WO2023074936A1 - Bifunctional compound using ubr box domain-binding ligand - Google Patents

Abstract

The present disclosure relates to a bifunctional compound using a UBR box domain-binding ligand. The UBR box domain is a domain commonly present in the UBR (Ubiquitin protein ligase E3 component n-recognin) protein of the N-end rule pathway. In this regard, the UBR box domain is known as a domain to which a substrate binds. The UBR box domain binds to the N-terminal residue of a substrate, playing an essential role in forming multi-ubiquitin chains in the substrate, and it is known that the substrate is degraded through this process. Employing the UBR box domain ligand that takes advantage of the UBR box domain function, the bifunctional compound can be used to induce a desired target protein (or peptide) to be more effectively degraded.

Description

Bifunctional compounds using UBR box domain binding ligands

The disclosure herein relates to bifunctional compounds using UBR box domain binding ligands. The UBR box domain is a domain commonly present in the UBR (Ubiquitin protein ligase E3 component n-recognin) protein of the N-end rule pathway. At this time, the UBR box domain is known as a substrate binding domain. It is known that the UBR box domain binds to the N-terminal residue of the substrate and is essential for forming multi-ubiquitin chains on the substrate, and the substrate is degraded through this process. A desired target protein (or peptide) can be induced to be degraded more effectively by using a bifunctional compound using a UBR box domain ligand that uses the function of the UBR box domain.

Cells regulate the amount and function of proteins in vivo through proteolysis. At this time, proteins in vivo can be degraded dependently on the N-terminal residue sequence, and this degradation pathway is known as the N-end rule pathway. That is, the N-terminal pathway rule is a protein degradation system that uses the N-terminus of a specific protein as a degradation signal. The N-terminal pathway rule may include the following protein degradation process.

In the case of eukaryotes, N-recognin recognizes the N-terminal degradation signal of a protein, and the N-recognin can degrade a protein by binding ubiquitin to a protein to be degraded. At this time, the N-terminal degradation signal has a positively charged residue (type 1; eg, arginine, lysine, histidine) or a large hydrophobic residue (type 2; phenylalanine, leucine, tryptophan, isoleucine, tyrosine) at the N-terminus. may include The present inventors first discovered or cloned N-lecognines, UBR1, UBR2, UBR3, and UBR5, and found that they had a UBR box domain as a substrate recognition domain (Tasaki et al. 2005). At this time, the ubiquitinated substrate produced by the binding of N-lecognin to the N-end rule ligand is delivered to the proteasome and degraded into short peptides. In this process, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Leu) are N-lecognin at the N-end It is an essential determinant for binding because it provides most of the hydrogen bonds necessary for targeting rule substrates (Sriram and Kwon, 2010).

The UBR is an abbreviation of Ubiquitin protein ligase E3 component n-recognin, and UBR is N-recognin that recognizes the N-terminal degradation signal of a protein. It is known that at least 7 types of UBRs 1 to 7 exist in mammals. In addition, the UBR box domain common to UBR is a zinc finger motif having a size of about 70 residues, and is known as a highly conserved substrate-binding domain. [Kwon et al., 1998; Xie and Varshavsky, 1999; Kwak et al., 2004; Varshavsky, 1996; Varshavsky, 1997; Kwon et al., 2011; and Zenker et al., 2014].

That is, UBR is N-lecognin associated with the N-terminal pathway law, which is a protein degradation pathway, and the UBR box domain in UBR is a substrate binding domain. In particular, among the UBRs 1 to 7, UBR1, UBR2, UBR3 and UBR5 act as ubiquitin protein ligase E3 and are known to have a RING domain or a HECT domain. Substrates of the N-terminal law binding to the UBR are degraded by the ubiquitin proteasome pathway. Specifically, the UBR box domain in the UBR recognizes the N-terminal amino acid of the substrate and ubiquitinates the substrate through the RING domain or the HECT domain, thereby degrading the substrate through the proteasome pathway. For example, misfolded proteins in cells are degraded through the ubiquitin proteasome pathway, as they can aggregate and block the proteasome or impair other cellular functions if left unattended for long periods of time. (Ji and Kwon, 2017).

That is, the UBR box domain plays an important role in the intracellular protein degradation pathway by recognizing the N-terminal degradation signal. Thus, ligands that bind to the UBR box domain can affect proteolytic pathways in cells. That is, the protein to be degraded (ie, the desired target protein (or peptide)) can be more effectively degraded by using a ligand that binds to the UBR box domain.

More specifically, the protein to be degraded (i.e., the desired target protein (or peptide)) is more specifically synthesized using a bifunctional compound comprising a ligand that binds to the UBR box domain and a ligand that binds to the target protein (or peptide). can be effectively decomposed. These bifunctional compounds position the protein of interest for degradation in proximity to the UBR, particularly the UBR box domain. At this time, the protein to be degraded located close to the UBR box domain is ubiquitinated by the UBR box domain and degraded through the proteasome pathway.

As described above, the present specification relates to a bifunctional compound comprising a ligand that binds to a UBR box domain associated with an intracellular protein degradation pathway and a ligand that binds to a target protein (or peptide).

One object of the present invention is to provide a bifunctional compound.

Another object of the present invention is to provide a composition for protein degradation comprising a bifunctional compound and a use thereof.

Another object of the present invention is to provide a pharmaceutical composition for treating a disease comprising a bifunctional compound and a use thereof.

The present application provides a bifunctional compound or a salt thereof. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL).

In one embodiment, the bifunctional compound may be a compound having a UBL (UBR box domain binding ligand)-TBL (target protein (or peptide) binding ligand) structure or a salt thereof.

The TBL (target protein (or peptide) binding ligand) may be a compound that binds to a target protein (or peptide) to be degraded. In this case, the TBL may be a known compound.

The UBL (UBR box domain binding ligand) may be a compound having a structure of Formula 1 or a salt thereof.

[Formula 1]

wherein X ₁ is phenyl, cycloalkyl or heterocyclyl optionally substituted or unsubstituted with one or more R ₂ ;

each R ₂ is independently alkyl, alkoxy, amino, aminoalkyl, -NO ₂ , =O, -NHC ₂ H ₄ OH, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)OH, phenyl or heterocycloalkyl is selected from;

X ₄ is phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R ₃ ;

each R ₃ is independently alkyl, alkoxy, amino, halo, hydroxyl, alkylamino, dialkylamino, -NO ₂ , -CONR'R'', -CO ₂ R', -NHCOR', phenyl or heterocyclo is selected from alkyl;

each R' and R'' is independently -H or alkyl;

X ₂ is SO ₂ , or CR _a R _b ;

R _a and R _b are each independently H or CH ₃ ;

X ₃ is NH or CH ₂ ;

B ₁ is CH ₂ or NH;

A ₁ is CH ₂ or NH.

At this time, the broken line (dotted line) represents a possible attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL).

At this time, in one embodiment, in Formula 1,

-X ₂ -B ₁ -X ₃ is selected from the group consisting of -SO ₂ -NH-NH, -SO ₂ -NH-CH ₂ , -SO ₂ -CH ₂ -NH and -CH ₂ -NH-NH;

X ₁ is phenyl, cycloalkyl or heterocyclyl optionally substituted or unsubstituted with one or more R ₂ ;

each R ₂ is independently alkyl, alkoxy, amino, aminoalkyl, -NO ₂ , =O, -NHC ₂ H ₄ OH, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C (=0)NHCH ₃ , -C(=0)OH, phenyl or heterocycloalkyl;

X ₄ is phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R 3 ;

each R ₃ is selected from alkyl, alkoxy, amino, halo, hydroxyl, alkylamino, dialkylamino, -NO ₂ , -CONR'R'', -CO ₂ R', -NHCOR', phenyl or heterocycloalkyl selected; wherein each R' and R'' is independently -H or alkyl;

A1 is CH ₂ or NH;

I is an integer of 0 or 1.

In specific embodiments, each of X ₁ and X ₄ is independently substituted or unsubstituted phenyl, cycloalkyl or heterocyclyl; In this case, each of X ₁ and X ₄ is independently substituted or unsubstituted phenyl, cyclohexyl, cyclopentyl, furanyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morphine polynyl, indolinyl, 1H-indolinyl, 1H-indolyl, 1H-indazolyl, isoindolinyl, indolin-2-oneyl, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyri denyl.

At this time, in one embodiment, each R ₂ is independently methyl, ethyl, amino, aminoalkyl, amino (hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH ₂ , -C( =O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl.

At this time, in one embodiment, each R ₃ is independently hydroxyl, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropyloxy, alkylamino, di selected from alkylamino, -NO ₂ , -C(=0)NH ₂ , -CO ₂ R', -NHCOR', -CONR'R'' and phenyl;

Each R' and R'' is independently -H or alkyl.

In one embodiment, Formula 1 provides a compound represented by Formula 1-1 or a salt thereof:

[Formula 1-1]

.

At this time, the A1 is CH ₂ or NH,

I is an integer of 0 or 1.

In another embodiment, Formula 1 provides a compound of Formula 1-2 or a salt thereof:

[Formula 1-2]

.

In another embodiment, Formula 1 provides a compound of Formula 1-3 or a salt thereof:

[Formula 1-3]

.

In another embodiment, Chemical Formula 1 provides a compound represented by Chemical Formulas 1-4 or a salt thereof.

[Formula 1-4]

At this time, in Formulas 1-1, 1-2, 1-3 and 1-4,

In one embodiment,

wherein X ₁ is phenyl, cycloalkyl or heterocyclyl optionally substituted or unsubstituted with one or more R ₂ , wherein each R ₂ is independently alkyl, alkoxy, amino, aminoalkyl, —NO ₂ , =O, -NHC ₂ H ₄ OH, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)OH, phenyl or heterocyclo is selected from alkyl;

X ₄ is phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R ₃ ; At this time, each R ₃ is independently alkyl, alkoxy, amino, halo, hydroxyl, alkylamino, dialkylamino, -NO ₂ , -CONR'R'', -CO ₂ R', -NHCOR', is selected from phenyl or heterocycloalkyl;

Each R' and R'' is independently -H or alkyl.

In this case, the broken lines (dotted lines) in Formulas 1-1, 1-2, 1-3, and 1-4 indicate possible attachment points of the linker (L) or the target protein (or peptide) binding ligand (TBL).

In specific embodiments, each of X ₁ and X ₄ is independently substituted or unsubstituted phenyl, cycloalkyl or heterocyclyl; In this case, each of X ₁ and X ₄ is independently substituted or unsubstituted phenyl, cyclohexyl, cyclopentyl, furanyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morphine polynyl, indolinyl, 1H-indolinyl, 1H-indolyl, 1H-indazolyl, isoindolinyl, indolin-2-oneyl, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyri denyl.

At this time, in one embodiment, each R ₂ is independently methyl, ethyl, amino, aminoalkyl, amino (hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH ₂ , -C( =O)NH ₂ , -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl.

In this case, as a specific embodiment, the R ₂ is amino, or a salt thereof.

At this time, in one embodiment, the X ₁ is

,

or

A phosphorus compound or a salt thereof is provided.

At this time, in one embodiment, each R ₃ is independently hydroxyl, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropyloxy, alkylamino, di selected from alkylamino, -NO ₂ , -C(=0)NH ₂ , -CO ₂ R', -NHCOR', -CONR'R'' and phenyl;

Each R' and R'' is independently -H or alkyl.

At this time, as a specific embodiment, the R ₃ is hydroxyl provides a compound or a salt thereof.

At this time, in one embodiment, the X ₄ is

,

or

A phosphorus compound or a salt thereof is provided.

At this time, in one embodiment, the UBL may be a compound or a salt thereof selected from the following:

N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide;

4-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide;

N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide;

N '-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide;

N '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide;

3-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

4-(1-aminoethyl) -N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

3,5-diamino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide;

4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide;

4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide;

6-amino- N '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonohydrazide;

4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide;

4-amino- N '-(1 H -indole-3-carbonyl)benzenesulfonohydrazide;

4-amino- N '-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide;

4-amino- N '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-1 H -pyrazole-4-sulfonohydrazide;

N '-(4-hydroxybenzoyl)indoline-4-sulfonohydrazide;

N '-(4-hydroxybenzoyl)-1 H -indole-4-sulfonohydrazide;

2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide;

4-amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide;

4-amino- N '-(indoline-4-carbonyl)benzenesulfonohydrazide;

4-amino- N '-(4-hydroxybenzoyl)-3-(peparazin-1-yl)benzenesulfonohydrazide;

4-amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl)benzenesulfonohydrazide;

4-amino- N '-(isoindoline-2-carbonyl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-1 H -indole-2-sulfonohydrazide;

4-amino- N '-(2-phenylacetyl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)-1 H -indazole-3-sulfonohydrazide;

4-amino- N '-(indoline-6-carbonyl)benzenesulfonohydrazide;

4-amino- N '-(indoline-3-carbonyl)benzenesulfonohydrazide;

N '-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide;

4-amino- N '-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide;

4-amino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide;

4-amino-3-morpholino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide;

N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide;

(1 S ,4 S )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide;

( 1R , 4R )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide;

4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid;

N '-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide;

N '-(4-hydroxybenzoyl)-1 H -pyrrolo[2,3- b ]pyridine-2-sulfonohydrazide;

2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide;

2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide;

N '-(4-aminobenzyl)-4-hydroxybenzohydrazide;

4-hydroxy-N'-(4-methoxybenzyl)benzohydrazide;

N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide;

4-amino- N- (2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide;

4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide;

3,5-diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide;

N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide;

4-hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide; and

N-(((4-aminophenyl)sulfonyl)methyl)-[1,1′-biphenyl]-4-carboxamide.

In another embodiment, the bifunctional compound may be a compound having a UBL (UBR box domain binding ligand) -L (linker) -TBL (target protein (or peptide) binding ligand) structure or a salt thereof.

The TBL (target protein (or peptide) binding ligand) may be a compound that binds to a target protein (or peptide) to be degraded. In this case, the TBL may be a known compound.

The L (linker) may be a compound that connects the UBL (UBR box domain binding ligand) and the TBL (target protein (or peptide) binding ligand) without affecting each function. In this case, L may be a known linker compound.

The UBL (UBR box domain binding ligand) is the same as described in one embodiment described above.

The present application provides a composition for protein degradation comprising a bifunctional compound and a use thereof. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL).

In one embodiment, the composition for protein degradation may include a bifunctional compound having a UBL-TBL structure or a UBL-L-TBL structure.

The UBL, L and TBL are as described above.

The present application provides a pharmaceutical composition containing a bifunctional compound and a method for treating a disease using the same. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL).

As one embodiment, the pharmaceutical composition may include a bifunctional compound having a UBL-TBL structure or a UBL-L-TBL structure.

The UBL, L and TBL are as described above.

The pharmaceutical composition may be used for treatment of disease. At this time, the disease is protein (or peptide) degradation, protein mutation, misfolded protein, protein accumulation, aggregated (aggregated) protein (and / or peptide), overexpressed protein, truncated protein, abnormal structure Eggplant may be a disease caused by protein problems, such as protein.

One invention disclosed herein relates to a bifunctional compound comprising a ligand binding to a UBR box domain associated with an intracellular protein degradation pathway and a ligand binding to a target protein (or peptide). More specifically, a bifunctional compound can position a protein of interest for degradation in proximity to a UBR, particularly a UBR box domain. At this time, the protein to be degraded located close to the UBR box domain is ubiquitinated by the UBR box domain and can be degraded through the proteasome pathway. Therefore, the protein to be degraded (ie, the desired target protein (or peptide)) can be more effectively degraded by using the bifunctional compound.

1 is an experimental result of confirming whether muscle actin is an Arg/N-degron pathway substrate using an immunoblotting method.

Figure 2 is an experimental result confirming whether the degradation of R-nsp4, which should be degraded by binding to UBR1, is inhibited by compounds ( Compounds 2, 3, 7, 12, 14, and 16) using an in vitro transcriptional translation method.

FIG. 3 is an experimental result using immunoblotting to confirm whether compounds (Compounds 2 and 3) bind to UBR1 and inhibit degradation of RGS4, a substrate for UBR proteins in embryonic kidney cells, which should be degraded.

4 is an experimental result confirming whether the compounds ( Compounds 1, 2, 3, 4, 5, 6, 7, and 16) inhibit the degradation of actin, a substrate for UBR proteins in muscle cells, using immunoblotting.

5 is an experimental result confirming whether the compounds (Compounds 35, 36, 37, 38, 39, 43, 44, 45, 46, 47) inhibit actin degradation in muscle cells using immunoblotting.

6 is an experimental result confirming whether the compounds ( Compounds 8, 9, 18, 19, 20, 21, 22, 23, and 24) inhibit actin degradation in muscle cells using immunoblotting.

7 shows experimental results confirming whether the compounds (Compounds 25, 26, 27, 28, 32, 33, and 34) inhibit actin degradation in muscle cells using immunoblotting.

8 is an experimental result confirming whether the compounds (Compounds 41 and 42) inhibit actin degradation in muscle cells using immunoblotting.

9 is an experimental result confirming whether the compounds (Compounds 12, 13, 14, 15, 17, 29, 30, 31) inhibit actin degradation in muscle cells using immunoblotting.

10 to 11 are experimental results confirming the intracellular UBR1, 2, 3, 5 binding efficacy of the compound (Compound 2) using immunoblotting.

12 to 19 are MST (Microscale thermophoresis) test results to determine whether compounds ( Compounds 1, 2, 5, 8, 9, 11, 12, and 13) and UBR1 bind to each other.

20 is an experimental result confirming the decomposition ability of AR, an androgen receptor, of Compound A, Compound B, and Compound C using immunoblotting.

21 is an experimental result confirming the PSA degradation ability of compounds A and compounds C, a prostate cancer-specific antigen, using immunoblotting.

22 is an experimental result confirming the estrogen receptor (ER-α) degradation ability of Compound D and Compound E using immunoblotting.

23 is an experimental result confirming the estrogen receptor (ER-β) degradation ability of Compound D using immunoblotting.

Hereinafter, with reference to the accompanying drawings, the content of the invention will be described in more detail through specific implementation examples and examples. It should be noted that the accompanying drawings include some, but not all, embodiments of the invention. The content of the invention disclosed by this specification may be implemented in various ways, and is not limited to the specific implementation described herein. Those skilled in the art to which the invention disclosed herein pertains will be able to come up with many modifications and other implementations of the subject matter of the invention disclosed herein. Accordingly, it should be understood that the content of the invention disclosed herein is not limited to the specific embodiments described herein, and that modifications and other embodiments thereof are included within the scope of the claims.

term definition

Definitions of key terms used in this specification are as follows.

UBR (Ubiquitin protein ligase E3 component n-recognin)

As used herein, the term UBR means an abbreviation for Ubiquitin protein ligase E3 component n-recognin. The UBR is N-lecognin that recognizes the N-terminal residue of a protein, and it is known that at least 7 types of UBRs 1 to 7 exist in mammals. The UBR is N-lecognin, which is involved in the N-terminal law pathway, which is a proteolytic pathway in vivo. Specifically, the UBR recognizes the N-terminal degradation signal (N-degron) of a protein and is involved in the process of degradation of a substrate protein through the ubiquitin proteasome pathway.

UBR box domain

As used herein, the term UBR box domain is a domain present in UBR protein and is a zinc finger motif. The UBR proteins include UBR 1 to 7 proteins. The UBR box domain is known as a domain to which a substrate protein binds. The compounds disclosed herein as UBR box domain ligands can bind to the UBR box domain and inhibit binding of the UBR box domain substrate. Furthermore, the compounds disclosed herein as UBR box domain ligands can affect proteolytic pathways in cells.

RING domain

As used herein, the term RING domain is known to be present in UBR 1, 2 and 3 proteins. The RING domain may also be used as the term RING ubiquitination domain. The RING domain is a domain present in a protein and is a zinc finger motif. The RING domain is a domain that plays an important role in the process of transferring ubiquitin from E2 to a substrate protein, and the RING domain serves to cause the process of transferring ubiquitin to a substrate protein in one step.

HECT domain

As used herein, the term HECT domain is known to exist within the UBR 5 protein. The HECT domain may also be used as the term HECT ubiquitination domain. The HECT domain is a domain that plays an important role in the process of transferring ubiquitin from E2 to a substrate protein. Ubiquitin in E2 is transferred to the HECT domain and then transferred to the substrate protein. That is, the HECT domain serves to make the process of transferring the ubiquitin to the substrate protein in two steps.

zinc finger motif

The term zinc finger motif used herein refers to a protein structural motif in which one or more zinc ions are present to stabilize the protein structure. The UBR box domain and RING domain of the present specification are zinc finger motifs.

N-end rule pathway

Cells regulate the amount of protein through proteolysis. At this time, it is known that the protein degradation process proceeds through a process of recognizing degron, which is a protein degradation signal. Specifically, protein degradation is regulated depending on the N-terminal residue sequence of the protein, and proteolysis signals present at the N-terminus are collectively referred to as N-degron. The N-degrons include those having a positively charged residue (eg, arginine, lysine, histidine) or a large hydrophobic residue (phenylalanine, leucine, tryptophan, isoleucine, tyrosine) at the N-terminus. In this way, the term N-end rule was used based on the relationship that the half-life of a protein is determined by what amino acid residue exists at the N-terminus of the protein.

PROTAC

PROTAC (proteolysis targeting chimera; PROTAC) is a heterobifunctional small molecule composed of two active domains and a linker, and is a technology capable of removing specific unwanted proteins. Instead of acting as an enzyme inhibitor, Protec works by inducing selective intracellular protein degradation. Protek consists of two covalently linked protein-binding ligands. One can bind the E3 ubiquitin ligase and the other binds to the target protein signifying degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. Since Protek only needs to bind to the target rather than inhibiting the activity of the target protein with high selectivity, many efforts are currently underway to convert previously ineffective inhibitor molecules of the target protein into Protek for next-generation drugs.

The protein disclosed herein is characterized by using a bifunctional compound including a ligand that binds to UBR, an E3 ligase, particularly a ligand that binds to the UBR box domain in UBR and a target protein (or peptide) binding ligand. . The UBR box domain binding ligand binds to the UBR box domain. The UBR box domain is known as a domain to which an N-terminal residue sequence or an N-terminal degradation signal binds. This domain is involved in the process of protein degradation by the N-terminal pathway rule. Thus, the UBR box domain binding ligand can affect the proteolytic process through the N-terminal law pathway.

In the N-terminal law pathway, N-degrons are recognized by N-recognin, and UBR (Ubiquitin protein ligase E3 component n-recognin) was discovered as N-recognin. It is known that the UBR recognizes an N-terminal residue sequence or an N-terminal degradation signal through the UBR box domain. That is, UBR recognizes a protein degradation signal through the UBR box domain, and through this, the protein degradation process proceeds.

The protein degradation process by the UBR may include the following. The UBR box domain recognizes a substrate having an N-terminal degradation signal, ubiquitin is bound to the substrate, and the substrate to which ubiquitin is bound can be degraded by the proteasome. That is, a substrate having an N-terminal degradation signal can be degraded by the ubiquitin proteasome system (UPS).

Therefore, the protein disclosed herein uses a bifunctional compound including a UBR box domain binding ligand and a target protein (or peptide) binding ligand to position a protein to be degraded in close proximity to the UBR, particularly the UBR box domain let it At this time, the protein to be degraded located close to the UBR box domain is ubiquitinated by the UBR box domain and degraded through the proteasome pathway.

At this time, in one embodiment, the protector may include a UBR box domain binding ligand and a target protein binding ligand. At this time, the protector may further include a linker, and the linker may serve to connect the two ligands by being present between the UBR box domain binding ligand and the target protein binding ligand.

ligand

The term ligand as used herein refers to a substance that specifically binds to a protein. The protein includes an enzyme or a receptor, and when the protein is an enzyme, the ligand may mean a substrate that binds to the enzyme, and when the protein is a receptor, the ligand may mean a hormone that binds to the receptor.

A compound as a UBR box domain ligand provided herein refers to a compound that binds to a UBR box domain, and is used interchangeably with UBR box domain binding ligand (UBL) in the present specification. In one embodiment, the UBL refers to a compound that binds to a UBR box domain in a UBR protein. In a specific embodiment, the UBL refers to a compound that binds to a UBR box domain present in one or more proteins of UBR1 to 7. However, it is not limited thereto. In addition, UBL can compete with substrates of the UBR box domain. That is, the UBL can inhibit the substrate of the UBR box domain from binding. In addition, the UBL may inhibit substrate degradation by inhibiting binding of the substrate.

A target protein (or peptide) binding ligand (TBL) provided herein refers to a compound that binds to a target protein (or peptide) to be degraded. In this case, the TBL may be a known compound.

As used herein, unless otherwise indicated, the term "compound" relates to any particular chemical compound described herein, and includes tautomers, regioisomers, geometric isomers and, where applicable, stereoisomers, which include: Optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives thereof (including prodrug forms) where applicable in the context are included. Within contextual usage, the term compound generally refers to a single compound or is also stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched compounds of the described compounds. It may contain other compounds such as mixtures. The term also, in context, refers to a prodrug form of a compound that has been modified to facilitate administration and delivery of the compound to the site of action. It will be appreciated by those skilled in the art that the molecules described herein are generally described stable compounds.

aminoalkyl

As used herein, the term aminoalkyl refers to an alkyl moiety substituted with an amino group. The aminoalkyl group includes -CH(NH ₂ )CH ₃ and -CH ₂ (NH ₂ ).

Cycloalkyl and Heterocycloalkyl

As used herein, the term cycloalkyl refers to a carbocyclic group containing one or more saturated ring structures and includes bicyclics. Cycloalkyl includes, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

Heterocycloalkyl refers to a ring structure containing at least one heteroatom selected from P, N, O and S in addition to the ring-carbon atom in the above cycloalkyl.

heterocyclyl

As used herein, the term heterocyclyl refers to an unsaturated, saturated or partially unsaturated monocyclyl, bicyclic, or tricyclic group of 2 to 14 ring carbon atoms, in addition to the ring-carbon atoms P, N, O and and one or more heteroatoms selected from S. The heterocyclyl includes heterocycloalkyl. In various embodiments the heterocyclic group is attached to another moiety through a carbon or heteroatom and is optionally substituted on the carbon or heteroatom. Examples of heterocyclyls include azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzo Thiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl ( indolinyl, isoindolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl ), isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl ( pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrothiopyranyl ), tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl , azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridine-2- oneyl (pyridin-2-only), pyrrolidinyl, pyrrolopyridinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzoimidazolyl, Hydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl , dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl ), dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl ( dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl ) and tetrahydrothienyl.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.

Hereinafter, specific details of the invention are disclosed.

<Bifunctional compound>

One aspect disclosed herein relates to a bifunctional compound or a salt, enantiomer, diastereomer, solvate, or polymorph thereof. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL).

The bifunctional compound is characterized by having the following structure:

UBR box domain binding ligand (UBL)-Target protein binding ligand (TBL); or

UBL (UBR box domain binding ligand)-L (linker)-TBL (Target protein binding ligand).

Each configuration is described in detail below.

1. UBR box domain binding ligand (UBL)

The UBR box domain-binding ligand disclosed herein was designed in consideration of the structure of the UBR box domain and the binding form between the UBR box domain and the N-terminal pathway substrate.

Various amino acids present in the UBR box domain interact and bind with amino acids of the N-terminal pathway substrate through ionic interaction, hydrogen bond, and hydrophobic interaction. By analyzing these binding modes, in the present specification, a low-molecular-weight compound capable of forming a suitable binding mode with the UBR box domain is synthesized and provided. Furthermore, in the present specification, compounds represented by Chemical Formulas 1 to 55 are provided.

The UBR box domain binding ligand (UBL) disclosed herein has a core structure that allows it to bind well to the UBR box domain. In the present specification, as described above, the binding mode between the UBR box domain and the amino acid of the N-terminal pathway substrate was analyzed to derive the core structure of the compound. UBL provided herein may have a structure of [Formula 1] below derived based on a core structure that binds well to the UBR box domain. [Formula 1] is as follows:

[Formula 1]

.

At this time, the dashed line (dotted line) in Chemical Formula 1 indicates the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL).

In the present specification, various compounds are designed and provided based on [Formula 1]. In this case, candidates for X ₁ , X ₂ , X ₃ , B ₁ , A ₁ , B _{1 , and} X ₄ are derived by considering the combination mode with the UBR box domain. A more detailed description of the various compounds based on [Formula 1] is described below.

1) Formula 1

[Formula 1]

i) X ₂ , B ₁ , X ₃ and A ₁

① X ₂

In Formula 1, X ₂ may be a structure that induces a folding structure in the compound disclosed herein. The folding structure may help X ₁ of the compound disclosed herein smoothly maintain charge-charge interaction or hydrogen bonding or hydrophobic action with the UBR box domain and increase bonding strength. Accordingly, in one embodiment, the X ₂ may be one of various structures that may cause a bending structure. In a specific embodiment, X ₂ may be SO ₂ or CR _a R _b . In this case, R _a and R _b may be each independently selected from H or CH ₃ . Furthermore, X ₂ may be CH ₂ , CH(CH ₃ ) or C(CH ₃ ) ₂ . In another specific embodiment, the X ₂ may be SO ₂ .

② B ₁ , X ₃ and A ₁

In Formula 1, A ₁ may be, in one embodiment, CH ₂ or NH.

In Formula 1, B ₁ may be, in one embodiment, CH ₂ or NH.

In Formula 1, X ₃ may be, in one embodiment, CH ₂ or NH.

In this case, as an embodiment, when B ₁ is CH ₂ in Formula 1, X ₃ may not be CH ₂ .

However, it is not limited thereto.

③ Examples of X ₂ , B ₁ , X ₃ and A ₁

As an example, Chemical Formula 1 may have a structure selected from the following:

In one embodiment, in Formula 1,

-X ₂ -B ₁ -X ₃ is selected from the group consisting of -SO ₂ -NH-NH, -SO ₂ -NH-CH ₂ , -SO ₂ -CH ₂ -NH and -CH ₂ -NH-NH;

A1 is CH ₂ or NH;

I is an integer of 0 or 1.

As a specific example, Chemical Formula 1 may have a structure selected from the following:

[Formula 1-1]

,

[Formula 1-2]

,

[Formula 1-3]

,

[Formula 1-4]

.

