Pyridazinedione-Based Heterobicyclic Covalent Linkers and Methods and Applications Thereof

Abstract

Disclosed are pyridazinedione-based heterobicyclic covalent linkers useful for forming protein-drug conjugates, protein-protein conjugates, and the like, methods for their preparation, and the use of the linkers in forming various protein conjugates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 271,692, filed on October 25, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present application relates to heterobicyclic covalent linkers useful for forming protein-drug conjugates and methods and applications thereof. [Background technology]

[0003] Previous approaches to prepare antibody-drug conjugates (ADCs) have typically used methods that generate heterogeneous products containing mixtures of species with different drug-antibody ratios (DARs), which can lead to different pharmacokinetic profiles and, consequently, altered efficacy in vivo. For example, IgG proteins contain four solvent-accessible interchain disulfide bridges in the protein hinge region that, when reduced, generate eight reactive thiols. Conjugation to these reduced species generates a heterogeneous mixture of conjugates with DAR loadings of 2–4 drugs per antibody. Cleavage and exchange of disulfide bridges in these heterogeneous ADCs can result in adverse pharmacokinetics and reduce the metabolic stability of IgG antibodies in plasma (Mauricio Morais et al., Drug Discovery Today: Technologies, 2018, 30, 91). In addition, some reduced thiol groups that do not participate in the bioconjugation reaction often undergo intramolecular oxidation reactions with other thiols to generate disulfide-scrambled products that disrupt the protein structure and function of the ADCs. The issue of uncontrolled conjugation is particularly problematic in therapeutic applications, where non-ideal drug loading and heterogeneous mixtures of bioconjugates can lead to poor pharmacokinetic properties and narrow Zelapiec-windows (Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27).

[0004] Cysteine ​​residues have been explored as a method to site-selectively modify proteins in ADC design, with many approved therapeutics using cysteine-targeted conjugation reagents (Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27). Cysteine ​​is the preferred residue over lysine for protein modification due to its lower abundance (1.9% for cysteine ​​vs. 5.9% for lysine) and the higher nucleophilicity of its sulfhydryl side chain (pKa=8.0 for cysteine ​​vs. 10.5 for lysine), which translates to higher selectivity and fewer by-products. Among the methods developed, including maleimide, pyridyldithiopropionate, methylsulfonylphenyloxadiazole, monobromomaleimide, and carbonylacrylic derivatives, the most widely used strategy for cysteine ​​modification of biomolecules is the use of maleimide reagents (Mauricio Morais et al., Drug Discovery Today: Technologies, 2018, 30, 91; Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27; Seah Ling Kuan et al., Chem. Eur. J. 2016, 22, 17112).

[0005] Maleimide derivatives are widely used to incorporate cysteine ​​thiols into antibody derivatives and proteins. However, maleimide conjugates suffer from instability, and the thioether can undergo a reverse Michael reaction back to the original thiol and maleimide. The maleimide moiety, still attached to the payload, reacts with low concentrations of endogenous thiols present in the blood. In addition, a key hydrolysis step can lead to the formation of two positional isomers and a total of four diastereomers, which can complicate plasma stability and pharmacokinetics (Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27; Archie Wall et al., Chem. Sci., 2020, 11, 11455; Vesela Kostova et al., Pharmaceuticals 2021, 14, 442).

[0006] To maintain protein structure and function and form recrosslinking covalent bonds that are non-reactive to serum thiols, it is important that the cysteine ​​recrosslinking reagent can react rapidly with the reduced thiols derived from disulfides to form plasma-stable conjugates to avoid the problem of sulfide scrambling in plasma. Various thiol-stable chemical techniques have been applied to the modification of disulfide bonds in mAbs and their derivatives. These include bissulfone derivatives, dibromoalkyloxetane derivatives, trivalent arsenic acid, vinylheteroaryl-backed divinylpyrimidines and divinyltriazines, monobromomaleimides, dibromopyridazinediones, and disulfide-substituted maleimides (Mauricio Morais et al., Drug Discovery Today: Technologies, 2018, 30, 91; Seah Ling Kuan et al., Chem. Eur. J. 2016, 22, 17112).

[0007] Despite their widespread use, disubstituted maleimides have several widely noted limitations: upon hydrolysis, two regioisomeric maleamic acids are generated that may exhibit different plasma stabilities and pharmacokinetics (Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27; Archie Wall et al., Chem. Sci., 2020, 11, 11455).

[0008] In particular, dibromopyridazinediones (diBrPD) have emerged as a class of disulfide cross-linking reagents with enormous potential because they do not require a hydrolysis step to result in serum stability and are resistant / compatible with common mild reducing agents. Moreover, diBrPD has enabled the site-selective addition of three functionalities to proteins with a single cysteine ​​residue (Calise Bahou et al., Org. Biomol. Chem., 2018, 16, 1359). Modification with diBrPD provides good homogeneity and long-term plasma stability with no detectable effect on the binding capacity of the parent antibody. Currently, the diBrPD linker platform is widely used to generate ADCs, antibody conjugates, and antibody-directed photosensitizers (Marcos Fernandez et al., Chem. Commun., 2020, 56, 1125). Although many reagents have been developed for cysteine-specific protein re-crosslinking modification, few reagents allow polyfunctionalization of single Cys residues and bioconjugation of disulfide bridges. Although protein derivatives using diBrPD moieties have achieved good levels of homogeneity and long-term plasma stability, several regioselectivity issues arise that may complicate bioconjugated compounds using this connector (Calise Bahou et al., Org. Biomol. Chem., 2018, 16, 1359). For example, different N-alkyl groups may result in two regioisomers of the thiol linker product, which are very difficult to separate. If these antibody regioisomers are carried into ADC products, they are expected to complicate plasma stability and lead to inconsistent pharmacokinetics, which may further hinder therapeutic efficacy and have several adverse effects. Furthermore, the extra N-methyl group of 3-(4,5-dibromo-2-methyl-3,6-dioxo-pyridazin-1-yl)propanoic acid (a suitable moiety for derivatization into protein-drug conjugates) may suffer from metabolic issues of demethylation, which may result in poor PK of the ADC and complex metabolic profile. Due to the asymmetric structural characteristics of diBrPD, it is difficult to establish high QC standards in the manufacture of ADC products using this connector. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides novel symmetrical bicyclic dibromopyridazinedione-based linkers conjugated to cysteine ​​to avoid or minimize the aforementioned drawbacks.

[0010] In one aspect, the present disclosure provides a compound of formula (I): [ka] (In the formula, Ring A is a 5- to 13-membered heterocycle; m and n are each independently 1, 2, 3, 4, or 5; [ka] represents a double bond or a single bond; X and X' are each independently O, S, or NR X and R X is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; Y is a bond, (CH2) i (i = an integer from 1 to 12), C(O), C(O)O, OC(O), C(O)NR a , N.R. a (CO), NR a , O, S, S(O), S(O)2, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5-12 membered heteroarylene, or combinations thereof; R 2a and R 3a are each independently hydrogen, halogen, -UR 2 Or -VR 3 and; U and V are independently a bond, S, S(O), S(O)2, O, NH, or CH2; L is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted cyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, O, S, NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O)2, (CH2CH2O) j , (OCH2CH2) j (PEG, j = 2 to 48), SS, hydrazone, substituted or unsubstituted oligopeptides (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein R ais hydrogen or C1-C6 alkyl, and the linker L optionally includes a non-self-immolative spacer; R 1 is a functional moiety selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted succinimidyl, or a leaving group (e.g., BSA, KLH, OVA), a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier (e.g., BSA, KLH, OVA), a radionuclide, a photosensitizer, cytotoxins and their prodrugs, an innate immune modulator, a biopolymer, an oligonucleotide, a PROTAC degrader, an antibiotic, and an exotoxin; R 2 and R 3 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, an antibody, an antigen, a liposome, a polymer moiety, a substituted or unsubstituted amino acid moiety, a substituted or unsubstituted peptide moiety, DNA, RNA, a virus or virus-like particle, and a ligand targeting small molecule having a nucleophilic group or moiety such as -SH, -OH, -NH2, guanidinyl, imidazolyl, indolyl, and carboxylic acid (-CO2H); Or, R 2 and R 3 Let's get together and 4 and U, V and [ka] together with the ring B as characterized in formula (II) [ka] (where R 4is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each of which is optionally substituted, or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand-targeting small molecule, wherein the ligand-targeting small molecule optionally contains a nucleophilic group selected from -SH, -OH, -NH2, guanidinyl, imidazolyl, indolyl, and carboxylic acid, or a combination thereof, or a salt thereof.

[0011] In one aspect, the present disclosure provides a compound of formula (II): [ka] (In the formula, Ring A is independently a 5- to 13-membered carbocyclic ring; m and n are each independently 1, 2, 3, 4, or 5; Ring B contains two non-nucleophilic groups (e.g., thiol groups from disulfide bridges in a peptide, protein, or antibody); X and X' are each independently O, S, or NR x and; Y is a bond, (CH2) i (i = an integer from 1 to 12), C(O), C(O)O, NR a , O, S, S(O), S(O)2, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5-12 membered heteroarylene, or combinations thereof; L is a bond, alkylene, O, S, NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O)2, (CH2CH2O) j , (OCH2CH2) j(PEGn, j = 2 to 48), SS, hydrazone, oligopeptide, (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein the linker optionally includes a self-immolative spacer; R 1 is a functional moiety selected from hydrogen, alkyl, cycloalkyl, aryl, succinimidyl, or leaving groups, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins; R x is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; R 4 is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each optionally substituted or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand targeting small molecule, the ligand targeting small molecule optionally containing a nucleophilic group selected from -SH, -OH, -NH2, guanidine, imidazole, indole, carboxylic acid, or combinations thereof; R a is hydrogen or C1-C6 alkyl; or a pharma- ceutically acceptable salt thereof.

[0012] In one aspect, the disclosure provides a pharmaceutical composition comprising a compound according to any one of the embodiments disclosed herein and a pharma- ceutically acceptable carrier.

[0013] In one aspect, the disclosure provides a method of treating a disease or disorder, comprising administering to a subject in need thereof a compound according to any one of the embodiments disclosed herein, or a pharma- ceutically acceptable salt or pharmaceutical composition thereof.

[0014] In one aspect, the disclosure provides the use of a compound according to any one of the embodiments disclosed herein, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or disorder.

