) was added a solution of compound A3(300mg, 0.280mmol) and triethylamine (56.7mg, 0.560mmol) in DCM (5.0 mL). The mixture was stirred at 8 ℃ for 15 min. The mixture was concentrated to give the crude product as a yellow solid (307mg, 99.9%) which was used directly in the next step. To a solution of the crude product (300.0mg, 0.270mmol) in DCM (10mL) was added a solution of MC _ VC _ PAB (156mg, 0.270mmol) and triethylamine (27.7mg, 0.270mmol) in DMF (6.0 mL). After the mixture was stirred at 8 ℃ for 12h, it was concentrated and purified by flash column chromatography (6% MeOH in DCM) to give the product compound a4 as a yellow solid (140mg, 31%). LCMS (5-95, AB,1.5min): RT 1.354min, M/z 836.4[ M/2+1] +.

A mixture of compound A4(140.0mg, 0.080mmol) in THF (2.0mL), water (2.0mL) and acetic acid (3.0mL) was stirred at 8 ℃ for 12 h. The mixture was diluted with EtOAc (60mL) and washed with water (3 × 50mL), saturated NaHCO mL₃(50mL), brine (50 mL). The organic layer was dried and concentrated to give the crude product compound a5(120mg, 99%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.983min, M/z 722.0[ M/2+ 1%]+。

To a solution of compound A5(140.0mg, 0.100mmol) in DMSO (4.0mL) at 18 ℃ was added IBX (108mg, 0.390 mmol). The reaction mixture was stirred at 40 ℃ for 8 h. The mixture was purified by preparative HPLC (ACN 40-70%/0.225% aqueous FA) to give the product as a white solid, PBD dimer disulfide prodrug 2(20mg, 14%) containing linker. LCMS (5-95, AB,1.5min): RT 0.760min, M/z 719.7[ M/2+1] +.

In some aspects of the disclosure, a PBD dimer disulfide prodrug 3 comprising a linker can be conjugated to an antibody to form a PBD dimer ADC disulfide prodrug 2A or a PBD dimer ADC disulfide prodrug 2B.

Example 21C: preparation of PBD dimer disulfide prodrug 2 comprising a linker

PBD dimer disulfide prodrug 2 comprising a linker was prepared according to the following reaction scheme:

to a solution of compound A8(3.18g, 10.24mmol) in DCM (25.0mL) was added compound A9(400mg, 5.12 mmol). The mixture was stirred at 8 ℃ for 12 h. Adding MnO to the mixture₂(100mg) and stirred for 10min and filtered. The filtrate was concentrated and purified by flash column chromatography (100% DCM) to give compound 2(620mg, 2.67mmol, 52.1%) as a yellow solid.

To a solution of triphosgene (191.6mg, 0.65mmol) in DCM (5.0mL) was added a solution of compound A2(300mg, 1.29mmol) and pyridine (306mg, 3.87mmol) in DCM (5.0 mL). The mixture was stirred at 8 ℃ for 10 min. The resulting mixture was added dropwise to a solution of compound a1(1.43g, 1.68mmol) and pyridine (306mg, 3.87mmol) in DCM (15.0 mL). After stirring the mixture at 8 ℃ for 30min, it was concentrated and purified by flash column chromatography (50% EtOAc in petroleum ether) to give the product compound a3(0.50g, 0.427mmol, 33.1%) as a yellow oil. LCMS (5-95, AB,1.5min): RT ═ 1.077min, M/z ═ 1111.7[ M +1] +.

To a solution of compound A3(300mg, 0.270mmol) in DCM (10mL) was added compound A4(205mg, 2.7 mmol). The mixture was stirred at 8 ℃ for 10 min. Adding MnO to the mixture₂(100mg), stirred for 10min and filtered. The filtrate was concentrated and purified by flash column chromatography (50% EtOAc in petroleum ether) to give the product compound a5(210mg, 0.204mmol, 75.4%) as a yellow solid.

To a solution of triphosgene (30.2mg, 0.100mmol) in DCM (5.0mL) was added a solution of compound A5(210mg, 0.200mmol) and triethylamine (61.7mg, 0.610mmol) in DCM (5.0 mL). The mixture was stirred at 8 ℃ for 30 min. The mixture was then added dropwise to a solution of MC _ VC _ PAB (139.8mg, 0.240mmol) and triethylamine (61.7mg, 0.610mmol) in DMF (5.0 mL). The mixture was stirred at 8 ℃ for 12 h. The mixture was concentrated and purified by flash column chromatography (8% MeOH in DCM) to give the product compound a6(140mg, 0.085mmol, 41.8%) as a yellow oil. LCMS (5-95, AB,1.5min): RT 1.248min, M/z 816.1[ M/2+1] +.

A mixture of Compound A6(140.0mg, 0.090mmol) in acetic acid (3.0mL), THF (2.0mL) and water (2.0mL) was stirred at 8 ℃ for 8 h. The mixture was diluted with EtOAc (60mL), and washed with water (3X50mL), saturated NaHCO₃(50mL), brine (50mL) and concentrated to give the product, Compound A7, as a white solid (120mg, 0.0856mmol, 99.7%). LCMS (5-95, AB,1.5min): RT 0.787min, M/z 701.6[ M/2+ 1%]+。

To a solution of compound A7(130.0mg, 0.090mmol) in DMSO (3.0mL) at 9 ℃ was added IBX (129.9mg, 0.460 mmol). The reaction mixture was stirred at 50 ℃ for 48 h. The mixture was purified by preparative HPLC (ACN 35-65%/0.225% aqueous FA) to give the product as a white solid, PBD dimer disulfide prodrug 4 containing linker (10mg, 0.0071mmol, 7.6%). LCMS (5-95, AB,1.5min): RT ═ 0.728min, M/z ═ 1397.9[ M +1] +.

In some aspects of the disclosure, PBD dimer disulfide prodrug 2 comprising a linker can be conjugated to an antibody to form PBD dimer ADC disulfide prodrug 4.

Example 21D: preparation of a PBD dimer disulfide prodrug comprising a linker for conjugation to form PBD dimer diaphorase prodrugs 1A and 1B, and having the following structure:

and name: 4- (tert-butyldisulfanyl) benzyl (11aS) -8- ((5- (((11aS) -10- (((4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoylamino) -3-methylbutanoylamino) -5-ureidopentanoylamino) benzyl) oxy) carbonyl) -11-hydroxy-7-methoxy-2-methylene-5-oxo-2, 3,5,10,11,11 a-hexahydro-1H-benzo [ e ] e]Pyrrolo [1,2-a][1,4]Diaza derivatives-8-yl) oxy) pentyl) oxy) -11-hydroxy-7-methoxy-2-methylene-5-oxo-2, 3,11,11 a-tetrahydro-1H-benzo [ e]Pyrrolo [1,2-a][1,4]Diaza derivatives-10(5H) -carboxylic acid ester.

PBD dimer disulfide prodrugs comprising a linker were prepared according to the following reaction scheme:

to a solution of A7(250mg, 1.78mmol) in 95% EtOH (10mL) was added A8(2.01mL, 17.85 mmol). The mixture was cooled to 0 ℃ and a solution of iodine (200mg, 0.79mmol) in 95% EtOH (10mL) was added dropwise until the color of the mixtureFrom colorless to brown. After stirring for 2h, saturated NaHCO was added at 0 deg.C₃(2.0mL) until the pH is greater than 7. The solution was concentrated in vacuo. EtOAc (20mL) was added and the organic layer was washed with 10% NaHCO3(3 × 15mL) and brine. Subjecting the organic layer to Na₂SO₄Dry, filter and concentrate, and purify by flash column chromatography (0-32% EtOAc in petroleum ether) to give compound a2(280mg, 68.8%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ 7.51(d, J ═ 8.4Hz,2H),7.23(d, J ═ 8.4Hz,2H),4.57(s,2H),2.40(br,1H),1.29(s, 9H).

At 0 ℃ and N₂To a solution of triphosgene (26mg, 0.090mmol) in DCM (1.0mL) was added a solution of compound A2(50.0mg, 0.220mmol) and pyridine (18.0mg, 0.230mmol) in DCM (4.0 mL). The reaction mixture was heated at 0 ℃ to N₂Stirring for 5min, and heating at 0 deg.C under N₂It was then added dropwise to a solution of pyridine (34.0mg, 0.430mmol) and A1(277mg, 0.320 mmol). The reaction mixture was heated at 20 ℃ N₂Stirring for 2 h. The solvent was removed and the residue was purified by preparative TLC (50% EtOAc in petroleum ether, Rf ═ 0.4) to give compound a3(70mg, 28.3%) as a yellow foam. LCMS (5-95, AB,1.5min): RT 1.328min, M/z 1108.7[ M + 1%]+。

At 0 ℃ and N₂To a solution of triphosgene (46.0mg, 0.160mmol) in DCM (7.0mL) was added a mixture of compound A3(400.0mg, 0.360mmol) and triethylamine (37.0mg, 0.370mmol) in DCM (3.0 mL). The reaction mixture was stirred at 0 ℃ for 30min, then at 20 ℃ N₂Next, a solution of MC-VC-PAB (247.0mg, 0.430mmol) and triethylamine (73.0mg, 0.720mmol) in DMF (3.0mL) was added. The reaction mixture was heated at 40 ℃ to N₂Stirring for 8 h. The mixture was concentrated and purified by column chromatography (0-6% MeOH in DCM) to give compound a4(190mg, 30.7%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.308min, M/z 854.2[ M/2+ 1[ ]]+。

To a solution of compound A4(415mg, 0.240mmol) in water (2.0mL) and THF (2mL) at 15 deg.C was added HOAc (6.47mL, 113 mmol). After stirring the reaction mixture at 15 ℃ for 7h, it was washed with EtOAcDiluted (30mL) and diluted with water (2 × 15mL), saturated NaHCO₃Aqueous solution (15mL) and brine (15 mL). It was dried and concentrated to give crude compound a5(150mg, 41.7%) as a yellow solid, which was used in the next step without further purification. LCMS (5-95, AB,1.5min): RT 0.997min, M/z 739.4[ M/2+ 1%]+。

To a solution of compound A5(50.0mg, 0.030mmol) in DMSO (3.0mL) at 18 ℃ was added IBX (38.0mg, 0.140 mmol). The reaction mixture was stirred at 37 ℃ for 8 h. The mixture was purified by preparative HPLC (ACN 40-70%/0.225% aqueous FA) to give PBD dimer disulfide prodrug 1(17.2mg, 33.1%) containing linker as a white solid. LCMS (5-95, AB,1.5min): RT 0.806min, M/z 737.1[ M/2+1] +.

In some aspects of the disclosure, a PBD dimer disulfide prodrug comprising a linker can be conjugated to an antibody to form PBD dimer ADC disulfide prodrug 1A or PBD dimer ADC disulfide prodrug 1B.

Example 21E: preparation of linker-containing PBD dimer disulfide prodrug 5

PBD dimer disulfide prodrug 5 comprising a linker was prepared according to the following reaction scheme:

to a solution of TBDPSCl (8.62g, 31.36mmol) in DMF (40mL) was added a solution of Compound A8(2.27g, 29.05mmol) in DMF (30 mL). After stirring the solution for 10min, imidazole (4.27g, 62.7mmol) in DMF (8.0mL) was added and the reaction mixture was stirred at 20 ℃ for 24 h. The mixture was concentrated and dissolved in DCM (30mL), filtered and washed with H2O (3 × 30 mL). Will be organicThe layers are over MgSO₄Dried, filtered, and the solvent removed. The residue was purified by flash column chromatography (3% EtOAc in petroleum ether) to give compound a9(7.0g, 76%) as a colorless oil.

To a solution of compound a9(1.00g, 3.16mmol) in DCM (10mL) was added dropwise a solution of compound a10(1.96mg, 6.32mmol) in DCM (10mL) over 15 min. After the mixture was stirred at 26 ℃ for a further 1h, manganese dioxide (1.00g, 11.5mmol) was added and stirred for 10min until the yellow solution became colourless. The manganese dioxide is filtered off and the filtrate is concentrated. MeOH (5.0mL) was added and the solid was filtered to remove compound a 10. The residue was purified by column chromatography (0-2.5% EtOAc in petroleum ether) to give compound a11(0.90g, 61%) as a yellow solid.

To a solution of compound a11(250mg, 1.89mmol) in DCM (6.0mL) was added dropwise a solution of compound a12(0.50g, 1.58mmol) in DCM (4.0mL) over 15 min. After addition, the mixture was stirred at 26 ℃ for a further 1 h. Manganese dioxide (1.0g, 11.5mmol) was added. The mixture was stirred for another 10min until the yellow reaction solution became colorless. Manganese dioxide was filtered off, the filtrate was concentrated and MeOH (5.0mL) was added. The solid was filtered off and the residue was purified by column chromatography (0-14% EtOAc in petroleum ether) to give compound a2(0.500g, 71%) as a yellow oil.

To a solution of triphosgene (695mg, 2.34mmol) in DCM (5.0mL) at 0 deg.C was added a solution of compound A1(2.0g, 2.34mmol) and triethylamine (711.0mg, 7.03mmol) in DCM (10 mL). The mixture was stirred at 0 ℃ for 30 min. The reaction mixture was concentrated and a solution of compound A2(680mg, 1.52mmol) and triethylamine (308mg, 3.04mmol) in DCM (4.0mL) was added. The mixture was stirred at 25 ℃ for 2h, concentrated, and purified by column chromatography (0-50% EtOAc in petroleum ether) to give a3(700mg, 33%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.401min, M/z 1326.0[ M +1] +.