At this time, the dashed lines (dotted lines) in Chemical Formulas 1-1 to 1-4 represent possible attachment points of the linker (L) or the target protein (or peptide) binding ligand (TBL).

ii) X ₁

As a result of performing a molecular docking study with a compound having a core structure of Formula 1 and analyzing the complex structure of the UBR box of UBR1 and UBR2 and the N-degron, X ₁ in Formula 1 is Corresponding to the side chain of the first residue (N1) of the N-degron, X ₁ is expected to bind to the negatively charged-surrounded region. Accordingly, in one embodiment, X ₁ in Chemical Formula 1 may have a ring structure including a moiety that has an electric charge or forms a hydrogen bond. In a specific embodiment, the X ₁ may have a ring structure having a planar structure including a moiety that has an electric charge or forms a hydrogen bond. Additionally, when the compound is used in combination with another substance, X ₁ in Chemical Formula 1 may include a structure capable of binding to a linker.

In one embodiment, X ₁ may be phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R ₂ . In a specific embodiment, X ₁ is _phenyl , cyclohexyl, cyclopentyl, furanyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, p Ferrazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indolyl, 1H-indazolyl, isoindolinyl, indolin-2-oneyl, 2,3-dihydro-1H-indenyl and 1H -pyrrolopyridinyl. At this time, each R ₂ is independently alkyl, alkoxy, amino, aminoalkyl, -NO ₂ , =O, -NHC ₂ H ₄ OH, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)OH, phenyl or heterocycloalkyl can be selected from. In one embodiment, each R ₂ is independently methyl, ethyl, amino, aminoalkyl, amino(hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl and morpholinyl. In a specific embodiment, R ₂ may be amino.

In a more specific embodiment, the X ₁ may be selected from the following structure:

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

.

In a more specific embodiment, the X ₁ may be selected from the following structure:

,

and

.

iii) X ₄

In Chemical Formula 1, X ₄ corresponds to the side chain of the second residue (N2) of the N-degron and may have a ring or chain structure to fill the bonding space when combined with the UBR box. . At this time, in one embodiment, the ring or chain structure may have a charge or a moiety forming a hydrogen bond may be introduced to increase bonding strength. Additionally, X ₄ may include a structure capable of serving to bind to a linker when the compound of the present specification is used in combination with another substance later.

In one embodiment, X ₄ may be phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R3. In a specific embodiment, X ₄ is phenyl, _cyclohexyl , cyclopentyl, furanyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, p Ferrazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indolyl, 1H-indazolyl, isoindolinyl, indolin-2-oneyl, 2,3-dihydro-1H-indenyl and 1H -pyrrolopyridinyl. In this case, each R ₃ is independently alkyl, alkoxy, amino, halo, hydroxyl, alkylamino, dialkylamino, -NO ₂ , -CONR'R'', -CO ₂ R', -NHCOR', phenyl or Heterocycloalkyl. In one embodiment, each R ₃ is independently hydroxyl, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropyloxy, alkylamino, dialkylamino, - NO ₂ , -C(=0)NH ₂ , -CO ₂ R', -NHCOR', -CONR'R'' and phenyl. In a specific embodiment, R ₃ may be hydroxyl. In this case, each of R' and R'' may independently be -H or alkyl.

In a more specific embodiment, the X ₄ may be selected from:

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

.

In a more specific embodiment, the X ₄ may be selected from:

,

,

.

The compounds disclosed herein may exist in the form of stereoisomers or salts thereof, and isomers or salts of such compounds are included in the scope of the present specification.

2) Specific examples of UBR box domain binding ligand (UBL)

i) A specific example of UBL

The following describes specific embodiments of UBL disclosed in this specification. The specific embodiments below are exemplary structures for easy understanding of UBL disclosed in this specification, and the scope of UBL disclosed in this specification is not limited to the following embodiments.

As a specific embodiment, the compound disclosed herein may have a structure of [Formula 1-1]:

[Formula 1-1]

.

At this time, in the UBL, A1 is CH ₂ or NH, and I is an integer of 0 or 1.

X ₁ and X ₄ are ii) of <UBR box domain binding ligand> described above X ₁ and iii) X ₄ are equally applicable.

As a more specific embodiment, a specific exemplary compound for [Formula 1-1] may be selected from the following. At this time, the broken line (dotted line) indicating the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL) is omitted.

Specific Exemplary Compounds for Formula [1-1]

compound number	compound
One	N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide
2	4-Amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
3	4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide
4	N '-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide
5	N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide
6	N '-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide
7	N '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide
8	3-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
9	4-(1-aminoethyl) -N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
10	3,5-diamino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
11	N '-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonohydrazide
13	N '-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide
14	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide
15	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide
17	6-Amino- N '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonohydrazide
18	4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide
20	4-amino- N '-(1 H -indole-3-carbonyl)benzenesulfonohydrazide
21	4-Amino- N '-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide
22	N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide
23	4-Amino- N '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydrazide
24	N '-(4-hydroxybenzoyl)-1 H -pyrazole-4-sulfonohydrazide
25	N '-(4-hydroxybenzoyl)indoline-4-sulfonohydrazide
26	N '-(4-hydroxybenzoyl)-1 H -indole-4-sulfonohydrazide
27	2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide
28	4-Amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide
29	4-amino- N '-(indoline-4-carbonyl)benzenesulfonohydrazide
30	4-amino- N '-(4-hydroxybenzoyl)-3-(peparazin-1-yl)benzenesulfonohydrazide
31	4-Amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl)benzenesulfonohydrazide
32	4-amino- N '-(isoindoline-2-carbonyl)benzenesulfonohydrazide
33	N '-(4-hydroxybenzoyl)-1 H -indole-2-sulfonohydrazide
34	4-Amino- N '-(2-phenylacetyl)benzenesulfonohydrazide
35	N '-(4-hydroxybenzoyl)-1 H -indazole-3-sulfonohydrazide
36	4-amino- N '-(indoline-6-carbonyl)benzenesulfonohydrazide
37	4-amino- N '-(indoline-3-carbonyl)benzenesulfonohydrazide
38	N '-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide
39	4-amino- N '-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide
40	4-Amino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide
41	4-Amino-3-morpholino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide
42	N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide
43	(1 S ,4 S )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide
44	( 1R , 4R )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide
45	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid
46	N '-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide
47	N '-(4-hydroxybenzoyl)-1 H -pyrrolo[2,3- b ]pyridine-2-sulfonohydrazide
52	2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide
53	2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide

In another specific embodiment, the UBL disclosed herein may have a structure of [Formula 1-2]:

[Formula 1-2]

.

In this case, in the UBL, X ₁ and X ₄ are ii) of <UBR box domain binding ligand> described above The same applies to X ₁ and iii) X ₄ .

Specific exemplary compounds for [Formula 1-2] may be selected from the following. At this time, the broken line (dotted line) indicating the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL) is omitted.

Specific Exemplary Compounds for Formula [1-2]

compound number	compound
19	N '-(4-aminobenzyl)-4-hydroxybenzohydrazide
48	4-hydroxy-N'-(4-methoxybenzyl)benzohydrazide
49	N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide

In another specific embodiment, UBL disclosed herein may have a structure of [Formula 1-3]:

[Formula 1-3]

.

In this case, in the UBL, X ₁ and X ₄ are ii) of <UBR box domain binding ligand> described above X ₁ and iii) X ₄ are equally applicable.

Specific exemplary compounds for [Formula 1-3] may be selected from the following. At this time, the broken line (dotted line) indicating the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL) is omitted.

Specific Exemplary Compounds for Formula [1-3]

compound number	compound
12	4-Amino- N- (2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide
50	4-Amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide
51	3,5-diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide

In another specific embodiment, UBL disclosed herein may have a structure of [Formula 1-4]:

[Formula 1-4]

.

In this case, in the UBL, X ₁ and X ₄ are ii) of <UBR box domain binding ligand> described above X ₁ and iii) X ₄ are equally applicable.

Specific exemplary compounds for [Formula 1-4] may be selected from the following. At this time, the broken line (dotted line) indicating the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL) is omitted.

Specific Exemplary Compounds for Formula [1-4]

compound number	compound
16	N -(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide
54	4-Hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide
55	N-(((4-aminophenyl)sulfonyl)methyl)-[1,1'-biphenyl]-4-carboxamide

At this time, the UBL may be considered in the form of one of its possible isomers or a form of a mixture thereof. For example, all stereoisomers, including enantiomers and diastereomers, or mixtures thereof (eg, racemic mixtures) are contemplated.

3) a salt of the UBL

The UBL disclosed herein may be considered in the form of a salt thereof. At this time, the salt includes a pharmaceutically acceptable salt. Salts disclosed herein include acid addition salts or basic addition salts. Exemplary acids forming the salt include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, glutaric acid, and the like, and exemplary bases forming the salt include lithium, sodium, potassium, calcium, magnesium , methylamine, trimethylamine, and the like. However, it is not limited thereto and can be easily selected by those skilled in the art.

2. Target protein (or peptide) binding ligand (TBL)

The target protein (or peptide) binding ligand disclosed herein is designed in various ways according to the target protein (or peptide) to be degraded. The target protein (or peptide) binding ligand (TBL) may be a known compound known to bind to a target protein (or peptide). For example, the TBL may be an inhibitor compound of a known target protein (or peptide). For example, the TBLs are Hsp90 inhibitors, kinase inhibitors, androgen receptor inhibitors, HDM2 & MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, nuclear It may be a hormone receptor compound, an immunosuppressive compound or a compound targeting the aryl hydrocarbon receptor (AHR), but is not limited thereto. The TBLs also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of known compounds, as well as other small molecules capable of targeting a protein of interest.

The target protein (or peptide), which is any protein that binds to the TBL of the bifunctional compound and is ubiquitinated by UBR and degraded by proteasome, is, for example, a structural protein, a receptor, an enzyme, a cell surface protein, or a cell surface protein. Proteins that act on various functions, such as catalytic activity, aromatase activity, locomotor activity, helicase activity, metabolic processes (anabolism and catabolism), antioxidant activity, proteins involved in proteolysis, biosynthesis, kinases activity, oxidoreductase activity, transferase activity, hydrolase activity, ligase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transducer activity, structural molecule activity, binding activity (protein, lipid carbohydrate) , receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimuli, behavior proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (protein transporters) activity, nuclear transport, including ion transporter activity, channel transporter activity, carrier activity, permease activity, secretory activity, electron transporter activity, pathogenesis, chaperone regulator activity, nucleic acid binding activity, transcriptional regulator activity, cell It may include proteins with exo-organizing and biogenic activities, translational regulator activities. The target proteins can be derived from eukaryotes and prokaryotes, including humans, other animals, including domestic animals, microorganisms and plants for the determination of targets for antibiotics and other antimicrobials, and even viruses, among others, as targets for drug therapy. May contain protein.

In certain embodiments, TBL can have the structure of Formula 2 below:

[Formula 2]

.

during the ceremony,

W ¹ is

or

is;

each R ₃ is independently H or -CN;

each R ₄ is independently H, halogen or -CF ₃ ;

Y ¹ and Y ² are each independently O or S;

R ¹ and R ² are each independently H or a methyl group;

W ² is a bond, C1-6 aryl, biphenyl, biphenylyl or heteroaryl, each optionally substituted with 1, 2 or 3 R ^W2 ; and

Each R ^W2 is independently H, OH, halogen, -C(=O)NHCH ₃ , C1-6 alkyl (optionally substituted with one or more F), or OC1-3 alkyl (optionally with one or more -F) is replaced).

The dashed line (dotted line) represents an attachment point with the UBR box domain binding ligand (UBL) or linker (L).

In a specific embodiment, the W ¹ is selected from the group consisting of:

.

In a specific embodiment, the W ² is selected from the group consisting of:

.

In this case, the TBL may be an androgen receptor binding compound.

In certain embodiments, TBL can be an androgen receptor binding compound.

As a specific example, the androgen receptor binding compound may be selected from the group consisting of:

4-(3-(4'-hydroxy-[1,1'-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl) -2-(trifluoromethyl)benzonithnyl;

4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro -N-methylbenzamide;

trans-2-chloro-4-[3-amino-2,2,4,4-tetramethylcyclobutoxy]benzonitrile;

cis-2-chloro-4-[3-amino-2,2,4,4-tetramethylcyclobutoxy]benzonitrile;

trans 6-amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridazine-3-carboxamide;

trans tert-butyl N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]carbamate;

trans 4-amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide;

trans 5-amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyrazine-2-carboxamide;

trans 2-amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyrimidine-5-carboxamide;

4-methoxy-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide;

trans 1-(2-hydroxyethyl)-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]-1H-pyrazole-4- carboxamide;

trans 6-amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide;

trans 4-[(5-hydroxypentyl)amino]-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide; and

trans tert-butyl 2-({5-[(4-{[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]carbamoyl}phenyl)aminophen ethyl} oxy)acetate; and

N-((1r,3r)-3-(4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-methylbenzamide.

In certain embodiments, TBL can have the structure of Formula 3 below:

[Formula 3]

.

during the ceremony,

X is O or C=0;

each X ₁ and X ₂ is independently selected from N or CH;

R ₁ is independently selected from OH, O(CO)R _a , O-lower alkyl, wherein R _a is an alkyl or aryl group in an ester;

R ₂ is selected from H, OH, halogen, CN, CF ₃ , SO ₂ -alkyl, O-lower alkyl;

R ₃ is selected from H, halogen; and

The dashed line (dotted line) represents an attachment point with the UBR box domain binding ligand (UBL) or linker (L).

In this case, the TBL may be an estrogen receptor binding compound.

The embodiments described herein are arbitrary embodiments, and TBLs can be designed in various ways according to various types of target proteins (or peptides). Also, since known compounds or inhibitor compounds known to bind target proteins (or peptides) can be used for TBL, TBL is not limited to any of the embodiments described above.

3. Linker (L)

The linker disclosed herein is a compound that chemically connects or binds a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL). In this case, the linker may be a compound that does not affect the functions of the UBR box domain binding ligand (UBL) and the target protein (or peptide) binding ligand (TBL). The functions of the UBR box domain binding ligand (UBL) and the target protein (or peptide) binding ligand (TBL) may be binding to the UBR box domain and binding to the target protein (or peptide), respectively.

As the linker, a known linker well known in the art may be used. For example, the linker may be arbitrarily selected from linkers described in Korean Patent Application No. 10-2020-7032733, US Patent Application No. 17/006193, and US Patent Application No. 17/082839.

The linker can be designed in various ways considering the structures of UBL and TBL.

In certain embodiments, the linker can be selected from the group consisting of:

.

At this time, n and m of the linker are each independently 0, 1, 2, 3, 4, 5 or 6.

At this time, the broken line (dotted line) or the wavy line represents an attachment point to the UBR box domain binding ligand (UBL) or target protein (or peptide) binding ligand.

In certain embodiments, the linker can be selected from the group consisting of:

-(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )m-;

-R ₁ -(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )pR ₂ -;

-O-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-; and

-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-.

At this time, in the linker,

m and p are each independently 0, 1, 2, 3, 4 or 5;

n is 0, 1, 2, 3, 4, 5, 6 or 7;

o is 0, 1, or 2;

R is H, methyl or ethyl;

R ₁ and R ₂ are each independently O or NH.

The embodiments described herein are arbitrary embodiments, and since the linker can be designed in various ways according to the structures of UBL and TBL, the linker is not limited to any of the embodiments described above.

4. Salts of bifunctional compounds

A bifunctional compound disclosed herein may be considered in the form of a salt thereof. At this time, the salt includes a pharmaceutically acceptable salt. Salts disclosed herein include acid addition salts or basic addition salts. Exemplary acids forming the salt include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, glutaric acid, and the like, and exemplary bases forming the salt include lithium, sodium, potassium, calcium, magnesium , methylamine, trimethylamine, and the like. However, it is not limited thereto and can be easily selected by those skilled in the art.

5. Examples of bifunctional compounds

Based on the foregoing description, examples of bifunctional compounds are described. Detailed descriptions of the components included in the following examples are the same as those described above for the corresponding components. The following example is a simple example, and is not limited thereto.

As an embodiment, the bifunctional compound may have a UBL-TBL or UBL-L-TBL structure.

The UBL may have a structure of Chemical Formula 1. At this time, the description of the structure of Formula 1 above <1. UBR box domain binding ligand> as described in the section.

The TBL may have a structure of Chemical Formula 2. At this time, the description of the structure of Formula 2 above <2. It is the same as described in the section Target Protein (or Peptide) Binding Ligand>.

The linker may be selected from the group consisting of:

-(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )m-;

-R ₁ -(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )pR ₂ -;

-O-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-; and

-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-.

At this time, in the linker,

m and p are each independently 0, 1, 2, 3, 4 or 5;

n is 0, 1, 2, 3, 4, 5, 6 or 7;

o is 0, 1, or 2;

R is H, methyl or ethyl;

R ₁ and R ₂ are each independently O or NH.

In one embodiment, the bifunctional compound can be selected from the group consisting of:

4-Amino- N '-(4-(2-(2-(2-(2-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5 -dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethoxy)benzoyl ) benzenesulfonohydrazide;

4-Amino- N '-(4-((5-((5-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4 -oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)pentyl)oxy)pentyl)oxy)benzoyl)benzenesulfonohydrazide; and

4-amino-3-(4-(14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thio Oxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)- N '-(4-Hydroxybenzoyl)benzenesulfonohydrazide.

As another embodiment, the bifunctional compound may have a UBL-TBL or UBL-L-TBL structure.

The UBL may have a structure of Chemical Formula 1. At this time, the description of the structure of Formula 1 above <1. UBR box domain binding ligand> as described in the section.

The TBL may have a structure of Chemical Formula 3. At this time, the description of the structure of Formula 3 above <2. It is the same as described in the section Target Protein (or Peptide) Binding Ligand>.

The linker may be selected from the group consisting of:

-(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )m-;

-R ₁ -(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )pR ₂ -;

-O-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-; and

-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-.

At this time, in the linker,

m and p are each independently 0, 1, 2, 3, 4 or 5;

n is 0, 1, 2, 3, 4, 5, 6 or 7;

o is 0, 1, or 2;

R is H, methyl or ethyl;

R ₁ and R ₂ are each independently O or NH.

In one embodiment, the bifunctional compound can be selected from the group consisting of:

2-((4-aminophenyl)sulfonyl) -N- (3-((3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene -3-carbonyl)phenoxy)-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)oxy)phenyl)hydrazine-1-carboxamide; and

4-amino-3-(4-(3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene-3-carbonyl)phenoxy)- 6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)piperazin-1-yl) -N '-(1 H -indole-4-carbonyl)benzene Sulfonohydrazide.

<Use of bifunctional compound>

One aspect disclosed herein relates to the use of a bifunctional compound.

1. Composition for protein degradation

The bifunctional compounds disclosed herein can be used to prepare compositions for target protein (or peptide) degradation. In one embodiment, the bifunctional compound disclosed herein can be used as PROTAC. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL). The bifunctional compound may use the property that UBL binds to the UBR box domain to position the target protein (or peptide) bound to the TBL of the bifunctional compound to be close to the UBR, particularly the UBR box domain. At this time, the target protein (or peptide) located close to the UBR box domain is ubiquitinated by the UBR box domain and can be degraded through the proteasome pathway. Therefore, the protein to be degraded (ie, the desired target protein (or peptide)) can be more effectively degraded by using the bifunctional compound. Therefore, the composition containing the bifunctional compound can be used for inducing or promoting the degradation of a target protein (or peptide) by the ubiquitin-proteasome pathway by binding to the UBR box domain.

Referring to Examples, it can be confirmed that the bifunctional compound disclosed herein induces or promotes degradation of a target protein (or peptide) through binding to UBR (FIGS. 20 to 23).

2. Treatment of diseases caused by protein problems

The bifunctional compound disclosed herein has a property of inducing or promoting target protein (or peptide) degradation. Therefore, by using such a bifunctional compound, it is possible to induce or promote degradation of problematic proteins in the body, and diseases caused by protein problems can be treated using this mechanism.

Diseases caused by the protein problem include abnormal protein (or peptide) degradation, protein mutation, misfolded protein, protein accumulation, aggregated protein (and/or peptide), overexpressed protein, truncated protein, abnormal It may be a disease or disorder caused by a protein problem, such as a protein having a structure. For example, diseases caused by these protein problems include asthma, autoimmune diseases such as multiple sclerosis, various cancers, chorionic diseases, cleft palate, diabetes, heart disease, hypertension, inflammatory bowel disease, mental retardation, mood disorders, obesity ( PKD1) or 4 (PKD2) Freder-Willi syndrome, sickle cell disease, Tay-Sarks disease, Turner syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), anorexia nervosa, anxiety disorders, atherosclerosis, attention deficit hyperactivity Disorders, autism, bipolar disorder, chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn's disease, coronary heart disease, dementia, depression, diabetes mellitus type 1, diabetes mellitus type 2, epilepsy, Guillain Barré syndrome, irritable bowel syndrome, lupus, metabolic syndrome, multiple sclerosis, myocardial infarction, obesity, obsessive-compulsive disorder, panic disorder, Parkinson's disease, psoriasis, rheumatoid arthritis, sarcoidosis, schizophrenia, stroke, thrombophlebitis obstructive, Tourette's syndrome, or vasculitis; It is not limited to this.

1) Pharmaceutical composition

The bifunctional compounds disclosed herein can be used in the manufacture of pharmaceutical compositions for treating a subject in need thereof.

In this case, the treatment includes an effect of improving symptoms of a specific medical condition or delaying the progression of a disease. In this case, the subject includes humans and non-human animals. In this case, the pharmaceutical composition may include a pharmaceutically acceptable carrier, excipients and/or additives together with the dual functional compound. Such pharmaceutically acceptable carriers, excipients and/or additives include, but are not limited to, water, saline, glycols, glycerol, animal and vegetable fats, oils, starches, etc. Pharmaceutically acceptable carriers, excipients known in the art and/or additives.

2) Treatment method

Provided herein are methods of treatment comprising administering a bifunctional compound disclosed herein or a pharmaceutically acceptable salt thereof to a subject in need thereof. At this time, administration of the bifunctional compound or a pharmaceutically acceptable salt thereof is

Hereinafter, the invention disclosed in this application will be described in detail through examples.

These examples are solely for explaining the invention disclosed in this application in more detail, and it is clear that the scope of the present invention is not limited by these examples to those skilled in the art. It will be self-explanatory for

Example

Example 1. Synthesis of compounds for UBR box domain binding ligands

List of UBL compounds

number	compound name
One	N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide
2	4-Amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
3	4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide
4	N '-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide
5	N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide
6	N '-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide
7	N '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide
8	3-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
9	4-(1-aminoethyl) -N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
10	3,5-diamino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide
11	N '-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonohydrazide
12	4-Amino- N- (2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide
13	N '-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide
14	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide
15	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide
16	N -(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide
17	6-Amino- N '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonohydrazide
18	4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide
19	N '-(4-aminobenzyl)-4-hydroxybenzohydrazide
20	4-amino- N '-(1 H -indole-3-carbonyl)benzenesulfonohydrazide
21	4-Amino- N '-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide
22	N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide
23	4-Amino- N '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydrazide
24	N '-(4-hydroxybenzoyl)-1 H -pyrazole-4-sulfonohydrazide
25	N '-(4-hydroxybenzoyl)indoline-4-sulfonohydrazide
26	N '-(4-hydroxybenzoyl)-1 H -indole-4-sulfonohydrazide
27	2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide
28	4-Amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide
29	4-amino- N '-(indoline-4-carbonyl)benzenesulfonohydrazide
30	4-amino- N '-(4-hydroxybenzoyl)-3-(peparazin-1-yl)benzenesulfonohydrazide
31	4-Amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl)benzenesulfonohydrazide
32	4-amino- N '-(isoindoline-2-carbonyl)benzenesulfonohydrazide
33	N '-(4-hydroxybenzoyl)-1 H -indole-2-sulfonohydrazide
34	4-Amino- N '-(2-phenylacetyl)benzenesulfonohydrazide
35	N '-(4-hydroxybenzoyl)-1 H -indazole-3-sulfonohydrazide
36	4-amino- N '-(indoline-6-carbonyl)benzenesulfonohydrazide
37	4-amino- N '-(indoline-3-carbonyl)benzenesulfonohydrazide
38	N '-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide
39	4-amino- N '-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide
40	4-Amino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide
41	4-Amino-3-morpholino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide
42	N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide
43	(1 S ,4 S )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide
44	( 1R , 4R )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide
45	4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid
46	N '-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide
47	N '-(4-hydroxybenzoyl)-1 H -pyrrolo[2,3- b ]pyridine-2-sulfonohydrazide
48	4-hydroxy-N'-(4-methoxybenzyl)benzohydrazide
49	N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide
50	4-Amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide
51	3,5-diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide
52	2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide
53	2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide
54	4-Hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide
55	N-(((4-aminophenyl)sulfonyl)methyl)-[1,1'-biphenyl]-4-carboxamide

¹ H NMR spectra were recorded on a Bruker Avance III 400 MHz and Bruker Fourier 300 MHz, with TMS used as an internal standard. LCMS was measured on a quadrupole mass spectrometer on an Agilent 1260HPLC and 6120MSD. (Column operating in ES (+) or (-) ionization mode: C18 (50 Х 4.6 mm, 5 μm); T = 30 ^o C; flow rate = 1.5 mL/min; detected wavelengths: 220 nm, 254 nm)

Experimental Example 1-1. compound 1 ( N Preparation of '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide)

Step 1) Synthesis of A2

A mixture of A1 (methyl 4-hydroxybenzoate, 2.00 g, 13 mmol, 1.0 eq) and hydrazine monohydrate (20 mL) was stirred at 100° C. for 16 h. The solvent was concentrated under reduced pressure to obtain a crude product, which was purified by flash column (DCM/MeOH=50/1~1/1) to obtain A2 (4-hydroxybenzohydrazide, 2.0 g, yield 60%) as a white solid. was obtained with

¹ H NMR (DMSO- d ₆ , 400 MHz): δ 9.49 (s, 1H), 7.67-7.69 (m, 2H), 6.76-6.79 (m, 2H), 4.38 (br s, 2H).

Step 2) Synthesis of Compound 1

A mixture of A2 (4-hydroxybenzohydrazide, 0.3 g, 1.97 mmol, 1.0 eq) and 4-methylbenzenesulfonyl chloride (0.3 g, 1.57 mmol, 0.8 eq) dissolved in pyridine (5 mL) at 80 °C. Stirred for 16 hours. 1N HCl was then added to the mixture and extracted with EA (20 mL x 3) until pH=3. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product (400 mg). About 130 mg of crude product was purified by prep-HPLC. The collected fractions were concentrated to remove most of the CH ₃ CN. The remaining fraction was lyophilized to obtain compound 1 ( N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide, 50 mg, 24.8% yield) as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.38 (s, 1H), 10.09 (s, 1H), 9.76 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 2.35 (s, 3H).

LCMS; Mass Calcd.:306.3; MS Found: 306.9.

Experimental Example 1-2. Compound 2 (4-amino- N Preparation of '-(4-hydroxybenzoyl) benzenesulfonohydrazide)

Step 1) Synthesis of A3

A mixture of A2 (4-hydroxybenzohydrazide, 0.3 g, 1.97 mmol, 1.0 eq) and 4-nitrobenzene-1-sulfonyl chloride (0.35 g, 1.57 mmol, 0.8 eq) in pyridine (5 mL) Stirred at 80° C. for 16 hours. 1N HCl was then added and extracted with EA (20 mL x 3) until the mixture was pH=3. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product A3 ( N ′-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 370 mg) as a yellow solid .

LCMS; Mass Calcd.:337.1; MS Found: 337.6 [MS], 359.6 [MS+22].

Step 2) Synthesis of Compound 2

A3 ( N ′-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 150 mg, 0.45 mmol, 1.0 eq) and 5% Pd/C (200 mg, 50%) in EtOH (10 mL) in water) was stirred with a H ₂ balloon at 10° C. for 4 hours. Then the mixture was filtered and the filtrate was concentrated to give a crude product which was purified by prep-HPLC. The collected fractions were concentrated to remove most of the CH ₃ CN. The remaining fractions were freeze-dried to give Compound 2 (4-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide), 50 mg, yield 36.6% as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.31 (s, 1H), 10.06 (s, 1H), 9.13 (s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.08 Hz, 2H), 6.50 (d, J = 8.8 Hz, 2H), 5.95 (s, 2H).

LCMS; Mass Calcd.:307.3; MS Found: 307.9.

Experimental Example 1-3. Compound 3 (4-amino- N Preparation of '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide)

Step 1) Synthesis of A4

3-fluoro-4-nitrobenzene-1-sulfonyl in pyridine (3 mL) in a mixture of A2 (4-hydroxybenzohydrazide, 500 mg, 3.29 mmol, 1.0 eq) in pyridine (5 mL) Chloride (790 mg, 3.29 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 25° C. for 3 hours. The solution was poured into water (30 mL). The mixture was extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1-30/1) to A4 (3-fluoro- N ' -(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 500 mg, 42.8%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.55 (s, 1H), 10.49 (s, 1H), 10.14 (s, 1H), 8.32 (t, J = 8.0 Hz, 1H), 7.98 (d, J = 10.4 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H).

Step 2) Synthesis of A5

A4 (3-fluoro- N '-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 500 mg, 1.41 mmol, 1.0 eq) and morpholine (184 mg, 2.11 mmol, 1.5 eq) was added K ₂ CO ₃ (486 mg, 3.52 mmol, 2.5 eq) at 25 °C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (30 mL). The mixture was extracted with EA (30 mL x 3). The combined organic layers were washed with water (50 mL x 3) and brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1-30/1) to A5 ( N ′-(4-hydroxybenzoyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 180 mg, 30.3%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.51 (s, 1H), 10.24 (s, 1H), 10.14 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.60-7.62 ( m, 3H), 7.52 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.4 Hz, 2H), 3.64 (t, J = 4.8 Hz, 4H), 2.90–2.94 (m, 4H).