[0015] Other aspects and advantages of the present disclosure will be better understood by those of ordinary skill in the art in view of the following detailed description, drawings, illustrative examples, and claims. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 illustrates the regioisomer problem of dibromopyridazinedione (diBrPD) connectors (FIG. 1a) and the disclosed solution (FIG. 1b). [Diagram 2] FIG. 2 shows the hydrophobic interaction chromatography (HIC) profile of the antibody-SBC-CL075(E13) conjugate corresponding to the drug loaded species, DAR4. [Diagram 3] FIG. 3 shows the HIC diagram of the antibody-SBC-MMAF (E14) conjugate corresponding to the drug-loaded species, DAR4. [Figure 4] Figure 4 shows the HIC diagrams of antibody-diBrPD-CL075 conjugates corresponding to drug-loaded species with DAR1, 2, 3, 4, and 5. [Diagram 5] FIG. 5 shows the HIC diagrams of antibody-diBrPD-MMAF conjugates corresponding to drug-loaded species with DARs of 3, 4, and 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The pyridazine diketone (PD) moiety can overcome many of the shortcomings associated with commonly used Michael acceptors (e.g., maleimides) for cysteine ​​modification purposes and has been proven to provide a stable linker for days in serum (i.e., PD-derived bioconjugates do not react with high concentrations of human serum albumin (HSA) and low concentrations of glutathione (GSH)). PD-protein constructs have also been proven to be cleavable with high concentrations of reactive thiols, providing a release mechanism in early endosomal conditions in cells (i.e., high GSH concentrations between 1-10 mM and a pH range of 6.8-5.9). Additionally, functional handles can be easily incorporated into the PD scaffold to link spacers and payloads, allowing their use in the production of antibody-drug conjugates (ADCs) (Peter A. Szijj et al., Drug Discovery Today: Technologies 2018, 30, 27; Calise Bahou et al., Org. Biomol. Chem., 2018, 16, 1359; Marcos Fernandez et al., Chem. Commun., 2020, 56, 1125; Calise Bahou et al., Org. Biomol. Chem., 2018, 16, 1359; Ofelia Feuillatre et al., ACS Omega 2020, 5, 1557-1565; WO2019034868). However, existing pyridazinedione-based linkers suffer from various limitations as mentioned above, one of which is shown in (Figure 1a).

[0018] The present invention provides novel linkers that overcome the limitations of existing pyridazinedione (PD)-based linkers, for example by having in one case a 2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-5,8-dione moiety, while maintaining the advantages of pyridazinedione (PD). These linkers can be synthesized in 1 to 3 steps from commercially available starting materials. The bromide on the dihydropyrazolopyridazinedione (6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine) is highly reactive (cysteine-specific modification) towards thiols obtained by reduction of disulfide bridges in proteins (a complete conversion in 16 h has been reported using diBrPD, which is longer than bromomaleimide). Compared with the monocyclic diBrPD linker reported by UCL, this structurally symmetric bicyclic heterocyclic scaffold is believed to solve the problem of regioselectivity brought by the monocyclic system. We believe that the Symmetric Bicyclic-dibromopyridazinedione Cysteine ​​(SBC) linker should have higher cysteine ​​specificity and be pharmacokinetically superior (N-methyl group removed). In addition, the additional functional groups, such as carboxylic acid, amino, and hydroxyl groups, introduced by this type of connector provide the opportunity for polyfunctionalization of single cysteines or bioconjugation of disulfide bridges. This method has a wide substrate scope, allowing the introduction of a wide range of synthetic modifications to various protein scaffolds, including antibodies, without compromising their native antigen-binding properties (Figure 1b).

[0019] In one aspect, the present disclosure provides a compound of formula (I): [ka] (In the formula, Ring A is a 5- to 13-membered heterocycle; m and n are each independently 1, 2, 3, 4, or 5; [ka] represents a double bond or a single bond; X and X' are each independently O, S, or NR X and; Y is a bond, (CH2) i (i = an integer from 1 to 12), C(O), C(O)O, C(O)NR a , N.R. a , O, S, S(O), S(O)2, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5-12 membered heteroarylene, or combinations thereof; R 2a and R 3a are each independently a halogen, -UR 2 Or -VR 3 and; U and V are independently a bond, S, O, NH, or CH2; L is a bond, alkylene, O, S, or NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O)2, (CH2CH2O) j , (OCH2CH2) j (PEGn, j = 2 to 48), SS, hydrazone, oligopeptide (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein the linker optionally includes a non-self-immolative spacer; R 1 is a functional moiety selected from hydrogen, alkyl, cycloalkyl, aryl, succinimidyl, or leaving groups, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins; R 2 and R 3are each independently selected from halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, antibody, antigen, liposome, polymer moiety, amino acid, peptide, DNA, RNA, virus or virus like particle, and ligand targeting small molecule having a nucleophilic group or moiety such as -SH, -OH, -NH2, guanidinyl, imidazolyl, indole, and carboxylic acid; Or, R 2 and R 3 Let's get together and 4 and U, V and [ka] together with the ring B as characterized in formula (II) [ka] (where R X is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; R 4 is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each of which may be optionally substituted, or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand targeting small molecule, where the ligand targeting small molecule optionally contains a nucleophilic group selected from -SH, -OH, -NH2, guanidinyl, imidazolyl, indole, and carboxylic acid, or combinations thereof; R a is hydrogen or C1-C6 alkyl, or a salt thereof.

[0020] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, ring A is a 5- to 9-membered heterocycle; m and n are each independently 1, 2, or 3.

[0021] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, ring A is a 5- to 7-membered heterocycle; m and n are each independently 1 or 2.

[0022] In some embodiments, in a compound of Formula (I) or (II), or a salt thereof, m=1, and n=1.

[0023] In some embodiments, in a compound of formula (I) or (II), or a salt thereof, [ka] is a double bond, and X and X' are each O.

[0024] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, U and V are each a bond, and R 2 and R 3 are each halogens.

[0025] In some embodiments, in the compound of formula (I) or (II), or a salt thereof, R 2 and R 3 are bromine (Br), respectively.

[0026] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, U and V are each sulfur (S); R 2 and R 3 are each independently an alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid moiety, each of which is optionally substituted.

[0027] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, U and V are each sulfur (S); R 2 and R 3are each independently or together selected from an antibody, an antigen, a liposome, a polymer moiety, an amino acid, an oligopeptide, DNA, RNA, a virus or virus-like particle, and a small molecule targeting ligand.

[0028] In some embodiments, in a compound of formula (I) or (II), or a salt thereof, Y is C(O)O, C(O)NH, CH2, O, S, NH; L is a bond, alkylene, -(CH2) k C(O)NH-, where k is an integer selected from 1 to 8; and R 1 is hydrogen, alkyl, cycloalkyl, aryl, or succinimidyl.

[0029] In some embodiments, in a compound of formula (I) or (II), or a salt thereof, Y is C(O)NH; L is a bond, -(CH2) k C(O)NH-, where k is an integer selected from 1 to 8, or an oligopeptide moiety, or a combination thereof; R 1 is selected from hydrogen, alkyl, cycloalkyl, aryl, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins.

[0030] In some embodiments, in the compound of Formula (I) or (II), or a salt thereof, the oligopeptide portion is selected from Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, and Val-Arg.

[0031] In some embodiments, in a compound of Formula (I) or (II), or a salt thereof, the self-immolative spacer comprises a para-aminobenzyloxycarbonyl (PABC) moiety or a PABC-type moiety that can lead to electronic cascade-mediated self-immolation or cyclization-mediated self-immolation.

[0032] In one aspect, the present disclosure provides a compound of formula (II): [ka] (In the formula, Ring A is independently a 5- to 13-membered carbocyclic ring; m and n are each independently 1, 2, 3, 4, or 5; Ring B contains two non-nucleophilic groups (e.g., thiol groups from disulfide bridges in a peptide, protein, or antibody); X and X' are each independently O, S, or NR x and; Y is a bond, (CH2) i (i = an integer from 1 to 12), C(O), C(O)O, NR a , O, S, S(O), S(O)2, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5-12 membered heteroarylene, or combinations thereof; L is a bond, alkylene, O, S, NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O)2, (CH2CH2O) j , (OCH2CH2) j (PEGn, j = 2 to 48), SS, hydrazone, oligopeptide, (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein the linker optionally includes a self-immolative spacer; R 1is a functional moiety selected from hydrogen, alkyl, cycloalkyl, aryl, succinimidyl, or leaving groups, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins; R x is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; R 4 is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each optionally substituted or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand targeting small molecule, the ligand targeting small molecule optionally containing a nucleophilic group selected from -SH, -OH, -NH2, guanidine, imidazole, indole, carboxylic acid, or combinations thereof; R a is hydrogen or C1-C6 alkyl; or a pharma- ceutically acceptable salt thereof.

[0033] In some embodiments, in the compound of formula (II), or a salt thereof, Ring A is a 5- to 9-membered heterocycle; m and n are each independently 1, 2, or 3.

[0034] In some embodiments, in the compound of formula (II), or a salt thereof, Ring A is a 5- to 7-membered heterocycle; m and n are each independently 1 or 2.

[0035] In some embodiments, in the compound of formula (II), or a salt thereof, m=1, and n=1.

[0036] In some embodiments, in the compound of formula (II), or a salt thereof, [ka] is a double bond, and X and X' are each O.

[0037] In some embodiments, in the compound of Formula (II), or a salt thereof, U and V are each a bond, and R 2 and R 3 are each halogens.

[0038] In some embodiments, in the compound of formula (II), or a salt thereof, R 2 and R 3 are bromine (Br), respectively.

[0039] In some embodiments, in the compound of formula (II), or a salt thereof, U and V are sulfur (S); R 2 and R 3 are each independently an alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid moiety, each of which is optionally substituted.

[0040] In some embodiments, in the compound of formula (II), or a salt thereof, U and V are sulfur (S); R 2 and R 3 are each independently or together selected from an antibody, an antigen, a liposome, a polymer moiety, an amino acid, an oligopeptide, DNA, RNA, a virus or virus-like particle, and a small molecule targeting ligand.

[0041] In some embodiments, in the compound of formula (II), or a salt thereof, Y is C(O)O, C(O)NH, CH2, O, S, or NH; L is a bond, alkylene, -(CH2) k C(O)NH-, where k is an integer selected from 1 to 8; R 1 is hydrogen, alkyl, cycloalkyl, aryl, or succinimidyl.

[0042] In some embodiments, in the compound of formula (II), or a salt thereof, Y is C(O)NH; L is a bond, -(CH2) k C(O)NH-, where k is an integer selected from 1 to 8, or an oligopeptide moiety, or a combination thereof; R 1 is selected from hydrogen, alkyl, cycloalkyl, aryl, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins.

[0043] In some embodiments, in the compound of Formula (II), or a salt thereof, the oligopeptide portion is selected from Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, and Val-Arg.

[0044] In some embodiments, in a compound of Formula (II), or a salt thereof, the self-immolative spacer comprises a para-aminobenzyloxycarbonyl (PABC) moiety or a PABC-type moiety (e.g., ortho-aminobenzyl, ortho-hydroxybenzyl, and para-hydroxybenzyl) that can undergo electronic cascade-mediated self-immolation or cyclization-mediated self-immolation.

[0045] In one aspect, the disclosure provides a pharmaceutical composition comprising a compound according to any one of the embodiments disclosed herein and a pharma- ceutically acceptable carrier.

[0046] In one aspect, the disclosure provides a method of treating a disease or disorder, comprising administering to a subject in need thereof a compound according to any one of the embodiments disclosed herein, or a pharma- ceutically acceptable salt or pharmaceutical composition thereof.

[0047] In one aspect, the disclosure provides the use of a compound according to any one of the embodiments disclosed herein, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or disorder.

[0048] In some embodiments, illustrative examples of compounds of the present invention include, but are not limited to, compounds E01 to E24 listed below. [ka] [ka] [ka]

[0049] In some embodiments, the disclosure provides a compound selected from compounds E1-E24, or a salt or stereoisomer thereof, or a pharmaceutical composition comprising any compound selected from compounds E1-E24.