At 0 ℃ and N₂To a solution of triphosgene (54.0mg, 0.180mmol) in DCM (10mL) was added compound a3(500mg, 0.450mmol) and triethylamine (50.0mg,0.490mmol) in DCM (5.0 mL). The reaction mixture was stirred at 0 ℃ for 30 min. To the reaction mixture was added a solution of Fmoc-VC _ PAB (260.0mg, 0.450mmol) and triethylamine (78.0mg, 0.770mmol) in DMF (3.0 mL). The mixture was stirred at 40 ℃ for 8h, concentrated, and purified by column chromatography (0-8% MeOH in DCM) to give compound a4(130mg, 14%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.474min, M/z 978.1[ M/2+ 1%]+。

To a solution of compound A4(130.0mg, 0.070mmol) in water (2.0mL) and THF (2.0mL) at 26 ℃ was added HOAc (3.0mL, 52.5mmol) and stirred at 26 ℃ for 7 h. The reaction mixture was diluted with EtOAc (20mL), diluted with water (2 × 15mL), saturated NaHCO₃(10mL) and brine (10 mL). It was dried and concentrated to give crude compound a5(130mg, 83%) as a yellow solid, which was used in the next step without further purification. LCMS (5-95, AB,1.5min): RT ═ 1.065min, M/z ═ 863.2[ M/2+1]+。

To a solution of compound A5(130.0mg, 0.080mmol) in DMSO (3.0mL) at 25 ℃ was added 2-iodoxybenzoic acid (84.4mg, 0.300 mmol). After the mixture was stirred at 40 ℃ for 10h, it was purified by preparative HPLC (ACN 85-100%, 0.225% aqueous FA) to give product a 6as a white solid (60mg, 45%). LCMS (5-95, AB,1.5min): RT 1.178min, M/z 861.4[ M/2+1] +.

To a solution of compound A6(60.0mg, 0.035mmol) in THF (3.0mL) at 26 deg.C was added TBAF (39.4mg, 0.150 mmol). The reaction mixture was stirred at 26 ℃ for 2 h. The reaction mixture was diluted with DCM (20mL), washed with water (3 × 15mL) and taken over Na₂SO₄Dried and concentrated to give crude compound a7(70mg, crude product) as a yellow oil.

To a stirred solution of compound a7(70.0mg, 0.060mmol) in DMF (2.0mL) at 26 ℃ was added MC _ OSu (51.4mg, 0.170 mmol). The mixture was stirred at 26 ℃ for 2 h. The reaction mixture was purified by preparative HPLC (ACN 35-55%/0.225% aqueous FA) to give as a white solid (5.5mg, 6.7%). LCMS (5-95, AB,1.5min): RT ═ 0.852min, M/z ═ 1453.5[ M +1] +.

In some aspects of the disclosure, PBD dimer disulfide prodrug 5 comprising a linker can be conjugated to an antibody to form PBD dimer ADC disulfide prodrug 5.

Example 22: preparation of PBD dimer boronic acid prodrugs comprising linkers for conjugation to antibodies

Example 22A: preparation of PBD dimer boronic acid prodrug 1 comprising linker

PBD dimer boronic acid prodrug 1 comprising linker was prepared according to the following reaction scheme:

each asterisk in the above structure and elsewhere depicted in example 22 represents a chiral center.

At 0 ℃ and N₂To a solution of triphosgene (228mg, 0.77mmol) in THF (15mL) was added a solution of A2(450mg, 1.92mmol) and pyridine (304mg, 3.84mmol) in THF (5.0 mL). Mixing the mixture in N₂Stirring at 0 deg.C for 20min, and stirring at 0 deg.C under N₂Add to A1(2.04g, 2.39mmol) and triethylamine (389mg, 3.84mmol) in DCM (20 mL). The reaction mixture was stirred at 10 ℃ for 2 h. The mixture was concentrated and purified by preparative TLC (30-60% EtOAc in petroleum ether) to give compound a3(500mg, 26%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.268min, M/z 1113.4[ M + 1%]+。

At 0 ℃ and N₂To a solution of triphosgene (53mg, 0.18mmol) in DCM (15mL) was added a mixture of compound A3(500mg, 0.39mmol) and triethylamine (45mg, 0.44mmol) in DCM (5.0 mL). The reaction mixture was stirred at 0 ℃ for 30 min. At 10 ℃ and N₂Down to the reaction mixtureTo this was added a solution of MC _ VC _ PAB (308mg, 0.54mmol) and triethylamine (90mg, 0.89mmol) in DMSO (4.0 mL). The reaction mixture was heated at 40 ℃ to N₂Stirring for 6 h. The mixture was diluted with DCM (30mL), washed with water (2 × 15mL) and the aqueous layer extracted with EtOAc (2 × 20 mL). The combined organic layers were passed over Na₂SO₄Dry, concentrate and purify through flash column (0-10% MeOH in DCM) to give compound 8 as a yellow solid (300mg, 34% yield). LCMS (5-95, AB,1.5min): RT 1.253min, M/z 857.1[ M/2+ 1[ ]]+。

To a solution of compound A4(100.0mg, 0.050mmol) in water (2.0mL) and THF (2.0mL) at 10 deg.C was added HOAc (3.0 mL). The reaction mixture was stirred at 10 ℃ for 6 h. The reaction mixture was diluted with EtOAc (20mL), diluted with water (2 × 15mL), saturated NaHCO₃Aqueous solution (15mL) and brine (15 mL). Subjecting it to Na₂SO₄Dried and concentrated to give crude compound a5 as a yellow solid (71mg, 99.8% yield). LCMS (5-95, AB,1.5min): RT 0.829min, M/z 857.1[ M/2-17%]+。

To a solution of compound a5(36mg, 0.030mmol) in DMSO (3.0mL) at 9 ℃ was added 2-iodoxybenzoic acid (22mg, 0.080 mmol). The reaction mixture was stirred at 40 ℃ for 6 h. The mixture was purified by preparative HPLC (ACN 27-47%/0.225% aqueous FA) to give PBD dimer boronic acid prodrug 1(2.0mg, 5.5%) containing linker as a white solid. LCMS (5-95, AB,1.5min): RT ═ 0.813min, HRMS: M/z ═ 1397.5906[ M +1] +.

In some aspects of the disclosure, the PBD dimer boronic acid prodrug 1 comprising a linker can be conjugated to an antibody to form PBD dimer ADC boronic acid prodrug 1A or PBD dimer ADC boronic acid prodrug 1B.

Example 22B: preparation of PBD dimer boronic acid control 1 containing linker

PBD dimer boronic acid control 1 containing linker was prepared according to the following reaction scheme:

to compound A1(500.0mg, 0.590mmol) in DMF (2.0mL) was added Cbz-OSu (161mg, 0.640 mmol). After stirring the mixture at 50 ℃ for 4h, another batch of Cbz-OSu (161mg, 0.640mmol) was added. The mixture was stirred at 50 ℃ for 16h and purified by preparative TLC (5% MeOH in DCM, Rf ═ 0.8) and then by preparative HPLC to give compound a2 as an orange oil (220mg, 35.2%). LCMS (5-95, AB,1.5min): RT ═ 1.097min, M/z ═ 987.5[ M +1] +.

Triphosgene (26.45mg, 0.090mmol) andMS (30mg) to a mixture of DCM (3.0mL) was added a solution of compound A2(220.0mg, 0.220mmol) and triethylamine (22.6mg, 0.220mmol) in DCM (2.0 mL). The mixture was stirred at 0 ℃ for 1h and concentrated. To a solution of isocyanate (225.0mg, 0.220mmol) in DCM (6.0mL) at 0 deg.C were added MC _ VC _ PAB (34.59mg, 0.060mmol), Et3N (23mg, 0.22mmol) anda solution of MS (30mg) in DMF (2.0 mL). After stirring the mixture at 20 ℃ for 16h, it was quenched with water. DCM (10mL) was added, separated and the DCM phase was concentrated and purified by preparative TLC (15% MeOH in DCM, Rf ═ 0.4) to give compound a3(70mg, 19.8%) as a light yellow oil. LCMS (5-95, AB,1.5min): RT 1.107min, M/z 793.9[ M/2+ 1%]+。

To compound A3(70.0mg, 0.040mmol) in a mixture of THF (3.0mL) and water (2.0mL) was added HOAc (1.0mL, 17.49mmol), and the mixture was stirred at 40 ℃ for 8 h. The mixture was concentrated and purified by preparative TLC (13% MeOH in DCM, Rf ═ 0.5) to give compound a4(40mg, 67.8%) as a yellow oil. LCMS (5-95, AB,1.5min): RT ═ 0.751min, M/z ═ 679.6[ M/2+1] +.

To a solution of compound a4(30.0mg, 0.020mmol) in DMSO (3.0mL) at 18 ℃ was added 2-iodoxybenzoic acid (30.9mg, 0.110 mmol). After stirring the reaction mixture at 40 ℃ for 16h, it was purified by preparative TLC (10% MeOH in DCM, Rf ═ 0.4) to give PBD dimer boronic acid control 1 containing linker as a white solid (13mg, 43.5%). LCMS (5-95, AB,1.5min): RT ═ 0.726min, M/z ═ 677.5[ M/2+1] +.

In some aspects of the disclosure, the PBD dimer boronic acid control 1 comprising a linker can be conjugated to an antibody to form the PBD dimer ADC boronic acid control 1A or the PBD dimer ADC boronic acid control 1B.

Example 23: preparation of PBD monomer and dimer diaphorase prodrug

Example 23A: preparation of PBD monomer diaphorase prodrug 2

PBD monomeric diaphorase prodrug 2 was prepared according to the following reaction scheme:

each asterisk in the above structure and elsewhere depicted in example 23 represents a chiral center.

In N₂To a solution of triphosgene (58.39mg, 0.200mmol) in DCM (2.0mL) at 0 ℃ was added slowly a solution of compound a1 in DCM (5.0mL) and the mixture was stirred at 0 ℃ for 1 h. Compound a2(100.0mg, 0.450mmol) and triethylamine (91.5mg, 0.900mmol) in DCM (2.0mL) were added dropwise to the above solution over 10min at 0 ℃. The mixture was stirred at 0 ℃ for 1 h. The mixture was quenched with water (5.0mL)Separated and concentrated. It was purified by preparative TLC (5% MeOH in DCM, Rf ═ 0.7) to give compound a3(63mg, 19%) as an orange solid. LCMS (5-95, AB,1.5min): RT 1.007min, M/z 654.3[ M + 1%]+。

To a solution of compound A3(63.0mg, 0.100mmol) in THF (2.0mL) and water (1.0mL) was added acetic acid (2.0mL, 2.1 mmol). The mixture was stirred at 15 ℃ for 16 h. The mixture was concentrated and purified by preparative TLC (5% MeOH in DCM, Rf ═ 0.3) to give a4(40mg, 77%) as an orange solid. LCMS (5-95, AB,1.5min): RT 0.761min, M/z 540.1[ M +1] +.

To compound a4(26.0mg, 0.050mmol) in DCM (3.0mL) was added DMP (40.0mg, 0.090mmol) and the mixture was stirred at 10 ℃ for 16 h. The mixture was saturated with Na₂SO₃And NaHCO₃The mixture of solutions (3.0mL/3.0mL) was quenched. The organic phase was separated, concentrated and purified by preparative HPLC (Diamonsil 150 x20 mm x 5um, 0.225% FA-ACN, ACN 23-53%) to give PBD monomeric diaphorase prodrug 2 as an orange solid (20mg, 74%). LCMS (5-95, AB,1.5min): RT 0.747min, M/z 538.1[ M +1]+。

1H NMR(400MHz,CDCl3)δ7.22(s,1H),6.64(s,1H),6.52(s,1H),5.69(s,1H),5.59-5.57(d,J＝9.6Hz,1H),5.30-5.27(d,J＝13.6Hz,1H),5.16-5.15(m,2H),4.94-4.90(d,J＝13.6Hz,1H),4.32-4.27(d,J＝16.4Hz,1H),4.17-4.13(d,J＝16.4,1H),3.93(s,3H),3.83(s,3H),3.78-3.76(m,6H),3.64-3.59(m,1H),2.47(m,1H),2.96-2.89(m,1H),2.73-2.69(m,1H)。

Example 23B: preparation of PBD dimer diaphorase prodrug 1

PBD dimer diaphorase prodrug 1 was prepared according to the following reaction scheme:

three lights at 0 deg.CTo a solution of gas (56.03mg, 0.190mmol) in DCM (2.0mL) was added dropwise a solution of compound A1(400mg, 0.420mmol) and triethylamine in DCM (5.0 mL). After the mixture was stirred at 19 ℃ for 1h, a solution of compound A2(92.8mg, 0.420mmol) and triethylamine (42.5mg, 0.420mmol) in DMSO (0.50mL)/DCM (2.50mL) was added. The mixture was stirred at 19 ℃ for 2.0 h. The mixture was diluted with DCM (20.0mL), washed with water (2X10.0mL), and separated. The aqueous layer was extracted with EtOAc (2 × 20mL) and the combined organic layers were filtered over Na₂SO₄Dry, concentrate and purify by preparative TLC (20% MeOH in DCM, Rf ═ 0.7) to give compound a3(150mg, 30%). LCMS (5-95, AB,1.5min): RT 1.118min, M/z 1201.7[ M +1 [)]+。

To a solution of compound A3(130.0mg, 0.110mmol) in THF (2.0mL) was added acetic acid (3.0mL) and water (1.0 mL). The mixture was stirred at 18 ℃ for 18 h. Addition of saturated NaHCO₃To adjust the pH to 8 and extract the mixture with DCM (2 × 50 mL). The organic layers were combined, washed with brine (30mL) and water (30mL), and Na₂SO₄And (5) drying. It was concentrated and purified by preparative TLC (6.7% MeOH in DCM) to give compound a4(72mg, 68%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.766min, M/z 972.3[ M + 1%]+。