Step 3) Synthesis of Compound 3

Pd/C in a mixture of A5 ( N '-(4-hydroxybenzoyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 180 mg, 0.427 mmol, 1.0 eq) dissolved in EtOH (10 mL). (200 mg) was added at 25°C. The mixture was then stirred under a H ₂ balloon at 25° C. for 4 hours. The solution was filtered and the filtrate was concentrated and purified by prep-HPLC to obtain compound 3 (4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide, 40 mg, 23.9% ) was obtained as a white solid. (TLC: N/A)

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.35 (d, J = 3.6 Hz, 1H), 10.06 (s, 1H), 9.20 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.23-7.28 (m, 2H), 6.76 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.4 Hz, 1H), 5.63 (s, 2H), 3.69 (t, J = 4.4 Hz, 4H), 2.62 (t, J = 4.4 Hz, 4H).

LCMS; Mass Calcd.: 392.4; MS Found: 393.

Experimental Example 1-4. compound 4 ( N Preparation of '-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide)

Step 1) Synthesis of A7

A mixture of chlorosulfonic acid (0.88 g, 7.52 mmol, 1.0 eq) and A6 (indolin-2-one, 1.0 g, 7.52 mmol, 1.0 eq) was stirred at 25 °C for 1.5 h followed by 68 °C for 1 h Stirred. The mixture was cooled and carefully poured into water. The precipitate formed was collected by filtration, washed with water and dried under vacuum to give A7 (2-oxoindoline-5-sulfonyl chloride, 0.7 g, crude) as a pink solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ10.47 (br s, 1H), 7.44-7.46 (m, 2H), 6.75 (d, J = 8.8 Hz, 1H), 3.47 (s, 2H).

Step 2) Synthesis of Compound 4

A2 (4-hydroxybenzohydrazide, 0.20 g, 1.32 mmol, 1.0 eq) and A7 (2-oxoindoline-5-sulfonyl chloride, 0.30 g, 1.32 mmol, 1.0 eq) in pyridine (10 mL) The mixture was stirred at 30 °C for 5 hours. The mixture was poured into water. The precipitate formed was collected by filtration, washed with water and dried under vacuum. The solid was stirred in DCM at 30 °C for 30 min. The mixture was filtered and the filtercake was dried to give compound 4 ( N ′-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide 50 mg, 11%) as a pink solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.79 (br s, 1H), 10.35 (br s, 1H), 10.11 (br s, 1H), 9.62 (br s, 1H), 7.63–7.66 (m, 2H), 7.57 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 3.51 ( s, 2H).

LCMS; Mass Calcd.: 347.3; MS Found: 347.8.

Experimental Example 1-5. compound 5 ( N' Preparation of (4-hydroxybenzoyl) indoline-5-sulfonohydrazide)

Step 1) Synthesis of A8

A mixture of A2 (4-hydroxybenzohydrazide, 293 mg, 1.93 mmol, 1.0 eq) and 1-acetylindoline-5-sulfonylchloride (500 mg, 1.93 mmol, 1.0 eq) in pyridine (10 mL) It was stirred for 5 hours at 30°C. After completion, the reaction mixture was diluted with H ₂ O (20 mL) and extracted with EA (20 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A8 (1-acetyl- N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide, 500 mg, crude) as yellow obtained as a solid.

Step 2) Synthesis of Compound 5

A8 (1-acetyl- N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide, 200 mg, 0.53 mmol, 1.0 eq) in THF (10 mL) and 2N HCl (6 mL) The mixture was stirred at 50 °C for 5 hours. After completion, the reaction mixture was diluted with H ₂ O (20 mL) and extracted with EA (20 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude product was purified by prep-HPLC and lyophilized to give compound 5 ( N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide, 50 mg, 28.2%) as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.30 (br s, 1H), 10.05 (br s, 1H), 9.14 (br s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.33-7.36 (m, 2H), 6.77 (d, J = 8.4 Hz, 2H), 6.37-6.40 (m, 2H), 3.51 (t, J = 8.4 Hz, 2H), 2.90 (t, J = 8.4 Hz, 2H).

LCMS; Mass Calcd.: 333.3; MS Found: 333.8.

Experimental Example 1-6. compound 6 ( N Preparation of '-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide)

Step 1) Synthesis of A10

Methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate ( A9 , 801 mg, 3.0 mmol), K ₃ PO ₄ (541 mg, 7.5 mmol) and Pd(dppf) Cl ₂ —CH ₂ Cl ₂ (208 mg, 0.25 mmol) were added at room temperature. The mixture was stirred at 100 °C for 12 hours. After the reaction was complete, the reaction mixture was cooled. The reaction mixture was filtered through celite and then extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (Hex/EA = 3/1) to give A10 (methyl [1,1'-biphenyl]-4-carboxylate, 160 mg, yield: 45%) as a white solid. got

1H NMR (DMSO-d ₆ , 600 MHz) δ (ppm): 8.05-8.03 (m, 2H), 7.84-7.83 (m, 2H), 7.76-7.74 (m, 2H), 7.52-7.50 (m, 2H) ), 7.50–7.42 (m, 1H), 3.87 (s, 3H); LCMS Calcd m/z for C ₁₄ H ₁₂ O ₂ [M+H] ⁺ 213.25 Found 213.

Step 2) Synthesis of A11

A solution of A10 (methyl [1,1'-biphenyl]-4-carboxylate, 160 mg, 0.70 mmol) in hydrazine monohydrate (8 mL) was stirred at 100 °C for 16 h. After the reaction was complete, the reaction mixture was cooled, then concentrated under reduced pressure and then purified by flash column chromatography (DCM/MeOH = 15/1) to obtain the crude product, A11 ([1,1'-biphenyl]-4-carb Bohydrazide, 152 mg, theoretical yield: 100%) was obtained as a white solid. LCMS Calcd m/z for C ₁₃ H ₁₂ N ₂ O [M+H] ⁺ 213.25 Found 213.

Step 3) Synthesis of A12

To a solution of A11 ([1,1'-biphenyl]-4-carbohydrazide, 152 mg, 0.70 mmol) in pyridine (5 mL) was added 4-nitrosulfonyl chloride (143 mg, 0.60 mmol) It became. The mixture was refluxed for 12 hours. After the reaction was complete, the resulting mixture was cooled and evaporated to remove pyridine and purified by flash column chromatography (DCM/MeOH = 15/1) to obtain the crude product, A12 ( N '-([1,1'-biphenyl ]-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 60 mg, yield: 21%) was obtained as a yellow solid. LCMS Calcd m/z for C ₁₉ H ₁₅ N ₃ O ₅ S [M+H] ⁺ 398.41 Found 398.

Step 4) Synthesis of Compound 6

A12 ( N '-([1,1'-biphenyl]-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 60 mg, 0.15 mmol) in THF:MeOH = 3:1 (12 mL) To a solution of Zn (99 mg, 1.5 mmol) and NH ₄ Cl (81 mg, 1.5 mmol) were added. The mixture was stirred at room temperature for 12 hours. Ethyl acetate was added after the reaction was complete. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 15/1) to give compound 6 (N'-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide , 10 mg, yield: 20%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.64 (br s, 1H), 9.32 (br s, 1H), 7.79-7.77 (m, 2H), 7.74-7.70 (m, 4H), 7.49 (t, 2H), 7.45-7.39 (m, 3H), 6.51 (d, J = 6.0 Hz, 2H), 5.96 (s, 1H), 6.52 (d, J = 5.0 Hz, 2H); LCMS Calcd m/z for C ₁₉ H ₁₇ N ₃ O ₃ S [M+H] ⁺ 368.42 Found 368.

Experimental Example 1-7. compound 7 ( N Preparation of '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide)

Step 1) Synthesis of A14

Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate ( A13 , 801 mg, 3.0 mmol), K ₃ PO ₄ (541 mg, 7.5 mmol) and Pd(dppf) Cl ₂ —CH ₂ Cl ₂ (208 mg, 0.25 mmol) were added at room temperature. The mixture was stirred at 100 °C for 12 hours. After the reaction was complete, the reaction mixture was cooled. The mixture was filtered through celite and then extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (Hex/EA = 3/1) to give A14 (methyl [1,1'-biphenyl] -3-carboxylate, 160 mg, yield: 49%) as a white solid. got

1H NMR (DMSO-d6, 600 MHz) δ (ppm): 8.18 (t, J = 1.8 Hz, 1H), 7.97-7.95 (m, 2H), 7.71-7.70 (m, 2H), 7.63 (t, J = 7.7 Hz, 1H), 7.52-7.49 (m, 2H), 7.43-7.41 (m, 1H), 3.89 (s, 3H); LCMS Calcd m/z for C ₁₄ H ₁₂ O ₂ [M+H] ⁺ 213.25 Found 213.

Step 2) Synthesis of A15

A solution of A14 (methyl [1,1'-biphenyl]-3-carboxylate, 160 mg, 7.5 mmol) in hydrazine monohydrate (8 mL) was stirred at 100 °C for 16 h. After the reaction was complete, the reaction mixture was cooled, then concentrated under reduced pressure and then purified by flash column chromatography (DCM/MeOH = 15/1) to obtain crude product A15 ([1,1'-biphenyl]-3-carbo Hydrazide, 200 mg, yield: 100%) was obtained as a yellow solid.

LCMS Calcd m/z for C ₁₃ H ₁₂ N ₂ O [M+H] ⁺ 213.25 Found 213.

Step 3) Synthesis of A16

To a solution of A15 ([1,1'-biphenyl]-3-carbohydrazide, 200 mg, 0.90 mmol) in pyridine (7 mL) was added 4-nitrosulfonyl chloride (2.5 mL). The mixture was refluxed for 12 hours. After the reaction was complete, the resulting mixture was cooled and evaporated to remove pyridine and then purified by flash column chromatography (DCM/MeOH = 15/1) to obtain the crude product, A16 ( N '-([1,1'-biphenyl) ]-3-carbonyl)-4-nitrobenzenesulfonohydrazide, 102 mg, yield: 53%) was obtained as an ivory solid.

LCMS Calcd m/z for C ₁₉ H ₁₅ N ₃ O ₅ S [M+H] ⁺ 398.41 Found 398.

step 4) Synthesis of Compound 7

A16 ( N '-([1,1'-biphenyl]-3-carbonyl)-4-nitrobenzenesulfonohydrazide, 102 mg, 0.25 mmol) in THF:MeOH = 3:1 (10 mL) To a solution of Zn (168 mg, 2.5 mmol) and NH ₄ Cl (137 mg, 2.5 mmol) were added. The mixture was stirred at room temperature for 12 hours. Ethyl acetate was added after the reaction was complete. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 15/1) to give compound 7 ( N '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide , 14 mg, yield: 14%, purity: 96.8%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.71 (br s, 1H), 9.37 (br s, 1H), 7.97 (s, 1H), 7.83 (d, J = 6.0 Hz, 1H) , 7.72 (d, J = 6.0 Hz, 2H), 7.65 (d, J = 6.0 Hz, 1H), 7.54–7.49 (m, 3H), 7.45 (d, J = 6.0 Hz, 2H), 7.42 (t, 1H), 6.52 (d, J = 6.0 Hz, 2H), 5.97 (s, 2H); ESI-MS Calcd m/z for C ₁₉ H ₁₇ N ₃ O ₃ S [M+H] ⁺ 368.42 Found 368.

Experimental Example 1-8. Compound 8 (3-amino- N Preparation of '-(4-hydroxybenzoyl)benzenesulfonohydrazide)

Step 1) Synthesis of A17

To a solution of A2 (200 mg, 0.90 mmol) in pyridine (8 mL) was added 3-nitrosulfonyl chloride (168 mg, 1.08 mmol). The mixture was refluxed for 12 hours. After the reaction was complete, the resulting mixture was cooled and evaporated to remove pyridine and then purified by flash column chromatography (DCM/MeOH = 15/1) to obtain the crude product, A17 ( N '-(4-hydroxybenzoyl)- 3-Nitrobenzenesulfonohydrazide, 129 mg, yield: 43%) was obtained as an ivory solid. LCMS Calcd m/z for C ₁₃ H ₁₁ N ₃ O ₆ S [M+H] ⁺ 338.31 Found 338.

Step 2) Synthesis of Compound 8

To a solution of A17 ( N ′-(4-hydroxybenzoyl)-3-nitrobenzenesulfonohydrazide, 129 mg, 0.38 mmol) in THF:MeOH = 3:1 (10 mL) was added Zn (250 mg, 3.8 mmol) and NH ₄ Cl (204 mg, 3.8 mmol) were added. The mixture was stirred at room temperature for 12 hours. Ethyl acetate was then added after the reaction was complete. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 15/1) to give compound 8 (3-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 25 mg, yield: 21%, purity: 98.0%) was obtained as a white solid. 1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.36 (br s, 1H), 10.06 (br s, 1H), 9.53 (br s, 1H), 7.59 (d, J = 12 Hz, 2H ), 7.11–7.08 (t, J = 9.0 Hz, 1H), 7.02 (s, 1H) 6.90 (d, J = 6.0 Hz, 1H), 6.77 (d, J = 6.0 Hz, 1H), 6.71 (d, J = 12 Hz, 1H), 5.49 (s, 2H); LCMS Calcd m/z for C ₁₃ H ₁₃ N ₃ O ₄ S [M+H] ⁺ 308.32 Found 308.

Experimental Example 1-9. Compound 9 (4-(1-aminoethyl)- N Preparation of '-(4-hydroxybenzoyl)benzenesulfonohydrazide)

Step 1) Synthesis of A18

A mixture of A2 (4-hydroxybenzohydrazide, 500 mg, 3.29 mmol, 1.0 eq) and 4-acetylbenzene-1-sulfonyl chloride (717 mg, 3.29 mmol, 1.0 eq) in pyridine (5 mL) was Stirred at 25° C. for 3 hours. The mixture was cooled and carefully poured into water. The mixture was extracted with EA (50 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give A18 (4-acetyl-N'-(4-hydroxybenzoyl)benzenesulfonohydrazide, 0.5 g, crude) as a brown solid. lost.

LCMS Calcd m/z for C ₁₅ H ₁₄ N ₂ O ₅ S [M+H] ⁺ 335.3 Found 335.

Step 2) Synthesis of Compound 9

A18 (4-acetyl-N'-(4-hydroxybenzoyl)benzenesulfonohydrazide, 200 mg, 0.60 mmol, 1.0 eq) in MeOH (5 mL), NH ₄ OAc (461 mg, 5.98 mmol, 10 eq) and sodium cyanoborohydride (188 mg, 2.99 mmol, 5 eq) was stirred at 60 °C for 16 h. The mixture was concentrated, purified by prep-HPLC and lyophilized to yield compound 9 (4-(1-aminoethyl) -N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 55 mg, 24%) as a white solid. was obtained with

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.37 (br s, 2H), 8.28 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.57 (dd, J = 11.2, 8.8 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 4.22 (q, J = 6.8 Hz, 1H), 1.33 (d, J = 6.4 Hz, 3H).

LCMS; Mass Calcd.: 335.3 (C ₁₅ H ₁₇ N ₃ O ₄ S); MS Found: 336.

Experimental Example 1-10. Compound 10 (3,5-diamino- N Preparation of '-(4-hydroxybenzoyl)benzenesulfonohydrazide)

Step 1) Synthesis of A20

To a mixture of 3,5-dinitroaniline ( A19 , 500 mg, 2.73 mmol, 1.0 eq) in concentrated HCl (10 mL) was added NaNO ₂ (226 mg, 3.28 mmol, 1.2 eq) in H ₂ O (2 mL). ) was added at 0 °C. The mixture was stirred at 0 °C for 0.5 h. To a mixture of CuCl (27 mg, 0.27 mmol, 0.1 eq) in H ₂ O (10 mL) was added SOCl ₂ (1.30 g, 10.9 mmol, 4.0 eq) at 0 °C. The diazo salt solution was then added dropwise at 0°C. The mixture was stirred at 0° C. for 3 hours and then poured into water. The precipitate that formed was collected by filtration and dried under vacuum to give A20 (3,5-dinitrobenzenesulfonyl chloride, 0.5 g, crude) as a yellow solid.

Step 2) Synthesis of A21

A mixture of A20 (3,5-dinitrobenzenesulfonyl chloride, 175 mg, 0.66 mmol, 1.0 eq) and A2 (4-hydroxybenzohydrazide, 100 mg, 0.66 mmol, 1.0 eq) in pyridine (5 mL). The mixture was stirred at 60° C. for 16 hours. The mixture was cooled and carefully poured into water. The mixture was extracted with EA (50 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give A21 ( N ′-(4-hydroxybenzoyl)-3,5-dinitrobenzenesulfonohydrazide, 0.1 g, crude) pink color. obtained as a solid.

Step 3) Compound 10 (3,5-diamino- N Synthesis of '-(4-hydroxybenzoyl)benzenesulfonohydrazide)

A21 ( N '-(4-hydroxybenzoyl)-3,5-dinitrobenzenesulfonohydrazide, 0.1 g, 0.26 mmol, 1.0 eq) and 10% Pd/C (0.1 g) dissolved in EtOH (5 mL). ) was stirred under a H ₂ balloon at room temperature for 3 hours. The reaction mixture was filtered and the filter cake was washed with EA (50 mL x 2). The combined filtrates were concentrated to give the crude product and stirred in MeOH (5 mL) for 10 min. The mixture was filtered and the filter cake was dried in vacuo to give compound 10 (3,5-diamino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 27 mg, 32%) as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.20 (br s, 1H), 10.04 (br s, 1H), 9.19 (br s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 6.26 (d, J = 1.6 Hz, 2H), 5.94 (s, 1H), 5.10 (s, 4H).

LCMS; Mass Calcd.: 322.3 (C ₁₃ H ₁₄ N ₄ O ₄ S); MS Found: 323 [MS+1].

Experimental Example 1-11. compound 11 ( N Preparation of '-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonohydrazide)

Compound 2 (4-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 500 mg, 1.63 mmol, 1.0 eq) and oxirane in AcOH/ _H 2 O=1:1 (4 mL) (72 mg, 1.63 mmol, 1.0 eq) was stirred at 25 °C for 4 h. NaHCO ₃ was then added and extracted with EA until the mixture was pH=8 (50 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by prep-TLC to compound 11 ( N ′-(4-hydroxybenzoyl)-4-((2-hydroxy oxyethyl)amino)benzenesulfonohydrazide, 50 mg, yield 8.8%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 7.56 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 6.56 (d, J = 8.8 Hz, 2H), 6.45 (t, J = 5.2 Hz, 1H), 3.53 (t, J = 6.0 Hz, 2H), 3.12 (q, J = 6.0 Hz, 2H).

LCMS; Mass Calcd.:351.3; MS Found: 351.8 [MS+1].

Experimental Example 1-12. Compound 12 (4-amino- N Preparation of -(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide)

Step 1) Synthesis of A23

To a mixture of A22 (1-(4-(benzyloxy)phenyl)ethan-1-one, 4.0 g, 17.7 mmol, 1.0 eq) in EtOH (40 mL) was added Br ₂ (2.83 g, 17.7 mmol, 1.0 eq). was added at 5 °C. The mixture was stirred at 30 °C for 30 minutes. The mixture was poured into PE and stirred for 30 minutes. The mixture was filtered and the filter cake was dried to give A23 (1-(4-(benzyloxy)phenyl)-2-bromoethane-1-one, 2.5 g, 46.3%) as a white solid.

Step 2) Synthesis of A24

A23 (1-(4-(benzyloxy)phenyl)-2-bromoethan-1-one, 2.0 g, 6.55 mmol, 1.0 eq) and HMTA (1.38 g, 9.83 mmol, 1.5 in DCM (20 mL)) The mixture of eq) was stirred at 10 °C for 2 h. The mixture was then filtered and the filter cake was dissolved in EtOH (15 mL) and concentrated HCl (5 mL). The mixture was stirred at 85 °C for 2 hours. The mixture is filtered and the filter cake is dried to give A24 (2-amino-1-(4-(benzyloxy)phenyl)ethan-1-one hydrochloride, 2.0 g, crude) was obtained as a white solid.

¹ HNMR (CD ₃ OD, 400 MHz): δ 8.03 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 7.2 Hz, 2H), 7.34-7.42 (m, 3H), 7.17 (d, J = 8.8 Hz, 2H), 5.23 (s, 2H), 4.55 (s, 2H).

Step 3) Synthesis of A25

A24 (2-amino-1-(4-(benzyloxy)phenyl)ethan-1-one hydrochloride in DCM (20 mL), 2.00 g, 7.20 mmol, 1.0 eq), a mixture of 4-nitrobenzene-1-sulfonyl chloride (1.60 g, 7.20 mmol, 1.0 eq) and TEA (2.19 g, 21.6 mmol, 3.0 eq) at 20 °C for 1 hour stirred while The mixture was poured into water and extracted with DCM. The organic layer was washed with water and brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was stirred in PE for 30 minutes. The mixture was filtered and a filter cake yielded A25 (N-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-4-nitrobenzenesulfonamide, 1.0 g, 35.8% for 2 steps) as a white solid. got

¹ HNMR (CD ₃ Cl, 400 MHz): δ 8.36 (d, J = 8.8 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.37- 7.43 (m, 5H), 7.03 (d, J = 8.4 Hz, 2H), 5.86 (t, J = 4.0 Hz, 1H), 5.16 (s, 2H), 4.49 (d, J = 4.0 Hz, 2H).

Step 4) Synthesis of Compound 12

A25 (N-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-4-nitrobenzenesulfonamide, 200 mg, 0.47 mmol, 1.0 eq) and Pd/C in EtOH (10 mL) (150 mg, 50% in water) was stirred under a H ₂ balloon at 25° C. for 4 hours. Then the mixture was filtered and the mixture was concentrated, purified by prep-HPLC and lyophilized to give compound 12 (4-amino- N- (2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide, 69 mg , yield 48.0%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.43 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.32 (t, J = 5.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 8.8 Hz, 2H), 5.91 (s, 2H), 4.19 (d, J = 5.6 Hz, 2H).

LCMS; Mass Calcd.:306.3; MS Found: 307 [MS+1].

Experimental Example 1-13. compound 13 ( N Preparation of '-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide)

To a solution of A2 (4-hydroxybenzohydrazide, 100 mg, 0.66 mmol) in DMF (6 mL) was added triethylamine (0.11 mL, 0.76 mmol) and 4-methoxybenzenesulfonyl chloride (124 mg, 0.60 mmol). mmol) was added. The mixture was kept at room temperature for 12 hours. After the reaction was complete, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 5/1) to give compound 13 (N'-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide, 25 mg, yield: 12% , purity: 96.9%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.37 (br s, 1H), 10.07 (br s, 1H), 9.64 (br s, 1H), 7.73 (d, J = 12 Hz, 2H ), 7.57 (d, J = 6.0 Hz, 2H), 7.03 (d, J = 6.0 Hz, 2H), 6.76 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H); LCMS Calcd m/z for C ₁₄ H ₁₄ N ₂ O ₅ S [M+H] ⁺ 323.3 Found 323.

Experimental Example 1-14. Preparation of compound 14 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide

Step 1) Synthesis of A26

To a mixture of A2 (4-hydroxybenzohydrazide, 1.00 g, 6.57 mmol, 1.0 eq) in pyridine (10 mL) was added 4-cyanobenzene-1-sulfonyl chloride (1.33 g in pyridine (3 mL)). , 6.57 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 25° C. for 3 hours. The solution was poured into water (50 mL). The mixture was extracted with EA (50 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (PE/EA=1/1) to A26 (4-cyano- N '-(4- Hydroxybenzoyl)benzenesulfonohydrazide, 1.40 g, 67.1%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.49 (s, 1H), 10.27 (s, 1H), 10.14 (s, 1H), 8.02-8.04 (m, 2H), 7.97-7.99 (m, 2H) ), 7.57 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H).

Step 2) Synthesis of Compound 14

A mixture of A26 (4-cyano- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 200 mg, 0.63 mmol, 1.0 eq) in HCl/EtOH (5 mL, 6 mol/L) was 25 It was stirred for 3 hours at °C. The solution was concentrated and MeOH was added. The mixture was concentrated again. The residue was added to MeOH (10 mL) followed by NH ₄ OAc (485 mg, 6.30 mmol, 10 eq). The mixture was stirred at 25 °C for 16 hours. The mixture was concentrated, purified by prep-HPLC and lyophilized to give compound 14 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide, 26 mg, 10.8%) as a white solid. lost.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 8.43 (s, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H).

LCMS; Mass Calcd.:334; MS Found: 334.8 [MS+1].

Experimental Example 1-15. Preparation of compound 15 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide)

To a cooled (0°C) solution of A26 (4-cyano- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide, 210 mg, 0.66 mmol) in DMSO was added hydrogen peroxide, 35% w/w aq. Soln., (0.42 mL, 4.8 mmol) and potassium carbonate (30 mg, 0.20 mmol) were added. The reaction was warmed to room temperature and stirred for 12 hours. After the reaction was complete, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 5/1) to give compound 15 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide, 20 mg, yield: 10% , purity: 96.5%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.44 (br s, 1H), 10.10 (br s, 1H), 10.03 (br s, 1H), 8.15 (br s, 1H), 7.96 ( d, J = 6.0 Hz, 2H), 7.87 (d, J = 10 Hz, 2H), 7.59 (br s, 1H), 7.57 (d, J = 6.0 Hz, 2H), 6.76 (d, J = 6.0 Hz) , 2H); LCMS Calcd m/z for C ₁₄ H ₁₃ N ₃ O ₅ S [M+H] ⁺ 336.33 Found 336.

Experimental Example 1-16. compound 16 ( N Preparation of -(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide)

Step 1) Synthesis of A28

To a mixture of A27 (4-hydroxybenzamide, 4.0 g, 29.2 mmol, 1.0 eq) in DMF (40 mL) was added K ₂ CO ₃ (6.05 g, 43.8 mmol, 1.5 eq) and BnBr (4.99 g, 29.2 mmol). , 1.0 eq) was added. The mixture was stirred at 60° C. for 16 hours. The mixture was poured into water and extracted with EA. The organic layer was washed with water and brine, dried over Na ₂ SO ₄ and concentrated to give A28 (4-(benzyloxy)benzamide, 6.0 g, 90.5%) as a white solid.

Step 2) Synthesis of A29

A28 (4-(benzyloxy)benzamide, 4.0 g, 17.6 mmol, 1.0 eq), K ₂ CO ₃ (0.24 g, 1.76 mmol) in THF/H ₂ O (40 mL, v/v=1/1) , 0.1 eq) and HCOH (1.43 g, 17.6 mmol, 37% in water, 1.0 eq) was stirred at 65° C. for 16 h. The mixture was then concentrated and filtered. The filter cake was dried to give A29 (4-(benzyloxy) -N- (hydroxymethyl)benzamide, 4.0 g, crude) as a white solid.

Step 3) Synthesis of A30

A29 (4-(benzyloxy) -N- (hydroxymethyl)benzamide, 4.00 g, 15.6 mmol, 1.0 eq) and 4-nitrobenzenethiol (2.41 g, 15.6 mmol, 1.0 eq) in TFA (20 mL) ) was stirred at 20 °C for 1 hour. The mixture was concentrated and added to water and aq. with NaHCO ₃ Adjusted to pH=8. The mixture was extracted with EA. The organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to give A30 (4-(benzyloxy) -N -(((4-nitrophenyl)thio)methyl)benzamide, 2.0 g, 32.6% for 2 steps ) was obtained as a white solid.

Step 4) Synthesis of A31

A30 (4-(benzyloxy) -N -(((4-nitrophenyl)thio)methyl)benzamide, 500 mg, 1.27 mmol, 1.0 eq) and m-CPBA (656 g, A mixture of 3.80 mmol, 3.0 eq) was stirred at 20 °C for 16 h. The mixture was aq. It was poured with Na ₂ O ₃ S ₂ and extracted. The organic layer was aq. washed with NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give A31 (4-(benzyloxy) -N -(((4-nitrophenyl)sulfonyl)methyl)benzamide, 300 mg, crude) It was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 9.39 (t, J = 6.4 Hz, 1H), 8.42 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.34–7.46 ( m , 5H), 7.08 (d, J = 8.8 Hz, 2H), 5.17 (s, 2H), 5.00 (d, J = 6.4 Hz, 2H) .

Step 5) Synthesis of Compound 16

A31 (4-(benzyloxy) -N -(((4-nitrophenyl)sulfonyl)methyl)benzamide, 300 mg, 0.47 mmol, 1.0 eq) and Pd/C (150 mg) in EtOH (10 mL) , 50% in water) was stirred under a H ₂ balloon at 25° C. for 2 hours. Then the mixture was filtered and the mixture was concentrated, purified by prep-HPLC and lyophilized to give compound 16 (N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide, 35 mg, yield 9.0% for 2 steps) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.15 (br s, 1H), 9.06 (t, J = 6.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.58 (d, J = 8.8 Hz, 2H), 6.12 (s, 2H), 4.65 (d, J = 6.4 Hz, 2H).

LCMS; Mass Calcd.:306; MS Found: 307 [MS+1].

Experimental Example 1-17. Compound 17 (6-amino- N Preparation of '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonohydrazide)

Step 1) Synthesis of A33

A32 (3-bromo-4-nitroaniline, 1 g, 4.61 mmol, 1.0 eq), phenylboronic acid (0.56 g, 4.61 mmol, 1.0 eq), Pd(dppf)Cl ₂ in toluene (10 mL) A mixture of 337 mg, 0.09 mmol, 0.1 eq) and AcOK (1.14 g, 13.8 mmol, 3.0 eq) was stirred for 16 h at 90° C. under N ₂ . The mixture was poured into water (30 mL) and extracted with EA (30 mL x 2). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude product was purified by flash column (PE/EA = 30/1 to 10/1) to yield A33 (6-nitro-[1,1'-biphenyl]-3-amine, 1 g, crude) as a yellow solid. got

Step 2) Synthesis of A34

Add _{H 2} O ( ₂ mL) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 0.5 h. To a mixture of CuCl (31.3 mg, 0.23 mmol, 0.1 eq) in H ₂ O (10 mL) was added SOCl ₂ (1.39 g, 11.6 mmol, 5.0 eq) at 0 ^° C. The diazo salt solution was then added dropwise at 0°C. The mixture was stirred at 0° C. for 3 hours and then poured into water. The precipitate that formed was collected by filtration and dried under vacuum to give A34 (6-nitro-[1,1′-biphenyl]-3-sulfonyl chloride, 400 mg, 57.6%) as a yellow solid.