[0050] definition As used herein, terms such as "moiety," "chemical moiety," "molecular moiety," and the like, refer to a characteristic (often major) portion of another molecule as an integral part of the defined subject structure, where the moiety is covalently attached to the remainder of the structure through one, two, or three positions on the molecule after removal of one, two, or three surrounding atoms or groups from one, two, or three positions on the molecule. For example, a peptide contains multiple amino acid moieties. An amino acid moiety within a peptide chain (if branched) may be covalently attached to two or three other amino acid moieties, but an amino acid moiety at the end of the peptide is covalently attached only to adjacent amino acid moieties.

[0051] As used herein, the term "detectable moiety" refers to a moiety capable of generating a detectable signal, and is also commonly known in the art as a "tag," "probe," and "label." Examples of detectable moieties include chromogenic moieties, fluorescent moieties, radioactive moieties, and electrochemically active moieties.

[0052] Chromogenic moieties are moieties that are incorporated into a conjugate and subsequently become colored or changed to color when the conjugate interacts with a secondary target species. Examples include porphyrins, polyenes, polyynes, and polyaryls.

[0053] A fluorescent moiety is a moiety that contains a fluorophore. Examples of fluorescent compounds include Alexa Fluor dyes, cyanines and merocyanines, boron dipyrromethene dyes, ATIO dyes, fluorescein and its derivatives (rhodamine, coumarin, sulforhodamine 101 acid chloride (Texas Red), and dansyl), rhodamine and its derivatives, naphthalene derivatives, pyridyloxazoles, nitrobenzoxadiazoles and benzoxadiazole derivatives, coumarins and their derivatives, pyrene derivatives, Oregon Green, eosin, Cascade Blue, Nile Red, etc.

[0054] Radioactive moiety refers to a "radionuclide", "radionucleon", "radioisotope", "radioisotope", "radioactive compound" or "radiolabel" and is the part that makes up a radionuclide, an atom with excess nuclear energy. Radionuclides can be used for their radiation (e.g., to damage or kill pathogenic cells) or can be used in combination with chemistry and radiation (e.g., as tracers or biopharmaceuticals). Non-limiting examples of radioisotopes include gallium-68, copper-64, lutetium-177, iodine-131, iodine-125, bismuth-212, yttrium-90, yttrium-88, technetium-99m, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114m, indium-114, boron-10, tritium (hydrogen-3), carbon-14, sulfur-35, fluorine-18, and carbon-11. For example, fluorine-18 and carbon-11 are commonly used in positron emission tomography.

[0055] An electrochemically active moiety is one that is capable of generating an electrochemical signal in the ampere or voltaic manner and can exist in at least two different redox states. Examples of electrochemically active moieties include dopamine hydrochloride, ascorbic acid, phenols and derivatives, benzoquinones and derivatives, etc.

[0056] The term "affinity tag" as used herein means a chemical moiety capable of interacting with an "affinity partner" (a second chemical moiety present in a single sample) such as between an enzyme and its substrate. Examples of affinity tag / affinity partner pairs that are particularly widely used in biochemistry are amylase / maltose binding protein, glutathione / glutathione-S-transferase, and metal (biotin / streptavidin, e.g. nickel or cobalt) / poly(His). As used herein, the term "hapten" refers to a low molecular weight non-proteinaceous moiety that contains an epitope and becomes an immunostimulatory agent when linked to an immunogenic carrier molecule.

[0057] The term "immunogenic carrier" as used herein refers to an antigen that can promote an immune response. Examples of immunogenic carriers include proteins, liposomes, synthetic or natural polymer moieties (such as dextran, agarose, polylysine, polyglutamic acid moieties), and synthetically designed organic moieties. Commonly used protein immunogenic carriers include keyhole limpet hemocyanin, bovine serum albumin, aminoethylated or cationized bovine serum albumin, thyroglobulin, ovalbumin, and various toxoid proteins such as tetanus toxoid and diphtheria toxoid. Synthetically designed organic molecular carriers include multiple antigentic peptides (MAPs).

[0058] As used herein, the term "photosensitizer" means a moiety that can absorb light and transfer the energy from the incident light to another nearby molecule. A vast number of photosensitizers are used in photoimmunotherapy, including porphyrins, chlorins, and phthalocyanine dyes.

[0059] The term "cytotoxin" as used herein refers to a moiety that can exert cytotoxicity on cells by disrupting tubulin, damaging DNA, inhibiting topoisomerase, and interfering with other important cellular processes. Exemplary cytotoxins and their prodrugs include maytansinoids, auristatins, dolastatins, tubulysins, eribulin, cryptomycins, topoisomerase inhibitors, durcarmycins, nemorubicins, pyrrolobenzodiazepines (PBDs), calicheamicins, camptothecins, amatoxins, antimitotic EG5 inhibitors, apoptosis inducers, tylanstatins, nicotinamide phosphoribosyltransferase inhibitors, and carmaficins. As used herein, the term "innate immune modulator" refers to portions of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) that bind to pattern recognition receptors (PRRs). Recognition of PAMPs or DAMPs by PRRs triggers inflammatory responses, such as secretion of cytokines / chemokines, induction of antimicrobial peptides, cell death by pyroptosis, and recruitment of phagocytes. Exemplary innate immune modulators include tumor necrosis factor (TNF) superfamily ligands, C-type lectin receptor (CLR) ligands, retinoic acid-inducible gene I (RIG-1)-like receptor (RLR) ligands, stimulator of interferon genes (STING) ligands, Toll-like receptor (TLR) ligands, cytoplasmic DNA sensor (CDS) ligands, and the like.

[0060] Innate immune modulators suitable for use in the compositions and methods described herein include CU-T12-9, Pam3CSK4, FSL-1 (Pam2CGDPKHPKSF), poly(I:C), LPS (lipopolysaccharide), MPLA (monophosphoryl lipid A), CRX-527, FLA (flagellin), CL075 (also known as 3M002, a thiazoloquinolone derivative), CL097, CL264, CL307, CL429, galdiquimod, R837 (imiquimod), R848 (resiquimod), loxoribine, TL8-506, CU-CPT9a, ODN2088 (CpG oligodeoxynucleotide 2088), ODN4084, ODN INH-18, ODN1585, ODN2216, ODN2336 , ODN1668, ODN2006, ODN1826, ODN BW006, ODN D-SL01, ODN2395, ODN M362, ODN SL03, C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, iE-Lys, Tri-DAP, MDP (muramyl dipeptide), L18-MDP, beta-glucan, curdlan, HKCA (C. albicans These include, but are not limited to, heat-killed preparations of , laminarin, pustulan, scleroglucan, zymosan, furfulmane, GlcC14C18, beta-glucosylceramide, TDB (trehalose-6,6-dibehenate), 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, 2'2'-cGAMP, DMXAA, dsDNA, G3-YSD (unpaired guanosine trimer-terminated Y-shaped short stranded DNA), HSV-60, ISD (interferon-stimulated DNA), etc.

[0061] The term "biopolymer" as used herein means a portion of a natural polymer produced by cells of a living organism. Biopolymers are covalently bonded to form larger molecules. Exemplary biopolymers include high molecular weight phosphorylcholine polymers.

[0062] As used herein, the term "enzymatically active moiety" refers to an enzyme, enzyme substrate, or enzyme cofactor that can act as a biological catalyst and / or therapeutic agent to modify microenvironmental conditions through the acceleration of a chemical reaction. An example of a microenvironment modifier and therapeutic enzyme is urease, a member of the amidohydrolase and phosphotriesterase superfamily.

[0063] As used herein, the term "oligonucleotide" refers to a short DNA or RNA molecule, an oligomer, that can be a therapeutic agent; exemplary therapeutic oligonucleotides include myotonic dystrophy type 1 antisense oligonucleotides that degrade DMPK transcripts and permanently reduce levels of DMPK mRNA.

[0064] "PROTAC degraders" form an enzyme complex within a cell and act as a catalyst to knock down a target protein. They usually contain a binding moiety for an E3 ubiquitin ligase and a binding moiety for a target protein joined by a linker. Exemplary protein degraders include bromodomain-containing protein 4 (BRD4) degraders.

[0065] As used herein, the term "antibiotic" refers to a chemical substance produced by living microorganisms that is harmful to other microorganisms. Antibiotics work by inhibiting the synthesis or function of bacterial cell walls or by inhibiting bacterial protein synthesis.

[0066] As used herein, the term "exotoxin" refers to a moiety secreted by a bacterium. Well-known exotoxins include botulinum toxin and Corynebacterium diphtheria toxin.

[0067] The term "antibody" as used herein refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) and more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to various cells of the immune system and to host tissues or factors, including the first component of the classical complement system (C1q). The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Antibodies may be of any isotype (IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, etc.), or subclass.

[0068] The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope. Examples of binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains), F(ab)2 fragments (bivalent fragments containing two Fab fragments linked by disulfide bridges at the hinge region), Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single arm of an antibody, dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), and isolated complementarity determining regions (CDRs).

[0069] Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker, allowing them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv), e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antibody fragment." These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0070] Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted onto scaffolds, such as polypeptides, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules that contain a pair of tandem Fv segments (VH-CH1-VH-CH1) to form a pair of antigen-binding regions with a light chain polypeptide complementary to the Fv segments (Zapata et al., (1995) Protein Eng. 8:1057-1062 and US Pat. No. 5,641,870).

[0071] The term "antigen" as used herein refers to a site on a polypeptide macromolecule to which an antibody binds to form an antibody-antigen complex. Proteins useful as antigens herein can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term "epitope" refers to a site on a proteinaceous or nonproteinaceous antigen to which an antibody binds. Epitopes may be formed from a contiguous amino acid sequence (linear epitopes) or may consist of discontinuous amino acids (conformational epitopes) that are brought into spatial proximity by antigen folding, i.e., tertiary folding of protein antigens. Linear epitopes usually remain bound by antibodies after exposure of the protein antigen to denaturing agents, whereas conformational epitopes are usually destroyed by treatment with denaturing agents.

[0072] The term "ligand" as used herein refers to a moiety that can interact with a biomolecule (e.g., a protein) to modify the functional properties of the biomolecule. Typically, a ligand is a moiety that binds to a site on a target protein. The interaction between the ligand and the biomolecule is typically non-covalent. For example, the interaction may occur via ionic bonds, hydrogen bonds, or van der Waals interactions. However, some ligands are also capable of forming covalent bonds with the biomolecule. Typically, a ligand can change the chemical conformation of the biomolecule upon interaction with the biomolecule.

[0073] As used herein, the term "liposome" refers to a structure composed of a phospholipid bilayer having amphiphilic properties. Liposomes suitable for use in accordance with the present invention include unilamellar and multilamellar vesicles.

[0074] As used herein, the term "polymer moiety" refers to a single polymer chain (branched or unbranched) derived from a single corresponding polymer molecule. A polymer moiety may be a natural polymer or a synthetic polymer. However, typically, the polymer molecule is not a polynucleotide.

[0075] As is well known in the biochemistry field, the creation of conjugates containing polymer moieties is useful in many in vivo and in vitro applications. For example, various properties of macromolecules such as proteins can be modified by attaching polymer moieties, including solubility properties, surface properties, and stability in solution or when frozen. Another common application is to attach polymer moieties to biologically active compounds such as drugs to enhance biocompatibility, reduce or eliminate immune responses upon administration, and / or improve in vivo stability.