To a solution of compound 4(50.0mg, 0.050mmol) in DCM (10mL) was added DMP (76.36mg, 0.180mmol) and the mixture was stirred at 18 ℃ for 18 h. The mixture was filtered and saturated Na was used₂CO₃The filtrate was washed (20.0 mL). The aqueous layer was extracted with DCM (2X20.0mL) and the organic layers were combined and taken over Na₂SO₄Dried and concentrated. It was purified by preparative TLC (6.7% MeOH in DCM, Rf ═ 0.5) to give compound a5(40mg, 74%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.731min, M/z 990.2[ M +23 ═ M]+。

TFA (1.0mL) was added dropwise to Compound A5(40.0mg, 0.040mmol) at 0 ℃. The mixture was stirred at 0 ℃ for 30 min. Saturated NaHCO at 0 deg.C₃Added dropwise to the mixture to adjust pH 7. It was extracted with DCM (3X20.0mL) and combinedWas washed with brine (20.0mL) and Na₂SO₄Dried and concentrated. The residue was purified by preparative TLC (6.7% MeOH in DCM, Rf ═ 0.6) to give PBD dimer diaphorase prodrug 1(4.9mg, 14%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.807min, M/z 850.2[ M + 23%]+。

Example 23C: preparation of PBD monomer myocardial xanthase prodrug 3

PBD monomeric diaphorase prodrug 3 was prepared according to the following reaction scheme:

to a solution of compound A1(280mg, 1.27mmol) in DMF (10mL) at 16 ℃ were added DIEA (490mg, 3.79mmol) and bis (4-nitrophenyl) carbonate (770mg, 2.53 mmol). The reaction mixture was heated at 16 ℃ and N₂Stirring for 2 h. The reaction solution was concentrated and washed with MTBE to give compound a2(500mg, 78%) as an orange solid. LCMS (5-95, AB,1.5min): RT ═ 0.726min, M/z ═ 491.0[ M + 23-]+。

To a solution of compound A2(500mg, 0.99mmol), compound A3(240mg, 1.95mmol) and HOBt (13mg, 0.10mmol) in DMF (8.0mL) at 16 ℃ was added DIEA (340mg, 2.63 mmol). The reaction mixture was heated at 50 ℃ to N₂Stirring for 2 h. The reaction was concentrated and the residue was washed by MeCN (3x8mL) to give compound a4(350mg, 95%) as an orange solid. LCMS (5-95, AB,1.5min): RT 0.745min, M/z 393.1[ M + 23%]+。

At 0 ℃ and N₂To a solution of triphosgene (58mg, 0.20mmol) in DCM (8.0mL) was added a solution of compound A5(200mg, 0.49mmol) and triethylamine (60.0mg, 0.59mmol) in DCM (1.5 mL). The reaction mixture was heated at 12 ℃ and N₂Stirred for 30min under N₂Next, compound A4(91mg, 0.25mmol), TEA (75mg, 0.74mmol) and DMAP (6mg, 0.05mmol) in DCM (1.5mL) and D at 0 deg.C were addedSolution in MSO (1.0 mL). The reaction mixture was heated at 12 ℃ and N₂After stirring for 6h, it was diluted with DCM (30mL) and washed with water (2 × 15 mL). The aqueous layer was extracted with EtOAc (2 × 20mL) and the combined organic layers were filtered over Na₂SO₄Dried, concentrated, and purified by preparative HPLC (ACN 66-86%/0.225% aqueous FA) to give compound a6(60mg, 12%) as an orange solid. LCMS (5-95, AB,1.5min): RT 1.051min, M/z 803.2[ M + 1%]+。

To a solution of compound A6(40mg, 0.05mmol) in water (1.0mL) and THF (1.0mL) at 10 deg.C was added HOAc (1.5mL, 26 mmol). The reaction mixture was stirred at 10 ℃ for 6 h. The reaction mixture was diluted with EtOAc (20mL) and washed with water (2 × 15mL), saturated NaHCO₃(15mL) and brine (15 mL). Subjecting it to Na₂SO₄Dried, concentrated, and purified by preparative TLC (6.25% MeOH in DCM) to give compound a7(35mg, 97%) as an orange solid. LCMS (5-95, AB,1.5min): RT 0.819min, M/z 689.1[ M + 1%]+。

To a solution of compound a7(35mg, 0.050mmol) in DCM (4.0mL) at 0 ℃ was added DMP (61mg, 0.14 mmol). The reaction mixture was stirred at 10 ℃ for 10 h. The reaction was quenched with saturated NaHCO₃/Na₂SO₃Quenched (4.0mL/4.0mL) and extracted with DCM (3X10 mL). The combined organic layers were washed with NaHCO₃/Na₂SO₃(4.0mL/4.0mL), brine (7.0 mL). Subjecting it to Na₂SO₄Dried, concentrated, and purified by preparative HPLC (ACN 30-60%/0.225% aqueous FA) to give PBD monomeric diaphorase prodrug 3(15mg, 45%) as an orange solid. LCMS (5-95, AB,1.5min): RT 0.806min, M/z 687.2[ M +1 [)]+；1H NMR(400MHz,CDCl3)δ7.30-7.25(m,2H),7.18-7.15(M,4H),6.77(s,1H),6.69(S,1H),6.50(s,1H),5.67(s,1H),5.56(d,J＝9.2Hz,1H),5.27(d,J＝12.4Hz,1H),5.17-5.12(m,4H),4.81(d,J＝12.4Hz,1H),4.27(d,J＝16.0Hz,1H),4.12(d,J＝16.0Hz,1H),3.33(s,3H),3.89(s,3H),3.81(s,3H),3.68-3.59(m,4H),2.93-2.87(m,1H),2.69(d,J＝15.6Hz,1H)。

Example 23D: preparation of PBD dimer diaphorase prodrug 2

PBD dimer diaphorase prodrug 2 was prepared according to the following reaction scheme:

at 0 ℃ and N₂To a solution of triphosgene (62.0mg, 0.210mmol) in DCM (12mL) was added a solution of compound A1(500.0mg, 0.520mmol) and triethylamine (63.0mg, 0.620mmol) in DCM (2.0 mL). The reaction mixture was heated at 12 ℃ and N₂Stirring for 30min at 0 deg.C, N₂Next, a solution of compound A2(97.0mg, 0.260mmol), DMAP (6.0mg, 0.050mmol) and triethylamine (79.0mg, 0.780mmol) in DCM (1.5mL) and DMSO (0.60mL) was added. The reaction mixture was heated at 12 ℃ and N₂Stirring for 6 h. It was diluted with DCM (30mL) and washed with water (2 × 15 mL). The aqueous layer was extracted with EtOAc (2 × 20mL) and the organic layer was washed with Na₂SO₄And (5) drying. It was purified by silica gel chromatography (3% MeOH in DCM) to give compound a3(200mg, 49%) as an orange solid. LCMS (5-95, AB,1.5min): RT 1.294min, M/z 1349.6[ M +1 [)]+。

To a solution of compound A3(206.9mg, 0.130mmol) in water (2.0mL) and THF (2.0mL) at 9 ℃ was added HOAc (3.0mL, 52.46 mmol). The reaction mixture was stirred at 9 ℃ for 10 h. It was diluted with DCM (20mL) and the mixture was washed with NaHCO₃(2 × 15mL), water (15 mL). Subjecting it to Na₂SO₄Dry, concentrate, and purify by preparative TLC (6.25% MeOH in DCM) to give compound a4(100mg, 66%) as a yellow solid. LCMS (5-95, AB,1.5min): RT ═ 0.932min, M/z ═ 1121.6[ M + 1-]+。

To a solution of compound A4(100.0mg, 0.090mmol) in DCM (10mL) at 0 deg.C was added DMP (113.0mg, 0.270 mmol). The reaction mixture was stirred at 9 ℃ for 10 h. NaHCO is used for reaction₃/Na₂SO₃Was quenched and extracted with DCM (3X10mL) (5.0mL/5.0mL)And (6) taking. The combined organic layers were washed with NaHCO₃/Na₂SO₃(5mL/5mL), brine (10mL), dried and concentrated. The residue was purified by preparative TLC (6.25% MeOH in DCM) to give compound a5(45mg, 46%) as an orange solid. LCMS (5-95, AB,1.5min): RT 0.884min, M/z 1140.0[ M + 23%]+。

Cold TFA (95% aq, 2.0mL) was added to Compound A5(35.0mg, 0.030mmol) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 15 min. The reaction mixture was added dropwise to saturated NaHCO at 0 deg.C₃Aqueous (4.0mL) and extracted with DCM (4X8.0 mL). The combined organic layers were washed with brine (15mL) and Na₂SO₄Dried and concentrated. It was purified by preparative HPLC (ACN 36-66/0.225% aqueous FA) to give PBD dimeric diaphorase prodrug 2 as an orange solid (6.1mg, 19%). LCMS (5-95, AB,1.5min): RT 0.831min, M/z 999.3[ M +1 [)]+。

Example 23E: preparation of PBD dimer diaphorase prodrug 3

PBD dimer diaphorase prodrug 3 was prepared according to the following reaction scheme:

to a solution of triphosgene (28.0mg, 0.090mmol) in DCM (5.0mL) at 0 deg.C was added a solution of compound A1(200mg, 0.210mmol) and triethylamine (42.0mg, 0.420mmol) in DCM (3.0 mL). Placing it in N₂Stirring was continued at 26 ℃ for 20 min. The mixture was concentrated and redissolved in DCM (3.0mL) and added to a stirred solution of triethylamine (38.0mg, 0.380mmol) and compound a2(51.0mg, 0.230mmol) in DCM (3.0mL) at 0 ℃. Placing it in N₂Stirring was continued at 26 ℃ for 2h and purification by column chromatography (0-40% EtOAc in petroleum ether) gave compound A3(240mg, 90%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.159min, M/z 1200.5[ M [)+1]+。

To a solution of compound A3(240.0mg, 0.200mmol) in THF (4.0mL) and water (4.0mL) at 25 deg.C was added HOAc (6.0mL, 153 mmol). The mixture was stirred at 25 ℃ for 10h, and EtOAc (100mL) was added. It was washed with water (50mL), saturated NaHCO₃(50mL) followed by a brine (50mL) wash. The organic layer was dried and concentrated to give the crude product compound a4(194mg, 99.8%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.767min, M/z 972.4[ M + 1%]+。

To a solution of compound A4(194.0mg, 0.200mmol) in DCM (15mL) was added DMP (211.6mg, 0.500 mmol). The mixture was stirred at 26 ℃ for 1h and saturated Na₂SO₃/NaHCO₃(10mL/10mL) quenching. It was diluted with DCM (2 × 30mL) and isolated. The DCM phase was washed with water (20mL) and Na₂SO₄Dried, concentrated and purified by preparative HPLC (ACN 43-63/0.225% aqueous FA) to give compound a5(70mg, 36% yield) as a yellow solid. LCMS (5-95, AB,1.5min): RT 0.733min, M/z 968.6[ M + 1%]+。

TFA (0.20mL, 2.68mmol) was added to Compound A5(40.0mg, 0.040mmol) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 30min and with cold saturated NaHCO₃(2.0mL) quench. DMSO (2.0mL) was added and the mixture was purified by preparative HPLC (ACN 36-66%/10 mM aqueous NH4HCO 3) to give PBD dimeric diaphorase prodrug 3 as a yellow solid (9.9mg, 28%). LCMS (5-95, AB,1.5min): RT ═ 0.695min, M/z ═ 850.3[ M +1]+。

Example 24: synthesis of quinones

Example 24A: synthesis of quinone 1

Quinone 1 was prepared according to the following reaction scheme:

a suspension of NaH (8.9g, 0.223mol) in DMF (300mL) was cooled in an ice bath. To this was added dropwise a solution of the starting amine (30g, 0.171mol) in DMF (150 mL). The reaction mixture was stirred at room temperature for 60 minutes. Methyl iodide (31.5g, 0.223mol) was then added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was then poured into 10% NaHCO₃In aqueous solution, extracted with EA. The combined organic phases were washed with 10% NaHCO₃Aqueous solution, brine and dried. The solution was concentrated to give the crude product, which was triturated from EA/Hex to give the product as a pale yellow solid (29.5g, 91.2%).

A solution of the starting aldehyde (29.5g, 156mmol, 1 eq.) 5-methoxy-1-methyl-1H-indole-3-carbaldehyde in acetic acid (300mL) was cooled to 10 ℃. To this was added a mixture of nitric acid (4.6mL) in acetic acid (20 mL). The reaction mixture was then stirred at room temperature for 16 h. A yellow suspension is obtained, which is poured into an ice-water mixture, and the resulting crystals are filtered off and dried. The crude product was triturated from EA/Hex to give the product as a yellow solid (30.0g, 82.1%).

To a suspension of the starting material (10g, 43mmol) in ethanol (600mL) was added tin powder (44.23g, 0.37 mol). 3N HCl (200mL) was then added. The reaction solution was stirred at room temperature for 2 hours. The solution was saturated with NaHCO₃And (5) diluting the aqueous solution. The mixture was filtered and washed with EA. The organic phase is separated and the aqueous phase is extracted with EA. The combined organic phases were washed with brine, dried and concentrated to give the crude product, which was triturated from EA/Hex ═ 1/20 to give the product as a grey solid (6.0g, 68.3%).