^1HNMR (DMSO- d ₆ , 400 MHz): δ 7.98 (d, J = 8.4 Hz, 1H), 7.78-7.80 (m, 1H), 7.64 (d, J = 1.2 Hz, 1H), 7.44-7.50 ( m, 3H), 7.32-7.34 (m, 2H).

Step 3) Synthesis of A35

A34 (6-nitro-[1,1'-biphenyl]-3-sulfonyl chloride, 200 mg, 0.67 mmol, 1.0 eq) and 4-hydroxybenzohydrazide (122 mg, A mixture of 0.81 mmol, 1.2 eq) was stirred at 30 °C for 0.5 h. The mixture was carefully poured into the water. The mixture was extracted with EA (50 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to A35 ( N ′-(4-hydroxybenzoyl)-6-nitro-[1,1′-biphenyl]-3-sulfonohydra Zide, 200 mg, crude) was obtained as a brown solid.

Step 4) Synthesis of Compound 17

A35 ( N ′-(4-hydroxybenzoyl)-6-nitro-[1,1′-biphenyl]-3-sulfonohydrazide, 200 mg, 0.48 mmol, 1.0 eq) in EtOH (10 mL) and 10% Pd/C (200 mg) was stirred under a H ₂ balloon at 30° C. for 3 hours. The reaction mixture was filtered and the filter cake was washed with EA (50 mL x 2). The combined filtrates were concentrated to give the crude product, which was purified by prep-HPLC and lyophilized to give compound 17 (6-amino- N '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3 -sulfonohydrazide, 45 mg, 17.5% for 2 steps) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.39 (s, 1H), 10.09 (s, 1H), 9.32 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.32–7.59 (m, 5H), 7.23 (d, J = 6.4 Hz, 2H), 6.73–6.79 (m, 3H), 5.64 (s, 2H).

LCMS; Mass Calcd.: 383; MS Found: 384 [MS+1].

Experimental Example 1-18. Preparation of compound 18 (4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide)

Step 1) Synthesis of A37

To a cooled (0 °C) solution of methyl 4-(chlorocarbonyl)benzoate (A36, 1.5 g, 7.5 mmol) in DCM was added ammonia solution (25-30%) (1.8 mL, 15 mmol). The reaction was warmed to room temperature and stirred for 4 hours. After the reaction was complete, the reaction mixture was evaporated and then water was added. The precipitate formed was collected by filtration. The filter cake was washed with water and dried to give A37 (methyl 4-carbamoylbenzoate, 1.0 g, 74%) as a white solid. LCMS Calcd m/z for C ₉ H ₉ NO ₃ [M+H] ⁺ 152.17 Found 152.

Step 2) Synthesis of A38

A solution of A37 (methyl 4-carbamoylbenzoate, 1 g, 6.6 mmol) and hydrazine monohydrate (10 mL) in MeOH (10 mL) was stirred at 80 °C for 16 h. The solvent was concentrated under reduced pressure to give a crude product which was purified by flash column (DCM/MeOH = 10/1) to give A38 (4-(hydrazinecarbonyl)benzamide, 600 mg, yield: 60%) as a white solid. got

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 9.87 (s, 1H), 8.06 (s, 1H), 7.92-7.91 (m, 2H), 7.87-7.86 (m, 2H), 7.48 ( s, 1H), 4.55 (brs, 2H).

Step 3) Synthesis of A39

To a solution of A38 (4-(hydrazinecarbonyl)benzamide, 600 mg, 3.3 mmol) in DMF (7 mL) was added TEA (0.55 mL, 3.9 mmol) and 4-methoxybenzenesulfonyl chloride (676 mg, 3.0 mmol). mmol) was added. The mixture was kept at room temperature for 12 hours. After the reaction was complete, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 10/1) to give A39 (4-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide, 366 mg, yield : 30%) was obtained as a white solid. LCMS Calcd m/z for C ₁₄ H ₁₂ N ₄ O ₆ S [M+H] ⁺ 365.33 Found 365.

Step 4) Synthesis of Compound 18

Zn in a solution of A39 (4-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide, 366 mg, 1.0 mmol) in THF:MeOH = 3:1 (10 mL) (657 mg, 10 mmol) and NH ₄ Cl (537 mg, 10 mmol) were added at 60° C. for 5 h. After the reaction was complete, the reaction mixture was cooled and then ethyl acetate was added. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 15/1) to give compound 18 (4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide, 10 mg, 4.51 mmol, yield: 3%, purity: 97.0%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.68 (br s, 1H), 9.38 (br s, 1H), 8.06 (s, 1H), 7.90 (d, J = 6.0 Hz, 2H) , 7.73 (d, J = 6.0 Hz, 2H), 7.05 (s, 1H), 7.44 (d, J = 6.0 Hz, 2H), 6.51 (d, J = 6.0 Hz, 2H), 5.97 (s, 2H) ; LCMS Calcd m/z for C ₁₄ H ₁₄ N ₄ O ₄ S [M+H] ⁺ 335.35 Found 335.

Experimental Example 1-19. compound 19 ( N Preparation of '-(4-aminobenzyl)-4-hydroxybenzohydrazide)

Step 1) Synthesis of A40

To a solution of A2 (500 mg, 3.2 mmol) in DMF (10 mL) was added triethylamine (0.46 mL, 3.2 mmol) and 1-(bromomethyl)-4-nitrobenzene (545 mg, 2.5 mmol). It became. The mixture was kept at room temperature for 12 hours. After the reaction was complete, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 5/1) to give A40 (4-hydroxy- N '-(4-nitrobenzyl)benzohydrazide, 410 mg, yield: 44%) as a yellow solid. was obtained with LCMS Calcd m/z for C ₁₄ H ₁₃ N ₃ O ₄ [M+H] ⁺ 288.28 Found 288.

Step 2) Synthesis of Compound 19

Zn (933 mg, 14 mmol) to a solution of A40 (4-hydroxy- N '-(4-nitrobenzyl)benzohydrazide, 410 mg, 1.4 mmol) in THF:MeOH = 3:1 (12 mL) and NH ₄ Cl (764 mg, 14 mmol) were added. The mixture was stirred at room temperature for 12 hours. Ethyl acetate was then added after the reaction was complete. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 15/1) to give compound 19 ( N '-(4-aminobenzyl)-4-hydroxybenzohydrazide, 6 mg, yield: 16%, purity: 95.0%) was obtained as a white solid.

1H NMR (DMSO-d6, 600 MHz) δ (ppm): δ 10.03 (br s, 1H), 9.80 (br s, 1H), 7.67 (d, J = 6.0 Hz, 2H), 7.00 (d, J = 6.0 Hz, 2H), 6.77 (d, J = 6.0 Hz, 2H), 6.51 (d, J = 6.0 Hz, 2H), 4.97 (bs, 3H), 3.72 (bs, 2H); LCMS Calcd m/z for C ₁₄ H ₁₅ N ₃ O ₂ [M+H] ⁺ 258.29 Found 258.

Experimental Example 1-20. Compound 20 (4-amino- N '-(One H Synthesis of -indole-3-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A42

To a mixture of A41 (1 H -indole-3-carbohydrazide, 500 mg, 2.85 mmol, 1.0 eq) in pyridine (5 mL) was added 4-nitrobenzene-1-sulfonyl chloride in pyridine (5 mL) ( 632 mg, 2.85 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 10° C. for 2 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1~30/1) to A42 ( N '-(1 H- Indole-3-carbonyl)-4-nitrobenzenesulfonohydrazide, 0.9 g, yield 87.5%) was obtained as a yellow solid. (TLC: DCM/MeOH =20/1, Rf=0.5)

Step 2) Synthesis of Compound 20

To a mixture of A42 ( N ′-(1 H- indole-3-carbonyl)-4-nitrobenzenesulfonohydrazide, 300 mg, 0.83 mmol, 1.0 eq) in MeOH (10 mL), Pd/C (100 mg) was added. The mixture was then stirred under a H ₂ balloon at 10° C. for 2 hours. The solution was filtered, and the filtrate was purified by prep-HPLC and lyophilized to obtain compound 20 (4-amino- N '-(1 H -indole-3-carbonyl)benzenesulfonohydrazide, 30 mg, yield 10.9% ) was obtained as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 11.62 (d, J = 2.8 Hz, 1H), 9.98 (s, 1H), 9.11 (d, J = 3.2 Hz, 1H), 8.00 (d, J = 2.8 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.40–7.46 (m, 3H), 7.05–7.16 (m, 2H), 6.51 (d, J = 8.8 Hz, 2H), 5.89 (br s, 2H).

LCMS; Mass Calcd.:330; MS Found: 331.1 [MS+1].

Experimental Example 1-21 and Experimental Example 1-22. Compound 21 (4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide) and Compound 22 ( N Preparation of '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide)

Step 1) Synthesis of Compound 22

A4 (3-fluoro- N '-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 400 mg, 1.13 mmol, 1.0 eq) and K ₂ CO ₃ (389 mg, 2.81 mmol, 2.5 eq) was added pyrrolidine (96 mg, 1.35 mmol, 1.2 eq) at 10 °C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1-30/1) to compound 22 ( N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide, 176 mg, yield 38.5%) was obtained as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.48 (s, 1H), 10.14 (s, 2H), 7.86 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H) , 7.37 (d, J = 1.6 Hz, 1H), 7.11–7.13 (m, 1H), 6.78 (d, J = 8.8 Hz, 2H), 3.64 (t, J = 6.0 Hz, 4H), 1.86 (t, J = 6.0 Hz, 4H).

Step 2) Synthesis of Compound 21

Compound 22 ( N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide, 100 mg, 0.54 mmol, 1.0 eq in MeOH (5 mL) ) was added Pd/C (30 mg). The mixture was then stirred under a H ₂ balloon at 10° C. for 16 hours. The solution was filtered and the filtrate was concentrated to give the crude product which was stirred in MeOH (5 mL) and DMSO (0.5 mL) for 5 min. The mixture was filtered and the filter cake was washed with MeOH and dried to obtain compound 21 (4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide, 30 mg , yield 32.4%) was obtained as an off-white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.34 (s, 1H), 10.06 (s, 1H), 9.13 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.17 (t, J = 1.6 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.4 Hz, 1H), 5.47 (s, 2H), 2.80 (s, 4H), 1.77 (s, 4H).

LCMS; Mass Calcd.:330; MS Found: 331.1 [MS+1].

Experimental Example 1-23. Compound 23 (4-amino- N Synthesis of '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydrazide)

Step 1) Synthesis of A43

A4 (3-fluoro- N '-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 400 mg, 1.13 mmol, 1.0 eq) and K ₂ CO ₃ (389 mg, 2.81 mmol, 2.5 eq) was added piperidine (115 mg, 1.35 mmol, 1.2 eq) at 10°C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1~30/1) to A43 ( N '-(4-hydroxy) Roxybenzoyl)-4-nitro-3-(piperidin-1-yl)benzenesulfonohydrazide, 240 mg, yield 50.7%) was obtained as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.53 (s, 1H), 10.21 (s, 1H), 10.15 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 1.6 Hz, 1H), 7.40–7.43 (m, 1H), 6.79 (d, J = 8.8 Hz, 2H), 2.88 (t, J = 5.2 Hz, 4H), 1.50-1.51 (m, 6H).

Step 2) Synthesis of Compound 23

A43 ( N ′-(4-hydroxybenzoyl)-4-nitro-3-(piperidin-1-yl)benzenesulfonohydrazide, 100 mg, 0.24 mmol, 1.0 eq) in MeOH (5 mL) To the mixture was added Pd/C (30 mg). The mixture was then stirred under a H ₂ balloon at 10° C. for 16 hours. The solution was filtered and the filtrate was purified by prep-HPLC and lyophilized to form compound 23 (4-amino- N '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydra Zide, 25 mg, yield 26.9%) was obtained as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ10.37 (d, J = 4.0 Hz, 1H), 10.07 (s, 1H), 9.17 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.2-7.25 (m, 2H), 6.77 (d, J = 7.2 Hz, 2H), 6.64 (d, J = 8.4 Hz, 1H), 5.51 (s, 2H), 2.55 (s, 4H), 1.55-1.60 (m, 4H), 1.45 (s, 2H).

LCMS; Mass Calcd.:390; MS Found: 390.8 [MS+1].

Experimental Example 1-24. compound 24 ( N '-(4-hydroxybenzoyl)-1 H Synthesis of -pyrazole-4-sulfonohydrazide)

Step 1) Synthesis of A44

A solution of 1 H -pyrazole (1 g, 14.7 mmol, 1.0 eq) in chlorosulfonic acid (5 mL) was stirred at 100 °C overnight. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A44 (1 H -pyrazole-4-sulfonyl chloride, 340 mg, yield 14%) as an off-white solid.

^1HNMR (CDCl3, 400 MHz): 8.22 (s, 2H), 7.92 (s, 1H).

Step 2) Synthesis of Compound 24

A44 (1 H -pyrazole-4-sulfonyl chloride, 100 mg, 0.6 mmol, 1.0 eq) and A2 (4-hydroxybenzohydrazide, 109 mg, 0.72 mmol, 1.2 eq) in pyridine (20 mL) The mixture was stirred overnight at 80 °C. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residue was purified by prep-HPLC to give compound 24 ( N ′-(4-hydroxybenzoyl)-1 H -pyrazole-4-sulfonohydrazide, 60 mg) as an off-white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): 13.4 (s, 1H), 10.39 (s, 1H), 10.1 (s, 1H), 9.5 (s, 1H), 8.47-7.63 (m, 2H), 7.62 (d, 2H), 6.78 (d, 2H).

Experimental Example 1-25 and Experimental Example 1-26. compound 25 ( N '-(4-hydroxybenzoyl)indoline-4-sulfonohydrazide) and compound 26 ( N '-(4-hydroxybenzoyl)-1 H -Indole-4-sulfonohydrazide) Preparation

Step 1) Synthesis of A46

NaH ( ₂₀₄ mg, 5.10 mmol, 60% in mineral oil, 1.0 eq) was added at 0 °C. After stirring for 15 min, the mixture was cooled to -78 °C and t-BuLi (7.9 mL, 10.2 mmol, 1.3 M in THF, 2.0 eq) was added slowly. After 30 min, SO ₂ (gas, 1 L) was added slowly at -78 °C. The mixture was warmed to room temperature and stirred overnight. A mixture of acetic acid (307 mg, 5.10 mmol, 1.0 eq) in Et ₂ O (15 mL) was added at 0 °C. The mixture was stirred at 0° C. for 30 min and then filtered. The filter cake was quickly washed with Et ₂ O. The solid was suspended in Et ₂ O (15 mL), cooled to 0 °C and NCS (682 g, 5.10 mmol, 1.0 eq) was carefully added. The resulting suspension was stirred rapidly for 30 minutes and then filtered. The filter cake was washed with Et ₂ O. The combined filtrates were concentrated to give A46 (1 H -indole-4-sulfonyl chloride, 420 mg, crude) as a brown solid.

Step 2) Synthesis of Compound 26

A mixture of A46 (1 H -indole-4-sulfonyl chloride, 420 mg, 1.95 mmol, 1.0 eq) and 4-hydroxybenzohydrazide ( A2 , 297 mg, 1.95 mmol, 1.0 eq) in pyridine (30 mL). The mixture was stirred at 25 °C for 30 minutes. The solution was poured into water (30 mL). The solid formed was collected by filtration and the filtercake was washed with water. The crude product was purified by prep-HPLC and lyophilized to give compound 26 ( N ′-(4-hydroxybenzoyl) -1H -indole-4-sulfonohydrazide, 200 mg, yield 31.0%) as a white solid. lost.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 11.48 (s, 1H), 10.30 (s, 1H), 10.04 (s, 1H), 9.52 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.49–7.52 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.85 (t, J = 2.0 Hz, 1H), 6.73 (dd, J = 6.8, 2.0 Hz, 2H).

LCMS; Mass Calcd.:331.3; MS Found: 331.9 [MS+1].

Step 3) Synthesis of Compound 25

A mixture of compound 26 ( N ′-(4-hydroxybenzoyl)-1 H -indole-4-sulfonohydrazide, 150 mg, 0.48 mmol, 1.0 eq) in TFA (5 mL) and DCM (5 mL). To NaBH ₃ CN (89 mg, 1.43 mmol, 3.0 eq) was added at 0 °C. The mixture was stirred at 10 °C for 30 minutes. The solution was poured into water (30 mL) and extracted with DCM (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by prep-HPLC and lyophilized to give compound 25 ( N ′-(4-hydroxybenzoyl)indoline-4- sulfonohydrazide, 30 mg, yield 20.0%) was obtained as a gray solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.35 (d, J = 2.8 Hz, 1H), 10.09 (s, 1H), 9.62 (d, J = 2.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 6.97–6.99 (m, 1H), 6.93 (d, J = 6.8 Hz, 1H), 6.76 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 7.2 Hz, 1H) , 3.39–3.44 (m, 2H), 3.27–3.32 (m, 2H).

LCMS; Mass Calcd.:333; MS Found: 333.8 [MS+1].

Experimental Example 1-27. Compound 27 (2-((4-aminophenyl)sulfonyl)- N -Preparation of phenylhydrazine-1-carboxamide)

Step 1) Synthesis of A47

To a mixture of 4-nitrobenzenesulfonyl chloride (3 g, 13.5 mmol, 1.0 eq) in pyridine (25 mL) was added tert-butyl hydrazinecarboxylate (1.8 g, 13.6 mmol, 1.0 eq) in pyridine (5 mL). has become an enemy. The mixture was then stirred at 10° C. for 2 hours. The solution was poured into water (100 mL) and the solution stirred for 1 hour. The formed solid was collected by filtration and dried to give A47 ( tert -butyl 2-((4-nitrophenyl)sulfonyl)hydrazine-1-carboxylate, 3.0 g, yield 69.7%) as a yellow solid.

¹ HNMR (CDCl ₃ , 400 MHz): δ 8.35 (d, J = 8.4 Hz, 2H), 8.13 (dd, J = 7.2, 2.0 Hz, 2H), 6.79 (s, 1H), 6.68 (s, 1H), 1.25 (s, 9H).

Step 2) Synthesis of A48

To a mixture of A47 ( tert -butyl 2-((4-nitrophenyl)sulfonyl)hydrazine-1-carboxylate, 3 g, 9.45 mmol, 1.0 eq) in MeOH (30 mL) was added with MeOH/HCl (30 mL). , 6 mol/L) was added. The mixture was then stirred at 10° C. for 2 hours. The solution was concentrated to give A48 (4-nitrobenzenesulfonohydrazide hydrochloride, 2.0 g, yield 83.4%) as a yellow solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 8.45-8.48 (m, 2H), 8.15 (d, J = 8.8 Hz, 2H).

Step 3) Synthesis of A49

To a mixture of A48 (4-nitrobenzenesulfonohydrazide hydrochloride, 500 mg, 1.97 mmol, 1.0 eq) in THF (20 mL) was added DIEA (764 mg, 5.91 mmol, 3.0 eq) and isocyanatobenzene (235 mg, 1.97 mmol, 1.0 eq) was added at 10 °C. The mixture was then stirred at 10° C. for 2 hours. The solution was poured into water (80 mL). The solid formed was filtered and the filter cake was stirred in EA (20 mL) for 30 min. Then the mixture was filtered again and the filtercake was dried to give A49 (2-((4-nitrophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide, 340 mg, yield 51.3%) as a white solid. .

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 10.09 (s, 1H), 8.69 (s, 1H), 8.53 (s, 1H), 8.40-8.43 (m, 2H), 8.09 (dd, J = 7.2 , 2.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.21 (t, J = 8.0 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H).

Step 4) Synthesis of Compound 27

A49 (2-((4-nitrophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide, 340 mg, 1.01 mmol, 1.0 eq) and Pd/C (200 mg) in MeOH (30 mL) The mixture was stirred at 10° C. for 15 hours under a H ₂ balloon. The solution was filtered and the filtrate was concentrated to give the crude product which was stirred in MeOH (5 mL) for 30 min. The mixture was filtered and the filter cake was dried in vacuo to give compound 27 (2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide, 50 mg, yield 16.2%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 9.12 (s, 1H), 8.41 (s, 1H), 8.15 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 8.0 Hz, 2H), 6.94 (t, J = 7.2 Hz, 1H), 6.60 (d, J = 8.8 Hz, 2H), 6.04 (s, 2H).

LCMS; Mass Calcd.:306; MS Found: 306.9.

Experimental Example 1-28. Compound 28 (4-amino- N '-(One H -Indole-4-carbonyl) -3-morpholinobenzenesulfonohydrazide) Preparation

Step 1) Synthesis of A51

A mixture of A50 (methyl 1 H -indole-4-carboxylate, 1.00 g, 5.71 mmol, 1.0 eq) in N ₂ H ₄ H ₂ O (10 mL) was stirred at 100 °C for 1 hour. The solution was poured into water and extracted with EA (30 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give A51 (1H-indole-4-carbohydrazide, 500 mg, crude) as a yellow solid.

Step 2) Synthesis of A52

To a mixture of A51 (1H-indole-4-carbohydrazide, 100 mg, 0.57 mmol, 1.0 eq) in pyridine (2 mL) was added 3-fluoro-4-nitrobenzene-1- in pyridine (2 mL). Sulfonyl chloride (175 mg, 0.57 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with 1N HCl (30 mL x 2) and brine, dried over Na ₂ SO ₄ and concentrated to A52 (3-fluoro- N '-(1 H -indole-4-carbonyl)-4- Nitrobenzenesulfonohydrazide, 200 mg, crude) was obtained as a yellow solid.

Step 3) Synthesis of A53

A52 (3-fluoro- N '-( 1H -indole-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 200 mg, 0.52 mmol, 1.0 eq) and K ₂ in DMF (5 mL) To a mixture of CO ₃ (183 mg, 1.30 mmol, 2.5 eq) was added morpholine (54 mg, 0.62 mmol, 1.2 eq) at 10 °C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A53 ( N ′-(1 H -indole-4-carbonyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 100 mg, crude) was obtained as a yellow solid.

Step 4) Synthesis of Compound 28

A mixture of A53 ( N ′-(1 H -indole-4-carbonyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 100 mg, 0.22 mmol, 1.0 eq) in EtOH (5 mL). to Fe (61.6 mg, 1.10 mmol, 5.0 eq) and sat. aq. NH ₄ Cl (3 mL) was added. The mixture was then stirred at 85° C. for 3 hours. The solution was filtered and the filtrate was purified by prep-HPLC and lyophilized to give compound 28 (4-amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide, 20 mg, yield 21.5%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ11.30 (s, 1H), 10.34 (s, 1H), 9.35 (d, J = 4.0 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H) ), 7.43 (t, J = 2.4 Hz, 1H), 7.30-7.35 (m, 3H), 7.11 (t, J = 7.6 Hz, 1H), 6.65-6.67 (m, 2H), 5.60 (br s, 2H), 3.64 (t, J = 4.0 Hz, 4H), 2.60 (t, J = 4.0 Hz, 4H).

LCMS; Mass Calcd.:415; MS Found: 415.9 [MS+1].

Experimental Example 1-29. Compound 29 (4-amino- N Preparation of '-(indoline-4-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A54

To a mixture of A51 (1 H -indole-4-carbohydrazide, 400 mg, 2.29 mmol, 1.0 eq) in pyridine (5 mL) was added 4-nitrobenzene-1-sulfonyl chloride in pyridine (5 mL) ( 505 mg, 2.29 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A54 ( N ′-(1 H -indole-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 300 mg, crude) as yellow obtained as a solid.

Step 2) Synthesis of A55

To a mixture of A54 ( N '-( 1H -indole-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 300 mg, 0.83 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1.5 mL). and NaBH ₃ CN (157 mg, 2.49 mmol, 3.0 eq) at 10 °C. The mixture was then stirred at 10° C. for 45 minutes. The solution was poured into water (30 mL) and sat. aq. with NaHCO ₃ Adjusted to pH = 7. The mixture was filtered and the filter cake was washed with MTBE and dried in vacuo to give A55 ( N ′-(indoline-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 150 mg, crude) as a yellow solid .

Step 3) Synthesis of Compound 29

A mixture of A55 ( N '-(indoline-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 150 mg, 0.41 mmol, 1.0 eq) and Pd/C (100 mg) in MeOH (5 mL). The mixture was stirred at 10° C. for 2 hours under a H ₂ balloon. The solution was filtered and the filtrate was concentrated to give the crude product which was stirred in MeOH (10 mL) for 30 min. The mixture was filtered and the filter cake was dried in vacuo to give compound 29 (4-amino- N '-(indoline-4-carbonyl)benzenesulfonohydrazide, 40 mg, yield 29.1%) as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ10.28 (d, J = 3.6 Hz, 1H), 9.28 (d, J = 3.6 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.03 (t, J = 8.0 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 6.75 (d, J = 7.2 Hz, 1H), 6.53 (d, J = 8.8 Hz, 2H), 3.42 (t, J = 8.4 Hz, 2H) , 2.96 (t, J = 8.4 Hz, 2H).

LCMS; Mass Calcd.:332; MS Found: 332.8 [MS+1].

Experimental Example 1-30. Preparation of compound 30 (4-amino-N'-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonohydrazide)

Step 1) Synthesis of A56

A4 (3-fluoro- N '-(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 500 mg, 1.41 mmol, 1.0 eq) and K ₂ CO ₃ (290 mg) in DMF (5 mL). mg, 2.10 mmol, 1.5 eq) was added tert-butyl piperazine-1-carboxylate (315 mg, 1.69 mmol, 1.2 eq) at 10°C. The mixture was then stirred at 10° C. for 16 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by flash column (DCM/MeOH=50/1-30/1) to yield A56 ( tert -butyl 4-(5 Obtained -((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-2-nitrophenyl)piperazine-1-carboxylate, 310 mg, crude) as a yellow solid.

Step 2) Synthesis of A57

A56 ( tert -butyl 4-(5-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-2-nitrophenyl)piperazine-1-carboxylate, 250 in EtOH (3 mL) mg, 0.48 mmol, 1.0 eq) sat. aq. NH ₄ Cl (3 mL) and Fe (135 mg, 2.41 mmol, 5.0 eq) were added. The mixture was then stirred at 85° C. for 1 hour. The solution was filtered and the filtrate was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A57 ( tert -butyl 4-(2-amino-5-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)phenyl) Piperazine-1-carboxylate, 210 mg, crude) was obtained as a yellow solid.

Step 3) Synthesis of Compound 30

A57 ( tert -butyl 4-(2-amino-5-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)phenyl)piperazine-1-carboxylate, 270 in DCM (5 mL) mg, 0.55 mmol, 1.0 eq) was added TFA (0.5 mL) at 10 °C. The mixture was then stirred at 10° C. for 3 hours. The solution was concentrated to obtain a crude product, purified by prep-HPLC and lyophilized to obtain a crude product. The crude product was stirred in MeOH (2.5 mL) and CH ₃ CN (2.5 mL) for 5 min. The mixture was filtered and the filtercake was washed with MeOH and dried to give compound 30 (4-amino- N '-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonohydrazide, 20 mg, yield 9.30%) was obtained as a white solid.

¹ HNMR (DMSO- d ₆ , 400 MHz): δ 7.57 (d, J = 8.0 Hz, 2H), 7.21-7.25 (m, 2H), 6.76 (d, J = 8.0 Hz, 2H), 6.64 (d, J = 8.4 Hz, 1H), 5.54 (s, 2H), 2.78 (s, 4H), 2.54 (s, 4H).

LCMS; Mass Calcd.:391; MS Found: 392.1 [MS+1].

Experimental Example 1-31. Compound 31 (4-amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl) benzenesulfonohydrazide) Preparation

Step 1) Synthesis of A59

To a mixture of A58 (2,3-dihydro-1 H -indene-2-carboxylic acid, 5.0 g, 30.8 mmol, 1.0 eq) in MeOH (50 mL) was added H ₂ SO ₄ (5 mL) dropwise. . The mixture was then stirred at 70° C. for 12 hours. The solution was poured into water (60 mL) and extracted with EA (60 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A59 (methyl 2,3-dihydro-1 H -indene-2-carboxylate, 5.3 g, yield 97.5%) as a yellow oil. lost.

Step 2) Synthesis of A60

N ₂ H ₄ .H ₂ O ( 8.56 g, 171 mmol, 10 eq) was added dropwise. The mixture was then stirred at 80° C. for 12 hours. The solution was concentrated to give A60 (2,3-dihydro-1 H -indene-2-carbohydrazide, 2.0 g, yield 87.5%) as a yellow solid.

^1HNMR (DMSO-d6, 400 MHz): δ 9.13 (s, 1H), 7.18-7.20 (m, 2H), 7.11-7.13 (m, 2H), 4.25 (br s, 2H), 3.01-3.11 (m, 5H).

Step 3) Synthesis of A61

To a mixture of A60 (2,3-dihydro- 1H -indene-2-carbohydrazide, 2 g, 11.4 mmol, 1.0 eq) in pyridine (20 mL) was added 4-nitrobenzene-1-sulfonyl chloride (2.52 g, 11.4 mmol, 1.0 eq) was added in portions. The mixture was then stirred at 10° C. for 2 hours. The solution was poured into water (100 mL) and extracted with EA (100 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to form A61 ( N ′-(2,3-dihydro-1 H -indene-2-carbonyl)-4-nitrobenzenesulfonohydrazide, 1.4 g, yield 34.1%) was obtained as a yellow solid. (TLC: DCM/MeOH =10/1, Rf=0.5)

Step 4) Synthesis of Compound 31

A solution of A61 ( N '-(2,3-dihydro- 1H -indene-2-carbonyl)-4-nitrobenzenesulfonohydrazide, 200 mg, 0.83 mmol, 1.0 eq) dissolved in MeOH (10 mL). To the mixture was added Pd/C (100 mg). The mixture was then concentrated under a H ₂ balloon at 10° C. for 12 hours. The solution was filtered and the filtrate was purified by prep-HPLC and lyophilized to yield compound 31 (4-amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl)benzenesulfonohydrazide , 80 mg, yield 43.6%) was obtained as a white solid.