[0076] Thus, one of skill in the art will recognize that the methods of the present invention can be used to prepare conjugates that include a polymer moiety, which can then be used for known applications of conjugates that include a polymer moiety. One of skill in the art will be able to readily select suitable polymer moieties for use in accordance with the present invention based on polymer moieties commonly used in the art.

[0077] Thus, the nature of the polymer moiety depends on the intended use of the conjugated molecule. Exemplary polymer moieties used according to the present invention include polysaccharides, polyethers, polyamino acids (e.g., polylysine), polyvinyl alcohols, polyvinylpyrrolidones, poly(meth)acrylic acids and their derivatives, polyurethanes, and polyphosphazenes. Typically, such polymers contain at least 10 monomer units. Thus, for example, polysaccharides typically contain at least 10 monosaccharide units.

[0078] Two particularly preferred polymer molecules are dextran and polyethylene glycol ("PEG"), as well as derivatives of these molecules (e.g., monomethoxypolyethylene glycol, "mPEG"). Preferably, the molecular weight of the PEG or its derivative is less than 20,000. Preferably, the molecular weight of the dextran or its derivative is between 10,000 and 500,000. In a preferred embodiment, the compound of the invention comprises a biologically active moiety (e.g., a drug) and PEG or its derivative.

[0079] The term "amino acid" as used herein refers to a molecule that contains both an amine function and a carboxyl function. However, preferably, the amino acid is an α-amino acid. Preferably, the amino acid is a proteinogenic amino acid, i.e., an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, phenylalanine, pyrrolidine, selenocysteine, serine, threonine, tyrosine, and valine. However, the amino acid may be a non-proteinogenic amino acid. Examples of non-proteinogenic amino acids include lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, ornithine, citrulline, canavanine, and mimosine. A particularly preferred amino acid in the present invention is cysteine.

[0080] The term "peptide" as used herein refers to a polymer moiety composed of amino acid residues. As will be appreciated by those skilled in the art, the term "peptide" is generally used in the art to refer to polymers of relatively short length, while the term "protein" is generally used in the art to refer to polymers of relatively long length. As used herein, by convention, peptides contain up to 50 amino acid residues, while proteins contain more than 50 amino acid residues. However, it will be understood that this distinction is not important, as the functional moieties identified in this application can generally refer to either peptides or proteins.

[0081] As used herein, the term "polypeptide" can be used interchangeably with "protein."

[0082] As used herein, a peptide or protein can include any natural or unnatural amino acid. For example, a peptide or protein may include only α-amino acid residues that correspond to natural α-amino acids. Alternatively, a peptide or protein may further include one or more chemical modifications that correspond to post-translational modifications that are modifications to proteins after translation in vivo, such as acylation (e.g., acetylation), alkylation (e.g., methylation), amidation, biotinylation, formylation, glycosylation, glycation, hydroxylation, iodination, oxidation, sulfation, or phosphorylation. Of course, a person skilled in the art will recognize that such post-translationally modified peptides or proteins still constitute a "peptide" or "protein" within the meaning of the present invention. For example, it is well established in the art that glycoproteins (proteins with one or more oligosaccharide side chains) are a type of protein.

[0083] As used herein, the term "DNA" refers to deoxyribonucleic acid composed of one or more nucleotides. DNA may be single-stranded or double-stranded. Preferably, DNA contains one or more nucleotides.

[0084] As used herein, the term "RNA" refers to a ribonucleic acid comprising one or more nucleotides. Preferably, the RNA comprises one or more nucleotides.

[0085] The term "virus-like particles (VLPs)" as used herein refers to multiprotein structures that mimic the organization and conformation of authentic native viruses, but lack the viral genome. VLPs are useful as vaccines. VLPs contain a repetitive and dense display of viral surface proteins that present conformational viral epitopes that can elicit strong T-cell and B-cell immune responses. The particle has a small radius of approximately 20-200 nm, allowing for efficient attraction to lymph nodes. VLPs cannot replicate, making them a safer alternative to attenuated viruses. VLPs have been used in the development of FDA-approved and currently commercially available vaccines for Hepatitis B and Human Papillomavirus.

[0086] As used herein, the term "targeting ligand small molecule" refers to organic compounds with low molecular weight (<900 Daltons) and sizes of about 1 nm that may regulate biological processes. Examples of ligand targeting small molecules include HSP90 binding small molecules.

[0087] "Linker" or "linker group" refers to a group capable of covalently linking one chemical moiety (e.g., an antibody) to another chemical moiety (e.g., a functional moiety). Two main categories of linkers have been described: non-cleavable linkers and cleavable linkers, such as disulfide-containing, hydrazone, and enzyme-cleavable linkers with self-immolative spacers. Examples of linker groups suitable for use according to the present invention are common knowledge in the art and are described in standard reference textbooks such as "Bioconjugate Technology" (Greg T. Hermanson, Academic Press Inc., 1996), the contents of which are incorporated herein by reference in their entirety.

[0088] An "antibody-drug conjugate" or "ADC" is an antibody, usually a monoclonal antibody specific for a cancer antigen, that is conjugated via a linker to one or more (usually 1-4) cytotoxins.

[0089] The antibody conjugation reactive end of the linker is typically a site that can be conjugated to an antibody via a thiol of a cysteine ​​or an amine group of a lysine on the antibody, and is therefore typically a thiol-reactive group such as a double bond (e.g., maleimide), or a leaving group such as chlorine, bromine or iodine, or an R-sulfanyl group, or an amine-reactive group such as a carboxyl group.

[0090] The term "alkyl" in the present invention refers to a linear or branched saturated hydrocarbon (i.e., does not contain double or triple bonds). The alkyl group can have 1 to 9 carbon atoms, preferably 1 to 6, more preferably 1 to 4 carbon atoms (in the present invention, the numerical range of "1 to 9" refers to any integer within this range, for example, "1 to 9 carbon atoms" means that the alkyl group can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, ..., up to 9 carbon atoms. At the same time, the definition of alkyl also includes alkyl groups with no chain length specified. The alkyl group can be a medium-sized alkyl group containing 1 to 9 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, 2-methylhexyl, n-octyl, 2,3-dimethylhexyl, etc.

[0091] The term "alkenyl" in the present invention refers to a straight or branched chain hydrocarbon containing one or more double bonds. The alkenyl group can have 2-9 carbon atoms and can also include alkenyl groups with no specific chain length. The alkenyl group can be a medium-sized alkenyl group containing 2-9, preferably 2-6, carbon atoms. The alkenyl group can also be a small alkenyl group containing 2-4 carbon atoms. The alkenyl group can be designed as "C2-4 alkenyl" or a similar design. For example, "C2-4 alkenyl" means that there are 2 to 4 carbon atoms in the alkenyl chain, i.e., the alkenyl chain can be selected from ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethenyl-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl, buta-1,2-dien-4-yl. Exemplary alkenyls include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0092] The term "alkynyl" in the present invention refers to a straight or branched chain hydrocarbon containing one or more triple bonds. An alkynyl group can have 2-9 carbon atoms and can also include alkynyls with no specific chain length. An alkynyl group can be a medium size alkynyl group containing 2-9 carbon atoms. An alkynyl group can also be a lower alkynyl group containing 2-4 carbon atoms. An alkynyl group can be designed as "C2-4 alkynyl" or similar design. For example, "C2-4 alkynyl" means that there are 2-4 carbon atoms in the alkynyl chain, that is, the alkynyl chain can be selected from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl, and 2-butynyl. Exemplary alkynyls include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0093] As used herein, the term "aryl" refers to a ring or ring system having a conjugated pi-electron system, and includes carbocyclic aryls (such as phenyl) and heterocyclic aryls (such as pyridine). The term includes groups having a single ring or multiple fused rings (i.e., rings which share a pair of adjacent atoms), where the entire ring system is aromatic.

[0094] The term "heteroaryl" in the present invention refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) that contains one or more heteroatoms. That is, the ring backbone includes, but is not limited to, elements such as nitrogen, oxygen, and sulfur in addition to carbon. When a heteroaryl is a ring system, each ring in the system is aromatic. A heteroaryl can have 5 to 18 ring members (i.e., the number of atoms that make up the ring backbone, including the number of carbon atoms and heteroatoms). The current definition also includes heteroaryl groups where the ring size is not specified. Examples of heteroaryl include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0095] As used herein, the term "cycloalkyl" refers to a fully saturated carbocyclic ring or ring system, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0096] The term "(heterocyclyl)alkyl" in the present invention refers to a heterocyclyl as a substituent linked to another group via an alkylene. Examples include, but are not limited to, imidazolinylmethyl, indolinylethyl. The term "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in its backbone. Heterocyclyls can be attached in the form of fused, bridged, or spiro rings. At least one ring in the heterocyclic ring system is non-aromatic and can have any degree of saturation. The heteroatom can be located on a non-aromatic or aromatic ring of the ring system. Heterocyclyls can have 3 to 20 ring atoms (i.e., the number of atoms that make up the ring backbone, including the number of carbon atoms and heteroatoms). This definition also includes heterocyclyl groups where the range of the number of rings is not specified. Heterocyclyl groups can be medium-sized heterocyclyl groups containing 3 to 10 ring atoms. Heterocyclyl groups can be small-sized heterocyclyl groups containing 3 to 6 ring atoms. Examples of heterocyclyl include azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thietanyl, piperidinyl, piperazinyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianilinyl, and the like. Examples of aryl groups include, but are not limited to, 1,4-oxathiyl, 1,4-oxathiyl, 2H-1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, oxazolidinone, thiazolidinyl, 1,3-oxathiolyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl and tetrahydroquinolinyl.

[0097] As used herein, "alkoxy" refers to a group of the formula -OR, where R is alkyl as defined above, such as "C1-9 alkoxy", including, but not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0098] The term "alkylthio" in the present invention refers to the formula SR, where R is alkyl as defined above, such as "C1-9 alkylthio", including, but not limited to, methyl mercapto, ethyl mercapto, n-propyl mercapto, 1-methylethyl mercapto (isopropyl mercapto), n-butyl mercapto, isobutyl mercapto, sec-butyl mercapto, tert-butyl mercapto, and the like.

[0099] As used herein, "aryloxy" and "arylthio" refer to RO- and RS- (wherein R is aryl as defined above), such as "C6-10 aryloxy" or "C6-10 arylthio", and are not limited to phenyloxy.

[0100] The term "halogen" or "halo" as used herein refers to any radiostable atom of column 7 of the Periodic Table of the Elements, such as fluorine, chlorine, bromine or iodine, with bromine and chlorine being preferred in some instances.

[0101] A "bond" represents a covalent bond using the "-" symbol.

[0102] "Hydroxy" refers to the group --OH.

[0103] "Amino" refers to the group -NH2.

[0104] "Cyano" refers to the group -CN.

[0105] "Nitro" refers to the -NO2 group.

[0106] An "oxo group" refers to the group =O.

[0107] "Carboxyl" refers to the group -C(=O)OH.

[0108] Any of the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl groups may be substituted or unsubstituted, either alone or as part of another group such as alkoxy, alkylthio, aryloxy, etc. If substituted, the substituent may be substituted at any available point of attachment and the substituent may be halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkylsulfo, C1-C6 alkylamino, thiol, hydroxy, nitro, cyano, amino, C3-C6 cycloalkyl, 5-10 membered heterocyclyl, C6 ... 10 It may be one or more, in some cases preferably 1 to 5, and in some cases more preferably 1 to 3 groups independently selected from aryl, 5- to 10-membered heteroaryl, oxo group, and the like.