The starting aldehyde (5.0g, 24.5mmol, 1 eq.) was dissolved in 100mL THF. Mixing LiAlH₄A suspension of (1.86g, 49mmol, 2 equiv.) in 200mL THF was cooled to 0 deg.C. Adding the aldehyde solution dropwise to LiAlH₄In solution. The reaction was allowed to reach room temperature and stirred at room temperature for 30 min. It was quenched with water, then filtered through celite, over MgSO₄Dried and evaporated. The residue is used directlyIn the next reaction. The residue was dissolved in 300mL of acetone. To 300mL of 0.3M NaH₂PO₄To the solution was added 19.7g (73.5mmol, 3 equivalents) of a friemi salt. The mixture was added to the residue in acetone and stirred at room temperature for 0.5 h. Excess acetone was removed in vacuo. The resulting residue was extracted with dichloromethane and washed with water. The organic layer was dried (MgSO₄) And evaporated. The crude product was triturated from EA/Hex to give the product as an orange solid (2.51g, 46.1%, two steps).

1H NMR(400MHz,CDCl3)δ6.70(s,1H),5.69(s,1H),4.64(d,J＝6.9Hz,2H),3.93(s,3H),3.84(s,3H),3.78(t,J＝7.0Hz,1H)。

Example 24B: synthesis of quinone 2

Quinone 2 was prepared according to the following reaction scheme:

4.6g of silica gel was added to ethyl 3-oxobutyrate (50g, 0.38 mmol). To this was added methylamine solution (aqueous solution; 40%, 35.7g, 0.46mol) and the mixture was stirred overnight. The reaction mixture was extracted with DCM and MgSO₄Dried, filtered and evaporated to give the product as a colourless oil (50g, 92%).

50g (0.35mol, 1 eq.) of imine and 37.7g (0.35mol, 1 eq.) of 1, 4-benzoquinone were dissolved in 400mL of nitromethane. The mixture was left to stand for 24h (without stirring). The product crystals precipitated. They were filtered, washed with nitromethane and recrystallized from EtOAc. Yellow solid, yield: 25g, 30.6%.

A solution of alcohol (25g, 0.11mol) and KOH (25.5g, 0.46mol) in DMSO (200mL) was stirred at room temperature for 30 minutes. Methyl iodide (62g, 0.44mol) was then added. The mixture was diluted with EA (700mL), washed with 1N HCl, brine and dried. The solution was concentrated to give the crude product, which was purified through a silica gel column to give the product as a grey solid (20g, 75.5%).

To a solution of 20g (81mmol, 1 eq) of 5-methoxy-1, 2-dimethyl-1H-indole-3-carboxylic acid ethyl ester in acetic acid (200mL) cooled to 0 deg.C was added a mixture of nitric acid (4.6mL) and acetic acid (20 mL). The mixture was then stirred at room temperature for 2 h. A yellow suspension is obtained, which is poured into an ice-water mixture, and the resulting crystals are filtered off and dried. The crude product was purified by flash chromatography to give the product as a yellow solid (14.0g, 59.3%).

To a suspension of the starting ester (8.0g, 27mmol) in ethanol (600mL) was added tin powder (14.6g, 0.123 mol). 3N HCl (200mL) was then added. The reaction solution was stirred at room temperature for 2 hours. The solvent was removed and the residue was diluted with water and saturated NaHCO₃And (4) neutralizing. The mixture was filtered and washed with EA. The organic phase is separated and the aqueous phase is extracted with EA. The combined organic phases were washed with brine, dried and concentrated to give the crude product, which was triturated from EA/Hex ═ 1/20 to give the product as a grey solid (5.0g, 70.6%).

5.0g (19.06mmol, 1 eq.) of the starting material was dissolved in 50mL of THF. 2.9g (76.25mmol, 4 equiv.) of LiAlH₄Dissolved in 250mL THF and cooled to 0 ℃. A solution of the starting material was added dropwise to the LiAlH4 solution. The reaction was allowed to reach room temperature and stirred at room temperature for 30 min. It was quenched with water, NaOH and silica gel. It was filtered through celite and MgSO₄Dried and evaporated. The residue was used directly in the next reaction. The residue was dissolved in 330mL of acetone. To 330mL of 0.3M NaH₂PO₄To the solution was added 15.32g (57.18mmol, 3 equivalents) of friemi salt. The mixture was added to hydroxymethylindole in acetone and stirred at room temperature for 1 h. Excess acetone was removed in vacuo. The resulting residue was extracted with dichloromethane and washed with water. The organic layer was dried (MgSO₄) And evaporated. The crude product was purified by flash chromatography to give the product as a red solid (2.51g, 56%).

1H NMR(400MHz,CDCl3)δ5.63(s,1H),4.61(s,2H),3.88(s,3H),3.82(s,3H),2.23(s,3H)。

Example 24C: synthesis of quinone 3

Quinone 3 was prepared according to the following reaction scheme:

4-Methoxyaniline (32g, 259.2mmol) was dissolved in HCl (37%, 64mL) and water (112 mL). Dropwise addition of NaNO at-5 deg.C₂(19.5g, 283.2mmol) in water (32 mL). After addition, the mixture was stirred at 0 ℃ for 15min and was purified by addition of CH₃COONa (16.8g, 204.8mmol) brought the pH to 3-4. Ethyl-2-acetoacetate (44.8g, 283.2mmol) was dissolved in ethanol (200mL) at 0 ℃. To this solution was added a solution of KOH (15.6g, 283.2mmol) in water (24 mL). The resulting solution was treated with 320g of ice. The diazonium salt of 4-methoxyaniline is added immediately. The mixture was then adjusted to pH 5-6 and stirred at 0 ℃ for 4 h. The solution was stored at 4 ℃ overnight and extracted with EA (4X 200 mL). The combined extracts were washed with brine and passed over NaSO₄And (5) drying. Most of the solvent was evaporated and the residue was used directly for the next reaction (180 mL).

Ethyl (Z) -2- (2- (4-methoxyphenyl) hydrazono) butyrate (180mL) was added dropwise to a solution of 3MHCl/EtOH (180mL) at 80 ℃. After addition, the mixture was kept at 80 ℃ for 3 h. The solvent was evaporated and the residue was treated with water (60mL) and DCM (300 mL). The aqueous layer was then extracted with DCM (3X100 mL). The combined organic layers were washed with brine (150mL) and Na₂SO₄Dried and evaporated to dryness to give crude compound, which was triturated from Hex to give the product as a yellow solid (32.0g, 53.2%).

5-methoxy-3-methyl-1H-indole-2-carboxylic acid ethyl ester (20.0g, 85.7mmol) was dissolved in DCM (200mL) and the mixture was cooled to-20 ℃ with the addition of HNO₃(70%, 9mL) and the mixture was stirred for 20 min. Using NaHO₃Neutralized and extracted with DCM (3 × 100mL) and NaSO₄Dried and concentrated to dryness. The crude product was purified by flash chromatography to give the product as a yellow solid (15.5g, 59.3%).

MeI (30mL) was added to ethyl 5-methoxy-3-methyl-4-nitro-1H-indole-2-carboxylate (14.5g, 52.1mmol) in acetone (500mL) containing KOH (10g, 178 mmol). After the addition, the mixture was stirred at room temperature for 1 h. The solvent was decanted from excess KOH and the mixture was neutralized with HCl. The mixture was then extracted with DCM (3X100 mL). The combined organic layers were washed with brine (150mL) and Na₂SO₄Dried and evaporated to dryness to give the crude compound, which was triturated from Hex and DCM to give the product as a yellow solid (14.0g, 53.2%).

Tin powder (9.14g, 77mol) was added to a suspension of ethyl 5-methoxy-1, 3-dimethyl-4-nitro-1H-indole-2-carboxylate (5.0g, 17.1mmol) in ethanol (500 mL). 3N HCl (130mL) was then added. The reaction solution was stirred at room temperature for 2 hours. The solvent was removed and the residue was diluted with water and saturated NaHCO₃And (4) neutralizing. The mixture was filtered and washed with DCM. The organic phase was separated and the aqueous phase was extracted with DCM. The combined organic phases were washed with brine, dried and concentrated to give the crude product, which was triturated from EA/Hex ═ 1/20 to give the product as a grey solid (4.0g, 80%).

4-amino-5-methoxy-1, 3-dimethyl-1H-indole-2-carboxylic acid ethyl ester (1.5g, 5.72mmol) was dissolved in 90mL THF. 0.88g (22.68mmol, 4 equivalents) of LiAlH4 was dissolved in 180mL THF and cooled to 0 ℃. Adding a solution of 4-amino-5-methoxy-1, 3-dimethyl-1H-indole-2-carboxylic acid ethyl ester dropwise to LiAlH₄In solution. The reaction was allowed to reach room temperature and stirred at room temperature for 30 min. It was then quenched with water, NaOH and silica gel. It was then filtered through celite over MgSO₄Dried and evaporated. The residue was dissolved in 170mL of acetone and used directly for the next reaction. To 180mL of 0.3M NaH₂PO₄Adding Flemide to the solutionSalt (4.6g, 17.16mmol, 3 equiv.). The mixture was added to hydroxymethylindole in acetone and stirred at room temperature for 1 h. Excess acetone was removed in vacuo. The resulting residue was extracted with dichloromethane and washed with water. The organic layer was dried (MgSO₄) And evaporated. The crude product was purified by flash chromatography to give the product as an orange solid (1.0g, 56%).

1H NMR(400MHz,CDCl3)δ5.63(s,1H),4.65(s,2H),4.02(s,3H),3.81(s,3H),2.34(s,3H)。

Example 24D: synthesis of quinone 4

Quinone 4 was prepared according to the following reaction scheme:

to a 250-mL round bottom flask was placed a solution of 5-methoxy-1H-indole-2-carboxylic acid (10g, 52.31mmol, 1.00 equiv.) in methanol (100mL) and thionyl chloride (12.5g, 105.07mmol, 2.00 equiv.) was then added dropwise with stirring. The resulting solution was heated to reflux for 4H, cooled to room temperature and concentrated in vacuo to give 11g (crude) of methyl 5-methoxy-1H-indole-2-carboxylate as a grey solid.

To a 5-L4-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed N, N-dimethylformamide (2L), and then sodium hydride (37.6g, 1.10mol, 1.50 equivalents, 70%) was added in several portions with stirring. To this was added 5-methoxy-1H-indole-2-carboxylic acid methyl ester (150g, 730.96mmol, 1.00 equiv.) dropwise with stirring at below 10 ℃. The mixture was stirred for 0.5 h. MeI (125g, 0.88mol, 1.20 equiv.) was added dropwise to the mixture with stirring. The resulting solution was stirred at room temperature overnight and diluted with 5L of water. The solid was collected by filtration, washed with 3 × 1L water and dried to give 163g (crude product) of methyl 5-methoxy-1-methyl-1H-indole-2-carboxylate as a yellow solid.

Into a 5000-mL 4-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed LiAlH₄(111g, 2.92mol, 4.00 equiv.) in tetrahydrofuran (1500mL) and then a solution of methyl 5-methoxy-1-methyl-1H-indole-2-carboxylate (160g, 729.81mmol, 1.00 equiv.) in tetrahydrofuran (1000mL) was added dropwise with stirring at 0 ℃ over 30 min. The mixture was stirred at 0 ℃ for 1h and at room temperature for 3 h. The mixture was then quenched by the addition of 111g of water, 333mL of aqueous NaOH (15%) and 111g of water at 0 ℃. The solid was filtered off. The filtrate was dried over anhydrous sodium sulfate and concentrated in vacuo to give 100g (72%) of (5-methoxy-1-methyl-1H-indol-2-yl) methanol as a yellow solid.

To a 3000-mL 4-necked round bottom flask purged with and maintained under a nitrogen inert atmosphere was placed a solution of (5-methoxy-1-methyl-1H-indol-2-yl) methanol (100g, 522.94mmol, 1.00 equiv.) in dichloromethane (2000mL), then triethylamine (61.6g, 608.76mmol, 1.50 equiv.) was added dropwise at room temperature with stirring. The mixture was stirred for 30 min. Acetyl chloride (79g, 1.01mol, 1.50 equivalents) was added dropwise thereto under stirring at room temperature. The resulting solution was stirred at room temperature for 3h and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate-petroleum ether (1:10-1:5) to give 75g (61%) of acetic acid (5-methoxy-1-methyl-1H-indol-2-yl) methyl ester as a yellow solid.

To a 250-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed N, N-dimethylformamide (20g, 273.64mmol, 6.00 equiv.), followed by dropwise addition of POCl under stirring at 0 deg.C₃(9.85g, 0.0642mol, 1.50 equiv.). The mixture was stirred at room temperature for 30 min. To this was added acetic acid (5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (10g, 42.87mmol, 1.00 eq) in portions with stirring at a temperature below 0 ℃. The resulting solution was stirred at room temperature for 2h and quenched by the addition of 100mL of water/ice. With sodium hydroxide solutionSolution (2N) the pH of the solution was adjusted to 7-8. The resulting solution was extracted with 3x200mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:3) to give 9g (80%) of acetic acid (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester as a yellow solid.

To a 250-mL 3-neck round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (9g, 3.60mmol, 1.00 equiv), AcOH (100mL), then a solution of HNO3(20mL) in AcOH (50mL) was added dropwise with stirring at a temperature below 5 ℃. The resulting solution was stirred at room temperature for 30min, diluted with 1000mL of water and stirred for 30 min. The solid was collected by filtration, washed with 3x100mL water and dried to give 8.6g (crude product) of acetic acid (3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl) methyl ester as a pale red solid.

To a 1000-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl) methyl ester (8g, 26.12mmol, 1.00 equiv.), ethanol (400mL), then Sn (34.1g, 11.00 equiv.) was added portionwise with stirring at 0 ℃. Hydrogen chloride (4N) (400mL) was added dropwise thereto under stirring. The resulting solution was stirred at 0 ℃ for 2h, concentrated in vacuo and diluted with 500mL of water. The pH of the solution was adjusted to 7-8 with saturated aqueous sodium bicarbonate. The solid was filtered off and washed with 3 × 50mL EA. The filtrate was extracted with 4x200mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:2) to give 6.5g (90%) of acetic acid (4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester as a yellow solid.