¹ HNMR (CD ₃ OD, 400 MHz): δ 7.55 (dd, J = 6.8, 2.0 Hz, 2H), 7.07-7.12 (m, 4H), 6.65 (dd, J = 6.8, 2.0 Hz, 2H), 2.92 -3.03 (m, 5H).

LCMS; Mass Calcd.:331; MS Found: 331.8 [MS+1].

Experimental Example 1-32. Preparation of compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A63

To a mixture of A62 (isoindoline, 1.00 g, 8.4 mmol, 1.0 eq) in dichloromethane (10 mL) was added triethylamine (2 mL) and 4-nitrophenyl chloroformate (1.68 g, 8.4 mmol). It became. The mixture was stirred at room temperature for 16 hours. The solution was poured into water (30 mL) and extracted with dichloromethane (30 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated. The product was added to tetrahydrofuran (10 mL) and N ₂ H ₄ H ₂ O (2 mL). The mixture was stirred at 60° C. for 16 hours. The solution was poured into water (50 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over sodium sulfate (Na ₂ SO ₄ ) and concentrated to give A63 (isoindoline-2-carbohydrazide, 600 mg, crude) as a yellow solid. (TLC: DCM/MeOH =10/1, Rf=0.6)

LCMS; Mass Calcd.: 177.2; MS Found: 178.2 [MS+1].

Step 2) Synthesis of A64

To a mixture of A63 (isoindoline-2-carbohydrazide, 600 mg, 3.39 mmol, 1.0 eq) in pyridine (10 mL) was added 4-nitrobenzene-1-sulfonyl chloride (750 mg, 3.39 mmol, 1.0 eq) was added dropwise. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (50 mL) and extracted with EA (50 mL x 3). The combined organic layers were washed with 1N HCl (50 mL x 2) and brine, dried over Na ₂ SO ₄ and concentrated to give A64 (N'-(isoindoline-2-carbonyl)-4-nitrobenzenesulfonohydrazide , 200 mg, crude) was obtained as a yellow solid. (TLC: DCM/MeOH =20/1, Rf=0.5)

LCMS; Mass Calcd.:362.3; MS Found: 363.1 [MS+1].

Step 3) Synthesis of compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonohydrazide)

To a mixture of A64 (200 mg, 0.55 mmol, 1.0 eq) in ethanol (10 mL) was added Fe (154 mg, 2.75 mmol, 5.0 eq) and sat. aq. NH4Cl (6 mL) was added. The mixture was then stirred at 85° C. for 3 hours. The solution was filtered and the filtrate was concentrated to give the crude product. The crude product was purified by prep-HPLC and lyophilized to give compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonohydrazide, 20 mg, yield 11.0%) as a white solid. lost.

1HNMR (DMSO-d6, 400 MHz): δ 8.64 (s, 1H), 8.53 (s, 1H), 7.41-7.44 (m, 2H), 7.29-7.31 (m, 4H), 6.51-6.54 (m, 2H) ), 5.93 (s, 2H), 4.51 (s, 4H).

LCMS; Mass Calcd.:332; MS Found: 333 [MS+1].

Experimental Example 1-33. Preparation of compound 33 (N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonohydrazide)

Step 1) Synthesis of A66

To a stirred solution of A65 (tert-butyl 1H-indole-1-carboxylate, 2.00 g, 9.2 mmol, 1.0 eq) in tetrahydrofuran (20 mL) was added n-BuLi (4.0 mL, 2.5M in hexanes, 10.0 mmol, 1.1 eq) was added dropwise at -70 °C. After 1 hour, SO ₂ (gas, 1 L) was added slowly at -70 °C. The reaction mixture was warmed to 10° C. over 2 hours. Solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (DCM, 20 mL). N-chlorosuccinimide (NCS, 1.84 g, 13.8 mmol, 1.5 eq) was added. The mixture was stirred at room temperature for 10 hours. The mixture was washed with water (2 x 20 mL) and brine (2 x 20 mL). The organic layer was dried and concentrated. The residue was purified by column chromatography (PE/EA=30/1-5/1) to give A66 (tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate, 1.0 g, yield 34.4 %) was obtained as a brown oil.

1HNMR (CDCl3, 400 MHz): δ 8.23-8.25 (m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.69 (s, 1H), 7.56-7.60 (m, 1H), 7.35-7.39 ( m, 1H), 1.75 (s, 9H).

Step 2) Synthesis of A67

To a stirred solution of 4-hydroxybenzohydrazide (481 mg, 3.17 mmol, 1.0 eq) in pyridine (10 mL) was added A66 (tert-butyl 2-(chlorosulfonyl)-1H- A solution of indole-1-carboxylate, 1.0 g, 3.17 mmol, 1.0 eq) was added dropwise at 0°C. The mixture was stirred at room temperature for 5 hours. The mixture was filtered. The filtrate was concentrated and purified by column chromatography to obtain A67 (tert-butyl 2-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-1H-indole-1-carboxylate, 500 mg, yield 36.5%) was obtained as a white solid.

Step 3) Synthesis of Compound 33

To a stirred solution of A67 (500 mg, 1.16 mmol, 1.0 eq) in methanol (MeOH, 5 mL) was added 4N HCl (g)/MeOH (2 mL) at 0 ^° C. The mixture was stirred at room temperature for 4 hours. The mixture was concentrated, purified by prep-HPLC and lyophilized to yield compound 33 (N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonohydrazide, 50 mg, yield 13%) as an off-white solid. got

¹ HNMR (DMSO-d6, 400 MHz): δ 11.94 (s, 1H), 10.42 (d, J = 1.6 Hz, 1H), 10.09 (s, 1H), 9.86 (d, J = 2.8 Hz, 1H), 7.61-7.64 (m, 3H), 7.45-7.47 (m, 1H), 7.24-7.28 (m, 1H), 7.07-7.11 (m, 1H), 6.99 (d, J = 1.2 Hz, 1H), 6.77 ( d, J = 8.8 Hz, 2H).

LCMS; Mass Calcd.:341; MS Found: 342 [MS+1].

Experimental Example 1-34. Preparation of compound 34 (4-amino-N'-(2-phenylacetyl)benzenesulfonohydrazide)

Step 1) Synthesis of A69

To a mixture of A68 (2-phenylacetohydrazide, 500 mg, 3.33 mmol, 1.0 eq) in pyridine (5 mL) was added 4-nitrobenzene-1-sulfonyl chloride (738 mg, 3.33 mmol) in pyridine (5 mL). , 1.0 eq) was added dropwise. The mixture was then stirred at 10° C. for 2 hours. The solution was poured into water (30 mL) and extracted with ethyl acetate (EA, 30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was stirred in DCM (30 mL) for 30 min. The mixture was filtered and the filtercake was dried to give A69 (4-nitro-N'-(2-phenylacetyl)benzenesulfonohydrazide, 1 g, yield 89.6%) as a yellow solid.

1HNMR (DMSO-d6, 400 MHz): δ 10.50 (s, 1H), 10.37 (s, 1H), 8.22 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.21 -7.29 (m, 3H), 7.10-7.12 (m, 2H), 3.30 (s, 2H).

Step 1) Synthesis of Compound 34

To a mixture of A69 (200 mg, 0.60 mmol, 1.0 eq) in MeOH (10 mL) was added Pd/C (50 mg). The mixture was then stirred under a H2 balloon at 10° C. for 2 hours. The solution was filtered and the filtrate was concentrated. The crude product was crystallized 3 times from MeOH (10 mL) to give compound 34 (4-amino-N'-(2-phenylacetyl)benzenesulfonohydrazide, 20 mg, yield 10.9%) as a gray solid. TLC: DCM/MeOH =10/1, Rf=0.3)

¹ HNMR (DMSO-d6, 400 MHz): δ 9.87 (s, 1H), 8.77 (s, 1H), 7.40 (d, J = 6.8 Hz, 2H), 7.18-7.29 (m, 3H), 7.13 (d , J = 6.8 Hz, 2H), 5.74 (s, 2H), 3.37 (s, 2H).

LCMS; Mass Calcd.:305; MS Found: 306.1 [MS+1].

Experimental Example 1-35. Preparation of compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonohydrazide)

Step 1) Synthesis of A70

acetic acid (16 mL), con. To a stirred solution of 1H-indazol-3-amine (1.00 g, 7.5 mmol, 1.0 eq) in hydrochloric acid (1.6 mL) and formic acid (1.6 mL) was added NaNO ₂ (0.62 g, 9.0 mmol, 1.2 eq) to 0 was added at °C. The mixture was stirred for 1 hour. SO ₂ (gas, 1 L) and CuCl ₂ (0.38 g, 2.3 mmol, 0.3 eq) were added slowly at 0 °C. The reaction mixture was warmed to 10 °C. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (PE/EA=30:1-1:1) to give A70 (1H-indazole-3-sulfonyl chloride, 0.5 g, yield 30.8%) as a brown solid.

1HNMR (DMSO-d6, 400 MHz): δ 14.13 (s, 2H), 7.94-7.92 (m, 1H), 7.46-7.38 (m, 2H), 7.14 (s, 1H).

Step 2) Synthesis of Compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonohydrazide)

To a stirred solution of 4-hydroxybenzohydrazide (225 mg, 1.48 mmol, 0.8 eq) in pyridine (5 mL) A70 (1H-indazole-3-sulfonyl chloride, 0.40 g in pyridine (5 mL) , 1.85 mmol, 1.0 eq) was added dropwise at 0 °C. The mixture was stirred at room temperature for 5 hours. The mixture was concentrated. The residue was purified by prep-HPLC and lyophilized to give compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonohydrazide, 60 mg, yield 9.77%) as a white solid. lost.

¹ HNMR (DMSO-d6, 400 MHz): δ 13.90 (s, 1H), 10.43 (s, 1H), 10.05-9.99 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.62 (d , J = 8.4 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.0 Hz, 1H), 7.22 (t, J = 7.0 Hz, 1H), 6.72 (d, J = 8.0 Hz, 2H).

LCMS; Mass Calcd.:332; MS Found: 333 [MS+1].

Experimental Example 1-36. Preparation of compound 36 (4-amino-N'-(indoline-6-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A72

A mixture of A71 (methyl 1H-indole-6-carboxylate, 500 mg, 2.86 mmol, 1.0 eq) and hydrazine monohydrate (10 mL) was stirred at 100 ^° C for 3 h. The mixture was then cooled to 0 °C and filtered. The filtercake was washed with ice water and dried in vacuo to give A72 (1H-indole-6-carbohydrazide, 300 mg, yield 60.0%) as a white solid.

LCMS; Mass Calcd.: 175.18; MS Found: 176.0 [MS+1].

Step 2) Synthesis of A73

To a mixture of A72 (300 mg, 1.71 mmol, 1.0 eq) in pyridine (5 mL) was added 4-nitrobenzenesulfonyl chloride (380 mg, 1.71 mmol, 1.0 eq) at 0 °C. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with 1N HCl (30 mL x 2) and brine, dried over Na ₂ SO ₄ and concentrated to give a crude product, which was purified by flash column (DCM/MeOH=50/1~30/1). A73 (N'-(1H-indole-6-carbonyl)-4-nitrobenzenesulfonohydrazide, 500 mg, yield 81.0%) was obtained as a yellow solid.

LCMS; Mass Calcd.:360.34; MS Found: 361.1 [MS+1].

Step 3) Synthesis of A74

To a mixture of A73 (400 mg, 1.11 mmol, 1.0 eq) in DCM (15 mL) and TFA (5 mL) was added NaBH ₃ CN (209 mg, 3.33 mmol, 3.0 eq) at 0 °C. The mixture was then stirred at 15° C. for 1 hour. The solution was poured into ice water (30 mL) and sat. aq. It was adjusted to pH=7-8 with NaHCO ₃ . The solution was filtered, and the filtercake was washed with PE and dried to give A74 (N'-(indoline-6-carbonyl)-4-nitrobenzenesulfonohydrazide, 200 mg crude) as a yellow solid.

LCMS; Mass Calcd.:362.36; MS Found: 363.1 [MS+1].

Step 3) Synthesis of Compound 36

A74 (200 mg, 0.55 mmol, 1.0 eq), NH ₄ Cl (146 mg, 2.75 mmol, 5.0 eq), Fe (154 mg, 2.75 mmol, 5.0 eq) in H ₂ O (10 mL) and EtOH (20 mL) The mixture of eq) was stirred at 85 °C for 2 h. The mixture was filtered and the filtrate was extracted with EA (20 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude product was recrystallized from methanol and the solid was lyophilized to give compound 36 (4-amino-N'-(indoline-6-carbonyl)benzenesulfonohydrazide, 60 mg, 32.7%) as a white solid. lost.

1HNMR (DMSO-d6, 400 MHz): δ 10.32 (s, 1H), 9.14 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 7.6 Hz, 1H), 6.87 (d, J = 7.2 Hz, 1H), 6.75 (s, 1H), 6.49 (d, J = 8.4 Hz, 2H), 5.95 (s, 2H), 5.67 (s, 1H), 3.41 (t, J = 8.2 Hz, 2H), 2.91 (t, J = 8.6 Hz, 2H).

LCMS; Mass Calcd.:332; MS Found: 333 [M+1].

Experimental Example 1-37. Preparation of compound 37 (4-amino-N'-(indoline-3-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A76

Boc ₂ was added to a mixture of A75 (methyl 1H-indole-3-carboxylate, 1.00 g, 5.71 mmol, 1.0 eq) and triethylamine (TEA, 1.16 g, 11.4 mmol, 2.0 eq) in DCM (10 mL). O (1.37 g, 6.28 mmol, 1.1 eq) was added dropwise at 20 °C. The mixture was then stirred at 20° C. for 12 hours. The solution was poured into water (60 mL) and extracted with DCM (60 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A76 (1-(tert-butyl) 3-methyl 1H-indole-1,3-dicarboxylate, 1.20 g, crude) as a yellow solid was obtained with

Step 2) Synthesis of A77

To a mixture of A76 (1.20 g, 4.36 mmol, 1.0 eq) in ethyl acetate (EA, 30 mL) was added Pd/C (0.65 g) and degassed. The mixture was then stirred at 60° C. for 12 h under H ₂ (50 psi). The solution was filtered and the filtrate was concentrated to give the crude product. The crude product was purified on silica gel (PE/EA=30:1-15:1) to obtain A77 (1-(tert-butyl) 3-methyl indoline-1,3-dicarboxylate, 0.80 g, yield 66.2% ) was obtained as a white solid.

Step 3) Synthesis of A78

To a mixture of A77 (800 mg, 2.89 mmol, 1.0 eq) in MeOH (20 mL) was added N ₂ H ₄ .H 2 O (1.6 mL). The mixture was then stirred at 80° C. for 4 hours. The reaction mixture was concentrated to give A78 (700 mg of tert-butyl 3-(hydrazinecarbonyl)indoline-1-carboxylate, yield 87.5%) as a white solid.

Step 3) Synthesis of A79

To a mixture of A78 (tert-butyl 3-(hydrazinecarbonyl)indoline-1-carboxylate, 300 mg, 1.08 mmol, 1.0 eq) in pyridine (5 mL) was added 4-nitrobenzene-1-sulfonyl chloride (0.24 g, 1.08 mmol, 1.0 eq) was added in portions. The mixture was then stirred at 10° C. for 4 hours. The solution was poured into water (100 mL) and extracted with EA (100 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A79 (tert-butyl 3-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)indoline-1-carb Voxylate, 200 mg, yield 39.7%) was obtained as a yellow solid.

Step 4) Synthesis of A80

To a mixture of A79 (200 mg, 0.43 mmol, 1.0 eq) in EA (10 mL) was added Pd/C (50 mg). The mixture was then stirred under a H2 balloon at 10° C. for 12 hours. The solution was filtered and the filtrate was concentrated to obtain A80 (tert-butyl 3-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)indoline-1-carboxylate, 200 mg, yield : 100%) was obtained as a yellow solid.

Step 5) Synthesis of compound 37 (4-amino-N'-(indoline-3-carbonyl)benzenesulfonohydrazide)

To a mixture of A80 (200 mg, 0.463 mmol, 1.0 eq) in DCM (5 mL) was added trifluoroacetic acid (TFA, 1 mL). The mixture was then stirred at 10° C. for 2 hours. The solution was concentrated and to the residue was added H ₂ O (5 mL) and aq. Adjusted to pH=9-10 with K ₂ CO ₃ . The solution was extracted with EA (20 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The crude product was purified by Prep-HPLC and lyophilized to give compound 37 (4-amino-N'-(indoline-3-carbonyl)benzenesulfonohydrazide, 30.0 mg, yield 19.6%) as an off-white solid. .

1HNMR (DMSO_d6, 400 MHz): δ 7.45 (d, J = 8.4 Hz, 2H), 6.91-6.95 (m, 2H), 6.48-6.59 (m, 4H), 5.72 (s, 2H), 5.23 (s, 1H), 3.93 (br s, 1H), 3.49 (d, J = 9.2 Hz, 2H).

LCMS; MS Found: 333.1 [MS+1].

Experimental Example 1-38. Preparation of compound 38 (N'-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide)

Step 1) Synthesis of A82

A mixture of 4-hydroxybenzohydrazide (A2, 268 mg, 1.77 mmol, 1.0 eq) in pyridine (2 mL) A81 (tert-butyl 4-(chlorosulfonyl)piperi in pyridine (1 mL) A solution of din-1-carboxylate, 500 mg, 1.77 mmol, 1.0 eq) was added dropwise at 0°C. The mixture was stirred at room temperature for 5 hours. The solution was filtered. The filtrate was concentrated and purified by column chromatography (DCM/MeOH=100:1-10:1) to obtain A82 (tert-butyl 4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)piperi din-1-carboxylate, 300 mg, yield 42%) was obtained as a yellow solid.

1HNMR (CDCl3, 400 MHz): δ 8.47 (br s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.30 (br s, 1H), 6.87 (d, J = 8.4 Hz, 2H), 3.15 -3.18 (m, 1H), 2.67-2.71 (m, 2H), 2.25 (d, J = 10.8 Hz, 2H), 1.72-1.78 (m, 2H), 1.61-1.63 (m, 2H), 1.45 (s) , 9H).

Step 2) Synthesis of Compound 38 (N'-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide)

To a mixture of A82 (300 mg, 0.75 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1.5 mL) at 0 °C. The mixture was stirred at room temperature for 4 hours. The solution was concentrated, purified by prep-HPLC, and lyophilized to obtain compound 38 (N'-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide, 50 mg, yield 22%) as a yellow solid. lost.

1HNMR (DMSO-d6, 400 MHz): δ 10.40 (br s, 1H), 8.34 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.17–3.24 (m, 3H), 2.69 (t, J = 12.0 Hz, 2H), 2.28 (d, J = 12.0 Hz, 2H), 1.64–1.74 (m, 2H).

LCMS; Mass Calcd.:299; MS Found: 300 [Ms+1].

Experimental Example 1-39. Preparation of compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide)

Step 1) Synthesis of A83

To a mixture of A72 (1H-indole-6-carbohydrazide, 500 mg, 2.86 mmol, 1.0 eq) in pyridine (5 mL) was added 3-fluoro-4-nitrobenzene-1- in pyridine (2 mL). Sulfonyl chloride (686 mg, 2.86 mmol, 1.0 eq) was added dropwise at 0°C. The mixture was then stirred at 10° C. for 3 hours. The solution was concentrated to give a crude product which was purified by column (DCM:MeOH=50:1---20:1) to give A83 (3-fluoro-N'-(1H-indole-6-carbonyl) -4-nitrobenzenesulfonohydrazide, 700 mg, yield: 64.8%) was obtained as a yellow solid.

LCMS; Mass Calcd.: 378.3; MS Found: 379.1 [Ms+1].

Step 2) Synthesis of A84

Morpholine (193 mg, 2.22 mmol, 1.2 eq) was added to a mixture of A83 (700 mg, 1.85 mmol, 1.0 eq) and K ₂ CO ₃ (640 mg, 4.64 mmol, 2.5 eq) in DMF (7 mL). was added at °C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A84 (N'-(1H-indole-6-carbonyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 600 mg , yield: 72.8%) was obtained as a yellow solid.

LCMS; Mass Calcd.:445.4; MS Found: 446.1 [Ms+1].

Step 3) Synthesis of A85

To a mixture of A84 (600 mg, 1.35 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1 mL) and NaBH ₃ CN (251 mg, 4.04 mmol, 3.0 eq) at 10 °C. The mixture was then stirred at 25° C. for 4 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give A85 (N′-(indoline-6-carbonyl)-3-morpholino-4-nitrobenzenesulfonohydrazide, 450 mg, crude) was obtained as a yellow solid.

LCMS; Mass Calcd.:447.4; MS Found: 448.2 [Ms+1].

Step 4) Synthesis of compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide)

To a mixture of A85 (450 mg, 1.01 mmol, 1.0 eq) in EtOH (5 mL) was added Fe (282 mg, 5.05 mmol, 5.0 eq) and sat. aq. NH ₄ Cl (1 mL) was added. The mixture was then stirred at 85° C. for 3 hours. The solution was concentrated then DMSO (5 mL) was added and filtered. The filtrate was purified by prep-HPLC and lyophilized to obtain compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide, 30 mg, yield 7.0%). It was obtained as an off-white solid.

1HNMR (DMSO-d6, 400 MHz): δ10.36 (d, J = 4.4 Hz, 1H), 9.21 (d, J = 4.4 Hz, 1H), 7.23-7.27 (m, 2H), 7.03 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.67 (s, 1H), 6.65 (d, J = 8.4 Hz, 1H), 5.63 (br s, 2H), 3.69 (t, J = 4.2 Hz, 4H), 3.42 (t, J = 8.6 Hz, 2H), 2.91 (t, J = 8.6 Hz, 2H), 2.63 (t, J = 4.2 Hz, 4H).

LCMS; Mass Calcd.:417.1; MS Found: 418.1 [MS+1].

Experimental Example 1-40. Preparation of compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A87

To a mixture of A86 (tert-butyl 4-(hydrazinecarbonyl)piperazine-1-carboxylate, 1.00 g, 4.09 mmol, 1.0 eq) in pyridine (10 mL), 4-nitrobenzene in pyridine (5 mL) -1-sulfonyl chloride (906 mg, 4.09 mmol, 1.0 eq) was added dropwise at 10°C. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (50 mL) and extracted with EA (50 mL x 3). The combined organic layers were washed with 1N HCl (50 mL x 2) and brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The crude product was washed with EA (5 mL), filtered and the solid dried in vacuo to yield A87 (tert-butyl 4-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine- 1-carboxylate, 800 mg, yield 45.5%) was obtained as a yellow solid.

¹ HNMR (DMSO-d6, 400 MHz): δ 9.74 (s, 1H), 9.08 (s, 1H), 8.38 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 3.17 (br s, 8H), 1.40 (s, 9H).

Step 2) Synthesis of A88

To a mixture of A87 (400 mg, 0.93 mmol, 1.0 eq) in THF (10 mL) was added Pd/C (40 mg). The mixture was then stirred under a H ₂ balloon at 10° C. for 16 hours. The solution was filtered and the filtrate was concentrated to give A88 (tert-butyl 4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 350 mg, yield 94%) was obtained as a yellow solid.

Step 3) Synthesis of compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide)

To a mixture of A88 (150 mg, 0.37 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1 mL). The mixture was then stirred at 25° C. for 3 hours. The solution was concentrated to give crude product. MeOH (5 mL) was added to the crude product, K ₂ CO ₃ (62 mg, 0.45 mmol) was added and stirred at room temperature for 1 hour. The solution was filtered, purified by prep-HPLC, and lyophilized to give compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide, 50 mg, yield 38.7%) as a white solid. lost.

1HNMR (DMSO-d6, 400 MHz): δ 8.80 (d, J = 2.8 Hz, 1H), 8.28-8.32 (m, 3H), 7.38 (d, J = 8.8 Hz, 2H), 6.56 (d, J = 8.8 Hz, 2H), 5.98 (s, 2H), 3.22 (s, 4H), 2.69 (s, 4H).

LCMS; Mass Calcd.:299; MS Found: 300 [MS+1].

Experimental Example 1-41. Preparation of compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide)

Step 1) Synthesis of A89

To a mixture of A86 (1.50 g, 6.14 mmol, 1.0 eq) in pyridine (10 mL) was added 3-fluoro-4-nitrobenzene-1-sulfonyl chloride (1.46 g, 6.14 mmol, 1.0 eq) in pyridine (5 mL). eq) was added dropwise. The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with 1N HCl (30 mL x 2) and brine, dried over Na ₂ SO ₄ and concentrated to give A89 (tert-butyl 4-(2-((3-fluoro-4-nitrophenyl)sulfonyl )Hydrazine-1-carbonyl)piperazine-1-carboxylate, 1.5 g, crude) was obtained as a yellow solid.

Step 2) Synthesis of A90

Morpholine (350 mg, 4.02 mmol, 1.2 eq) was added to a mixture of A89 (1.50 g, 3.35 mmol, 1.0 eq) and K ₂ CO ₃ (1.16 g, 8.37 mmol, 2.5 eq) in DMF (15 mL). was added at °C. The mixture was then stirred at 25° C. for 16 hours. The solution was poured into water (50 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give a crude product which was purified by column (DCM/MeOH =50/1-10/1) A90 (tert-butyl 4-(2- ((3-morpholino-4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 700 mg, yield:40.6%) was obtained as a yellow solid.

LCMS; Mass Calcd.:514.55; MS Found: 516.2 [MS+2].

Step 3) Synthesis of A91

To a mixture of A90 (700 mg, 1.36 mmol, 1.0 eq) in THF (10 mL) was added Pd/C (200 mg). The mixture was then stirred at room temperature for 15 minutes under H ₂ (50 psi). The solution was filtered and the filtrate was concentrated to give A91 (tert-butyl 4-(2-((4-amino-3-morpholinophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxyl rate, 300 mg, crude) was obtained as a yellow solid.

Step 4) Synthesis of compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide)

To a mixture of A91 (200 mg, 0.41 mmol, 1.0 eq) in DCM (10 mL) was added TFA (2 mL). The mixture was then stirred at room temperature for 3 hours. The mixture was concentrated, purified by prep-HPLC, and lyophilized to give compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonohydrazide, 20.0 mg, yield 12.6% ) was obtained as a pink solid.

1HNMR (DMSO-d6, 400 MHz): δ 8.92 (d, J = 5.2 Hz, 1H), 8.69-8.75 (m, 2H), 7.23 (d, J = 5.6 Hz, 2H), 6.70 (t, J = 8.6Hz, 1H), 5.68 (s, 2H), 3.78 (s, 4H), 3.39 (s, 4H), 2.98 (s, 4H), 2.76 (s, 4H).

LCMS; Mass Calcd.:384; MS Found: 384.9 [MS+1].

Experimental Example 1-42. Preparation of compound 42 (N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide)

Step 1) Synthesis of A92

A stirred mixture of 2,2,2-trichloroacetaldehyde (20 g, 0.13 mmol), acetamide (7 g, 0.118 mmol), and concentrated sulfuric acid (1.2 g) was heated at 100° C. for 1 hour. The reaction mixture cooled and crystallized. The mixture was triturated with deionized water, filtered, washed with plenty of water, and recrystallized from ethanol to give A92 (N-(2,2,2-trichloro-1-hydroxyethyl)acetamide, 15 g) as a white solid. was obtained with

1HNMR (DMSO_d6, 400 MHz): 8.72 (d, 1H), 7.64 (d, 1H), 5.74-5.70 (m, 1H), 1.92 (s, 3H).

Step 2) Synthesis of A93

Zinc powder (5 g, 78 mmol) was added slowly over 3 hours to a stirred suspension of A92 (8 g, 39 mmol) in glacial acetic acid (50 mL). During the zinc addition, the temperature of the reaction mixture was maintained below 40 ^° C. The reaction mixture was stirred at room temperature for 24 hours. The precipitated zinc salt was then filtered off and washed with glacial acetic acid. Acetic acid was removed under reduced pressure. The solid residue was triturated with deionized water and recrystallized to give A93 (N-(2,2-dichlorovinyl)acetamide, 3 g) as a white solid.

1HNMR (DMSO_d6, 400 MHz): 9.87 (d, 1H), 7.21 (d, 1H), 2.03 (s, 3H).

Step 3) Synthesis of A94

Benzylthiol (4 g, 32 mmol) and triethylamine (3.29 g, 32.6 mmol) were added to a stirred solution of A93 (2 g, 13 mmol) in 2-propanol (25 mL). The reaction mixture was stirred at room temperature for 48 hours. The solvent was then removed under reduced pressure and the residue was triturated with water to give a crystalline solid. The crude product was purified by recrystallization from 2-propanol or ethanol to obtain A94 (N-(1-(benzylthio)-2,2-dichloroethyl)acetamide, 2.5 g) as a white solid.

1HNMR (DMSO_d6, 400 MHz): 8.72 (d, 1H), 7.23-7.25 (m, 5H), 6.42 (d, 1H), 5.40 (dd, 1H), 3.87 (q, 2H), 1.93 (S, 3H) ).

Step 4) Synthesis of A95

Lawesson's reagent (7.6 g, 18.8 mmol) was added to a stirred solution of A94 (5 mmol) in toluene (30 mL). The reaction mixture was refluxed for 8 hours and the solvent was removed under reduced pressure. The residue was triturated with 10% aqueous NaOH and adjusted to pH 9. The crude product was filtered, dried and recrystallized from 2-propanol. The liquid product was extracted with dichloromethane to give A95 (4-(benzylthio)-2-methylthiazole, crude, 2.5 g) as a yellow oil.

1HNMR (DMSO_d6, 400 MHz): 7.44 (s, 1H), 7.30-7.20 (m, 5H), 4.04 (s, 2H), 2.59 (S, 3H).

Step 5) Synthesis of A96

To a solution of A95 (crude, 1 g) in acetic acid (10 ml) was added NCS (3 g) and water (2 mL) at 0 ^° C. The reaction mixture was stirred overnight at room temperature. The reaction mixture was triturated with 10% aqueous NaHCO ₃ adjusted to pH =8 and extracted with DCM (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residue was purified through silica gel to give A96 (2-methylthiazole-4-sulfonyl chloride, 100 mg) as a yellow solid.