[0109] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, including cases where the event or circumstance may or may not occur. For example, "a heterocyclic group optionally substituted with alkyl" means that an alkyl group may, but need not, be present, and includes cases where the heterocyclic group is either substituted or unsubstituted with alkyl. The phrases "optionally substituted" and "substituted or unsubstituted" are sometimes used interchangeably.

[0110] "Substituted" refers to one or more, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group being independently replaced with a corresponding number of substituents. Those skilled in the art can determine whether or not substitution is possible without undue effort by experiment or theory. For example, a combination of an amino group or a hydroxyl group having free hydrogen and a carbon atom having an unsaturated bond (e.g., olefinic) may be unstable.

[0111] A "pharmaceutical composition" refers to a mixture of one or more of the compounds described in the present disclosure or their physiologically / pharmaceutical acceptable salts or prodrugs with other chemical components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism and promote absorption of the active ingredient to exert its biological activity.

[0112] "Pharmaceutically acceptable salt" refers to salts of the compounds of the present disclosure, which are safe and effective when used in mammals and have the corresponding biological activity. These salts can be prepared during the final isolation and purification of the compounds, or can be prepared separately by reacting a suitable nitrogen atom with a suitable acid. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, hydrogen disulfide, and organic acids such as paratoluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, parabromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and related inorganic and organic acids.

[0113] Base addition salts can be prepared during the final isolation and purification of the compounds by reacting the carboxyl group with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a metal cation, or ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salt cations include, but are not limited to, lithium, sodium, potassium, calcium, magnesium, aluminum, and non-toxic quaternary amine cations, such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, and the like.

[0114] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a patient, free from excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio, and effective for the intended use.

[0115] The term "therapeutically effective amount" as used herein refers to the total amount of each active ingredient sufficient to show a meaningful benefit to the patient, such as a sustained reduction in viral load. When applied to an individual active ingredient administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0116] The terms "treat," "treating," "treatment," and the like refer to (i) inhibiting a disease, disorder, or condition, i.e., arresting its progression, and (ii) alleviating a disease, disorder, or condition, i.e., causing regression of the disease, disorder, and / or condition. Additionally, the compounds of the disclosure can be used for a prophylactic effect to prevent a disease, disorder, or condition from occurring in a subject who may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed.

[0117] As used herein, the singular forms "a," "an," and "the" include plural references and vice versa unless the context clearly dictates otherwise.

[0118] The term "about" when applied to parameters such as pH, concentration, temperature, etc., indicates that the parameter may vary within ±10%, more preferably ±5%. As will be appreciated by those of skill in the art, when a parameter is not critical, the numerical value is often given for illustrative purposes only, rather than as a limitation.

[0119] Abbreviation Common organic abbreviations used herein are defined as follows: Ac Acetyl CAN Acetonitrile Ala Alanine Asn Asparagine aq. aqueous BOC or Boc tert-butoxycarbonyl BSA Bovine Serum Albumin ℃ Temperature (degrees Celsius) Cit Citrulline DCM Dichloromethane DIEA Diisopropylethylamine DMF N,N'-Dimethylformamide EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Et Ethyl EtOAc Ethyl acetate Eq equivalent Fmoc 9-Fluorenylmethoxycarbonyl g grams GSH Glutathione H Time (hours) HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluranium hexafluorophosphate HOBt N-Hydroxybenzotriazole HPLC High Performance Liquid Chromatography KLH Keyhole limpet hemocyanin LC / MS Liquid Chromatography Mass Spectrometry Lys Lysine Me Methyl Mg Milligrams MeOH Methanol mL Milliliters μL, uL microliter mol mmol μmol, umol micromol MS mass spectrometry NHS N-hydroxysuccinimide OVA egg albumin PAB p-aminobenzyl Pip Piperidine PyBOP Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate RP-HPLC Reversed Phase HPLC RT / rt room temperature t-Bu tert-Butyl Tert, t tertiary TFA Trifluoroacetic acid THF Tetrahydrofuran Val Balin

[0120] Synthesis method Synthesis and Preparation Methods Compounds of formula I and II can be prepared by processes known to those skilled in the art, according to the reactions in Scheme 1 and Scheme 2, and in the Examples section.

[0121] Preparation of SBC framework VI and its disubstituted alternatives IX Synthesis of Core Skeleton IX (Scheme 1) From commercially available starting materials, such as di-tert-butylhydrazine-1,2-dicarboxylate, 3,4-dibromofuran-2,5-dione (IV), and 4,5-dibromo-1,2-dihydropyridazine-3,6-dione (I), the cycloalkyl 4,5-dibromo-1,2-dihydropyridazine-3,6-dione scaffold IX can be prepared in steps 1-3. Treatment of the dibromo precursor (II) bearing a functional group (such as a carboxylate group) with bis-Boc-protected hydrazine produces the Boc-protected hydrazine V. Heating of intermediate V with 3,4-dibromofuran-2,5-dione (IV) or furan-2,5-dione (VII) then gives the cycloalkyl dibromopyridazine dione scaffold VI and pyridazine dione scaffold VIII, respectively. The dibromopyridazine dione scaffold VI can be further reacted with thiols and phenols to produce the derivatives IX. Both compounds VI and IX can be further derivatized (see Figure 2). Alternatively, 4,5-dibromo-1,2-dihydropyridazine-3,6-dione (I) can be directly alkylated with dibromoalkyl ester II to give the dibromopyridazinedione scaffold III as the t-butyl ester. Scheme 1 [ka]

[0122] Functionalization and derivatization of disubstituted cycloalkylpyridazinedione scaffolds IX. (1) Functionalization of the core scaffold IX (Scheme 2) A common method for re-bridging to add functionality to scaffold IX is to couple a spacer linker R1 bearing an amine function using various amide coupling reagents such as DCC, EDCI, HATU, PyBOP, or via an activated HOSu ester. The resulting amide bond shows excellent stability in vivo, and many toxic payloads, fluorescent dyes, and imaging agents are commercially available as amines. The functionalized intermediate X can selectively react with alkylthiols, arylthiols, cysteine, GSH, or thiols derived from reduced disulfide bonds of proteins and antibodies. Sequential introduction of different nucleophiles affords the mono-substituted product XI and the di-substituted product XIII. Furthermore, the functionalized intermediate X is ready to form disulfide bonds to re-bridging thiol groups derived from peptides or reduced antibodies.

[0123] Scheme 2 [ka] EXAMPLES

[0124] The present invention is further illustrated, but not limited, by the following examples which illustrate certain aspects of the invention including the preparation of the compounds.

[0125] Example 1 Synthesis of tri-tert-butyl diazepane-1,2,5-tricarboxylate (E01) [ka] A biphasic reaction mixture of di-tert-butylhydrazine-1,2-dicarboxylate (2.3 g, 1.0 mmol), TEAB (0.1 g, 0.7 mmol) and tert-butyl 4-bromo-2-(2-bromoethyl)butanoate (5.0, 1.5 mmol) was vigorously stirred in 2 / 1 toluene / 50% aqueous sodium hydroxide (15 mL) and heated to 100° C. A thick white solid formed. After 6 h, the reaction was cooled to room temperature, diluted with ethyl acetate (50 mL) and the organic phase was washed with 10% sodium bicarbonate (20 mL), water (20 mL) and brine (20 mL), dried (Na2SO4) and concentrated in vacuo to give 3.4 g of tri-tert-butyldiazepane-1,2,5-tricarboxylate as a white solid (86%). LCMS: 401.3 [M+H + ].

[0126] Example 2 Synthesis of tert-butyl 2-[[tert-butoxycarbonyl-(tert-butoxycarbonylamino)amino]methyl]prop-2-enoate (3) [ka] To a solution of di-tert-butylhydrazine-1,2-dicarboxylate (1) (76.8 mg, 0.33 mmol) and tert-butyl 3-bromo-2-(bromomethyl)propanoate (2) (200 mg, 0.66 mmol) in 3 mL of anhydrous THF was added NaH (60% in oil, 80 mg, 2.0 mmol). The mixture was stirred at room temperature for 15 min and then quenched with a solution of 60 μL of AcOH in 1 mL of water. The mixture was then purified by preparative HPLC. The pure fractions were lyophilized to give 204 mg of the title compound 3 as a white solid. LCMS: 373.6 [M+H + ].

[0127] Example 3 Synthesis of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (E03) [ka] To a solution of tert-butyl 2-[[tert-butoxycarbonyl-(tert-butoxycarbonylamino)amino]methyl]prop-2-enoate (3) (204 mg, 0.55 mmol) in 8.0 mL of glacial acetic acid was added 3,4-dibromofuran-2,5-dione (4) (140 mg, 0.55 mmol). The mixture was stirred at reflux under an argon gas atmosphere for 11 days, concentrated to 3 mL, and purified by preparative HPLC. The pure fractions were lyophilized to give 43 mg of the title compound E03 as a white solid (22%). LCMS: 354.8 [M+H + ].

[0128] Example 4 Synthesis of 5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (E02) [ka] To a solution of tert-butyl 2-[[tert-butoxycarbonyl-(tert-butoxycarbonylamino)amino]methyl]prop-2-enoate (3) (372 mg, 1.0 mmol) in 15 mL of glacial acetic acid was added furan-2,5-dione (5) (98 mg, 1.0 mmol). The mixture was stirred at reflux under an argon gas atmosphere for 7 days, concentrated to 3 mL, and purified by preparative HPLC. The pure fractions were lyophilized to give 136 mg of the title compound E02 as a white solid (69%). LCMS: 197.2 [M+H + ].

[0129] Example 5 Synthesis of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (E03) [ka] 5,8-Dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (186 mg, 0.95 mmol, 1.0 eq) and sodium acetate (171 mg, 2.08 mmol, 2.2 eq) were dissolved in acetic acid (2.7 mL) in a test tube at 0 °C. Bromine (107 μL, 2.08 mmol, 2.2 eq) was added, the test tube was sealed and the mixture was stirred at 135 °C for 4 h. After cooling to 0 °C, water (10 mL) was added and the solution was extracted with ethyl acetate (3 x 15 mL). The organic phases were combined, washed with sodium thiosulfate (2 x 15 mL), dried over magnesium sulfate and concentrated under reduced pressure. Residual acetic acid was coevaporated with toluene under reduced pressure. After purification by flash chromatography (SiO2, cyclohexane / ethyl acetate, 40:60), 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[l,2-a]pyridazine-2-carboxylic acid (273 mg; 78%) was obtained as a white solid. LCMS: 354.8 [M+H + ].

[0130] Example 6 Synthesis of tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E04) [ka] The title compound was prepared by treating 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid with t-butyl acrylate (25 eq) in a stoppered flask containing 3 drops of 60% HClO4 at 25°C for 48 h, followed by careful neutralization (10% NaHCO3) and extraction (dichloromethane, 3 times) followed by drying and evaporation of the organic phase (86%). LCMS: 408.9 [M+H + ].