To a 2000-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (6g, 21.72mmol, 1.00 equiv.), acetone (600mL), then stirred gradually at a temperature of less than 10 deg.CDropwise addition (KO)₃S)₂NO (17.48g, 65.2mol, 3.00 eq.) in NaH₂PO₄(0.4M) (1200 mL). The resulting solution was stirred at room temperature for 2h and concentrated in vacuo. The residue was extracted with 3x300mL dichloromethane. The organic layers were combined, washed with 3x300mL water, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:2) to give 3.5g (55%) of acetic acid (3-formyl-5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-2-yl) methyl ester as a yellow solid.

To a 500-mL 3-necked round bottom flask was placed acetic acid (3-formyl-5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-2-yl) methyl ester (3.5g, 12.02mmol, 1.00 eq.), CH₃CN(200mL)、NaH₂PO₄(0.6g, 4mmol, 0.30 equiv.), H₂O₂(2g, 59mmol, 5.00 equiv.), NaClO₂(2.5g, 28mmol, 2.41 equiv.) and H₂O (50 mL). The resulting solution was stirred at room temperature for 2h and quenched by the addition of 500mL of water. The pH of the solution was adjusted to 2 with HCl (2 mol/L). The resulting solution was extracted with 4x300mL ethyl acetate. The organic layers were combined, washed with 2 × 200mL brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give 3.5g (95%) of 2- [ (acetoxy) methyl group as a red solid]-5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indole-3-carboxylic acid.

To a 250-mL 3-neck round-bottom flask purged and maintained with a nitrogen inert atmosphere was placed 2- [ (acetoxy) methyl ] -5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indole-3-carboxylic acid (3.8g, 12.37mmol, 1.00 eq.), methanol (100mL) and hydrogen chloride (20 mL). The resulting solution was stirred at 60 ℃ for 4h, concentrated in vacuo, quenched by addition of 500mL water and extracted with 4x200mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1: 3). The crude product was purified by preparative HPLC to give 0.3g (9%) of methyl 2- (hydroxymethyl) -5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indole-3-carboxylate as a yellow solid.

LC-MS(ES,m/z):280[M+H]+。1H-NMR-300MHz,CDCl3,ppm):δ5.73(s,1H),4.77(s,2H),4.09(s,3H),3.95(s,3H),3.85(s,3H)。

Example 24E: synthesis of quinone 5

Quinone 5 was prepared according to the following reaction scheme:

to a 250-mL round bottom flask was placed a solution of 5-methoxy-1H-indole-2-carboxylic acid (10g, 52.31mmol, 1.00 equiv.) in methanol (100mL) and thionyl chloride (12.5g, 105.07mmol, 2.00 equiv.) was then added dropwise with stirring. The resulting solution was heated to reflux for 4H, cooled to room temperature and concentrated in vacuo to give 11g (crude) of methyl 5-methoxy-1H-indole-2-carboxylate as a grey solid.

To a 5-L4-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed N, N-dimethylformamide (2L), and then sodium hydride (37.6g, 1.10mol, 1.50 equivalents, 70%) was added in several portions with stirring. To this was added 5-methoxy-1H-indole-2-carboxylic acid methyl ester (150g, 730.96mmol, 1.00 equiv.) dropwise with stirring at a temperature below 10 ℃. The mixture was stirred for 0.5 h. MeI (125g, 0.88mol, 1.20 equiv.) was added dropwise to the mixture with stirring. The resulting solution was stirred at room temperature overnight and diluted with 5L of water. The solid was collected by filtration, washed with 3 × 1L water and dried to give 163g (crude product) of methyl 5-methoxy-1-methyl-1H-indole-2-carboxylate as a yellow solid.

Into a 5000-mL 4-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed LiAlH₄(111g, 2.92mol, 4.00 equiv.) in tetrahydrofuran (1500mL) and then 5-methoxy-1-methyl-1H-indole-2-carboxylic acid methyl ester (160g, 729.81mmol, 1.00 equiv.) in tetrahydrofuran (1.00 equiv.) was added dropwise over 30min with stirring at 0 deg.C000 mL). The mixture was stirred at 0 ℃ for 1h and at room temperature for 3 h. The mixture was then quenched by the addition of 111g of water, 333mL of aqueous NaOH (15%) and 111g of water at 0 ℃. The solid was filtered off. The filtrate was dried over anhydrous sodium sulfate and concentrated in vacuo to give 100g (72%) of (5-methoxy-1-methyl-1H-indol-2-yl) methanol as a yellow solid.

To a 3000-mL 4-necked round bottom flask purged with and maintained under a nitrogen inert atmosphere was placed a solution of (5-methoxy-1-methyl-1H-indol-2-yl) methanol (100g, 522.94mmol, 1.00 equiv.) in dichloromethane (2000mL), then triethylamine (61.6g, 608.76mmol, 1.50 equiv.) was added dropwise at room temperature with stirring. The mixture was stirred for 30 min. Acetyl chloride (79g, 1.01mol, 1.50 equivalents) was added dropwise thereto under stirring at room temperature. The resulting solution was stirred at room temperature for 3h and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate-petroleum ether (1:10-1:5) to give 75g (61%) of methyl (5-methoxy-1-methyl-1H-indol-2-yl) acetate as a yellow solid.

To a 250-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed N, N-dimethylformamide (20g, 273.64mmol, 6.00 equiv.), followed by dropwise addition of POCl under stirring at 0 deg.C₃(9.85g, 0.0642mol, 1.50 equiv.). The mixture was stirred at room temperature for 30 min. To this was added acetic acid (5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (10g, 42.87mmol, 1.00 eq) in portions with stirring at a temperature below 0 ℃. The resulting solution was stirred at room temperature for 2h and quenched by the addition of 100mL of water/ice. The pH of the solution was adjusted to 7-8 with aqueous sodium hydroxide (2N). The resulting solution was extracted with 3x200mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:3) to give 9g (80%) of methyl (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) acetate as a yellow solid.

To a 250-mL 3-neck round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (9g, 3.60mmol, 1.00 equiv), AcOH (100mL), then a solution of HNO3(20mL) in AcOH (50mL) was added dropwise with stirring at a temperature below 5 ℃. The resulting solution was stirred at room temperature for 30min, diluted with 1000mL of water and stirred for 30 min. The solid was collected by filtration, washed with 3x100mL water and dried to give 8.6g (crude product) of acetic acid (3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl) methyl ester as a pale red solid.

To a 1000-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl) methyl ester (8g, 26.12mmol, 1.00 equiv.), ethanol (400mL), then Sn (34.1g, 11.00 equiv.) was added portionwise with stirring at 0 ℃. Hydrogen chloride (4N) (400mL) was added dropwise thereto under stirring. The resulting solution was stirred at 0 ℃ for 2h, concentrated in vacuo and diluted with 500mL of water. The pH of the solution was adjusted to 7-8 with saturated aqueous sodium bicarbonate. The solid was filtered off and washed with 3 × 50mL EA. The filtrate was extracted with 4x200mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:2) to give 6.5g (90%) of acetic acid (4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester as a yellow solid.

To a 2000-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed acetic acid (4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl) methyl ester (6g, 21.72mmol, 1.00 equiv.), acetone (600mL), and then added dropwise with stirring (KO) at a temperature of less than 10 ℃. (KO)₃S)₂NO (17.48g, 65.2mol, 3.00 eq.) in NaH₂PO₄(0.4M) (1200 mL). The resulting solution was stirred at room temperature for 2h and concentrated in vacuo. The residue was extracted with 3x300mL dichloromethane. The organic layers were combined, washed with 3x300mL water, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:2) to give 3.5g (55%) of (3-formyl-5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indole as a yellow solidIndole-2-yl) acetic acid methyl ester.

To a 250-mL 3-neck round bottom flask was placed acetic acid (3-formyl-5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-2-yl) methyl ester (3.5g, 12.02mmol, 1.00 equiv.), dichloromethane (47mL) and then a solution of LiOH (380mg, 15.87mmol, 1.30 equiv.) in methanol (40mL) was added dropwise with stirring. The resulting solution was stirred at room temperature for 30min, diluted with 80mL of DCM and washed with 3 × 100mL water. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:2) to give 1.1g (37%) of 2- (hydroxymethyl) -5-methoxy-1-methyl-4, 7-dioxo-4, 7-dihydro-1H-indole-3-carbaldehyde as a yellow solid.

LC-MS(ES,m/z):250[M+H]+。1H-NMR(CDCl3,300MHz,ppm):10.55(s,1H),5.79(s,1H),4.83(s,2H),4.09(s,3H),3.89(s,3H)。

Example 24F: synthesis of quinone 6

Quinone 6 was prepared according to the following reaction scheme:

to a stirred solution of compound 1(30mg, 0.135mmol) in anhydrous DMF (2.0mL) at 0 deg.C was added bis (4-nitrophenyl) carbonate (82.5mg, 0.271mmol) and DIEA (87.5mg, 0.678mmol) then under N₂The mixture was stirred at 25 ℃ for 2 h. The mixture was used in the next step without further purification.

To the above mixture was added compound 3(100.2mg, 0.814mmol), DIEA (87.6mg, 0.678mmol), DMF (2.0mL) and catalytic amount of HOBt (5.0mg) at 0 ℃. Then in N₂The mixture was stirred at 25 ℃ for 15 h. It was diluted with water (15mL) and extracted with EtOAc (30 mL. times.4). The combined organic phases were washed with brine (30mL) and Na₂SO₄Drying and concentratingTo give the desired product as a red solid (26mg, 52%). LCMS (5-95AB,1.5min),0.714min, MS 392.8[ M +23 ]]。

To a solution of compound 4(26mg, 0.070mmol) in anhydrous DMF (1.0mL) at 0 deg.C was added bis (4-nitrophenyl) carbonate (42.7mg, 0.140mmol) and DIEA (45.4mg, 0.351 mmol). Then in N₂The mixture was stirred at 25 ℃ for 15 h. The mixture was used in the next step without further purification.

To a solution of compound 5(37.59mg, 0.070mmol) in anhydrous DMF (1.0mL) at 0 deg.C was added norfloxacin (44.83mg, 0.140mmol) and DIEA (45.36mg, 0.351 mmol). In N₂The mixture was stirred at 25 ℃ for 2 h. It was filtered and the filtrate was purified by preparative hplc (fa) to give the product as a yellow solid (30mg, 59.7%). LCMS (5-95, AB,1.5min), RT 0.862min, MS 715.9[ M +1 ═]+；1H NMR(400MHz,DMSO-d6)δ9.86(s,1H),8.95(s,1H),8.28(s,1H),7.95-7.93(d,J＝8.0Hz,1H),7.46-7.44(d,J＝8.0Hz,2H),7.33-7.31(d,J＝8.0,2H),7.19(s,1H),6.68(s,1H),5.85(s,1H),5.22(s,2H),5.04(s,2H),4.57(s,2H),3.96(s,3H),3.77(s,3H),3.60(s,8H),1.40(s,3H)。

Example 24G: synthesis of quinone 7

Quinone 7 was prepared according to the following reaction scheme:

to a solution of compound 1(100mg, 0.45mmol) in anhydrous DMF (10mL) at 30 ℃ was added bis (4-nitrophenyl) carbonate (280mg, 0.9mmol) and DIEA (175mg, 1.36 mmol). In N₂The mixture was then stirred at 30 ℃ for 16 h. It was used in the next step without further purification.

To a solution of compound 2, norfloxacin (288mg, 0.9mmol) in anhydrous DMF (10.0mL) at 30 ℃ was added DIEA (116mg, 0.90 mmol). After the mixture was stirred at 30 ℃ for 2h, it was filtered and the filter cake was washed with DCM/MeOH (10/1) and then concentrated to give the desired product as a yellow solid (150mg, 59%).

LCMS:(5-95,AB,1.5min),RT＝0.824min,MS＝566.9[M+1]；1H NMR(400MHz,DMSO-d6)δ8.89(s,1H),7.94(d,J＝13.2Hz,1H),7.20(d,J＝7.2Hz,1H),6.63(s,1H),5.83(s,1H),5.22(s,2H),4.57(d,J＝6.8Hz,2H),3.97(s,2H),3.80(s,2H),3.63(s,3H),3.37(s,3H),3.21(s,4H),1.46(d,J＝7.2Hz,3H)。

Example 24H: synthesis of quinone 8

Quinone 8 was prepared according to the following reaction scheme:

the alcohol (100mg, 0.4mmol) was dissolved in DMF (2 mL). P-nitrophenyl carbonate (610mg, 2.0mmol, 5 equiv.) followed by DIPEA (0.35mL, 2.0mmol, 5 equiv.) was added and the reaction was stirred at room temperature for 3 h. The reaction solution was concentrated and carried out on the crude product.

Norfloxacin (130mg, 0.4mmol, 1 eq) was added to the vial followed by the addition of the starting carbonate (200mg, 0.4mmol) followed by DMF (3 mL). HOBt (10mg, 0.08mmol, 0.2 equiv.) was added followed by pyridine (0.3mL, 4mmol, 10 equiv.) and the reaction was stirred at room temperature for 19 h. The reaction solution was purified by HPLC to obtain 136mg of the product (57%, two steps).