1HNMR (CDCl3, 400 MHz): 8.33 (s, 1H), 2.86 (S, 3H).

Step 6) Synthesis of Compound 42 (N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide)

A mixture of A96 (100 mg, 0.5 mmol) and A2 (4-hydroxybenzohydrazide, 80 mg, 0.5 mmol) in pyridine (20 mL) was stirred at 80° C. overnight. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residue was purified by prep-HPLC to obtain compound 42 (N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide, 30 mg) as an off-white solid.

1HNMR (DMSO-d6, 400 MHz): 10.53 (s, 1H), 10.27 (s, 1H), 10.13 (s, 1H), 8.02 (s, 1H), 7.63 (d, 2H), 6.80 (d, 2H) ), 2.70 (s, 3H).

LCMS; Mass Calcd.:313.3; MS Found: 314.0 [MS+1].

Experimental Example 1-43. Preparation of compound 43 ((1S,4S)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide)

Step 1) Synthesis of A98

4-Methylbenzenesulfonyl chloride ( 28.6 g , 151 mmol, 1.2 eq) was added in portions and the mixture was stirred overnight at room temperature. Pyridine was removed in vacuo and the residue was purified by silica gel column chromatography to give A98 ((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl 4-methylbenzenesulfonate, 40 g, 86.4%) was obtained as a white solid.

Step 2) Synthesis of A99

A solution of A98 (10.0 g, 27.1 mmol, 1.0 eq) in DMF (100 mL) was treated with potassium thioacetate (9.3 g, 81.3 mmol, 3.0 eq) and the reaction mixture was stirred at 60 °C under nitrogen for 4 h. The reaction mixture was quenched with brine (200 mL) and extracted with EtOAc (100 mL x 2). The combined organics were dried and concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to give A99 (S-((1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexyl)ethanethioate , 3.0 g, 40.5%) was obtained as a white solid.

Step 3) Synthesis of A100

A solution of A99 (2.50 g, 9.16 mmol, 1.0 eq) in DCM (30 mL) and water (30 mL) was bubbled with chlorine gas at 0 °C for 30 min. The two layers were separated and the DCM layer was aq. Washed with sodium thiosulfate and brine, dried over sodium sulfate, filtered and concentrated to yield crude A100 (tert-butyl ((1s,4s)-4-(chlorosulfonyl)cyclohexyl)carbamate) as a brown solid. got

Step 4) Synthesis of A101

To a solution of 4-hydroxybenzohydrazide (A2, 2.76 g, 18.2 mmol, 2.0eq) in pyridine (10 mL) was added dropwise a solution of A100 (crude) in DCM (5 mL), and the mixture was stirred at room temperature. Stirred for 2 hours. The solvent was removed in vacuo and the crude product was purified by silica gel column chromatography to obtain A101 (tert-butyl ((1s,4s)-4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)cyclohexyl) Carbamate, 0.2 g, 5.3% for 2 steps) was obtained as a white solid.

Step 5) Synthesis of compound 43 ((1s,4s)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide)

A101 (200 mg, 4.2 mmol, 1.0 eq) was dissolved in a mixture of TFA (1 mL) and DCM (5 mL) and stirred for 2 h. The mixture was concentrated in vacuo and dissolved in MeOH, NH3/MeOH was added to pH=9, the solvent was concentrated in vacuo, the residue was dissolved in MeOH, purified by prep-HPLC and lyophilized to give compound 43 ((( 1s,4s)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide, 20 mg, 13.2%) was obtained as a yellow solid.

1HNMR (CD3OD, 400 MHz): δ 7.749 (d, J=8.8Hz, 2H), 6.862 (d, J=8.4Hz, 2H), 3.286-3.331 (m, 2H), 2.315-2.350 (m, 2H) , 1.995–2.153 (m, 4H), 1.855–1.918 (m, 2H).

LCMS; MS Calcd.:313.11; MS Found: 313.9 ([M+1]+).

Experimental Example 1-44. Preparation of compound 44 ((1R,4R)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide)

Compound 44 was synthesized as a white solid (26.7% yield) in the same manner as in Experimental Example 1-45 using tert-butyl ((1s,4s)-4-hydroxycyclohexyl)carbamate instead of A97 as a starting material.

1HNMR (CD3OD, 400 MHz): δ 7.750 (d, J=8.8Hz, 2H), 6.868 (d, J=8.4Ha, 2H), 3.042-3.174 (m, 2H), 2.567 (d, J=12.4Hz , 2H), 2.180 (d, J = 12.4 Hz, 2H), 1.680-1.784 (m, 2H), 1.410-1.514 (m, 2H).

LCMS; MS Calcd.:313.11; MS Found: 313.9 ([M+1]+).

Experimental Example 1-45. Preparation of compound 45 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid)

Step 1) Synthesis of A102

A stirred mixture of 5-methylfuran-2-carboxylic acid (10 g, 80 mmol) and chlorosulfonic acid (30 mL) was stirred at 50° C. for 3 h before quenching with ice water. The aqueous layer was extracted with DCM and the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give compound A102 (14 g) as a yellow solid.

1HNMR (DMSO_d6, 400 MHz): 13.95 (s, 1H), 6.97 (s, 1H), 2.50 (s, 3H).

Step 2) Synthesis of Compound 45 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid)

A mixture of Compound A102 (5 g, 22.3 mmol) and Compound A2 (3.4 g, 22.3 mmol) in pyridine (50 mL) was stirred at 60° C. overnight. The solution was poured into water (30 mL) and extracted with EA (30 mL x 3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residue was purified by prep-HPLC to yield compound 45 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid, 1.3 g) as a yellow solid. got

1HNMR (DMSO-d6, 400 MHz): 13.5 (brs, 1H), 10.47 (s, 1H), 10.14 (s, 1H), 10.03 (s, 1H), 7.63 (d, 2H), 7.18 (s, 1H) ), 6.79 (s, 2H), 3.17 (s, 3H)

Experimental Example 1-46. Preparation of compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide)

Step 1) Synthesis of A104

To a stirred solution of A103 (5.00 g, 26.7 mmol, 1.0 eq) and TEA (5.40 g, 53.4 mmol, 2.0 eq) in DCM (50 mL) was added methanesulfonyl chloride (4.59 g, 40.1 mmol, 1.5 eq). It was added dropwise at 0°C. The mixture was stirred at room temperature for 2 hours. The mixture was quenched with H ₂ O (100 mL) and extracted with DCM (100 mLx2). The combined organic layers were washed with brine (100 mL), dried and concentrated. The residue was purified by column chromatography (PE/EA=100:1-10:1) to give A104 (5.0 g, yield 70.6%) as a yellow oil. (PE/EA = 10:1, Rf = 0.6)

Step 2) Synthesis of A105

A mixture of A104 (5.00 g, 18.9 mmol, 1.0 eq) and potassium thioacetate (4.30 g, 37.7 mmol, 2.0 eq) in DMF (50 mL) was stirred at 70 °C for 16 h. The mixture was treated with H ₂ O (200 mL) and extracted with EA (200 mL x 2). The combined organic layers were washed with H ₂ O (100 mL x 3), brine (100 mL), dried and concentrated. The residue was purified by column chromatography (PE/EA=50:1-5:1) to give A105 (2.0 g, yield 43.4%) as a brown solid.

Step 3) Synthesis of A106

To a stirred solution of A105 (2.00 g, 8.16 mmol, 1.0 eq) in acetic acid (AcOH 30 mL) and H ₂ O (30 mL) was added N-chlorosuccinimide (5.45 g, 40.8 mmol, 5.0 eq). It became. The mixture was stirred at room temperature for 16 hours. The mixture was concentrated and purified by column chromatography (PE/EA=50:1-1:1) to give A106 (1.0 g, yield 45.6%) as a yellow oil. (PE/EA = 3:1, Rf = 0.5)

¹ HNMR (CDCl3, 400 MHz): δ 4.28-4.31 (m, 1H), 4.00-4.02 (m, 1H), 3.85-3.95 (m, 1H), 3.66-3.75 (m, 1H), 3.52-3.58 ( m, 1H), 2.63 (br s, 1H), 2.44–2.54 (m, 1H), 1.49 (s, 9H).

Step 4) Synthesis of A107

A solution of A106 (1.00 g, 3.71 mmol, 1.0 eq) in pyridinium (10 mL) to a stirred solution of 4-hydroxybenzohydrazide (0.56 g, 3.71 mmol, 1.0 eq) in pyridinium (30 mL). was added dropwise at 0°C. The mixture was stirred at room temperature for 6 hours. The mixture was concentrated and purified by column chromatography (DCM/MeOH=100:1-10:1) to give A107 (0.50 g, yield 34.9%) as a yellow oil. (DCM/MeOH = 10:1, Rf = 0.4)

Step 5) Synthesis of Compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide)

To a mixture of A107 (500 mg, 1.30 mmol, 1.0 eq) in DCM (10 mL) was added TFA (4 mL) at 0 °C. The mixture was stirred at room temperature for 4 hours. The solution was concentrated, purified by prep-HPLC, and lyophilized to yield compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide, 30 mg, yield 7.0%) as a pale yellow solid. got (TLC: N/A)

1HNMR (DMSO-d6, 400 MHz): δ 10.47 (br s, 2H), 8.25 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.67-3.72 (m, 1H), 3.19-3.21 (m, 2H), 2.89-2.91 (m, 1H), 2.83-2.85 (m, 1H), 2.07-2.10 (m, 2H).

Experimental Example 1-47. Preparation of compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonohydrazide)

Step 1) Synthesis of A109

NaH (2.44 g (60% w /w), 60.9 mmol, 1.2 eq) was added at 0 °C and stirred at 0 °C for 1 hour. To the solution was then added dropwise TsCl (9.65 g, 50.8 mmol, 1.0 eq) in THF (20 mL). The solution was stirred for 16 hours. The solution was poured into water (200 mL) and extracted with EA (100 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give the crude product, washed with PE (30 mL) for 1 hour, filtered and the solid collected. The solid was dried in vacuo to give A109 (11.0 g, yield 79.5%) as a white solid. (TLC: N/A)

LCMS; Mass Calcd.:272.32; MS Found: 273.1 [MS+1].

Step 2) Synthesis of A110

To a mixture of A109 (2.00 g, 7.35 mmol, 1.0 eq) in THF (20 mL) was added n-BuLi (3.24 mL, 8.08 mmol, 1.1 eq) dropwise at -76 °C. The mixture was stirred at -76 °C for 1 hour. The mixture was then stirred under a SO ₂ balloon at −76° C. to 10° C. for 1 hour and the solution was concentrated. To the residue in DCM (30 mL) was added NCS (1.58 g, 11.7 mmol, 1.6 eq) at 20 °C, then the mixture was stirred at 20 °C for 1 hour. The solution was poured into water (50 mL) and extracted with DCM (50 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give A110 (1.30 g, yield 47.8%) as a yellow solid.

LCMS; Mass Calcd.:370.82; MS Found: 371.0 [MS+1].

Step 3) Synthesis of A111

To a mixture of A110 (1.30 g, 3.51 mmol, 1.0 eq) in pyridine (10 mL) was added 4-hydroxybenzohydrazide (A2, 587 mg, 3.86 mmol, 1.1 eq) in pyridine (5 mL) at 10 °C. became enemies The mixture was then stirred at 10° C. for 3 hours. The solution was poured into water (50 mL) and extracted with EA (50 mL x 3). The combined organic layers were washed with 1N HCl (50 mL x 2) and brine (50 mL), dried over Na ₂ SO ₄ and concentrated to give the crude product. The crude product was washed with EA (5 mL), filtered and dried in vacuo as a solid to give A111 (600 mg, yield 35.3%) as a yellow solid. (TLC: N/A)

LCMS; Mass Calcd.:486.51; MS Found: 487.1 [MS+1].

Step 4) Synthesis of Compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonohydrazide)

Con . HCl (2 mL) was added. The mixture was then stirred at 60° C. for 3 hours. The solution was concentrated, and the crude product was purified by prep-HPLC and lyophilized to compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonohydra Zide, 20 mg, yield 8.36%) was obtained as a white solid. (TLC: N/A)

¹ HNMR (DMSO-d6, 400 MHz): δ 12.61 (s, 1H), 10.45 (s, 1H), 10.11 (br s, 1H), 9.90 (d, J=2 Hz, 1H), 8.40-8.42 ( m, 1H), 8.08-8.10 (m, 1H), 7.62 (d, J=8.8 Hz, 2H), 7.16-7.19 (m, 1H), 7.13 (s, 1H), 6.77 (d, J=8.8 Hz) , 2H).

LCMS; Mass Calcd.:332; MS Found: 333 [MS+1].

Experimental Example 1-48. Compound 48 (4-hydroxy- N Preparation of '-(4-methoxybenzyl)benzohydrazide)

Compound 48 was obtained by the same preparation method as in Experimental Example 1-19, using 1-(bromomethyl)-4-methoxybenzene instead of 1-(bromomethyl)-4-nitrobenzene.

Experimental Example 1-49. compound 49 ( N '-(4-aminobenzyl)-2,3-dihydro-1 H -Indene-2-carbohydrazide) Preparation

Compound 49 was obtained by the same preparation method as in Experimental Example 1-19 using A60 (2,3-dihydro-1H-indene-2-carbohydrazide) as a starting material instead of A2 .

Experimental Example 1-50. Compound 50 (4-amino- N Preparation of -(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide)

4-nitrobenzene-1-sulfonyl chloride in step 3 of Experimental Example 1-12 Instead, compound 50 was obtained by a preparation method similar to Experimental Example 1-12 using 3-fluoro-4-nitrobenzenesulfonyl chloride.

Experimental Example 1-51. Compound 51 (3,5-diamino- N Preparation of -(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide)

4-nitrobenzene-1-sulfonyl chloride in step 3 of Experimental Example 1-12 Instead, compound 51 was obtained by a preparation method similar to Experimental Example 1-12 using 3,5-dinitrobenzenesulfonyl chloride (A20).

Experimental Example 1-52. Compound 52 (2-((4-aminophenyl)sulfonyl)- N Preparation of (3-hydroxyphenyl)hydrazine-1-carboxamide)

Compound 52 was obtained by the same preparation method as in Experimental Example 1-27 using 3-isocyanatophenol instead of isocyanatobenzene in step 3 of Experimental Example 1-27.

Experimental Example 1-53. Compound 53 (2-((4-amino-3-morpholinophenyl)sulfonyl)- N -Preparation of phenylhydrazine-1-carboxamide)

Compound 53 was obtained by a preparation method similar to Experimental Example 1-27 using 3-fluoro-4-nitrobenzenesulfonyl chloride instead of 4-nitrobenzenesulfonyl chloride as a starting material.

Experimental Example 1-54. Compound 54 (4-hydroxy- N Preparation of -(((4-methoxyphenyl)sulfonyl)methyl)benzamide)

In the same preparation method as in Experimental Example 1-16, compound 54 was obtained by using 4-methoxybenzenethiol instead of 4-nitrobenzenethiol in synthesis step 3.

Experimental Example 1-55. compound 55 ( N Preparation of (((4-aminophenyl)sulfonyl)methyl)-[1,1′-biphenyl]-4-carboxamide)

Compound 55 was obtained by a similar preparation method as in Experimental Example 1-16 using [1,1`-biphenyl]-4-carboxamide as a starting material instead of A28, which is an intermediate of Experimental Example 1-16.

Example 2. Binding assay experiment

Example 2-1. Determining whether muscle actin is an Arg/N-degron pathway substrate

After culturing the L6 cell line, which is a rat muscle-derived cell, in a DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained with 5% carbon dioxide, each cell was dispensed into a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. To confirm whether MG132 increases UBR1 binding, cells were harvested after treatment with MG132 (10 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in FIG. 1 . Immunoblotting was plotted representatively from at least three independent experiments.

Referring to Figure 1, it was confirmed that the levels of ACTA1, ACTC1, ACTG2 increased more by MG132 than the control group. In addition, it was confirmed that the levels of ACTA1 and ACTG2 increased when the UBR protein was knocked down. That is, it was confirmed that muscle actin is an Arg/N-degron pathway substrate.

Example 2-2. Confirmation of inhibition of R-nsP4 degradation through in vitro transcription-translation method

To confirm the R-nsP4 expression of the compounds, TnT® Quick Coupled Transcription/Translation System kit was used. Pre-mix was prepared using Transcend Biotin-Lysyl-tRNA, Methionine, Bestatin, TnT quick Master mix and DHFR-Ub-R-nsP4 Plasmid and mixed with the compound (1 μM). After each sample was reacted at 30 ° C. for 40 minutes, 5X SDS loading dye was added. After reacting at 95 ° C. for 2 minutes, 5 μL was taken and dispensed into each well of an acrylamide gel, and then immunoblotting was performed. The results of the experiment are shown in FIG. 2 . In vitro transcription-translation methods are schematically representative from at least three independent experiments.

Referring to Figure 2, it can be confirmed that the level of R-nsP4 is further increased by Compound 2, Compound 3, Compound 7, Compound 12, Compound 14, and Compound 16 compared to the control group (Control). That is, when the compound according to the present invention was treated, it was confirmed that the level of R-nsP4 increased through binding to UBR1.

Example 2-3. Evaluation of inhibition of RGS4 degradation in cells through transfection

L6 cell line derived from rat muscle was cultured in a DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained at 5% carbon dioxide. In order to measure the binding force of UBR1 according to treatment with a representative compound selected from among these compounds, each cell was seeded in a 6-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. React with Opti-MEM, Lipofectamin, and RGS4 plasmid for transfection. After the reaction is over, the cells are treated so that the DNA is expressed in the cells. After 24 hours, cells were harvested after treatment with the compound (5 μM) alone for 24 hours to confirm whether the compound increases UBR1 binding. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in FIG. 3 . Immunoblotting was plotted representatively from at least three independent experiments.

Referring to FIG. 3, it was confirmed that the level of RGS4 was further increased by compounds 2 and 3 compared to the control group (Control). That is, it was confirmed that the level of RGS4 further increased through binding to UBR1 when the compound according to the present invention was treated.

Example 2-4. Evaluation of inhibition of muscle cell actin degradation through immunoblotting

In order to evaluate actin degradation in muscle cells by the compounds, L6 cell line, which is a rat muscle-derived cell, was cultured using DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained with 5% carbon dioxide. In order to measure the binding force of UBR1 according to treatment with a representative compound selected from among these compounds, each cell was seeded in a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. In order to confirm whether the compound increases UBR1 binding, cells were harvested after treatment with the compound (5 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in FIGS. 4, 5, 6, 7, 8, and 9 . Immunoblotting was plotted representatively from at least three independent experiments.

4 and 5, compared to the control group (Control), compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 16, compound 35, compound 36, compound 37, compound 38 , Compound 39, Compound 43, Compound 44, Compound 45, Compound 46 and Compound 47 can confirm that the level of ACTA1 is further increased. That is, it was confirmed that when the compound according to the present invention was treated, degradation of ACTA1, an intramuscular protein, was inhibited through binding to UBR1.

6 and 7, compared to the control (Control), compound 8, compound 9, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27 , It can be confirmed that the level of ACTA2 is further increased by compound 28, compound 32, compound 33, and compound 34. That is, it was confirmed that when the compound according to the present invention was treated, degradation of ACTA2, an intramuscular protein, was inhibited through binding to UBR1.

Referring to Figure 8, it can be confirmed that the level of ACTC1 is further increased by Compound 41 and Compound 42 compared to the control group (Control). That is, it was confirmed that when the compound according to the present invention was treated, degradation of ACTC1, an intramuscular protein, was inhibited through binding to UBR1.

Referring to FIG. 9, it was confirmed that the level of ACTG2 was further increased by Compound 12, Compound 13, Compound 14, Compound 15, Compound 17, Compound 29, Compound 30, and Compound 31 compared to the control group. That is, when the compound according to the present invention was treated, it was confirmed that the degradation of ACTG2, an intramuscular protein, was inhibited through binding to UBR1.

Example 2-5. Evaluation of UBR box domain binding ability through immunoprecipitation assay

In order to evaluate the binding ability of the compounds to UBR1, UBR2, UBR3 and UBR5 through the UBR box domain, L6 cell line, a rat muscle-derived cell, was maintained in 5% carbon dioxide in an incubator containing 10% FBS and 1% streptomycin/penicillin. It was cultured using a DMEM medium. In order to measure the binding force of UBR1 according to treatment with a representative compound selected from among these compounds, each cell was dispensed into a 100pi dish. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. In order to confirm whether the compound increases UBR1 binding, cells were harvested after treatment with the compound (5 μM), proteasome inhibitor MG132 (10 μM), or positive control (5 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, each sample was reacted with UBR1 antibody for 16 hours, and then Protein A/G beads were reacted for 3 hours. Sample buffer was added to the sample after the reaction and reacted at 100°C for 5 minutes. After taking 20 μL of the sample after the reaction was dispensed into each well of an acrylamide gel, immunoblotting was performed, and the results of the experiment are shown in FIGS. 10 and 11 . Immunoblotting was plotted representatively from at least three independent experiments.

Referring to Figures 10 and 11, when the DMSO control (DMSO control) and the proteasome inhibitor MG132 used as a negative control were treated, the binding force of UBR1 and the substrate ACTA1 was maintained, while the compound was treated. Similar to the positive control, the binding force of ACTA1, UBR2 and ACTA1, UBR3 and ACTA1, and UBR5 and ACTA1 decreased, confirming that compound 2 actually binds to the UBR box domain of UBR proteins. That is, it was confirmed that when the compound according to the present invention was treated, degradation of ACTG2, an intramuscular protein, was inhibited through binding to UBR1, UBR2, UBR3 or UBR5.

Example 2-6. Evaluation of UBR box domain binding force via MST

1) UBR1 protein preparation

The Gln97-Pro168 part corresponding to the UBR box of Human UBR1 (UniProt ID: Q8IWV7) was cloned into a modified expression vector and then expressed in E.coli. After using affinity chromatography and removing the tag with protease, Gly-His-Met was added to the N-terminus. After performing ion chromatography, the UBR box protein of the final UBR1 was purified using gel filtration chromatography in a buffer composition of 10 mM NaCl, 20 mM Tris-HCl, 2 mM beta-mercaptoethanol, pH 7.5.

2) UBR1 UBR box protein labeling

The monolith protein labeling kit RED-NHS 2nd generation (Cat# MO-L011) dye has an NHS-ester group that forms a covalent bond with primary amines (lysine residues). This dye is optimized for Monolith-series equipment with RED detector. The UBR1 UBR box protein purified using this kit was labeled according to the proposed protocol.

3) Measurement of UBR1 and ligand binding using MST

Thermophoresis refers to a phenomenon in which particles move due to a temperature gradient. Particles in high-temperature regions have greater kinetic energy than particles in low-temperature regions, and collide with neighboring particles more often with greater energy. As a result, particles move from a high-temperature region to a low-temperature region.

The thermophoresis of proteins is typically different from that of protein-ligand complexes. This is because the size, charge, and solvation energy change when the ligands bind. Even though ligand binding does not significantly change the protein's size or charge, MST can detect changes in the solvation entropy of protein molecules due to ligand binding. Therefore, the binding between the UBR1 UBR box protein and the ligand compound was measured using MST, and it was confirmed that the presented ligand binds to the UBR1 UBR box (see FIGS. 12 to 19).

Example 3. Preparation of bifunctional compounds

List of bifunctional compounds

Example No.	ID	compound name
Experimental Example 3-1	compound A	4-Amino- N '-(4-(2-(2-(2-(2-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5 -dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethoxy)benzoyl ) Benzenesulfonohydrazide
Experimental Example 3-2	compound B	4-Amino- N '-(4-((5-((5-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4 -oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)pentyl)oxy)pentyl)oxy)benzoyl)benzenesulfonohydrazide
Experimental Example 3-3	compound C	4-amino-3-(4-(14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thio Oxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)- N '-(4-Hydroxybenzoyl)benzenesulfonohydrazide
Experimental Example 3-4	compound D	2-((4-aminophenyl)sulfonyl) -N- (3-((3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene -3-carbonyl)phenoxy)-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)oxy)phenyl)hydrazine-1-carboxamide
Experimental Example 3-5	compound E	4-amino-3-(4-(3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene-3-carbonyl)phenoxy)- 6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)piperazin-1-yl) -N '-(1 H -indole-4-carbonyl)benzene Sulfonohydrazide

¹ H NMR spectra were recorded on a Bruker Avance III 400 MHz and Bruker Fourier 300 MHz, with TMS used as an internal standard.

LCMS was measured on a quadrupole mass spectrometer on an Agilent 1260HPLC and 6120MSD. (Column operating in ES (+) or (-) ionization mode: C18 (50 Х 4.6 mm, 5 μm); T = 30 ^o C; flow rate = 1.5 mL/min; detected wavelengths: 220 nm, 254 nm)

Experimental Example 3-1. 4-Amino- N '-(4-(2-(2-(2-(2-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5 -dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethoxy)benzoyl ) Preparation of benzenesulfonohydrazide (Compound A)

Step 1) Synthesis of A1

A mixture of 4-hydroxybenzohydrazide (5.00 g, 32.9 mmol, 1.0 eq) and 4-nitrobenzene-1-sulfonyl chloride (5.81 g, 26.3 mmol, 0.8 eq) in pyridine (50 mL) was heated to 80 °C. was stirred for 16 hours. Then, 1N HCl was added to the mixture until pH=3 and extracted with EA (20 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude A1 ( N′ -(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 6.50 g) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 10.53 (s, 1H), 10.37 (s, 1H), 10.14 (s, 1H), 8.36-8.38 (d, J = 8.0 Hz, 2H), 8.06–8.08 (d, J = 8.0 Hz, 2H), 7.56–7.59 (d, J = 12 Hz, 2H), 6.76–6.78 (d, J = 8.0 Hz, 2H).

LCMS; Chemical Formula: C ₁₃ H ₁₁ N ₃ O ₆ S; Mass Calcd.:337.3; MS Found: 337.8 [MS].

Step 2) Synthesis of A2

A mixture of Al (6.85 g, 20.3 mmol, 1.0 eq) and 5% Pd/C (500 mg, 50% in water) in EtOH (10 mL) was stirred at 10 °C for 4 h using a H ₂ balloon. Then, the mixture was filtered and the filtrate was concentrated to obtain a crude product, which was purified by prep-HPLC. The collected fractions were concentrated to remove most of the CH ₃ CN. The remaining fractions were freeze-dried to obtain A2 (4-amino- N' -(4-hydroxybenzoyl)benzenesulfonohydrazide, 2.30 g, yield 36.6%) as a white solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 10.31 (s, 1H), 10.06 (s, 1H), 9.13 (s, 1H), 7.55-7.58 (d, J = 12.0 Hz, 2H), 7.40–7.42 (d, J = 8.0 Hz, 2H), 6.75–6.77 (d, J = 8.0 Hz, 2H), 6.49–6.51 (d, J = 12.0 Hz, 2H).

LCMS; Chemical Formula: C ₁₃ H ₁₃ N ₃ O ₄ S; Mass Calcd.:307.3; MS Found: 307.9 [MS] and 329.0 [MS+22].

Step 3) Synthesis of A4

A3 (2,2′-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-1-ol), 10.0 g, 51.5 mmol, 1.0 eq) in DCM (100 mL) To a solution of DMAP (6.30 mg, 5.15 mmol, 0.1 eq) and TEA (7.80 g, 77.3 mmol, 1.5 eq) were added at 25 °C. The mixture was then added dropwise with TsCl (11.8 g, 61.8 mmol, 1.2 eq) in DCM (20 mL) at 0-5 °C. The mixture was stirred overnight at 25 °C. The reaction was extracted with ethyl acetate then brine, concentrated and purified by column chromatography (PE/EA=100/1-20/1) to yield A4 (2-(2-(2-(2-hydroxy) as a yellow oil ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate, 4.00 g, 22.3%) was obtained.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.80-7.82 (dd, J = 4.0 Hz, J = 4.0 Hz 2H), 7.35-7.37 (d, J = 8 Hz, 2H), 4.12-4.19 (m , 2H), 3.57–3.73 (m, 14H), 2.46 (s, 3H).

LCMS; Chemical Formula: C ₁₅ H ₂₄ O ₇ S; Mass Calcd.:348.4; MS Found: 349.2 [MS+1].

Step 4) Synthesis of A5

K ₂ CO ₃ (4Х265 mg, 1.92 mmol, 2.0 eq) and 4-(3-(4′-hydroxy-[1 ,1′-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (CAS number : 1973408-76-2, (4Х461 mg, 0.960 mmol, 1.0 eq) was added The mixture was stirred overnight at 60 ° C. The solid was collected by filtration and the reaction mass was quenched by adding 150 mL of water and extracted with 3Х100 mL of ethyl acetate. The organic layers were mixed and washed with 4Х200 mL of brine. The residue was applied to a silica gel column (PE/EA=100/1 to 5/1) with ethyl acetate to form A5 (4- (3-(4′-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethoxy)-[1,1′-biphenyl]-4-yl)-4 ,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile, 2.00 g, 74.0%) was obtained as a yellow solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 8.00-8.02 (m, 2H), 7.87-7.90 (m, 1H), 7.81-7.83 (m, 1H), 7.71-7.73 (m, 2H) 7.56- 7.58 (m, 2H) 7.34-7.37 (m, 3H), 7.03-7.05 (d, J = 8.0 Hz, 2H), 4.17-4.22 (m, 3H), 3.90-3.92 (m, 2H), 3.72-3.78 (m, 9H), 3.62–3.70 (m, 7H), 1.64 (s, 6H).

LCMS; Chemical Formula: C ₃₃ H ₃₄ F ₃ N ₃ O ₆ S; Mass Calcd.:657.7; MS Found: 658.3 [MS+1].