[0131] Example 7 Synthesis of tert-butyl 6-bromo-5,8-dioxo-7-phenylsulfanyl-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (6) [ka] To a solution of thiophenol (0.05 mL, 0.5 mmol) and triethylamine (0.18 mL, 1.3 mmol) in dichloromethane (6 mL) at 21° C., a solution of tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (185 mg, 0.45 mmol) in dichloromethane (6 mL) was added and the reaction mixture was stirred for 30 min. The reaction mixture was subsequently diluted with dichloromethane (20 mL) and washed with water (3×15 mL) and brine (15 mL). The organic phase was dried over MgSO4, concentrated in vacuo and the crude residue was purified by flash column chromatography (15-85% ethyl acetate / hexanes). The appropriate fractions were combined and concentrated in vacuo to give tert-butyl 6-bromo-5,8-dioxo-7-phenylsulfanyl-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (176 mg, 0.40 mmol, 89%) as a green solid. LRMS: 439.0 [M+H + ].

[0132] Example 8 Synthesis of tert-butyl 5,8-dioxo-6,7-bis(phenylsulfanyl)-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E07) [ka] To a solution of thiophenol (0.10 mL, 0.97 mmol) and triethylamine (0.39 mL, 2.80 mmol) in dichloromethane (6 mL) at 25° C., a solution of tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (127 mg, 0.31 mmol) in dichloromethane (6 mL) was added and the reaction mixture was stirred for 30 min. The reaction mixture was subsequently diluted with dichloromethane (20 mL) and washed with water (3×15 mL) and brine (15 mL). The organic phase was dried over MgSO4, concentrated in vacuo and the crude residue was purified by flash column chromatography (15-80% ethyl acetate / hexanes). The appropriate fractions were combined to give tert-butyl 5,8-dioxo-6,7-bis(phenylsulfanyl)-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (108 mg, 0.23 mmol, 76%) as a yellow solid. LCMS: 469.1 [M+H + ].

[0133] Example 9 tert-Butyl 6,7-bis(2-hydroxyethylsulfanyl)-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E05) [ka] To 2-mercaptoethanol (70 μL, 1 mmol) in buffer (10 ml, 150 mM NaCl, 100 mM sodium phosphate, pH 8.0, 5.0% DMF) was added tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (160 mg, 0.39 mmol) in DMF (0.25 ml). The reaction was stirred at room temperature for 30 min and lithium chloride (2 g) was added. The aqueous reaction mixture was extracted with ethyl acetate (7×15 ml). The organic layers were combined, the solvent removed in vacuo, and the residue purified by flash chromatography on silica gel (hexane:ethyl acetate 1:1 to 1:9). Fractions containing the product were collected and the solvent removed in vacuo to give the title compound as a yellow solid (83 mg, 53%). LCMS: 405.1 [M+H + ].

[0134] Example 10 Synthesis of tert-butyl 6,7-bis[[(2R)-3-methoxy-2-(methylamino)-3-oxopropyl]sulfanyl]-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E09) [ka] To a solution of tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (0.16 g, 0.40 mmol) and N-(tert-butoxycarbonyl)-L-cysteine ​​methyl ester (0.47 g, 2.0 mmol) in dichloromethane (10 mL) was added triethylamine (0.07 mL, 0.5 mmol) and the reaction mixture was stirred at 25 °C for 65 h. The reaction mixture was subsequently diluted with dichloromethane (20 mL) and washed with water (3x 20 mL) and brine (15 mL). The organic phase was dried over MgSO4, concentrated in vacuo and the crude residue was purified by flash column chromatography (15-60% ethyl acetate / hexanes). The appropriate fractions were combined and concentrated in vacuo to give a yellow oil (0.13 g, 0.24 mmol, 59%). LCMS: 547.2 [M+H + ].

[0135] Example 11 Synthesis of tert-butyl 6-bromo-7-[(2R)-3-methoxy-2-(methylamino)-3-oxopropyl]sulfanyl-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (7) [ka] To a solution of tert-butyl 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (0.25 g, 0.61 mmol) in dichloromethane (6 mL) at 25° C. was added a solution of N-(tertbutoxycarbonyl)-L-cysteine ​​methyl ester (0.14 g, 0.59 mmol) and triethylamine (0.13 mL, 0.93 mmol) in dichloromethane (6 mL) dropwise over 30 min. The reaction mixture was then diluted with dichloromethane (12 mL) and washed with water (3×15 mL) and brine (15 mL). The organic phase was dried over MgSO4, concentrated in vacuo, and the crude residue was purified by flash column chromatography (0-30% ethyl acetate / hexanes). The appropriate fractions were combined and concentrated in vacuo to give the title compound (0.17 g, 0.35 mmol, 60%) as a yellow oil. LCMS: 478.1 [M+H + ].

[0136] Example 12 Synthesis of tert-butyl 7-[(2R)-3-methoxy-2-(methylamino)-3-oxopropyl]sulfanyl-5,8-dioxo-6-phenylsulfanyl-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E08) [ka] To a solution of tert-butyl 6-bromo-7-[(2R)-3-methoxy-2-(methylamino)-3-oxopropyl]sulfanyl-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (0.45 g, 0.94 mmol) in dichloromethane (8 mL) at 25° C. was added a solution of thiophenol (0.10 mL, 0.98 mmol) and triethylamine (0.20 mL, 1.4 mmol) in CHCl (8 mL) and the reaction was stirred for 30 min. The reaction mixture was subsequently diluted with dichloromethane (16 mL) and washed with water (3×15 mL) and brine (15 mL). The organic phase was dried over MgSO, concentrated in vacuo and the crude residue was purified by flash column chromatography (0-40% ethyl acetate / hexanes). The appropriate fractions were combined and concentrated in vacuo to give the title compound (0.24 g, 0.47 mmol, 50%) as a yellow oil. LCMS: 508.2 [M+H + ].

[0137] Example 13 Synthesis of Ethyl 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (8) [ka] Under an inert atmosphere, 6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (212 mg, 0.6 mmol) was dissolved in dichloromethane (10 mL). Azabenzotriazole tetramethyluranium hexafluorophosphate (HATU; 300 mg, 0.8 mmol) and 2,6-lutidine (167 μL, 1.4 mmol) were then added and the mixture was stirred at room temperature for 10 min. Ethyl 4-aminobutanoate (105 mg, 0.8 mmol) was added and the resulting solution was stirred at room temperature for 18 h, then diluted with dimethylsulfoxide and purified by HPLC to give ethyl 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (227 mg, 81%) as a yellow solid. LCMS: 466.0 [M+H + ].

[0138] Example 14 Synthesis of 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoic acid (E06) [ka] To a solution of ethyl 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (233 mg, 0.5 mmol, 1 eq) in THF (5 mL) was added LiOH.H2O (42 mg, 1 mmol, 2 eq) in water (0.5 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (ACN / H2O condition) to give 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoic acid (176 mg, 80% yield) as a pale yellow solid. LCMS: 437.9 (M+H + ).

[0139] Example 15 Synthesis of (2,5-dioxopyrrolidin-1-yl) 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (E10) [ka] To a solution of 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoic acid (307 mg, 0.7 mmol) in THF (10 mL) cooled to 0° C. was added N,N′-dicyclohexylcarbodiimide (160 mg, 0.8 mmol). The homogeneous solution was stirred at 0° C. for 30 min. After this time, N-hydroxysuccinimide (89.0 mg, 0.8 mmol) was added and the reaction was stirred at 25° C. for an additional 16 h. The newly formed heterogeneous mixture was then filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (20% to 100% ethyl acetate / hexanes) to give (2,5-dioxopyrrolidin-1-yl) 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (268 mg, 72%) as a yellow solid. LCMS: 535 [M+H + ].

[0140] Example 16 Synthesis of 6,7-dibromo-5,8-dioxo-N-[4-oxo-4-[(2-propylthiazolo[4,5-c]quinolin-4-yl)amino]butyl]-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide (E11, SBC linker-CL-075 payload) [ka] To a solution of (2,5-dioxopyrrolidin-1-yl) 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (118 mg, 0.22 mmol) in THF (10 mL) was added 2-propylthiazolo[4,5-c]quinolin-4-amine (innate immune modulator CL-075, 58 mg, 0.24 mmol) and the reaction mixture was stirred at 25° C. for 16 h. After this time, the reaction was concentrated in vacuo and the crude residue was dissolved in dichloromethane (50 mL) and washed with water (2×30 mL) and saturated aqueous K2CO3 (30 mL). The organic layer was then dried (MgSO4) and concentrated in vacuo. The crude residue was purified by flash column chromatography (0% to 10% MeOH / ethyl acetate) to give the title amide product (102 mg, 70%) as a pale yellow solid. LCMS: 663.0 [M+H + ].

[0141] Example 17 Antibody / SBC linker / CL-075 conjugate (E13) [ka] The purified antibody was buffer exchanged into PBS, pH 7.4. Five eq of tris(2-carboxyethyl)phosphine (TCEP·HCl, 50 mM in deionized water) was freshly prepared and added to a solution of human antibody IgG1 kappa (in-house, 5 mg / mL, 1 eq) in reduction / conjugation buffer (25 mM sodium borate, 25 mM NaCl, 1 mM diethylenetriamine pentaacetate (DTPA) pH 8.0), and the solution was incubated at 37°C for 2 h and cooled to room temperature. A freshly prepared stock solution of 6,7-dibromo-5,8-dioxo-N-[4-oxo-4-[(2-propylthiazolo[4,5-c]quinolin-4-yl)amino]butyl]-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide (E11, 2 mM in DMSO, 10 eq) was added and the solution was incubated overnight at 4 °C. Excess reagent was removed by ultrafiltration (6X, 10000 MWCO) into PBS (pH = 7.4). The conjugate was characterized by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (see Figure 2). A homogeneity of 85% of the DAR4 ratio was demonstrated.

[0142] Example 18 Synthesis of SBC linker-MMAF(E12) [ka] To a solution of (2,5-dioxopyrrolidin-1-yl) 4-[(6,7-dibromo-5,8-dioxo-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-2-carbonyl)amino]butanoate (118 mg, 0.22 mmol) in THF (10 mL) was added the cytotoxic agent monomethylauristatin F (MMAF) (161 mg, 0.22 mmol) and the reaction mixture was stirred at 25° C. for 16 h. After this time, the reaction was concentrated in vacuo and the crude residue was dissolved in dichloromethane (50 mL) and washed with 10% aqueous citric acid (30 mL) and water (2×30 mL). The organic layer was then dried (MgSO4) and concentrated in vacuo. The crude residue was purified by preparative HPLC to afford the SBC linker-MMAF payload (E12) as a white solid (165 mg, 65%). LCMS: 1151.4 (M+H + ).

[0143] Example 19 Antibody / SBC linker / MMAF conjugate (E14) [ka] The purified antibody was buffer exchanged into PBS, pH 7.4. 5 eq of TCEP·HCl (50 mM in deionized water) was freshly prepared and added to a solution of human antibody IgG1 kappa (in-house, 5 mg / mL, 1 eq) in reduction / conjugation buffer (25 mM sodium borate, 25 mM NaCl, 1 mM diethylenetriamine pentaacetate (DTPA) pH 8.0), the solution was incubated for 2 h at 37 °C and cooled to room temperature. A freshly prepared stock solution of SBC linker-MMAF (E12, 2 mM in DMSO, 6 eq) was added and the solution was incubated overnight at 4 °C. Excess reagents were removed by ultrafiltration (6X, 10000 MWCO) into PBS (pH = 7.4). The HIC-HPLC profile showed that more than 90% of the conjugated payload was DAR4 (see Figure 3).