1H NMR(400MHz,DMSO-d6)δ10.40(s,1H),8.96(s,1H),7.94(d,J＝13.2Hz,1H),7.20(d,J＝7.2Hz,1H),5.99(s,1H),5.49(s,2H),4.58(q,J＝7.1Hz,2H),4.02(s,3H),3.83(s,3H),3.63-3.48(m,5H),2.07(s,3H),1.41(t,J＝7.1Hz,3H)。

Example 24I: synthesis of quinone 9

Quinone 9 was prepared according to the following reaction scheme:

the alcohol (200mg, 0.9mmol) was dissolved in DMF (4 mL). P-nitrophenyl carbonate (284mg, 1mmol, 1.1 equiv) was added followed by DIPEA (0.3mL, 1.7mmol, 2 equiv) and the reaction was stirred at room temperature for 19 h. The reaction solution was concentrated and carried out on the crude product.

Norfloxacin (290mg, 0.9mmol, 1 eq) was added to the vial followed by the addition of the starting carbonate (350mg, 0.9mmol) followed by DMF (4 mL). HOBt (25mg, 0.18mmol, 0.2 equiv.) was added followed by pyridine (0.74mL, 9mmol, 10 equiv.) and the reaction was stirred at room temperature for 24 h. The reaction solution was purified by HPLC to obtain 103mg of the product (20%, two steps).

1H NMR(400MHz,DMSO-d6)δ8.99(s,1H),7.98(d,J＝12.8Hz,1H),7.31(s,1H),7.24(d,J＝7.0Hz,1H),5.86(s,1H),5.19(s,2H),4.61(d,J＝9.9Hz,4H),3.92(s,3H),3.80(s,3H),3.63(s,6H),1.58-1.33(m,3H)。

Example 24J: synthesis of quinone 10

Quinone 10 was prepared according to the following reaction scheme:

the alcohol (100mg, 0.43mmol) was dissolved in DMF (2 mL). P-nitrophenyl carbonate (142mg, 0.47mmol, 1.1 equiv) was added followed by DIPEA (0.15mL, 0.85mmol, 2 equiv) and the reaction was stirred at room temperature for 19 h. The reaction solution was concentrated and carried out on the crude product.

Norfloxacin (136mg, 0.43mmol, 1 eq) was added to the vial followed by the addition of the starting carbonate (170mg, 0.43mmol) followed by DMF (7 mL). HOBt (12mg, 0.09mmol, 0.2 equiv.) was added followed by pyridine (0.35mL, 4.3mmol, 10 equiv.) and the reaction was stirred at room temperature for 24 h. The reaction solution was purified by HPLC to give 18mg of the product (7%, two steps).

1H NMR(400MHz,DMSO-d6)δ8.95(s,1H),8.48(s,1H),7.94(d,J＝13.0Hz,1H),7.20(d,J＝7.1Hz,1H),6.80(s,1H),5.78(s,1H),5.17(s,2H),4.58(d,J＝7.7Hz,2H),3.86(s,3H),3.76(s,4H),3.53(s,5H),2.28(s,3H),1.40(t,J＝7.0Hz,3H)。

Example 24K: synthesis of quinone 11

Quinone 11 was prepared according to the following reaction scheme:

the alcohol (100mg, 0.43mmol) was dissolved in DMF (2 mL). P-nitrophenyl carbonate (142mg, 0.47mmol, 1.1 equiv) was added followed by DIPEA (0.15mL, 0.85mmol, 2 equiv) and the reaction was stirred at room temperature for 19 h. The reaction solution was concentrated and carried out on the crude product.

Norfloxacin (136mg, 0.43mmol, 1 eq) was added to the vial followed by the addition of the starting carbonate (170mg, 0.43mmol) followed by DMF (7 mL). HOBt (12mg, 0.09mmol, 0.2 equiv.) was added followed by pyridine (0.35mL, 4.3mmol, 10 equiv.) and the reaction was stirred at room temperature for 24 h. The reaction solution was purified by HPLC to obtain 31mg of the product (12%, two steps).

1H NMR(400MHz,DMSO-d6)δ8.95(s,1H),8.49(s,2H),7.94(d,J＝13.1Hz,1H),6.76(s,2H),5.19(s,2H),4.58(d,J＝7.5Hz,2H),3.96(s,3H),3.83-3.69(m,5H),3.58(t,J＝5.1Hz,4H),2.30(s,3H),1.40(t,J＝7.1Hz,3H)。

Example 24L: synthesis of quinone 12

Quinone 12 was prepared according to the following reaction scheme:

the alcohol (100mg, 0.36mmol) was dissolved in DMF (2 mL). P-nitrophenyl carbonate (545mg, 1.8mmol, 5 equiv.) was added followed by DIPEA (0.31mL, 1.8mmol, 5 equiv.) and the reaction was stirred at room temperature for 3 h. The reaction solution was concentrated and carried out on the crude product.

Norfloxacin (110mg, 0.36mmol, 1 eq) was added to the vial followed by the addition of the starting carbonate (160mg, 0.36mmol) followed by DMF (3 mL). HOBt (10mg, 0.07mmol, 0.2 equiv.) was added followed by pyridine (0.29mL, 3.6mmol, 10 equiv.) and the reaction was stirred at room temperature for 19 h. More norfloxacin (110mg, 0.36mmol, 1 eq) was added and the reaction was stirred for 2.5 days. The reaction solution was purified by HPLC to give 89mg of the product (40%, two steps).

1H NMR(400MHz,DMSO-d6)δ8.96(s,1H),7.94(d,J＝13.1Hz,1H),7.20(d,J＝7.2Hz,1H),5.93(s,1H),5.32(s,2H),4.58(q,J＝7.1Hz,2H),4.00(s,3H),3.80(d,J＝3.3Hz,6H),3.64-3.49(m,8H),1.41(t,J＝7.1Hz,3H)。

Example 25: synthesis of PBD dimer diaphorase prodrug 1 comprising linker for conjugation to antibody

PBD dimer diaphorase prodrug 1 comprising a linker was prepared according to the following reaction scheme:

each asterisk in the above structure and elsewhere depicted in example 25 represents a chiral center.

To a solution of Compound A1(1.00g, 1.17mmol) in DCM (30mL) at 30 deg.C was added triphosgene (347mg, 1.17mmol) and Et₃A solution of N (356mg, 3.52mmol) in DCM (10 mL). After the mixture was stirred at 30 ℃ for 30min, it was concentrated, dibutyltin diacetate (0.32mL, 1.22mmol) was added, followed by the addition of Compound A2(215mg, 0.97mmol) and Et₃A solution of N (369mg, 3.65mmol) in DMF (15 mL). The mixture was stirred at 25 ℃ for 1 h. The mixture was diluted with water (10mL) and stirred for 20 min. The mixture was then concentrated and purified by flash column chromatography (0-5% MeOH in DCM) to give compound a3(800mg, 26.9%) as a yellow solid. LCMS (5-95, AB,1.5min): RT ═ 1.032min, M/z ═ 1100.5[ M + 1-]+。

To a solution of triphosgene (97.1mg, 0.33mmol) in DCM (15mL) was added a solution of compound A3(800.0mg, 0.33mmol) and Et3N (99.31mg, 0.98mmol) in DCM (5.0 mL). After stirring the mixture at 30 ℃ for 30min, it was concentrated. To the above residue were added MC _ VC _ PAB (368.0mg, 0.33mmol) and Et₃A solution of N (0.14mL, 0.98mmol) in DMF (15 mL). The mixture was stirred at 25 ℃ for 12 h. The mixture was concentrated and purified by flash column chromatography (5-10% MeOH in DCM) to give a4(300mg, 37%) as a yellow solid. LCMS (5-95, AB,1.5min): RT 1.023min, M/z 850.5[ M/2+ 1%]+。

To a solution of compound A4(300.0mg, 0.18mmol) in THF (1.0mL) was added water (1.0mL) and HOAc (1.5mL) and stirred at 25 ℃ for 12 h. The mixture was diluted with EtOAc (80mL) and saturated NaHCO₃(3 × 40mL) and concentrated to give compound a5 as a red solid (160mg, 61.7%).

To a solution of compound A5(140.0mg, 0.10mmol) in DMSO (5.0mL) was added IBX (266mg, 0.95 mmol). After stirring the mixture at 38 ℃ for 12h, it was purified by preparative HPLC (ACN 37-67%/0.225% aqueous FA) and then by preparative TLC (10% MeOH in DCM, Rf ═ 0.5) to give the linker-containing PBD dimeric diaphorase prodrug 1as a yellow solid (15mg, 10.5%). LCMS (5-95, AB,1.5min): RT ═ 0.712min, M/z ═ 734.2[ M/2+1] +.

Example 26: synthesis of PBD dimer prodrug antibody-drug conjugates (ADCs)

ADC 1A and 1B were prepared by conjugation of an antibody and the PBD dimer ADC disulfide prodrug linker-drug intermediate of example 21D, and have the following structures:

ADC 2A and 2B were prepared by conjugation of an antibody and the PBD dimer ADC disulfide prodrug linker-drug intermediate of example 21B and have the following structures:

ADC 3 was prepared by conjugation of an antibody and the PBD dimer ADC disulfide prodrug linker-drug intermediate of example 21A, and has the following structure:

ADC 4 was prepared by conjugation of an antibody and the PBD dimer ADC disulfide prodrug linker-drug intermediate of example 21C, and has the following structure:

ADC 5 was prepared by conjugation of an antibody and the PBD dimer ADC disulfide prodrug linker-drug intermediate of example 21E, and has the following structure:

PBD dimer ADC boronic acid prodrugs 1A and 1B were prepared by conjugation of an antibody and the PBD dimer ADC boronic acid prodrug linker-drug intermediate of example 22A and have the following structure:

PBD dimer ADC diaphorase prodrugs 1A and 1B were prepared by conjugation of an antibody and the PBD dimer ADC diaphorase prodrug linker-drug intermediate of example 25 and have the following structure:

when introducing elements of the present disclosure or the preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims (105)

1. A pyrrolobenzodiazepine of formula (I)Prodrug dimer-antibody conjugate compounds comprising a first pyrrolobenzodiazepineProdrug monomer M1 and a second pyrrolobenzodiazepineAntibody monomer M2

Wherein:

(I) for M1

(a)R²Is selected from-H, ═ CH₂-CN, -R, ═ CH-R, aryl, heteroaryl, bicyclic, and heterobicyclic;

(b)R³is H;

(c)R⁶、R⁷and R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH, and SR;

(d) x is selected from S, O and NH;

(e)R¹⁰is a prodrug moiety comprising (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester;

(f)R¹¹selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S;

(g) r is selected from a lower alkyl group having 1 to 10 carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i) wherein the alkyl group optionally contains one or more carbon-carbon double or triple bonds, or an aryl group of up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms; and is

(h) The dotted line represents an optional double bond between two of the following: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R²，

(II) for M2

(a)R^2’、R^3’、R^6’、R^7’、R^9’、R^11’And X' respectively correspond to R²、R³、R⁶、R⁷、R⁹、R¹¹And X;

(b) l is a self-immolative linker comprising at least one of a disulfide moiety, a peptide moiety, and a peptidomimetic moiety; and is

(c) The dotted line represents an optional double bond between two of the following: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’，

(III) M1 and M2 are bonded at the C8 position by a moiety-Q-T-Q '-wherein Q and Q' are independently selected from O, NH and S, and wherein T is optionally substituted C_1-12An alkylene group which is further optionally interrupted by one or more heteroatoms and/or aromatic rings,

(IV) p is 1,2, 3, 4,5, 6,7 or 8,

(V) Ab is an antibody, and

(VI) each asterisk independently represents a chiral center.

2. The compound of claim 1, wherein C of M1₁And C₂The bond between (A) and (B) is a single bond; c of M1₂And C₃The bond between (A) and (B) is a single bond; c of M2_1’And C_2’The bond between (A) and (B) is a single bond; c of M2_2’And C_3’The bond between (A) and (B) is a single bond; c of M1₃Is divided into two R³Is substituted by the radicals R³Each of the groups is H; c of M2_3’Is divided into two R^3’Is substituted by the radicals R^3’Each of the groups is H; c of M1₂And R²The bond between is a double bond; and C of M2_2’And R^2’The bond between is a double bond.

3. A compound as claimed in claim 1 or claim 2 wherein R is²And R^2’Is ═ CH₂。

4. The compound of any one of claims 1 to 3, wherein R⁶、R^6’、R⁹And R^9’Is H.

5. The compound of any one of claims 1 to 4, wherein R⁷And R^7’Is OCH₃。

6. The compound of any one of claims 1 to 5, wherein Q and Q' are O and wherein T is C₃Alkylene or C₅An alkylene group.

7. The compound of any one of claims 1 to 6, wherein p is 1,2, 3, or 4.

8. The compound of any one of claims 1 to 6, comprising a mixture of conjugate compounds, wherein the average drug loading per antibody in the mixture of conjugate compounds is from about 2 to about 5.

9. The compound of claim 8, wherein the antibody comprises at least one cysteine thiol moiety engineered for conjugation, wherein the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from:

(1) BMPR1, (2) E, (3) STEAP, (4) MUC, (5) MPF, (6) Napi2, (7) Sema5b, (8) PSCAhlg, (9) ETBR, (10) MSG783, (11) STEAP, (12) TrpM, (13) CRIPTO, (14) CD, (15) CD79, (16) FcRH, (17) HER, (18) NCA, (19) MDP, (20) IL20, (21) curdlan, (22) EphB2, (23) ASLG659, (24) PSCA, (25) GEDA, (26) BADA-R, (27) CD, (28) CD79, (29) CXCR, (30) HLA-DOB, (31) P2X, (32) CD, (33) LY 34, (FcRH, (35) FcRH, (LGH, (36) TENB 37), (38) TMF, (39) LyEFR-R, (40) TMR 6, (4) MUC, (5) MPF, (6) TMGPR (48) TZGPR (23), (48) TRPR (23) TMGPR 52) (TMGPR (23) TRPR 52) (TMGPR 52) and (TMGPR 52) TRPR (23).