Step 5) Synthesis of A6

To a solution of A5 (2 g, 3.04 mmol, 1.0 eq) in DCM (20 mL) was added DMAP (36.6 mg, 0.30 mmol, 0.1 eq) and TEA (615.2 mg, 6.08 mmol, 2.0 eq) at 25 °C. Subsequently, TsCl (1.16 g, 6.08 mmol, 2.0 eq) dissolved in DCM (10 mL) was added dropwise to the mixture at 0-5 °C. The mixture was stirred overnight at 25 °C. The reaction was extracted with 3Х40 mL of ethyl acetate, concentrated with brine, and purified by column chromatography (PE/EA=100/1~20/1) to obtain A6 (2-(2-(2-(2-((4'- (3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1′- Biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate, 1.00 g, 40.5%) was obtained as a yellow oil.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.99-8.01 (m, 2H), 7.79-7.81 (d, J = 8.0 Hz, 1H), 7.71-7.73 (d, J = 8.0 Hz, 2H), 7.55-7.57 (d, J = 8.0 Hz, 2H) 7.33-7.37 (m, 4H), 7.02-7.04 (d, J = 8.0 Hz, 2H), 4.12-4.21 (m, 4H), 3.89-3.91 (dd , J = 4.0 Hz, J = 4.0 Hz, 2H), 3.74–3.75 (m, 2H), 3.67–3.71 (m, 4H), 3.61 (s, 4H), 2.45 (s, 3H), 1.64 (s, 6H).

Step 6) Synthesis of Compound A

To a solution of A6 (50.0 mg, 0.061 mmol, 1.0 eq) in acetonitrile (8 mL) was added DBU (10.0 mg, 0.0610 mmol, 1.0 eq) and A2 (57.1 mg, 0.186 mmol, 3.0 eq). The mixture was stirred overnight at 60 °C. The solid was collected by filtration. Then, the reaction was quenched by adding 20 mL of water and extracted with 3Х20 mL of ethyl acetate. The organic layers were mixed and washed with 2Х20mL of brine. The residue was purified by silica gel column chromatography (PE/EA=100/1-5/1) to obtain compound A (4-amino- N' -(4-(2-(2-(2-(2-((( 4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1, 1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethoxy)benzoyl)benzenesulfonohydrazide, 30 mg, 55.5%) as a white solid.

^1H -NMR (CDCl ₃ , 400 MHz): δ 8.68 (s, 1H) 7.99-8.00 (d, J = 4.0 Hz, 2H), 7.87-7.89 (d, J = 8.0 Hz, 1H), 7.68-7.70 (d, J = 8.0 Hz, 3H), 7.60-7.62 (d, J = 8.0 Hz, 2H) 7.51-7.53 (d, J = 8.0 Hz, 2H) 7.34-7.36 (d, J = 8.0 Hz, 2H) , 6.98–7.00 (d, J = 8.0 Hz, 2H), 6.79–6.81 (d, J = 8.0 Hz, 2H), 6.64–6.66 (d, J = 8.0 Hz, 2H), 4.16 (s, 2H), 3.79–3.83 (d, J = 12 Hz, 6H), 3.62–3.68 (m, 8H), 1.64 (s, 6H).

LCMS; Chemical Formula: C ₄₆ H ₄₅ F ₃ N ₆ O ₉ S ₂ ; Mass Calcd.:947.0; MS Found: 949.5 [MS+2].

Experimental Example 3-2. 4-Amino- N '-(4-((5-((5-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4 -Oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)pentyl)oxy)pentyl)oxy)benzoyl)benzenesulfonohydrazide (compound B) manufacture of

Step 1) Synthesis of B1

To a solution of pentane-1,5-diol (60.0 g, 576 mmol, 1.0 eq) in ethyl acetate (300 mL) was added triethylamine (12.2 g, 242 mmol, 0.42 eq) and triphenylchloromethane (33.7 g, 121 mmol, 0.21 eq) was added continuously under stirring. The solution was slowly heated to reflux and stirred for 5 hours. After cooling, the reaction solution was poured into 100 mL of water to quench and the aqueous phase was extracted with ethyl acetate (200 mLХ3). The combined organic phases were washed with 60 mL of water and 60 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography (PE/EA=20/1~3/1) to give B1 (5-(trityloxy)pentan-1-ol, 40.0 g, 20.0%) as a colorless oil.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.48-7.46 (m, 5H), 7.34-7.23 (m, 10 H), 3.70-3.62 (m, 2H), 3.11-3.08 (t, J = 6.4 Hz, 2H), 1.72–1.40 (m, 6H).

Step 2) Synthesis of B2

To a solution of B1 (40.0 g, 115 mmol, 1.0 eq) in DCM (400 mL) was added triethylamine (23.3 g, 231 mmol, 2.0 eq), DMAP (2.8 g, 23.1 mmol, 0.2 eq) and p-toluenesulfonyl chloride. (44.0 g, 231 mmol, 2.0 eq) was added. The mixture was stirred overnight at room temperature. TLC showed the reaction to be complete. The reaction was then quenched by adding 500 mL of water and extracted with 2Х200 mL of DCM. The organic layers were mixed and washed with brine. The residue was applied to a silica gel column (PE/EA=50/1~10/1) to obtain B2 (5-(trityloxy)pentyl 4-methylbenzenesulfonate, 40.0 g, 69.5%) as a white solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.81-7.79 (d, J = 8.4 Hz, 2H), 7.44-7.42 (m, 5H), 7.34-7.23 (m, 12H), 4.04-4.01 (t , J = 6.4 Hz, 2H) 3.05–3.02 (t, J = 6.4 Hz, 2H) 2.44 (s, 3H), 1.65–1.56 (m, 4H), 1.44–1.39 (m, 2H).

LCMS; Chemical Formula: C ₃₁ H ₃₂ O ₄ S; Mass Calcd.:500.6; MS Found: 523.2 [MS+23].

Step 3) Synthesis of B3

To a solution of NaH (6.08 g in 50 mL THF, 60% dispersed in mineral oil, 25.3 mmol) was added a solution of B1 (31.5 g, 90.9 mmol, 1.2 eq) in THF (150 mL) at 0 °C under N ₂ . did The mixture was stirred at room temperature for 30 min and a solution of B2 (38.0 g, 76 mmol, 1 eq) in DMF (150 mL) was added at this temperature. The resulting mixture was stirred at room temperature for 12 hours. The reaction was quenched with brine and extracted with EtOAc (300 mL, twice). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (PE/EA=50/1-15/1) to B3 ((((oxybis(pentane-5,1-diyl))bis(oxy))bis(methanetetrayl) )) Hexabenzene, 32.0 g, 65%) was obtained as a colorless oil.

^1H -NMR (CDCl ₃ , 400 MHz): δ 7.49-7.47 (m, 12H), 7.34-7.23 (m, 18H), 3.41-3.38 (t, J = 6.8 Hz, 4H), 3.11-3.08 (t , J = 6.4 Hz, 4H), 1.70–1.65 (m, 4H), 1.59–1.54 (m, 4H), 1.48–1.42 (m, 4H).

Step 4) Synthesis of B4

To a solution of B3 (32.0 g, 47.4 mmol, 1 eq) in THF/MeOH (150 mL/150 mL) was added p-toluenesulfonic acid monohydrate (1.80 g, 9.48 mmol, 0.2 eq). The mixture was stirred overnight at room temperature. The reaction was quenched with a solution of NaOH (0.35 g in 5 mL water, 8.75 mmol) and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/MeOH=100/1-15/1) to give B4 (5,5'-oxybis(pentan-1-ol), 4.5 g, 50%) as a colorless oil. was obtained with

¹ H-NMR (CDCl ₃ , 400 MHz): δ 3.64-3.60 (m, 4H), 3.43-3.40 (t, J = 6.4 Hz, 4H), 2.49 (m, 2H), 1.63-1.54 (m, 8H) ), 1.46–1.40 (m, 4H).

Step 5) Synthesis of B5

NaOH (0.94g, 23.6mmol, 1eq) was added to a solution of B4 (4.50g, 23.6mmol, 1eq) in THF/H ₂ O (100mL/100mL). Subsequently, a solution of p-toluenesulfonyl chloride (2.25 g, 11.8 mmol, 0.5 eq) in THF (100 mL) was added dropwise at this temperature for 2 hours. TLC showed the reaction to be complete. The reaction was quenched with ice water and extracted with EtOAc (100 mL, 2 times). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (PE/EA=50/1-5/1) to give B5 (5-((5-hydroxypentyl)oxy)pentyl 4-methylbenzenesulfonate, 1.8 g, 22.1 % ) was obtained as a white solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.81-7.79 (d, J = 8.4 Hz, 2H), 7.37-7.35 (d, J = 8.0 Hz, 2H), 4.06-4.02 (m, 2H), 3.67-3.60 (m, 2H) 3.41-3.35 (m, 3H) 2.46 (s, 3H), 1.71-1.37 (m, 14H).

Step 6) Synthesis of B6

K ₂ CO ₃ (1.44 g, 10.4 mmol, 2.0 eq) and 4-(3-(4′-hydroxy-[1 ,1′-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (CAS number : 1973408-76-2, 3.0 g, 6.2 mmol, 1.2eq) was added. The mixture was stirred overnight at 60 °C. The solid was collected by filtration. The reaction was then quenched by adding 150 mL of water and extracted with 3Х300 mL of ethyl acetate. The organic layers were mixed and washed with 4Х200 mL of brine. The residue was applied to a silica gel column (PE/EA=50/1~5/1) with ethyl acetate to obtain B6 (4-(3-(4′-((5-((5-hydroxypentyl)oxy )) pentyl)oxy)-[1,1'-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(tri Fluoromethyl)benzonitrile, 1.50 g, 44.2%) was obtained as a yellow solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 8.01 (m, 2H), 7.99 (m, 1H), 7.87-7.71 (m, 2H), 7.57-7.55 (m, 2H) 7.36-7.34 (m, 2H) 7.01-6.99 (m, 2H), 4.09-4.05 (m, 2H), 3.68-3.65 (m, 2H), 3.48-3.41 (m, 4H), 1.88-1.84 (m, 2H), 1.70-1.55 (m, 16H).

LCMS; Chemical Formula: C ₃₅ H ₃₈ F ₃ N ₃ O ₄ S; Mass Calcd.:653.7; MS Found: 654.3 [MS+1].

Step 7) Synthesis of B7

To a solution of B6 (1.50 g, 2.29 mmol, 1.0 eq) in DCM (20 mL) was added DMAP (55.8 mg, 0.46 mmol, 0.2 eq) and TEA (463 mg, 4.58 mmol, 2.0 eq) at 25 °C. Then, TosCl (873 mg, 4.58 mmol, 2.0eq) dissolved in DCM (5 mL) was added dropwise to the mixture at 0-5 °C. The mixture was stirred overnight at 25 °C. The reaction was extracted with 3Х100mL of ethyl acetate, concentrated with brine, and purified by column chromatography (PE/EA=50/1~10/1) to obtain B7 (5-((5-((4'-(3-(4- Cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1′-biphenyl]-4- yl)oxy)pentyl)oxy)pentyl 4-methylbenzenesulfonate, 1.20 g, 64.8%) was obtained as a yellow solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 8.02-8.00 (m, 2H), 7.90-7.87 (m, 1H), 7.81-7.79 (m, 2H), 7.73-7.71 (m, 2H) 7.58- 7.56 (m, 2H) 7.37-7.34 (m, 4H), 7.02-7.00 (m, 2H), 4.06-4.02 (m, 4H), 3.45-3.37 (m, 4H), 2.46 (s, 3H), 1.88 -1.84 (m, 2H), 1.71-1.41 (m, 16H).

Step 8) Synthesis of Compound B

To a solution of B7 (1.20g, 1.48mmol, 1.0eq) in acetonitrile (15mL) was added DBU (226mg, 1.48mmol, 1.0eq) and A2(4-amino- N' -(4-hydroxybenzoyl)benzene Phonohydrazide, 1.37g, 4.45mmol, 3.0eq) was added. The mixture was stirred overnight at 60 °C. The solid was collected by filtration. The reaction was then quenched by adding 200 mL of water and extracted with 3Х100 mL of ethyl acetate. The organic layers were mixed and washed with 4Х100mL of brine. The crude product was then purified by preparative HPLC to compound B (4-amino- N' -(4-((5-((5-((4'-(3-(4-cyano-3-(tri Fluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)pentyl)oxy )pentyl)oxy)benzoyl)benzenesulfonohydrazide, 30 mg, 2.1%) as a white solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 8.06-8.05 (m, 1H), 8.02-7.99 (m, 2H), 7.89-7.86 (m, 1H), 7.70-7.66 (m, 4H), 7.59 -7.52 (m, 4H), 7.42 (m, 1H), 7.36-7.34 (m, 2H) 6.98-6.96 (d, J = 8.4 Hz, 2H), 6.86-6.84 (d, J = 8.8 Hz, 2H) , 6.58 (br s, 2H), 4.05–3.96 (m, 4H), 3.49–3.45 (m, 4H), 1.89–1.79 (m, 4H), 1.72–1.51 (m, 14H).

LCMS; Chemical Formula: C ₄₈ H ₄₉ F ₃ N ₆ O ₇ S ₂ ; Mass Calcd.: 943.0; MS Found: 944.3 [MS+1].

Experimental Example 3-3. 4-amino-3-(4-(14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thio Oxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)- N Preparation of '-(4-hydroxybenzoyl)benzenesulfonohydrazide (Compound C)

Step 1) Synthesis of C1

A mixture of 4-hydroxybenzohydrazide (316 mg, 2.08 mmol, 1.0 eq) in pyridine (10 mL) was added with 3-fluoro-4-nitrobenzenesulfonyl chloride (498 mg, 2.08 mmol, 1.0 eq) in pyridine (3 mL). eq) was added dropwise. The mixture was then stirred at 10 °C for 3 hours. The solution was poured into water (10 mL) and extracted with EA (30 mL). The combined organic layers were washed with 1N HCl (30 mL) and brine, dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography (DCM/MeOH=50/1~30/1) to C1 (3-fluoro Rho- N' -(4-hydroxybenzoyl)-4-nitrobenzenesulfonohydrazide, 300 mg, 40.5 %) was obtained as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 10.56 (s, 1H), 10.51 (s, 1H), 10.17 (s, 1H), 8.30-8.34 (m, 1H), 7.96–7.99 (m, 1H), 7.84–7.86 (m, 1H), 7.60 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H).

Step 2) Synthesis of C3

4-dimethylaminopyridine (256mg, 2.10mmol, 0.1eq) and triethylamine (3.18g, 31.5mmol, 1.5eq) were added at 25°C. Subsequently, p-toluenesulfonyl chloride (4.81 g, 25.2 mmol, 1.2 eq) dissolved in DCM (10 mL) was added dropwise to the mixture at 0-5 °C. The mixture was stirred overnight at 25 °C. The reaction was poured into water (50 mL), extracted with DCM (10 mL) then brine, concentrated and purified by column chromatography (PE/EA=100/1-20/1) to C3 (14-hydroxy-3 ,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate, 2.50 g, 30.3%) was obtained as a yellow oil.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.79 (d, J =8.4 Hz, 2H), 7.34 (d, J =8 Hz, 2H), 4.14-4.17 (m, 2H), 3.57-3.72 ( m, 18H), 2.74 (s, 1H), 2.44 (s, 3H)

LCMS; Chemical Formula: C ₁₇ H ₂₈ O ₈ S; Mass Calcd.: 392.4; MS Found: 393.1 [MS+1].

Step 3) Synthesis of C4

K ₂ CO ₃ (1.17g, 8.50mmol, 2.0eq) and 4- ( 3-(4′- Hydroxy-[1,1'-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl) Benzonitrile (CAS number: 1973408-76-2, 2.05g, 4.25mmol, 1.0eq) was added. The mixture was stirred overnight at 60 °C. The solid was collected by filtration. The reaction was then quenched with water (30 mL) and extracted with ethyl acetate (10 mLХ2). The organic layers were mixed and washed with brine. The residue was applied to a silica gel column (PE/EA=100/1~5/1) to C4 (4-(3-(4′-((14-hydroxy-3,6,9,12-tetraoxatetra Decyl)oxy)-[1,1'-biphenyl]-4-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoro methyl)benzonitrile, 1.50 g, 50.3%) was obtained as a yellow solid.

LCMS; Chemical Formula: C ₃₅ H ₃₈ F ₃ N ₃ O ₇ S; Mass Calcd.: 701.7; MS Found: 703.2 [MS+2].

Step 4) Synthesis of C5

4-dimethylaminopyridine (8.69 mg, 0.071 mmol, 0.1 eq) and triethylamine (144 mg, 1.42 mmol, 2.0 eq) were added to a solution of C4 (500 mg, 0.71 mmol, 1.0 eq) in DCM (5 mL) at 25 °C. added in. Subsequently, p-toluenesulfonyl chloride (272 mg, 1.42 mmol, 2.0eq) dissolved in DCM (2 mL) was added dropwise to the above mixture at 0-5°C. The mixture was stirred overnight at 25 °C. The reaction was washed with water (10 mL) then brine and concentrated. Another two batches were run with this procedure. It was then purified by column chromatography (PE/EA=100/1 to 20/1) to form C5 (14-((4′-(3-(4-isocyano-3-(trifluoromethyl)phenyl)) -5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- Tetraoxatetradecyl 4-methylbenzenesulfonate, 0.50 g, 82.1%) was obtained as a yellow oil.

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.97-7.99 (m, 2H), 7.85-7.88 (m, 1H), 7.79 (d, J =8.4 Hz, 2H), 7.70 (d, J =8.8 Hz, 2H), 7.55 (d, J =8.8 Hz, 2H), 7.32-7.35 (m, 4H), 7.01 (d, J =8.8 Hz, 2H), 4.14-4.20 (m, 4H), 3.87-3.90 (m, 2H), 3.73-3.76 (m, 2H), 3.62-3.70 (m, 8H), 3.58 (s, 4H), 2.44 (s, 3H), 1.63 (s, 6H)

LCMS; Chemical Formula: C ₄₂ H ₄₄ F ₃ N ₃ O ₉ S ₂ ; Mass Calcd.: 855.9; MS Found: 856.2 [MS+1] and 878.2 [MS+23].

Step 5) Synthesis of C6

K ₂ CO ₃ to a solution of C5 (500 mg, 0.58 mmol, 1.0 eq) and tert-butylpiperazine-1-carboxylate (131 mg, 0.70 mmol, 1.2 eq) in N,N-dimethylformamide (5 mL). (242mg, 1.74mmol, 3.0eq) was added at 25°C. The mixture was stirred overnight at 25 °C. The reaction was poured into cold water (5 mL), extracted with EA (5 mLХ2), then brine, and concentrated. Another two batches were run with this procedure. The residue was purified by column chromatography (DCM/MeOH=100/1-30/1), diluted with DCM (10 mL) and trifluoroacetic acid (5 mL) was added at 0 °C. The mixture was stirred at 25 °C for 3 hours. The reaction was concentrated to give C6 (400 mg, crude) as a yellow oil.

LCMS; Chemical Formula: C ₃₉ H ₄₆ F ₃ N ₅ O ₆ S; Mass Calcd.: 769.8; MS Found: 770.3 [MS+1].

Step 6) Synthesis of Compound C

C1 (408 mg, 0.52 mmol, 1.0 eq) and K ₂ CO ₃ (215 mg, 1.56 mmol, 3.0 eq) were added to a solution of C6 (400 mg, crude) in N,N- dimethylformamide (10 mL) at 25 °C. added. The mixture was stirred overnight at 25 °C. The reaction was poured into cold water (10 mL), extracted with EA (5 mLХ2), then brine, concentrated, and purified by column chromatography (DCM/MeOH=100/1-30/1) to give 3-(4-( 14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl) -[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)-N'-(4-hydroxybenzoyl)- 4-Nitrobenzenesulfonohydrazide (91 mg, 16% yield) was obtained as a yellow solid. It was then diluted with DCM (10 mL) and 10% Pd/C (110 mg, 10%) was added to the solution at 25° C. under H ₂ . The mixture was stirred at 25° C. under H ₂ for 1 hour. The reaction was filtered and concentrated. Another 300 mg batch was run as per the procedure above. Crude was purified by HPLC to obtain compound C (4-amino-3-(4-(14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5 -Dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl )piperazin-1-yl)-N'-(4-hydroxybenzoyl)benzenesulfonohydrazide, 50 mg) as a white solid.

¹ H-NMR (CD ₃ OD, 400 MHz): δ 8.15-8.20 (m, 2H), 8.00-8.04 (m, 1H), 7.74 (d, J =8.4 Hz, 2H), 7.57-7.64 (m, 4H), 7.43 (d, J =8.4 Hz, 4H), 7.05 (d, J =8.8 Hz, 2H), 6.80 (d, J =2.0 Hz, 2H), 6.72 (d, J =8.4 Hz, 1H) , 4.17-4.19 (m, 2H), 3.86-3.89 (m, 2H), 3.60-3.74 (m, 14H), 2.75-2.77 (m, 4H), 2.64-2.67 (m, 6H), 1.60 (s, 6H).

LCMS; Chemical Formula: C ₅₂ H ₅₇ F ₃ N ₈ O ₁₀ S ₂ ; Mass Calcd.: 1075.1; MS Found: 1076.6 [MS+1].

Experimental Example 3-4. 2-((4-aminophenyl)sulfonyl) -N- (3-((3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene -3-carbonyl)phenoxy)-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)oxy)phenyl)hydrazine-1-carboxamide (compound D) manufacture of

Step 1) Synthesis of D1

To a solution of hydrazine hydrate (11.3 g, 225.6 mmol, 2.5 eq) in THF (200 mL) was added a solution of 4-nitrobenzenesulfonyl chloride (20 g, 90.3 mmol, 1.0 eq) in THF (100 mL) at 0 °C. did The reaction mixture was stirred overnight at room temperature. The solution was poured into water (500 mL) and extracted with EA (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give D1 (4-nitrobenzenesulfonohydrazide, 15 g, yield: 76.5%) as a yellow solid.

¹ H-NMR (CD ₃ OD, 400 MHz): δ 8.77 (s, 1H), 8.41-8.44 (m, 2H), 8.03-8.07 (m, 2H), 4.34 (s, 2H)

LCMS; Chemical Formula: C ₆ H ₇ N ₃ O ₄ S; Mass Calcd.: 217.1; MS Found: 218.0 [MS+1].

Step 2) Synthesis of D2

Et ₃ N (34 g, 0.34 mol, 1.5 eq), DMAP (2.7 g, 0.022 mol, 0.1eq) and Boc ₂ O (57 g, 0.26 mol, 1.2eq) were added at 0° C. under N ₂ . The reaction mixture was stirred overnight at room temperature. The solution was poured into water (500 mL) and extracted with DCM (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to obtain D2 (15 g of tert-butyl ethyl(2-hydroxyethyl)carbamate, yield: 35.3%) as a colorless oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 4.65 (brs, 1H), 3.42-3.46 (m, 2H), 3.15-3.22 (m, 4H), 1.41 (s, 9H), 1.02 (s , 3H).

Step 3) Synthesis of D3

(4-fluorophenyl)(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophen-3-yl)methanone in DMF (220mL) (CAS number: 202336-25-2 , 22 g, 60.4 mmol, 1.0 eq) was added K ₂ CO ₃ (33 g, 241.6 mml, 4.0 eq) and benzyl bromide (31 g, 181.3 mmol, 3.0 eq) at 0 °C. The reaction mixture was stirred overnight at room temperature. The solution was poured into cold water (500 mL) and extracted with EA (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to D3 ((6-(benzyloxy)-2-(4-(benzyloxy)phenyl)benzo[b]thiophen-3-yl)(4-fluorophenyl)methane on, 22 g, yield: 66.9%) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.74-7.79 (m, 3H), 7.12-7.50 (m, 16H), 6.94-6.95 (m, 2H), 5.21 (s, 2H), 5.06 (s, 2H).

Step 4) Synthesis of D4

To a solution of D2 (17 g, 45.9 mmol, 1.0 eq) in DMF (250 mL) was added NaH (3.9 g, 96.4 mmol, 2.1 eq) at 0 °C. The reaction mixture was stirred for 1 hour. A solution of D3 (25 g, 45.9 mmol, 1.0 equiv) in DMF (100 mL) was added at 0 °C overnight. The solution was poured into cold water (600 mL) and extracted with EA (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residual crude product was purified by column chromatography to obtain D4 (tert-butyl(2-(4-(6-(benzyloxy)-2-(4-(benzyloxy)phenyl)benzo[b]thiophene-3-carb Bornyl)phenoxy)ethyl)(ethyl)carbamate, 20 g, yield: 61.0%) was obtained as a colorless oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.76-7.69 (m, 3H), .750-7.48 (m, 2H), 7.43-7.31 (m, 10H), 7.18-7.13 (m, 1H) ), 7.09-7.06 (m, 1H), 6.98-6.93 (m, 4H), 5.20 (s, 2H), 5.06 (s, 2H), 4.09 (s, 2H), 3.47-3.49 (m, 2H), 3.18-3.22 (m, 2H), 1.23-1.37 (m, 9H), 1.00-1.06 (m, 3H).

Step 5) Synthesis of D5

To a solution of D4 (17 g, 23.8 mmol, 1.0 eq) in EA (200 mL) was added EA/HCl (170 mL, 6 mol/L) at 0 °C. The reaction mixture was stirred overnight at room temperature. It was then concentrated and the solution was poured into cold water (500 mL), adjusted to pH=8 with NaHCO ₃ and extracted with EA (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residual crude product was purified by column chromatography to D5 ((6-(benzyloxy)-2-(4-(benzyloxy)phenyl)benzo[b]thiophen-3-yl)(4-(2-(ethyl) Amino)ethoxy)phenyl)methanone, 13 g, yield: 88.9%) was obtained as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.30-7.75 (m, 21H), 5.20 (s, 2H), 5.06 (s, 2H), 4.02-4.04 (m, 2H), 2.82-2.89 (m, 2H), 2.55–2.57 (m, 2H), 0.99 (t, 3H).

Step 6) Synthesis of D7

To a solution of D6 (30 g, 81 mmol, 1.0 eq) and imidazole (11 g, 162 mmol, 2.0 eq) in DCM (500 mL) was added TBDPSCl (24.4 g, 89 mmol, 1.1 eq) at 0 °C under N2. The reaction mixture was stirred overnight at room temperature. It was then concentrated and the solution was poured into cool water (500 mL) and extracted with DCM (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residual crude product was purified by column chromatography to give D7 (26 g, yield: 52.7%) as a colorless oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.1-7.69 (m, 5H), 7.44-7.39 (m, 6H), 3.82-3.81 (m, 2H), 3.74-3.72 (m, 2H) , 3.68-3.60 (m, 37H).

LCMS; Chemical Formula: C ₃₂ H ₅₂ O ₉ Si; Mass Calcd.: 608.8; MS Found: 609.3 [MS+1].

Step 7) Synthesis of D8

To a solution of D7 (6 g, 9.87 mmol, 1.0 eq) in DCM (60 mL) was added Et ₃ N (1.9 g, 19.72 mmol, 2.0 eq), DMAP (120 mg, 0.986 mmol, 0.1 eq) and Tos-Cl (2.8 g). , 14.8 mmol, 1.5 eq) was added at 0° C. under N ₂ . The reaction mixture was stirred overnight at room temperature. The solution was poured into water (50 mL) and extracted with DCM (30 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D8 (6 g, yield: 80%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.78 (d, 2H), 7.63-7.66 (m, 4H), 7.40-7.46 (m, 8H), 4.09-4.11 (m, 2H), 3.73 -3.75 (m, 2H), 3.50-3.56 (m, 28H), 2.29 (s, 3H), 0.99 (s, 9H).

Step 8) Synthesis of D9

To a solution of D8 (12 g, 15.7 mmol, 1.0 eq) in DMF (120 mL), K ₂ CO ₃ (4.3 g, 31.4 mmol, 2.0 eq) and 3-nitrophenol (2.6 g, 18.8 mmol, 1.2 eq) were added. did The reaction mixture was stirred overnight at 80 °C. The solution was poured into water (400 mL) and extracted with EA (100 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D9 (8 g, yield: 69.7%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.40-7.82 (m, 14H), 4.21-4.23 (m, 2H), 3.72-3.77 (m, 4H), 3.33-3.47 (m, 26H) , 0.98 (s, 9H).

Step 9) Synthesis of D10

To a solution of D9 (8 g, 10.9 mmol, 1.0 eq) in THF (80 mL) was added TBAF (22 mL, 21.9 mmol, 1 mol/L in THF, 2.0 eq) at 0 °C. The reaction mixture was stirred overnight at room temperature. It was then concentrated and the solution was poured into cool water (500 mL) and extracted with DCM (50 mLХ10). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residual crude product was purified by column chromatography to give D10 (4 g, yield: 74.3%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.82 (d, 1H), 7.73 (s, 1H), 7.58 (t, 1H), 7.43 (d, 1H), 4.24-4.23 (m, 2H) ), 3.78–3.76 (m, 2H), 3.76–3.43 (m, 28H).

Step 10) Synthesis of D11

To a solution of D10 (4 g, 8.15 mmol, 1.0 eq) in DCM (50 mL), Et ₃ N (1.2 g, 12.2 mmol, 1.5 eq), DMAP (99 mg, 0.82 mmol, 0.1 eq) and Tos-Cl were added to N ₂ was added at 0 °C under The reaction mixture was stirred overnight at room temperature. The solution was poured into water (50 mL) and extracted with DCM (30 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D11 (5 g, yield: 95.2%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.80-7.73 (m, 4H), 7.57 (t, 1H), 7.49-7.44 (m, 3H), 4.23-4.25 (m, 2H), 4.10 -4.12 (m, 2H), 3.77-3.79 (m, 2H), 3.49-3.61 (m, 26H), 2.42 (s, 3H).

Step 11) Synthesis of D12

NaI (2.2g, 14.3mmol, 1.5eq), DIEA (3.8g, 29.37mmol, 3.0eq), D5 (7.6 g, 11.7mmol) were added to a solution of D11 (6g, 9.79mmol, 1.0eq) in DMF (60mL). , 1.2eq) was added. The reaction mixture was stirred overnight at 60 °C. The solution was poured into water (300 mL) and extracted with EA (100 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D12 (4 g, yield: 39.6%) as a yellow oil.

LCMS; Chemical Formula: C ₆₁ H ₇₀ N ₂ O ₁₄ S; Mass Calcd.: 1087.2; MS Found: 543.9 [MS/2].