[0144] Example 20 Synthesis of 3-(4,5-dibromo-2-methyl-3,6-dioxopyridazin-1-yl)-N-(2-propylthiazolo[4,5-c]quinolin-4-yl)propanamide (10, diBrPD-CL075 payload) [ka] 2,5-Dioxopyrrolidin-1-yl 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoate (9, diBrPD-OSu) was prepared according to the procedure reported in Org. Biomol. Chem., 2018, 16, 1359, Supporting Information. To a solution of 2,5-dioxopyrrolidin-1-yl 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoate (diBrPD-OSu, 100 mg, 0.22 mmol) in THF (10 mL) was added 2-propylthiazolo[4,5-c]quinolin-4-amine (CL-075, 58 mg, 0.24 mmol) and the reaction mixture was stirred at room temperature for 16 h. After this time, the reaction was concentrated in vacuo and the crude residue was dissolved in dichloromethane (50 mL) and washed with water (2x30 mL) and saturated aqueous K2CO3 (30 mL). The organic layer was then dried (MgSO4) and concentrated in vacuo. The crude residue was purified by flash column chromatography (0%-10% MeOH / ethyl acetate) to give the title amide compound as a pale yellow solid (84 mg, 66%). LCMS: 580.1 [M+H + ].

[0145] Example 21 Antibody / diBrPD linker / CL075 conjugate (11) [ka] The purified antibody was buffer exchanged into PBS, pH 7.4. 6-10 eq of TCEP·HCl (50 mM in deionized water) was freshly prepared and added to a solution of human antibody IgG1 kappa (in-house, 5 mg / mL, 1 eq) in reduction / conjugation buffer (25 mM sodium borate, 25 mM NaCl, 1 mM diethylenetriamine pentaacetate (DTPA) pH 8.0), the solution was incubated for 2 h at 37 °C and cooled to room temperature. A freshly prepared stock solution of 3-(4,5-dibromo-2-methyl-3,6-dioxopyridazin-1-yl)-N-(2-propylthiazolo[4,5-c]quinolin-4-yl)propanamide (diBrPD-CL075 payload, 2 mM in DMSO, 4-20 eq) was added, and the solution was incubated overnight at 4 °C. Excess reagent was removed by ultrafiltration (6X, 10000 MWCO) into PBS (pH=7.4).

[0146] Table 1. Comparison of uniform DAR4 ratios obtained by conjugating a human IgG1 antibody with SBC linker-CL075 or diBrPD linker-CL075 under mild conditions. Due to the conjugation conditions, antibody-SBC-CL075 used less linker-payload reagent and achieved a higher uniform DAR4 ratio than antibody-diBrPD-CL075. [Table 1] HIC-HPLC diagrams demonstrated that when the diBrPD linker was used, the human IgG1-CL075 conjugate only achieved a homogeneity rate of 55.97% for DAR4, 19.68% for DAR3, 11.2% for DAR5, 10.05% for DAR2, and 3.07% for DAR1 (see FIG. 4). In contrast, the human IgG1 antibody with the SBC linker of FIG. 2 in Example 17 achieved a homogeneity rate of 85% (see FIG. 2).

[0147] Example 22 Antibody / diBrPD linker / MMAF conjugate (13) [ka] The purified antibody was buffer exchanged into PBS, pH 7.4. 6–10 eq of TCEP·HCl (50 mM in deionized water) was freshly prepared and added to a solution of human antibody IgG1 kappa (5 mg / mL, 1 eq) in reduction / conjugation buffer (25 mM sodium borate, 25 mM NaCl, 1 mM diethylenetriamine pentaacetate (DTPA) pH 8.0), the solution was incubated for 2 h at 37 °C and cooled to room temperature. A freshly prepared stock solution of diBrPD-MMAF payload (12, 2 mM in DMSO, 6–8 eq) was added, and the solution was incubated overnight at 4 °C. Excess reagent was removed into PBS (pH = 7.4) by ultrafiltration (6X, 10000 MWCO).

[0148] The HIC-HPLC diagram showed that the antibody-diBrPD-MMAF evenly distributed DAR3 and DAR4, and also added DAR5 (see FIG. 5). However, in contrast, the antibody with SBC-MMAF in FIG. 3 of Example 19 obtained a homogeneity rate of 90% DAR4.

[0149] Example 23 Synthesis of 2,5-dioxopyrrolidin-1-yl 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylate (E15) [ka] To a stirred solution of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (350 mg) in anhydrous dichloromethane (10 mL) was added N-hydroxysuccinimide (230 mg) followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (400 mg). The mixture was stirred at room temperature for 30 min and the reaction was concentrated to dryness under reduced pressure. The residue was directly purified by RP-HPLC to give the title compound as a white solid (337 mg) after lyophilization. MS detection: 452.0 [M+H + ].

[0150] Example 24 Synthesis of 4-((S)-2-((S)-2-(3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-propylthiazolo[4,5-c]quinolin-4-yl)carbamate (E16) [ka] To a solution of compound 14 (40 mg) in anhydrous DMF (1 mL) was added CL075 (10 mg), followed by DIEA (10 mL) and HOBt (2 mg). The reaction mixture was stirred at room temperature (22° C.). After 48 h, piperidine (50 mL) was added and the reaction was stirred at room temperature for 1 h. The mixture was directly purified by RP-HPLC to give compound 15 as a white solid (18 mg) after lyophilization. To a solution of compound 15 (15 mg) in DMF (1 mL) was added diBrPD-OSu(9) (10 mg) followed by DIEA (8 mL). The reaction mixture was stirred at room temperature. After 3 h, the crude mixture was purified by RP-HPLC to give compound E16 as a white solid (12 mg) after lyophilization. MS detection: 987.5 [M+H + ]

[0151] Example 25 Synthesis of 4-((S)-2-((S)-2-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-propylthiazolo[4,5-c]quinolin-4-yl)carbamate (17) [ka] To a solution of compound 15 (15 mg) in DMF (1 mL) was added E15 (10 mg), followed by DIEA (8 mL). The reaction mixture was stirred at room temperature. After 3 h, the crude mixture was purified by RP-HPLC to give compound E17 as a white solid (13 mg) after lyophilization. MS detection: 985.2 [M+H+ ].

[0152] Example 26 Synthesis of (14S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxiran-8(1,3)-benzenecyclotetradecaphane-10,12-dien-4-yl N-(6-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide)hexanoyl)-N-methyl-L-alaninate (E18) [ka] To a solution of compound 16 (65 mg) in anhydrous DMF (2 mL) was added Fmoc-aminohexanoic acid (38 mg), followed by DIEA (40 mL) and HATU (42 mg). The reaction mixture was stirred at room temperature (22° C.). After 10 min, piperidine (100 mL) was added and the reaction was stirred at room temperature for 30 min. The mixture was directly purified by RP-HPLC to give compound 17 as a white solid (72 mg, TFA salt) after lyophilization. To a solution of compound 17 (17 mg) in DMF (1 mL) was added E15 (10 mg), followed by DIEA (7 mL). The reaction mixture was stirred at room temperature. After 15 min, the crude mixture was purified by RP-HPLC to give compound E18 as a white solid (16 mg) after lyophilization. MS detection: 1099.4 [M+H + ].

[0153] Example 27 4-((S)-2-((S)-2-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide)propanamide)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydro Synthesis of (methoxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (E19) [ka] To a solution of compound 19 (35 mg) in anhydrous DMF (1 mL) was added monomethylauristatin E (35 mg), followed by DIEA (10 mL) and HOBt (2 mg). The reaction mixture was stirred at room temperature (22° C.). After 24 h, piperidine (100 mL) was added and the reaction was stirred at room temperature for 15 min. The mixture was directly purified by RP-HPLC to give compound 20 as a white solid (43 mg, TFA salt) after lyophilization. To a solution of compound 20 (11 mg) in DMF (1 mL) was added E15 (6 mg), followed by DIEA (5 mL). The reaction mixture was stirred at room temperature. After 1 h, the crude mixture was purified by RP-HPLC to give compound E19 as a white solid (10 mg) after lyophilization. MS detection: 1345.6 [M+H + ].

[0154] Example 28 4-((S)-2-((S)-2-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide)-3-methylbutanamide)propanamide)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy Synthesis of cis-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (E20) [ka] To a solution of compound 21 (39 mg) in anhydrous DMF (1 mL) was added monomethyl dolastatin 10 (38 mg), followed by DIEA (10 mL) and HOBt (2 mg). The reaction mixture was stirred at room temperature (22 °C). After 24 h, piperidine (100 mL) was added and the reaction was stirred at room temperature for 15 min. The mixture was directly purified by RP-HPLC to give compound 22 as a white solid (49 mg, TFA salt) after lyophilization. To a solution of compound 22 (12 mg) in DMF (1 mL) was added E15 (6 mg), followed by DIEA (5 mL). The reaction mixture was stirred at room temperature. After 3 h, the crude mixture was purified by RP-HPLC to give compound E20 as a white solid (11 mg) after lyophilization. MS detection: 1426.6 [M+H + ].

[0155] Example 29 Synthesis of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(((4-((S)-2-((S)-2-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (E21) [ka] To a solution of compound 23 (43 mg) in anhydrous DMF (2 mL) was added monomethylauristatin F (TFA salt, 41 mg), followed by DIEA (15 mL) and HOBt (2 mg). The reaction mixture was stirred at room temperature (22° C.). After 24 h, piperidine (100 mL) was added and the reaction was stirred at room temperature for 15 min. The mixture was directly purified by RP-HPLC to give compound 24 as a white solid (47 mg, TFA salt) after lyophilization. To a solution of compound 24 (12 mg) in DMF (1 mL) was added E15 (6 mg), followed by DIEA (5 mL). The reaction mixture was stirred at room temperature. After 3 h, the crude mixture was purified by RP-HPLC to give compound E21 as a white solid (10 mg) after lyophilization. MS found: 1473.8 [M+H + ].

[0156] Example 30 Synthesis of 6,7-dibromo-N-(2-((2-(((S)-1-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamide (E22) [ka] To a suspension of exatecan mesylate (53 mg) in anhydrous DMF (2 mL) was added Fmoc-Gly-Gly-Phe-Gly-OH (compound 25, 62 mg), followed by DIEA (54 mL) and PyAOP (60 mg). The reaction mixture was stirred at room temperature (22 °C). After 10 min, piperidine (100 mL) was added and the reaction was stirred at room temperature for 30 min. The mixture was directly purified by RP-HPLC to give compound 26 as a yellow solid (62 mg, TFA salt) after lyophilization. To a solution of compound 26 (17 mg) in DMF (1 mL) was added E15 (10 mg), followed by DIEA (7 mL). The reaction mixture was stirred at room temperature. After 10 min, the crude mixture was purified by RP-HPLC to give compound E22 as a yellow solid (14 mg) after lyophilization. MS detection: 1090.3 [M+H + ].