10. The compound of any one of claims 1 to 9, wherein the antibody is a cysteine engineered antibody.

11. The compound of claim 10, wherein the cysteine engineered antibody comprises LC K149C, HCA118C, HC a140C, or LC V205C as sites for linker conjugation.

12. The compound of claim 11, wherein the antibody is selected from anti-HER 2, anti-CD 22, anti-CD 33, anti-Napi 2b, anti-Ly 6E, and anti-CLL-1.

13. The compound of any one of claims 1 to 12, wherein the linker comprises a disulfide moiety or a peptide moiety.

14. The compound of any one of claims 1 to 12, wherein (i) the antibody comprises at least one reactive thiol moiety prior to conjugation, and (ii) the linker comprises a reactive sulfur atom, maleimide, bromoacetamide, and iodoacetamide, or an alkene prior to conjugation, wherein the antibody is conjugated to the linker by a covalent bond formed by reaction of the antibody reactive thiol with the linker reactive sulfur atom, maleimide, bromoacetamide, iodoacetamide, or alkene.

15. The compound of any one of claims 1 to 14, wherein the linker-antibody moiety has the structure:

wherein

(I) Ab is an antibody;

(II)S_Cis an antibody cysteine sulfur atom;

(III)R⁷⁰and R⁷¹Independently selected from H and C_1-3Alkyl radical, wherein R⁷⁰And R⁷¹Only one of which may be H, or R⁷⁰And R⁷¹Together with the carbon atom to which they are bonded form a four-to six-membered ring optionally containing oxygen heteroatoms;

(IV) wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M2; and is

(V) p is as defined in claim 1.

16. The compound of claim 15, wherein R⁷⁰And R⁷¹Independently selected from H, -CH₃and-CH₂CH₃Wherein R is⁷⁰And R⁷¹Only one of which may be H, or R⁷⁰And R⁷¹Together with the carbon atom to which they are bonded, form a ring selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuran and tetrahydropyran.

17. The compound of claim 15, wherein the linker-antibody moiety has the structure:

wherein L is a self-immolative linker comprising a peptide moiety and/or a peptidomimetic moiety, and wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M2.

18. The compound of any one of claims 1 to 17, wherein R¹⁰Is a quinone selected from the group consisting of formulas (IIIa) and (IIIb):

wherein

(I) A, D, E, G and J are independently selected from C and N, where N is a secondary amine, a tertiary amine or an imine having the structure ═ N-;

(II) each m is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker covalently bonded to the oxygen atom of the carbamate moiety of M1 of formula (I).

19. The compound of claim 18, wherein R^A、R^DAnd R^EOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

20. The compound of claim 18 or claim 19, wherein A- (R)^A)_m、D-(R^D)_mAnd E- (R)^E)_mOne of them is

21. The method of any one of claims 18 to 20Compound (I) wherein A- (R)^A)_mIs composed ofD is C and R^DIs C₁A joint; e- (R)^E)_mIs composed ofWherein the bond between D and E is a double bond.

22. The compound of any one of claims 18 to 21, wherein G and J are C.

23. The compound of any one of claims 18 to 21, wherein G and J are C, and R is^GAnd R^JOne of them is-O-CH₃。

24. The compound of any one of claims 18 to 23, wherein the quinone is selected from formulas (IIIc) and (IIId):

wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (I).

25. The compound of any one of claims 1 to 17, wherein R¹⁰Is a quinone selected from formulas (IIIe) and (IIIf):

wherein

(I) A, D, E, F, G and J are independently selected from C and N, wherein at least one of A, D, E and F is C and at least one of G and J is C, and wherein N is a secondary amine, a tertiary amine, or an imine having the structure ═ N-;

(II) each n is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^F、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^F、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker covalently bonded to the oxygen atom of the carbamate moiety of M1 of formula (I).

26. The compound of claim 25, wherein R^A、R^D、R^EAnd R^FOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

27. The compound of claim 25 or claim 26, wherein D-R^DOr E-R^ETo have a bond to said C_1-4An optionally substituted alkyl or heteroalkyl linker, and A, F and at least one of the other of D and E is N.

28. The compound of any one of claims 25 to 27, wherein R^A、R^EAnd R^FIs independently selected from H, optionally substituted C_1-4Alkyl or heteroalkyl, and optionally substituted C_1-4Alkoxy or heteroalkoxy, and wherein D is C and R^DIs C₁And (4) a joint.

29. The compound of any one of claims 25 to 28, wherein G- (R)^G)_nAnd J- (R)^J)_nAt least one of which is

30. The compound of any one of claims 25 to 29, wherein G- (R)^G)_nAnd J- (R)^J)_nOne of them is CO-CH₃，G-(R^G)_nAnd J- (R)^J)_nIs CH, wherein the bond between G and J is a double bond.

31. The compound of any one of claims 25 to 30, wherein the ring formed by A, D, E and F is unsaturated or partially saturated.

32. The compound of any one of claims 1 to 17, wherein R¹⁰An arylboronic acid or arylboronic acid ester of formula (IVa):

(I) wherein R is²⁰And R²¹Independently selected from H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, and optionally substituted aryl or heteroaryl, or

(II) wherein R²⁰And R²¹Together are an optionally substituted moiety- (CH)₂)_n-, wherein n is 2 or 3, said moieties, together with the O atom to which they are bonded, forming a heterocycloalkyl ring together with the B atom, wherein said heterocycloalkyl ring may optionally comprise a fused heteroalkyl ring, a fused aryl ring, or a fused heterocycloalkyl ringAryl ring, and

(III) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (I).

33. The compound of claim 32, wherein R¹⁰Selected from formulae (IVb) and (IVc):

(I) wherein R is³⁰、R³¹、R³²、R³³、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴And R⁴⁵Independently selected from H, halogen, -CN, -OH, -NH₂、-COOH、-CONH₂、-NO₂、-SH、-SO₂Cl、-SO₃H、-SO₄H、-SO₂NH₂、-NHNH₂、-ONH₂、-NHC＝(O)NHNH₂Optionally substituted C_1-8Alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl containing from 2 to 7 carbon atoms, optionally substituted aryl or heteroaryl, or

(II) wherein (i) R³⁰Or R³¹And R³²Or R³³(iii) R⁴⁰Or R⁴¹And R⁴²Or R⁴³And/or (iii) R⁴²Or R⁴³And R⁴⁴Or R⁴⁵Form an optionally substituted fused cycloalkyl ring, fused heterocycloalkyl ring, fused aryl ring, or fused heteroaryl ring having 2 to 7 carbon atoms.

34. The compound of claim 32 or claim 33, wherein R¹⁰Selected from formulae (IVd) to (IVi):

35. the compound of claim 34, wherein R¹⁰Is represented by formula (IVd).

36. The compound of any one of claims 1 to 17, wherein R¹⁰Is a disulfide of formula (V):

(I) wherein R is⁵⁰Is optionally substituted C_1-8Alkyl or heteroalkyl, and R⁵¹Is optionally substituted C₂Alkylene or optionally substituted benzylidene; and is

(II) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (I).

37. The compound of claim 36, wherein R⁵⁰Is C optionally substituted by OH_2-6Alkyl, and R⁵¹Having formula (Va):

(I) wherein R is⁶¹And R⁶²Independently selected from H and CH₃Or R is⁶¹And R⁶²Together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl group containing 2 to 6 carbon atoms;

(II) wherein R⁶³And R⁶⁴Independently selected from H and CH₃(ii) a And is

(III) with R therein⁶³And R⁶⁴Indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (I).

38. The compound of claim 37, wherein R⁶³And R⁶⁴Is H.

39. The compound of any one of claims 36 to 38, wherein:

(I)R⁵⁰is selected from CH₃CH₂-、HOCH₂CH₂-、(CH₃)₂CH-and (CH)₃)₃C-; and is

(II)R⁵¹Selected from formulae (Vb) to (Vf):

40. the compound of any one of claims 1 to 39, having the formula:

wherein R is¹⁰As defined in any one of claims 1 or 18 to 39, L as defined in any one of claims 1 or 15 to 17, Ab as defined in any one of claims 9 to 14 and p as defined in any one of claims 1,7 or 8.

41. A pyrrolobenzodiazepine of formula (II)Prodrug compounds:

wherein:

(a)R²is selected from-H, ═ CH₂-CN, -R, ═ CH-R, aryl, heteroaryl, bicyclic, and heterobicyclic;

(b)R³is H;

(c)R⁶、R⁷、R⁸and R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH and SR, OR R⁷And R⁸Together with the carbon atom to which they are bonded form a group-O- (CH)₂)_n-O-, wherein n is 1 or 2;

(d) x is selected from S, O and NH;

(e)R¹⁰is a prodrug moiety selected from the group consisting of (i) glutathione-activated disulfide, (ii) DT-diaphorase-activated quinone, and (iii) active oxygen species-activated arylboronic acid or arylboronic ester;

(f)R¹¹selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S;

(g) r is selected from a lower alkyl group having 1 to 10 carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i) wherein the alkyl group optionally contains one or more carbon-carbon double or triple bonds, or an aryl group of up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms;

(h) the dotted line represents (i) C₁And C₂，(ii)C₂And C₃And (iii) C₂And R²An optional double bond between two of one of (a); and is

(i) Each asterisk independently represents a chiral center.

42. The compound of claim 41, wherein C₁And C₂The bond between (A) and (B) is a single bond; c₂And C₃The bond between (A) and (B) is a single bond; c₃Is divided into two R³Is substituted by the radicals R³Each of the groups is H; and C₂And R²The bond between is a double bond.

43. The compound of claim 41 or claim 42, wherein R²Is ═ CH₂。

44. The compound of any one of claims 41 to 43, wherein R⁶And R⁹Is H.

45. The compound of any one of claims 41-44, wherein R⁷Is OCH₃。

46. The compound of any one of claims 41 to 45, wherein R¹⁰Is a quinone selected from the group consisting of formulas (IIIa) and (IIIb):

wherein

(I) A, D, E, G and J are independently selected from C and N, where N is a secondary amine, a tertiary amine or an imine having the structure ═ N-;

(II) each m is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker covalently bonded to the oxygen atom of the carbamate moiety of formula (II).

47. The compound of claim 46, wherein R^A、R^DAnd R^EOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

48. The compound of claim 46 or claim 47, wherein A- (R)^A)_m、D-(R^D)_mAnd E- (R)^E)_mOne of them is

49. The compound of any one of claims 46 to 48, wherein A- (R)^A)_mIs composed ofD is C and R^DIs C₁A joint; e- (R)^E)_mIs composed ofWherein the bond between D and E is a double bond.

50. The compound of any one of claims 46-49, wherein G and J are C.

51. The compound of any one of claims 46 to 49, wherein G and J are C, and R is^GAnd R^JOne of them is-O-CH₃。

52. The compound of any one of claims 46 to 51, wherein the quinone is selected from formulas (IIIc) and (IIId):

wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of formula (II).

53. The compound of any one of claims 41 to 45, wherein R¹⁰Is a quinone selected from formulas (IIIe) and (IIIf):

wherein

(I) A, D, E, F, G and J are independently selected from C and N, wherein at least one of A, D, E and F is C and at least one of G and J is C, and wherein N is a secondary amine, a tertiary amine, or an imine having the structure ═ N-;

(II) each n is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^F、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^F、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker covalently bonded to the oxygen atom of the carbamate moiety of formula (II).

54. The compound of claim 53, wherein R^A、R^D、R^EAnd R^FOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

55. The method of claim 53 or claim 54Compound (I) wherein D-R^DOr E-R^ETo have a bond to said C_1-4An optionally substituted alkyl or heteroalkyl linker, and A, F and at least one of the other of D and E is N.

56. The compound of any one of claims 53 to 55, wherein R^A、R^EAnd R^FIs independently selected from H, optionally substituted C_1-4Alkyl or heteroalkyl, and optionally substituted C_1-4Alkoxy or heteroalkoxy, and wherein D is C and R^DIs C₁And (4) a joint.

57. The compound of any one of claims 53 to 56, wherein G- (R)^G)_nAnd J- (R)^J)_nAt least one of which is

58. The compound of any one of claims 53 to 57, wherein G- (R)^G)_nAnd J- (R)^J)_nOne of them is CO-CH₃，G-(R^G)_nAnd J- (R)^J)_nIs CH, wherein the bond between G and J is a double bond.

59. The compound of any one of claims 53 to 58, wherein the ring formed by A, D, E and F is unsaturated or partially saturated.

60. The compound of any one of claims 41 to 45, wherein R¹⁰An arylboronic acid or arylboronic acid ester of formula (IVa):

(I) wherein R is²⁰And R²¹Independently selected from H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, and optionally substituted aryl or heteroaryl, or

(II) wherein R²⁰And R²¹Together are an optionally substituted moiety- (CH)₂)_n-, wherein n is 2 or 3, said moieties, together with the O atom to which they are bonded, form a heterocycloalkyl ring, wherein said heterocycloalkyl ring may optionally include a fused heterocycloalkyl ring, a fused aryl ring, or a fused heteroaryl ring, and

(III) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of formula (II).