Step 12) Synthesis of D13

_Iron powder (1.8g, 31.3mmol, 5.0eq), NH ₄ Cl (1.7g, 31.3mmol, 5.0 eq) was added. The reaction mixture was stirred at 60 °C for 3 hours. The solution was poured into water (300 mL) and extracted with EA (100 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D13 (4 g, yield: 60.5%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.78 (s, 1H), 7.69-7.67 (m, 2H), 7.51-7.49 (m, 2H), 7.31-7.43 (m, 10H), 7.06 -7.09 (m, 1H), 6.85-6.98 (m, 5H), 6.05-6.15 (m, 3H), 5.01-5.05 (m, 6H), 3.93-4.05 (m, 4H), 3.54-3.67 (m, 29H), 3.31–3.32 (m, 2H), 2.53–2.63 (m, 4H), 0.92 (t, 3H).

LCMS; Chemical Formula: C ₆₁ H ₇₂ N ₂ O ₁₂ S; Mass Calcd.: 1057.3; MS Found: 1058.8 [MS+1].

Step 13) Synthesis of D14

To a solution of D13 (3g, 2.84mmol, 1.0eq) in DCM (30mL) was added Et ₃ N (860mg, 8.52mmol, 3.0eq) and Triphosgene (421mg, 1.42mmol, 0.5eq) at 0°C under N2. . The mixture was stirred for 1 hour. Compound 2 (739 mg, 3.4 mmol, 1.2 eq) was then added. The reaction mixture was stirred overnight at room temperature. The solution was poured into water (50 mL) and extracted with DCM (30 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give D14 (1.3 g, yield: 35.2%) as a yellow oil.

¹ HNMR (DMSO-d ₆ , 400 MHz): δ 8.64 (S, 1H), 8.54 (s, 1H), 8.42-8.40 (m, 2H), 8.10-8.07 (m, 2H), 7.75-7.67 (m , 3H), 7.49-7.31 (m, 13H), 7.09-6.91 (m, 8H), 6.52-6.50 (m, 1H), 5.19 (s, 2H), 5.06 (s, 2H), 3.96-4.04 (m , 4H), 3.68–3.96 (m, 29H), 2.67–2.69 (m, 6H), 0.94 (t, 3H).

LCMS; Chemical Formula: C ₆₈ H ₇₇ N ₅ O ₁₇ S ₂ ; Mass Calcd.: 1300.5; MS Found: 1300.7 [MS].

Step 14) Synthesis of Compound D

Ammonium formate (1.9g, 29.7mmol, 30.0eq), H ₂ O (712.8mg, 39.6mmol, 40eq) and Pd/C ( 5 g) was added. The mixture was stirred at 50° C. under N ₂ overnight and concentrated to give the crude product. The residue was purified by preparative HPLC to compound D (2-((4-aminophenyl)sulfonyl)-N-(3-((3-ethyl-1-(4-(6-hydroxy-2-( 4-hydroxyphenyl) benzo [b] thiophene-3-carbonyl) phenoxy) -6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)oxy )phenyl)hydrazine-1-carboxamide, 120 mg, yield: 11.0%) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 8.45 (br s, 1H), 8.25-8.19 (m, 2H), 7.67-7.64 (d, 2H), 7.48-7.45 (d, 2H), 7.35 (s, 1H), 7.26-7.24 (m, 1H), 7.18-7.08 (m, 4H), 6.94-6.83 (m, 4H), 6.69-6.67 (d, 2H), 6.61-6.58 (m, 2H) ), 6.53-6.50 (m, 1H), 6.04 (s, 2H), 4.02-4.04 (m, 4H), 3.71-3.72 (m, 2H), 3.48-3.58 (m, 26H), 2.81-2.50 (m , 6H), 0.94 (t, 3H).

LCMS; Chemical Formula: C ₅₄ H ₆₇ N ₅ O ₁₅ S ₂ ; Mass Calcd.: 1090.2; MS Found: 1091.7 [MS+1].

Experimental Example 3-5. 4-amino-3-(4-(3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)- 6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)piperazin-1-yl)-N'-(1H-indole-4-carbonyl)benzene Preparation of phonohydrazide (Compound E)

Step 1) Synthesis of E1

Methyl 1 H -indole-4-carboxylate (10 g, 0.057 mol, 1 eq) dissolved in hydrazinium hydroxide solution (30 ml, 0.57 mol, 10 eq) was stirred at 80 °C overnight, concentrated, washed with water, Filtration and concentration in vacuo gave E1 (1 H -indole-4-carbohydrazide, 8.5 g, yield: 85.0%) as a yellow solid.

¹ H-NMR (DMSO_d ₆ , 400 MHz): δ 11.26 (s, 1H), 9.48 (s, 1H), 7.52 (d, 1H), 7.43-7.35 (m, 2H), 7.11 (t, 1H), 6.84 (s, 1H), 4.47 (s, 2H).

Step 2) Synthesis of E2

To a solution of E1 (5 g, 0.028 mol, 1 eq) and pyridine (6.75 g, 0.085 mol, 3 eq) in chloroform (50 mL) was added 3-fluoro-4-nitrobenzenesulfonyl chloride (8.13 g, 0.034 mol, 1.2 eq). ) was added at 0° C. under N ₂ . The reaction mixture was stirred at room temperature for 1 hour. The solution was poured into water (200 mL) and extracted with EA (300 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to obtain E2 (3-fluoro- N' -(1 H -indole-4-carbonyl)-4-nitrobenzenesulfonohydrazide, 3.2 g, yield: 29.6%). Obtained as a yellow solid.

¹ HNMR (DMSO_d ₆ , 400 MHz): δ 11.34 (s, 1H), 10.61 (s, 2H), 8.33 (t, 1H), 8.01 (d, 1H), 7.91 (d, 1H), 7.60-7.56 ( m, 1H), 7.44–7.43 (m, 1H), 7.35 (d, 1H), 7.15–7.11 (m, 1H), 6.56 (s, 1H).

LCMS; Chemical Formula: C ₁₅ H ₁₁ FN ₄ O ₅ S; Mass Calcd.: 378.3; MS Found: 380.9 [MS+2].

Step 3) Synthesis of E3

E2 (2g, 0.005mol, 1eq), tert-butylpiperazine-1-carboxylate (1.476g, 0.008mol, 1.5eq) and K ₂ CO ₃ (1.434g, 0.01mol, 2eq) in DMF (20mL) ) was stirred overnight at room temperature. The solution was poured into water (200 mL) and extracted with EA (200 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to obtain E3 (tert-butyl 4-(5-((2-(1 H -indole-4-carbonyl)hydrazinyl)sulfonyl)-2-nitrophenyl)piperazine- 1-carboxylate, 1.2 g, yield: 44.1%) was obtained as a yellow solid.

¹ H-NMR (DMSO_d ₆ , 400 MHz): δ 11.32 (s, 1H), 10.51 (s, 1H), 10.14 (s, 1H), 8.28 (d, 1H), 7.91-7.89 (m, 1H), 7.55 (d, 1H), 7.41 (d, 1H), 7.36-7.30 (m, 2H), 7.12-7.09 (m, 1H), 6.53 (s, 1H), 3.44 (t, 4H), 3.15 (t, 4H), 1.41 (s, 9H).

Step 4) Synthesis of E4

To a solution of E3 (2 g, 3.7 mmol, 1 eq) in EA (200 mL) was added EA hydrochloric acid gas (20 mL, 6 mol/L) at 0 °C and the reaction mixture was stirred at room temperature overnight. Then concentrated, poured the solution into cold water (200 mL), adjusted pH=8 with NaHCO ₃ , filtered, and concentrated in vacuo to give E4 (1 g, yield: 61%) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 11.3 (s, 1H), 7.88 (d, 1H), 7.68 (d, 1H), 7.54 (d, 1H), 7.48-7.43 (m, 2H) ), 7.33 (d, 1H), 7.13-7.09 (m, 1H), 6.62 (s, 1H), 2.89-2.76 (m, 4H), 2.69-2.65 (m, 4H).

Step 5) Synthesis of E5

A solution of E4 (0.7g, 0.22mmol, 1eq), Fe (0.43g, 1.1mmol, 5eq) and NH ₄ Cl (0.43g, 1.1mmol, 5eq) in MEOH/H ₂ O (20ml/10ml) was dissolved in 60 Stir overnight at °C. The reaction mixture was diluted with MEOH (200 ml), filtered, and concentrated in vacuo to give E5 (0.44 g, yield: 67.7%) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 11.3 (s, 1H), 10.7 (br s, 1H), 7.54 (d, 1H), 7.43-7.41 (m, 1H), 7.34-7.29 ( m, 3H), 7.12-7.09(m, 1H), 6.65(d, 2H), 5.56(s, 2H), 3.2(s, 2H), 2.86-2.84(m, 4H), 2.62(s, 4H) .

Step 6) Synthesis of E6

DIEA (0.6g, 4.8mmol, 3eq) and D6 (1g, 1.6mmol, 1eq) were added to a solution of D8 (2.48g, 3.2mmol, 2eq) in DMF. The reaction mixture was stirred overnight at 80 °C. The solution was poured into water (100 mL) and extracted with EA (100 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give E6 (1.35 g, yield: 34.6%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.66-7.64 (m, 1H), 7.64-7.63 (m, 6H), 7.50-7.31 (m, 19H), 7.08-7.06 (m, 1H) , 6.98-6.91(m, 4H), 5.2(s, 2H), 5.06(s, 2H), 4.03(t, 2H), 3.74-3.72(m, 2H)，3.53-3.45(m, 28H), 2.89 (s, 1H), 2.80 (s, 2H), 2.73 (s, 1H), 2.62 (s, 2H), 0.99-0.91 (m, 12H).

LCMS; Chemical Formula: C ₇₁ H ₈₅ NO ₁₂ SSi; Mass Calcd.: 1204.6; MS Found: 1205.1 [MS+1].

Step 7) Synthesis of E7

TBAF (0.78 g, 2.98 mmol, 2 eq) was added to a solution of E6 (1.8 g, 1.49 mmol, 1 eq) in THF at 0 °C. The reaction mixture was stirred overnight at room temperature. It was then concentrated, and the solution was poured into cool water (100 mL) and extracted with DCM (50 mLХ10). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residual crude product was purified by column chromatography to give E7 (1.2 g, yield: 83%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.76 (s, 1H), 7.75-7.67 (m, 5H), 7.49 (d, 2H), 7.43-7.31 (m, 10H), 7.09-7.06 (m, 1H), 6.99-6.91 (m, 4H), 6.55 (s, 1H), 5.20 (s, 2H), 5.06 (s, 2H), 4.57 (t, 1H), 4.03 (t, 1H), 3.51-3.38 (m, 28H), 2.81 (t, 2H), 2.63 (t, 2H), 0.94 (t, 3H).

LCMS; Chemical Formula: C ₅₅ H ₆₇ NO ₁₂ S; Mass Calcd.: 966.1; MS Found: 967.2 [MS+1].

Step 8) Synthesis of E8

To a solution of E7 (1.2 g, 1.2 mmol, 1 eq) in DCM, Et ₃ N (0.25 g, 2.4 mmol, 2 eq), DMAP (20 mg, 0.17 mmol, 0.14 eq) and Tos-Cl (0.484 g, 2.55 mmol, 1.5eq) was added at 0° C. under N ₂ . The reaction mixture was stirred overnight at room temperature. The solution was poured into water (50 mL) and extracted with DCM (30 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give E8 (0.9 g, yield: 66.9%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 7.78-7.75 (m, 3H), 7.68 (d, 2H), 7.49-7.30 (m, 15H), 7.09-7.06 (m, 1H), 6.98 -6.91 (m, 4H), 5.75 (s, 2H), 5.19 (s, 2H), 5.06 (s, 2H), 4.10-4.02 (m, 4H), 3.56-3.54 (m, 2H), 3.50-3.42 (m, 26H), 2.80 (s, 2H), 2.63–2.56 (m, 2H), 2.40 (s, 3H), 0.93 (t, 3H).

LCMS; Chemical Formula: C ₆₂ H ₇₃ NO ₁₄ S ₂ ; Mass Calcd.: 1120.3, Mass found: 1121.1 [MS+1].

Step 9) Synthesis of E9

NaI (0.072g, 0.48mmol, 1eq), DIEA (0.3g, 2.4mmol, 5eq), and E5 (0.11g, 0.53mmol, 1.1eq) were added to a solution of E8 (0.54g, 0.48mmol, 1eq) dissolved in DMF. added. The reaction mixture was stirred overnight at 60 °C. The solution was poured into water (30 mL) and extracted with EA (50 mLХ3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product. The residual crude product was purified by column chromatography to give E9 (0.28 g, yield: 42.6%) as a yellow oil.

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 11.28 (s, 1H), 10.31 (s, 1H), 9.31 (s, 1H), 7.76 (d, 1H), 7.68 (d, 2H), 7.54-7.34 (m, 2H), 7.11-7.06 (m, 2H), 6.98-6.91 (m, 4H), 6.65 (d, 2H), 5.48 (s, 2H), 5.20 (s, 2H), 5.06 ( s, 2H), 4.04 (s, 2H), 3.52-3.39 (m, 28H), 2.81 (s, 2H), 2.64 (s, 6H), 2.57-2.50 (m, 8H), 0.95 (t, 3H) .

LCMS; Chemical Formula: C ₇₄ H ₈₇ N ₇ O ₁₄ S ₂ ; Mass Calcd.: 1362.6, Mass found: 681.8 [MS+1]/2 and 1364.6 [MS+2].

Step 10) Synthesis of Compound E

Ammonium formate (1.16g, 18.5mmol, 50eq), H ₂ O (333mg, 18.5mmol, 50eq), and Pd/C (1g) were added to a solution of E9 (500mg, 0.37mmol, 1eq) dissolved in DMF at 50℃ under N ₂ . was added and stirred overnight. It was then concentrated to give the crude product. The residue was purified by flash chromatography on a silica board to compound E (4-amino-3-(4-(3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b] Thiophene-3-carbonyl) phenoxy) -6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl) piperazin-1-yl) -N ' -(1 H -indole-4-carbonyl)benzenesulfonohydrazide, 120 mg, yield: 27.9%) as a yellow solid.

¹ H-NMR (DMSO-d ₆ , 400 MHz): 11.28 (s, 1H), 10.31 (s, 1H), 9.75 (d, 2H), 9.32 (s, 1H), 7.66 (d, 2H), 7.53 (d, 1H), 7.42 (d, 1H), 7.34-7.29 (m, 4H), 7.25 (d, 1H), 7.18 (d, 2H), 7.10 (t, 1H), 6.91 (d, 2H), 6.91 (d, 1H), 6.69-6.64 (m, 4H), 5.48 (s, 2H), 4.03 (t, 2H), 3.52-3.42 (m, 29H), 2.80 (t, 2H), 2.6 3(s) , 6H), 2.57–2.52 (m, 2H), 0.94 (t, 3H).

LCMS; Chemical Formula: C ₆₀ H ₇₅ N ₇ O ₁₄ S ₂ ; Mass Calcd.: 1182.4, Mass found: 591.8 [MS+1]/2 and 1183.5 [MS+1].

Example 4. Evaluation of target protein degradation efficiency using bifunctional compounds

Example 4-1. Evaluation of androgen receptor degrading efficacy of compounds A to C through immunoblotting

Experiment method

In order to evaluate androgen receptor degradation by the compounds, human prostate cancer-derived cells, the LNCaP cell line, were cultured using RPMI1640 medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained at 5% carbon dioxide. In order to measure the androgen receptor degradation ability according to the treatment of a representative compound selected from among the present compounds, each cell was seeded in a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. Cells were harvested after treatment with the compound (1, 10, 100 μM) alone for 24 hours to confirm whether the compound reduces the expression of the androgen receptor. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed.

Experiment result

The results of the experiment are shown in FIG. 20 . As a result, it was confirmed that the level of AR was reduced by treatment with Compound A, Compound B, and Compound C, particularly at high concentrations, compared to Control. Through this, when the protective compound according to the present invention was treated, it was confirmed that AR, an androgen receptor, was degraded (FIG. 20). Immunoblotting was plotted representatively from at least three independent experiments.

Example 4-2. Evaluation of prostate cancer-specific antigen degrading efficacy of compounds A and C through immunoblotting

Experiment method

In order to evaluate the ability of the compounds to degrade prostate cancer-specific antigens, human prostate cancer-derived cells, the LNCaP cell line, were cultured using RPMI1640 medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained at 5% carbon dioxide. In order to measure the degradation of prostate cancer-specific antigens according to treatment with a representative compound selected from among these compounds, each cell was seeded in a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. In order to confirm whether the compound reduces the expression of prostate cancer-specific antigen, cells were harvested after treatment with the compound (0.001, 0.01, 0.1, 1, 10, 100 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed.

Experiment result

the result of the experiment 21 shows. As a result, it was confirmed that the level of PSA was decreased by Compound A and Compound C compared to Control. Through this, it was confirmed that PSA degradation, a prostate cancer-specific antigen, was confirmed when the protective compound according to the present invention was treated (FIG. 21). Immunoblotting was plotted representatively from at least three independent experiments.

Example 4-3. Evaluation of Estrogen Receptor (ER-α) Degradation Efficacy of Compounds D and E through Immunoblotting

Experiment method

To evaluate estrogen receptor degradation by the compounds, MCF-7 cell line derived from human breast cancer was cultured using RPMI1640 medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained at 5% carbon dioxide. In order to measure the estrogen receptor degradation ability according to the treatment of a representative compound selected from among the present compounds, each cell was seeded in a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. In order to confirm whether the compound reduces the expression of the androgen receptor, the cells were harvested after treatment with the compound (100 or 300 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed.

Experiment result

the result of the experiment 22. As a result, it was confirmed that the level of ER-α was decreased by Compound D and Compound E compared to Control. Through this, it was confirmed that ER-α degradation, which is an estrogen receptor, was confirmed when the protective compound according to the present invention was treated (FIG. 22). Immunoblotting was plotted representatively from at least three independent experiments.

Example 4-4. Evaluation of Estrogen Receptor (ER-β) Degradation Efficacy of Compound D by Immunoblotting

Experiment method

To evaluate estrogen receptor degradation by the compounds, MCF-7 cell line derived from human breast cancer was cultured using RPMI1640 medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained at 5% carbon dioxide. In order to measure the estrogen receptor degradation ability according to the treatment of a representative compound selected from among the present compounds, each cell was seeded in a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate. In order to confirm whether the compound reduces androgen receptor expression, cells were harvested after treatment with the compound (3, 10 μM) alone for 24 hours. To extract proteins from the pooled cells, 50 μL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, a sample buffer was added to each sample and reacted at 100 ° C for 5 minutes. 5 μL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed.

Experiment result

the result of the experiment 23. As a result, it was confirmed that the level of ER-β was decreased by Compound D compared to Control. Through this, it was confirmed that ER-β degradation, which is an estrogen receptor, was confirmed when the protective compound according to the present invention was treated (FIG. 23). Immunoblotting was plotted representatively from at least three independent experiments.

Claims (25)

1. A bifunctional compound or a salt thereof comprising a UBR box domain binding ligand (UBL) and a target protein binding ligand (TBL), wherein the bifunctional compound has a UBL-TBL structure or a UBL-L-TBL structure,

   The UBL is a compound having the structure of Formula 1 or a salt thereof:

   [Formula 1]

   

   ,

   wherein X ₁ is phenyl, cycloalkyl or heterocyclyl optionally substituted or unsubstituted with one or more R ₂ ;

   each R ₂ is independently alkyl, alkoxy, amino, aminoalkyl, -NO ₂ , =O, -NHC ₂ H ₄ OH, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)OH, phenyl or heterocycloalkyl is selected from;

   X ₄ is phenyl, cycloalkyl or heterocyclyl optionally substituted with one or more R ₃ ;

   each R ₃ is independently alkyl, alkoxy, amino, halo, hydroxyl, alkylamino, dialkylamino, -NO ₂ , -CONR'R'', -CO ₂ R', -NHCOR', phenyl or heterocyclo is selected from alkyl;

   each R' and R'' is independently -H or alkyl;

   X ₂ is SO ₂ , or CR _a R _b ;

   R _a and R _b are each independently H or CH ₃ ;

   X ₃ is NH or CH ₂ ;

   B ₁ is CH ₂ or NH;

   A ₁ is CH ₂ or NH; and

   The dashed line (dotted line) in Formula 1 represents the attachment point of the linker (L) or the target protein (or peptide) binding ligand (TBL),

   The TBL is a compound that binds to a target protein or peptide,

   The L is a linker, chemically linking or combining UBL and TBL,

   A bifunctional compound or a salt thereof.
2. According to claim 1,

   In Formula 1,

   -X2-B1-X3 is a bifunctional compound selected from the group consisting of -SO ₂ -NH-NH, -SO ₂ -NH-CH ₂ , -SO ₂ -CH ₂ -NH, and -CH ₂ -NH-NH or a salt thereof.
3. According to claim 1,

   Formula 1 is a bifunctional compound of Formula 1-1 or a salt thereof:

   [Formula 1-1]

   

   .
4. According to claim 1,

   Formula 1 is a bifunctional compound of Formula 1-2 or a salt thereof:

   [Formula 1-2]

   

   .
5. According to claim 1,

   Formula 1 is a bifunctional compound of Formula 1-3 or a salt thereof:

   [Formula 1-3]

   

   .
6. According to claim 1,

   Formula 1 is a bifunctional compound of Formula 1-4 or a salt thereof:

   [Formula 1-4]

   

   .
7. According to any one of claims 1 to 6,

   each of X ₁ and X ₄ is independently substituted or unsubstituted phenyl, cycloalkyl or heterocyclyl;

   wherein each X ₁ and X ₄ are independently substituted or unsubstituted phenyl, cyclohexyl, cyclopentyl, furanyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morphopoly Nyl, indolinyl, 1H-indolinyl, 1H-indolyl, 1H-indazolyl, isoindolinyl, indolin-2-oneyl, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyridinyl A bifunctional compound selected from or a salt thereof.
8. According to any one of claims 1 to 6,

   Each R ₂ is independently selected from methyl, ethyl, amino, aminoalkyl, amino(hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH ₂ , -C(=O)NH ₂ , -C (=O)NHCH ₃ , -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl, a bifunctional compound selected from morpholinyl, or a salt thereof.
9. According to claim 8,

   Wherein R ₂ is amino, a bifunctional compound or a salt thereof.
10. According to any one of claims 1 to 6,

    The X ₁ is

    

    ,

    

    or

    

    A phosphorus bifunctional compound or a salt thereof.
11. According to any one of claims 1 to 6,

    Each R ₃ above is independently hydroxyl, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropyloxy, alkylamino, dialkylamino, -NO ₂ , - selected from C(=0)NH ₂ , -CO ₂ R', -NHCOR', -CONR'R'' and phenyl;

    A bifunctional compound or salt thereof, wherein each R' and R'' is independently -H or alkyl.
12. According to claim 11,

    Wherein R ₃ is hydroxyl, a bifunctional compound or a salt thereof.
13. According to any one of claims 1 to 6,

    wherein X ₄ is phosphorus

    

    ,

    

    or

    

    A phosphorus bifunctional compound or a salt thereof.
14. According to claim 3,

    The UBL is a bifunctional compound selected from: or a salt thereof:

    N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonohydrazide;

    N '-(4-hydroxybenzoyl)indoline-5-sulfonohydrazide;

    N '-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonohydrazide;

    N '-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonohydrazide;

    3-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

    4-(1-aminoethyl) -N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

    3,5-diamino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-4-methoxybenzenesulfonohydrazide;

    4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzimidamide;

    4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide;

    6-amino- N '-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonohydrazide;

    4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide;

    4-amino- N '-(1 H -indole-3-carbonyl)benzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-1 H -pyrazole-4-sulfonohydrazide;

    N '-(4-hydroxybenzoyl)indoline-4-sulfonohydrazide;

    N '-(4-hydroxybenzoyl)-1 H -indole-4-sulfonohydrazide;

    2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide;

    4-amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide;

    4-amino- N '-(indoline-4-carbonyl)benzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)-3-(peparazin-1-yl)benzenesulfonohydrazide;

    4-amino- N '-(2,3-dihydro-1 H -indene-2-carbonyl)benzenesulfonohydrazide;

    4-amino- N '-(isoindoline-2-carbonyl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-1 H -indole-2-sulfonohydrazide;

    4-amino- N '-(2-phenylacetyl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)-1 H -indazole-3-sulfonohydrazide;

    4-amino- N '-(indoline-6-carbonyl)benzenesulfonohydrazide;

    4-amino- N '-(indoline-3-carbonyl)benzenesulfonohydrazide;

    N '-(4-hydroxybenzoyl)piperidine-4-sulfonohydrazide;

    4-amino- N '-(indoline-6-carbonyl)-3-morpholinobenzenesulfonohydrazide;

    4-amino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide;

    4-amino-3-morpholino- N '-(piperazine-1-carbonyl)benzenesulfonohydrazide;

    N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonohydrazide;

    (1 S ,4 S )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide;

    ( 1R , 4R )-4-amino- N '-(4-hydroxybenzoyl)cyclohexane-1-sulfonohydrazide;

    4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)-5-methylfuran-2-carboxylic acid;

    N '-(4-hydroxybenzoyl)pyrrolidine-3-sulfonohydrazide;

    N '-(4-hydroxybenzoyl)-1 H -pyrrolo[2,3- b ]pyridine-2-sulfonohydrazide;

    2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide; and

    2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide.
15. According to claim 14,

    The UBL is a bifunctional compound selected from: or a salt thereof:

    4-amino- N '-(4-hydroxybenzoyl)benzenesulfonohydrazide;

    2-((4-aminophenyl)sulfonyl) -N -phenylhydrazine-1-carboxamide;

    4-amino- N '-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonohydrazide;

    4-amino- N '-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonohydrazide; and

    4-Amino- N '-(1 H -indole-4-carbonyl)-3-morpholinobenzenesulfonohydrazide.
16. According to claim 4,

    The UBL is a bifunctional compound selected from: or a salt thereof:

    N '-(4-aminobenzyl)-4-hydroxybenzohydrazide;

    4-hydroxy-N'-(4-methoxybenzyl)benzohydrazide; and

    N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide.
17. According to claim 5,

    The UBL is a bifunctional compound selected from: or a salt thereof:

    4-amino- N- (2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide;

    4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide; and

    3,5-diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide.
18. According to claim 6,

    The UBL is a bifunctional compound selected from: or a salt thereof:

    N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide;

    4-hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide; and

    N-(((4-aminophenyl)sulfonyl)methyl)-[1,1′-biphenyl]-4-carboxamide.
19. According to claim 1,

    The TBL is a bifunctional compound or a salt thereof, which is a compound that binds to androgen receptors.
20. The method of claim 1 or 19,

    The TBL is a compound having the structure of Formula 2:

    [Formula 2]

    

    ,

    At this time,

    W ¹ is

    

    or

    

    is;

    each R ₃ is independently H or -CN;

    each R ₄ is independently H, halogen or -CF ₃ ;

    Y ¹ and Y ² are each independently O or S;

    R ¹ and R ² are each independently H or a methyl group;

    W ² is a bond, C1-6 aryl, biphenyl, biphenylyl or heteroaryl, each optionally substituted with 1, 2 or 3 R ^W2 ;

    Each R ^W2 is independently H, OH, halogen, -C(=O)NHCH ₃ , C1-6 alkyl (optionally substituted with one or more F), OC1-3 alkyl (optionally with one or more -F) substituted with); and

    The dotted line (dotted line) indicates the attachment point to the UBR box domain binding ligand (UBL) or linker (L). A bifunctional compound or salt thereof.
21. According to claim 1,

    The TBL is a bifunctional compound or a salt thereof, which is a compound that binds to an estrogen receptor.
22. The method of claim 1 or 21,

    The TBL is a compound having the structure of Formula 3:

    [Formula 3]

    

    ,

    At this time,

    X is O or C=0;

    each X ₁ and X ₂ is independently selected from N or CH;

    R ₁ is independently selected from OH, O(CO)R _a , O-lower alkyl, wherein R _a is an alkyl or aryl group in an ester;

    R ₂ is selected from H, OH, halogen, CN, CF ₃ , SO ₂ -alkyl, O-lower alkyl;

    R ₃ is selected from H, halogen; and

    The dotted line (dotted line) indicates the attachment point to the UBR box domain binding ligand (UBL) or linker (L). A bifunctional compound or salt thereof.
23. According to claim 1,

    L is selected from the group consisting of:

    -(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )m-;

    -R ₁ -(CH ₂ )m-(OCH ₂ CH ₂ )nO-(CH ₂ )pR ₂ -;

    -O-(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-; and

    -(CH ₂ )m-NR-(CH ₂ )p-(OCH ₂ CH ₂ )nO-(CH ₂ )o-.

    At this time, in the linker,

    m and p are each independently 0, 1, 2, 3, 4 or 5;

    n is 0, 1, 2, 3, 4, 5, 6 or 7;

    o is 0, 1, or 2;

    R is H, methyl or ethyl;

    A bifunctional compound or salt thereof, wherein R ₁ and R ₂ are each independently O or NH.
24. According to claim 20,

    The bifunctional compound is a bifunctional compound selected from the group consisting of or a salt thereof:

    4-Amino- N '-(4-(2-(2-(2-(2-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5 -dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethoxy)benzoyl ) benzenesulfonohydrazide;

    4-Amino- N '-(4-((5-((5-((4'-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4 -oxo-2-thioxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)pentyl)oxy)pentyl)oxy)benzoyl)benzenesulfonohydrazide; and

    4-amino-3-(4-(14-((4′-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thio Oxoimidazolidin-1-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)- N '-(4-Hydroxybenzoyl)benzenesulfonohydrazide.
25. According to claim 22,

    The bifunctional compound is a bifunctional compound selected from the group consisting of or a salt thereof:

    2-((4-aminophenyl)sulfonyl) -N- (3-((3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene -3-carbonyl)phenoxy)-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)oxy)phenyl)hydrazine-1-carboxamide; and

    4-amino-3-(4-(3-ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[ b ]thiophene-3-carbonyl)phenoxy)- 6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)piperazin-1-yl) -N '-( 1H -indole-4-carbonyl)benzene Sulfonohydrazide.

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