[0157] Example 31 Synthesis of 4-((S)-2-(6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxamido)propanamido)benzyl((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (E23) [ka] To a solution of 10-TBDMS-SN38 (compound 27, 50 mg) in anhydrous DCM (2 mL) was added triphosgene (15 mg) followed by DMAP (60 mg). The mixture was stirred at room temperature for 5 min and then Fmoc-Ala-PAB-OH (42 mg) was added. The reaction was maintained at room temperature for 30 min. The crude reaction was diluted with DCM (30 mL) and washed with water (20 mL). The organic layer was concentrated to dryness and the residue (compound 28) was redissolved in DMF (2 mL). Piperidine (100 mL) was added and after 15 min TBAF (1 M solution in THF, 0.2 mL) was added. The reaction was stirred at room temperature for 15 min and the crude product was directly purified by RP-HPLC to give compound 29 as a yellow solid (34 mg) after lyophilization. To a solution of compound 29 (14 mg) in DMF (1 mL) was added E15 (10 mg), followed by DIEA (7 mL). The reaction mixture was stirred at room temperature. After 30 min, the crude mixture was purified by RP-HPLC to give compound E23 as a yellow solid (12 mg) after lyophilization. MS detection: 949.2 [M+H + ].

[0158] Example 32 Synthesis of 6-monobromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (E24) [ka] 6,7-Dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (0.50 g, 1.4 mmol) was dissolved in methanol (5 mL) and treated with ammonium chloride (0.21 g, 2.9 eq) followed by Zn powder (0.27 g, 3.0 eq) with stirring. The mixture was heated at 40° C. for 4.5 h, the reaction was quenched with aqueous NH4Cl and extracted with dichloromethane (15 mL×3). The combined extracts were washed with brine, dried over Na2SO4, filtered and evaporated to dryness. The residue was purified on a SiO2 pad to give 0.23 g (80% purity by HPLC) of 6-bromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid as an off-white solid (60% yield). This product was used without further purification. MS (M+H): 275.0, 277.1.

[0159] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be within the spirit and scope of this application and the appended claims of the invention. All patent or non-patent references mentioned herein are incorporated by reference in their entirety without any admission as prior art.

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, Ring A is a 5- to 13-membered heterocycle; m and n are each independently 1, 2, 3, 4, or 5; 【Chemistry 2】 represents a double bond or a single bond; X and X' are each independently O, S, or NR X and R X is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; Y is a bond, (CH 2 ) i (i = integer from 1 to 12), C(O), C(O)O, OC(O), C(O)NR a , N.R. a (CO), NR a , O, S, S(O), S(O) 2 , substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5- to 12-membered heteroarylene, or a combination thereof; R 2a and R 3a are each independently hydrogen, halogen, or -UR 2 or -VR 3 and U and V are independently a bond, S, S(O), S(O) 2 , O, NH or CH 2 and L is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted cyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, O, S, NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O) 2 , (CH 2 CH 2 O) j , (OCH 2 CH 2 ) j (PEGn, j = 2 to 48), SS, hydrazone, substituted or unsubstituted oligopeptides (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein R a is hydrogen or C 1 ~C 6 alkyl, and the linker L optionally comprises a non-self-immolative spacer; R 1 is a functional moiety selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted succinimidyl, or a leaving group (e.g., BSA, KLH, OVA), a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier (e.g., BSA, KLH, OVA), a radionuclide, a photosensitizer, a cytotoxin and its prodrug, an innate immune modulator, a biopolymer, an oligonucleotide, a PROTAC degrader, an antibiotic, and an exotoxin; R 2 and R 3 are each independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, an antibody, an antigen, a liposome, a polymer moiety, a substituted or unsubstituted amino acid moiety, a substituted or unsubstituted peptide moiety, DNA, RNA, a virus or virus-like particle, and -SH, -OH, -NH 2 , guanidinyl, imidazolyl, indolyl, and carboxylic acids (-CO 2 H), a ligand-targeting small molecule having a nucleophilic group or moiety; Or, R 2 and R 3 Let's get together and 4 and U, V and 【Transformation 3】 together to form ring B characterized in formula (II) 【Chemistry 4】 (where R 4 is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each of which is optionally substituted, or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand-targeting small molecule, wherein the ligand-targeting small molecule is selected from -SH, -OH, -NH 2 , optionally containing a nucleophilic group selected from guanidinyl, imidazolyl, indolyl and carboxylic acid, or a combination thereof) or a salt thereof.

2. Ring A is a 5- to 9-membered heterocycle; 2. The compound or salt thereof according to claim 1, wherein m and n are each independently 1, 2, or 3.

3. Ring A is a 5- to 7-membered heterocycle; 2. The compound or salt thereof according to claim 1, wherein m and n are each independently 1 or 2.

4. 2. The compound or salt thereof according to claim 1, wherein m=1 and n=1. 【Request Item 5】 【Chemistry 5】 The compound or salt thereof according to claim 1 , wherein X is a double bond, and X and X′ are each O.

6. U and V are bonds, and R 2 and R 3 and each represents a halogen atom, or a salt thereof.

7. R 2 and R 3 and each represents bromine (Br), or a salt thereof.

8. U and V are sulfur (S); R 2 and R 3 and R are each independently an alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid moiety, each optionally substituted, or a salt thereof.

9. U and V are sulfur (S); R 2 and R 3 are each independently or together selected from an antibody, an antigen, a liposome, a polymer moiety, an amino acid, an oligopeptide, DNA, RNA, a virus or virus-like particle, and a ligand-targeting small molecule, or a salt thereof.

10. Y is C(O)O, C(O)NH, CH 2 , O, S, or NH; L is a bond, alkylene, or -(CH 2 ) k C(O)NH—, where k is an integer selected from 1 to 8; R 1 2. The compound of claim 1, or a salt thereof, wherein is hydrogen, alkyl, cycloalkyl, aryl, or succinimidyl.

11. Y is C(O)NH; L is a bond, -(CH 2 ) k C(O)NH-, or an oligopeptide moiety, or a combination thereof, wherein k is an integer selected from 1 to 8; R 1 is selected from hydrogen, alkyl, cycloalkyl, aryl, a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, a radionuclide, a photosensitizer, a cytotoxin and its prodrugs, an innate immune modulator, a biopolymer, an oligonucleotide, a PROTAC degrader, an antibiotic, and an exotoxin, or a salt thereof.

12. 12. The compound or salt thereof according to claim 11, wherein the oligopeptide moiety is selected from Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, and Val-Arg.

13. 2. The compound or salt thereof of claim 1, wherein the self-immolative spacer comprises a para-aminobenzyloxycarbonyl (PABC) moiety or a PABC-type moiety (e.g., ortho-aminobenzyl, ortho-hydroxybenzyl, and para-hydroxybenzyl) capable of undergoing electron cascade-mediated or cyclization-mediated self-immolation.

14. Formula (II): 【Transformation 6】 (In the formula, Ring A is independently a 5- to 13-membered carbocyclic ring; m and n are each independently 1, 2, 3, 4, or 5; Ring B contains two non-nucleophilic groups (e.g., thiol groups from disulfide bridges in a peptide, protein, or antibody); X and X' are each independently O, S, or NR x and Y is a bond, (CH 2 ) i (i = integer from 1 to 12), C(O), C(O)O, NR a ,O,S,S(O),S(O) 2 , substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5- to 12-membered heteroarylene, or a combination thereof; L is a bond, alkylene, O, S, or NR a , C(O), C(O)O, OC(O), C(O)NH, NHC(O), S(O), S(O) 2 , (CH 2 CH 2 O) j , (OCH 2 CH 2 ) j (PEGn, j = 2 to 48), SS, hydrazone, oligopeptide, (e.g., Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, or Val-Arg), and combinations thereof, wherein the linker optionally includes a self-immolative spacer; R 1 is hydrogen, alkyl, cycloalkyl, aryl, succinimidyl, or a functional moiety selected from leaving groups, detectable moieties, enzymatically active moieties, affinity tags, haptens, immunogenic carriers, radionuclides, photosensitizers, cytotoxins and their prodrugs, innate immune modulators, biopolymers, oligonucleotides, PROTAC degraders, antibiotics, and exotoxins; R x is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; R 4 is selected from alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclylene, and combinations thereof, each of which is optionally substituted, or a moiety of an antibody, antigen, liposome, polymer, amino acid, peptide, DNA, RNA, virus, virus-like particle, or ligand-targeting small molecule, wherein the ligand-targeting small molecule is selected from -SH, -OH, -NH 2 , optionally containing a nucleophilic group selected from guanidine, imidazole, indole, carboxylic acid, or a combination thereof; R a is hydrogen or C 1 -C 6 or a pharmaceutically acceptable salt thereof.

15. Ring A is a 5- to 9-membered heterocycle; 15. The compound or salt thereof according to claim 14, wherein m and n are each independently 1, 2, or 3.

16. Ring A is a 5- to 7-membered heterocycle; 15. The compound or salt thereof according to claim 14, wherein m and n are each independently 1 or 2.

17. 15. The compound or salt thereof according to claim 14, wherein m=1 and n=1. 【Request Item 18】 【Chemistry 7】 is a double bond; 15. The compound or salt thereof according to claim 14, wherein X and X' are each O.

19. U and V are bonds, and R 2 and R 3 and each represents a halogen atom, or a salt thereof.

20. R 2 and R 3 and each represents bromine (Br), or a salt thereof.

21. U and V are sulfur (S); R 2 and R 3 and R are each independently an alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid moiety, each optionally substituted, or a salt thereof.

22. U and V are sulfur (S); R 2 and R 3 and each independently or together are selected from an antibody, an antigen, a liposome, a polymer moiety, an amino acid, an oligopeptide, DNA, RNA, a virus or virus-like particle, and a ligand-targeting small molecule, or a salt thereof.

23. Y is C(O)O, C(O)NH, CH 2 , O, S, or NH; L is a bond, alkylene, -(CH 2 ) k C(O)NH—, where k is an integer selected from 1 to 8; R 1 15. The compound of claim 14, or a salt thereof, wherein is hydrogen, alkyl, cycloalkyl, aryl, or succinimidyl.

24. Y is C(O)NH; L is a bond, -(CH 2 ) k C(O)NH-, where k is an integer selected from 1 to 8, or an oligopeptide moiety, or a combination thereof; R 1 is selected from hydrogen, alkyl, cycloalkyl, aryl, a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, a radionuclide, a photosensitizer, a cytotoxin and its prodrugs, an innate immune modulator, a biopolymer, an oligonucleotide, a PROTAC degrader, an antibiotic, and an exotoxin.

25. 25. The compound or salt thereof according to claim 24, wherein the oligopeptide moiety is selected from Val-Cit, Gly-Gly-Phe-Gly, Val-Ala, Ala-Ala, Ala-Ala-Asn, Phe-Lys, Val-Lys, and Val-Arg.

26. 15. The compound of claim 14, wherein the self-immolative spacer comprises a para-aminobenzyloxycarbonyl (PABC) moiety or a PABC-type moiety (e.g., ortho-aminobenzyl, ortho-hydroxybenzyl, and para-hydroxybenzyl) that can undergo electron cascade-mediated or cyclization-mediated self-immolation.

27. A compound selected from compounds E1 to E24, or a salt or stereoisomer thereof.

28. A pharmaceutical composition comprising a compound according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition for treating a disease or disorder, comprising a compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof.

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