61. The compound of claim 60, wherein R¹⁰Selected from formulae (IVb) and (IVc):

(I) wherein R is³⁰、R³¹、R³²、R³³、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴And R⁴⁵Independently selected from H, halogen, -CN, -OH, -NH₂、-COOH、-CONH₂、-NO₂、-SH、-SO₂Cl、-SO₃H、-SO₄H、-SO₂NH₂、-NHNH₂、-ONH₂、-NHC＝(O)NHNH₂Optionally substituted C_1-8Alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl containing from 2 to 7 carbon atoms, optionally substituted aryl or heteroaryl, or

(II) wherein (i) R³⁰Or R³¹And R³²Or R³³(iii) R⁴⁰Or R⁴¹And R⁴²Or R⁴³One of, and/or (iii) R⁴²Or R⁴³And R⁴⁴Or R⁴⁵Form an optionally substituted fused cycloalkyl ring, fused heterocycloalkyl ring, fused aryl ring, or fused heteroaryl ring having 2 to 7 carbon atoms.

62. The compound of claim 60 or claim 61, wherein R¹⁰Selected from formulae (IVd) to (IVi):

63. the compound of claim 62, wherein R¹⁰Is represented by formula (IVd).

64. The compound of any one of claims 41 to 45, wherein R¹⁰Is a disulfide of formula (V):

(I) wherein R is⁵⁰Is optionally substituted C_1-8Alkyl or heteroalkyl, and R⁵¹Is optionally substituted C₂Alkylene or optionally substituted benzylidene; and is

(II) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of formula (II).

65. The compound of claim 64, wherein R⁵⁰Is C_2-6Alkyl and R⁵¹Having formula (Va):

(I) wherein R is⁶¹And R⁶²Independently selected from H and CH₃Or R is⁶¹And R⁶²Together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl group containing 2 to 6 carbon atoms;

(II) wherein R⁶³And R⁶⁴Independently selected from H and CH₃(ii) a And is

(III) with R therein⁶³And R⁶⁴Indicates the point of attachment to the oxygen atom of the carbamate moiety of formula (II).

66. The compound of claim 65, wherein R⁶³And R⁶⁴Is H.

67. The compound of any one of claims 64 to 66, wherein:

(I)R⁵⁰is selected from CH₃CH₂-、(CH₃)₂CH-and (CH)₃)₃C-; and is

(II)R⁵¹Selected from formulae (Vb) to (Vf):

68. the compound of any one of claims 41 to 67, having the formula:

69. a pyrrolobenzodiazepine of formula (VIII)Prodrug dimer compounds comprising a first pyrrolobenzodiazepineProdrug monomer M1 and a second pyrrolobenzodiazepineMonomer M2:

wherein

(I) For M1

(a)R²Is selected from-H, ═ CH₂-CN, -R, ═ CH-R, aryl, heteroaryl, bicyclic, and heterobicyclic;

(b)R³is H;

(c)R⁶、R⁷and R⁹Independently selected from H, R, OH, OR, halo, amino, nitro, SH, and SR;

(d) x is selected from S, O and NH;

(e)R¹⁰is a prodrug moiety comprising (i) a glutathione-activated disulfide, (ii) a DT-diaphorase-activated quinone, or (iii) an active oxygen species-activated arylboronic acid or arylboronic ester;

(f)R¹¹selected from (i) H and R, when X is O or NH, and (ii) H, R and O_zU, wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptable cation when X is S;

(g) r is selected from a lower alkyl group having 1 to 10 carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i) wherein the alkyl group optionally contains one or more carbon-carbon double or triple bonds, or an aryl group of up to 12 carbon atoms, and (ii) wherein R is optionally substituted with one or more halo, hydroxyl, amino, or nitro groups, and optionally contains one or more heteroatoms; and is

(h) The dotted line represents an optional between two ofDouble bond: (i) c₁And C₂；(ii)C₂And C₃(ii) a And (iii) C₂And R²，

(II) for M2

(a)R^2’、R^3’、R^6’、R^7’、R^9’、R^11’And X' respectively correspond to R²、R³、R⁶、R⁷、R⁹、R¹¹And X;

(b) m2 comprises an optional double bond between two of: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’；

(c)R¹²Is absent or selected from-C (O) O-L and-C (O) O-R¹⁰And when R is¹²In the absence, N10' and C_11’The bond between is a double bond; and is

(d) The dotted line represents an optional double bond between two of the following: (i) c_1’And C_2’；(ii)C_2’And C_3’(ii) a And (iii) C_2’And R^2’；

(III) L is a self-immolative linker comprising at least one of a disulfide moiety, a peptide moiety, and a peptidomimetic moiety,

(IV) M1 and M2 are bonded at the C8 position by a moiety-Q-T-Q '-wherein Q and Q' are independently selected from O, NH and S, and wherein T is optionally substituted C_1-12An alkylene group further optionally interrupted by one or more heteroatoms and/or aromatic rings; and is

Each asterisk independently represents a chiral center.

70. The compound of claim 69, wherein C of M1₁And C₂The bond between (A) and (B) is a single bond; c of M1₂And C₃The bond between (A) and (B) is a single bond; c of M2_1’And C_2’The bond between (A) and (B) is a single bond; c of M2_2’And C_3’The bond between (A) and (B) is a single bond; c of M1₃Is divided into two R³Is substituted by the radicals R³In the groupEach of (a) is H; c of M2_3’Is divided into two R^3’Is substituted by the radicals R^3’Each of the groups is H; c of M1₂And R²The bond between is a double bond; and C of M2_2’And R^2’The bond between is a double bond.

71. The compound of claim 69 or claim 70, wherein R²And R^2’Is ═ CH₂。

72. The compound of any one of claims 69 to 71, wherein R⁶、R^6’、R⁹And R^9’Is H.

73. The compound of any one of claims 69 to 72, wherein R⁷And R^7’Is OCH₃。

74. The compound of any one of claims 69 to 73, wherein Q and Q' are O and wherein T is C₃Alkylene or C₅An alkylene group.

75. The compound of any one of claims 69 to 74, wherein R¹⁰Is a quinone selected from the group consisting of formulas (IIIa) and (IIIb):

wherein

(I) A, D, E, G and J are independently selected from C and N, where N is a secondary amine, a tertiary amine or an imine having the structure ═ N-;

(II) each m is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker covalently bonded to the oxygen atom of the carbamate moiety of M1 of formula (VIII).

76. The compound of claim 75, wherein R^A、R^DAnd R^EOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

77. The compound of claim 75 or claim 76, wherein A- (R)^A)_m、D-(R^D)_mAnd E- (R)^E)_mOne of them is

78. The compound of any one of claims 75-77, wherein A- (R)^A)_mIs composed ofD is C and R^DIs C₁A joint; e- (R)^E)_mIs composed ofWherein the bond between D and E is a double bond.

79. The compound of any one of claims 75-78, wherein G and J are C.

80. The compound of any one of claims 75-78, wherein G and J are C, and R is^GAnd R^JOne of them is-O-CH₃。

81. The compound of any one of claims 75-80, wherein the quinone is selected from formulas (IIIc) and (IIId):

wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (VIII).

82. The compound of any one of claims 69 to 74, wherein R¹⁰Is a quinone selected from formulas (IIIe) and (IIIf):

wherein

(I) A, D, E, F, G and J are independently selected from C and N, wherein at least one of A, D, E and F is C and at least one of G and J is C, and wherein N is a secondary amine, a tertiary amine, or an imine having the structure ═ N-;

(II) each n is independently selected from 0 and 1;

(III) the dotted line represents an optional double bond;

(IV)R^A、R^D、R^E、R^F、R^Gand R^JIndependently selected from H, OH and optionally substituted C_1-4Alkyl or heteroalkyl, C_1-4Alkoxy or heteroalkoxy, and halogen; and is

(V)R^A、R^D、R^E、R^F、R^GAnd R^JOne of them is C_1-4An optionally substituted alkyl or heteroalkyl linker with said M1 of formula (VIII)The oxygen atom of the carbamate moiety is covalently bonded.

83. The compound of claim 82, wherein R^A、R^D、R^EAnd R^FOne of them is said C_1-4An optionally substituted alkyl or heteroalkyl linker.

84. The compound of claim 82 or claim 83, wherein D-R^DOr E-R^ETo have a bond to said C_1-4An optionally substituted alkyl or heteroalkyl linker, and A, F and at least one of the other of D and E is N.

85. The compound of any one of claims 82 to 84, wherein R^A、R^EAnd R^FIs independently selected from H, optionally substituted C_1-4Alkyl or heteroalkyl, and optionally substituted C_1-4Alkoxy or heteroalkoxy, and wherein D is C and R^DIs C₁And (4) a joint.

86. The compound of any one of claims 82 to 85, wherein G- (R)^G)_nAnd J- (R)^J)_nAt least one of which is

87. The compound of any one of claims 82-86, wherein G-R^GAnd J-R^JOne of them is C-O-CH₃，G-R^GAnd J-R^JIs CH, wherein the bond between G and J is a double bond.

88. The compound of any one of claims 82-87, wherein the ring formed by A, D, E and F is unsaturated or partially saturated.

89. The compound of any one of claims 69 to 74, wherein R¹⁰An arylboronic acid or arylboronic acid ester of formula (IVa):

(I) wherein R is²⁰And R²¹Independently selected from H, optionally substituted alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, and optionally substituted aryl or heteroaryl, or

(II) wherein R²⁰And R²¹Together are an optionally substituted moiety- (CH)₂)_n-, wherein n is 2 or 3, said moieties, together with the O atom to which they are bonded, form a heterocycloalkyl ring, wherein said heterocycloalkyl ring may optionally include a fused heterocycloalkyl ring, a fused aryl ring, or a fused heteroaryl ring, and

(III) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (VIII).

90. The compound of claim 89, wherein R¹⁰Selected from formulae (IVb) and (IVc):

(I) wherein R is³⁰、R³¹、R³²、R³³、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴And R⁴⁵Independently selected from H, halogen, -CN, -OH, -NH₂、-COOH、-CONH₂、-NO₂、-SH、-SO₂Cl、-SO₃H、-SO₄H、-SO₂NH₂、-NHNH₂、-ONH₂、-NHC＝(O)NHNH₂Optionally substituted C_1-8Alkyl or heteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl containing from 2 to 7 carbon atoms, optionally substituted aryl or heteroaryl, or

(II) wherein (i) R³⁰Or R³¹And R³²Or R³³(iii) R⁴⁰Or R⁴¹And R⁴²Or R⁴³And/or (iii) R⁴²Or R⁴³And R⁴⁴Or R⁴⁵Form an optionally substituted fused cycloalkyl ring, fused heterocycloalkyl ring, fused aryl ring, or fused heteroaryl ring having 2 to 7 carbon atoms.

91. The compound of claim 89 or claim 90, wherein R¹⁰Selected from formulae (IVd) to (IVi):

92. the compound of claim 91, wherein R¹⁰Is represented by formula (IVd).

93. The compound of any one of claims 69 to 74, wherein R¹⁰Is a disulfide of formula (V):

(I) wherein R is⁵⁰Is optionally substituted C_1-8Alkyl or heteroalkyl, and R⁵¹Is optionally substituted C₂Alkylene or optionally substituted benzylidene; and is

(II) wherein the wavy line indicates the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (VIII).

94. The compound of claim 93, wherein R⁵⁰Is C_2-6Alkyl and R⁵¹Having formula (Va):

(I) wherein R is⁶¹And R⁶²Independently selected from H and CH₃Or R is⁶¹And R⁶²Together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl group containing 2 to 6 carbon atoms;

(II) wherein R⁶³And R⁶⁴Independently selected from H and CH₃(ii) a And is

(III) with R therein⁶³And R⁶⁴Represents the point of attachment to the oxygen atom of the carbamate moiety of M1 of formula (VIII).

95. The compound of claim 94, wherein R⁶³And R⁶⁴Is H.

96. The compound of any one of claims 93 to 95, wherein:

(I)R⁵⁰is selected from CH₃CH₂-、(CH₃)₂CH-and (CH)₃)₃C-; and is

(II)R⁵¹Selected from formulae (Vb) to (Vf):

97. the compound of any one of claims 69 to 96, wherein R¹²Is-c (O) O-L and L comprises a reactive sulfur atom, maleimide, bromoacetamide, iodoacetamide, or a conjugated alkene suitable for use in an antibody reactive thiol moiety.

98. The compound of any one of claims 69 to 97, selected from the following formulae:

99. a pharmaceutical composition comprising the pyrrolobenzodiazepine of any one of claims 1 to 40A prodrug dimer-antibody conjugate compound and a pharmaceutically acceptable diluent, antibody or excipient.

100. A method of treating cancer comprising administering to a patient in need of treatment the pharmaceutical composition of claim 99.

101. Use of an antibody-drug conjugate compound of any one of claims 1 to 40 in the manufacture of a medicament for treating cancer in a mammal.

102. An antibody-drug conjugate compound according to any one of claims 1 to 40 for use in a method of treating cancer.

103. An article of manufacture comprising the pharmaceutical composition of claim 99, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat cancer.

104. The compound of claim 1, selected from:

105. the compound of claim 104, wherein Ab is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from the group consisting of:

(1) BMPR1, (2) E, (3) STEAP, (4) MUC, (5) MPF, (6) Napi2, (7) Sema5b, (8) PSCAhlg, (9) ETBR, (10) MSG783, (11) STEAP, (12) TrpM, (13) CRIPTO, (14) CD, (15) CD79, (16) FcRH, (17) HER, (18) NCA, (19) MDP, (20) IL20, (21) curdlan, (22) EphB2, (23) ASLG659, (24) PSCA, (25) GEDA, (26) BADA-R, (27) CD, (28) CD79, (29) CXCR, (30) HLA-DOB, (31) P2X, (32) CD, (33) LY 34, (FcRH, (35) FcRH, (LGH, (36) TENB 37), (38) TMF, (39) LyEFR-R, (40) TMR 6, (4) MUC, (5) MPF, (6) TMGPR (48) TZGPR (23), (48) TRPR (23) TMGPR 52) (TMGPR (23) TRPR 52) (TMGPR 52) and (TMGPR 52) TRPR (23).