Compounds acting as UBR box domain ligands

Abstract

This specification relates to a compound that acts as a ligand for a UBR box domain. This specification provides a small molecule compound that binds to a UBR box domain. Furthermore, this specification provides a composition for inhibiting substrate binding of a UBR box domain, a pharmaceutical composition for treating UBR-related diseases, and uses thereof, wherein the composition comprises a ligand compound that binds to a UBR box domain.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63 / 015,945, filed April 27, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The disclosure herein relates to compounds that are ligands for UBR box domains. UBR box domains are ubiquitin protein ligase E3 component n-recognin (UBR) proteins of the N-end rule pathway. In this context, the UBR box domain is referred to as a substrate-binding domain. The UBR box domain is essential for binding to the N-terminal residue of a substrate to form a polyubiquitin chain in the substrate, and substrates are known to be degraded by this process.

[0004] The present description relates to compounds that act as ligands that bind to a UBR box domain. Background Art

[0005] Cells regulate the amount and function of proteins in the body by degrading them. In this case, proteins in the body can be degraded based on the sequence of their N-terminal residues, and this degradation pathway is called the N-end rule pathway. In other words, the N-end rule pathway is a proteolytic system that uses the N-terminus of a specific protein as a degradation signal. The N-end rule pathway can include the following proteolytic processes.

[0006] In the case of eukaryotic organisms, N-recognition proteins (N-recognin) recognize the N-terminal degradation signal of proteins, and N-recognition proteins are able to degrade proteins by binding ubiquitin to the protein to be degraded. In this case, N-terminal degradation signals may include those having a positively charged residue at the N-terminus (Type 1: for example, arginine, lysine and histidine) or a large hydrophobic residue (Type 2: phenylalanine, leucine, tryptophan, isoleucine and tyrosine). The present inventors first discovered or cloned the N-recognition proteins UBR1, UBR2, UBR3 and UBR5, and revealed that the N-recognition proteins have a UBR box domain as a substrate recognition domain (Tasaki et al., 2005). In this case, the ubiquitinated substrate generated by the binding of the N-recognition protein to the N-end rule ligand is delivered to the proteasome and degraded into short peptides. In this process, when N-recognition proteins target N-end rule substrates, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Leu) provide the majority of the required hydrogen bonds, and therefore specific N-terminal residues are essential determinants for binding ( Sriram and Kwon, 2010 ).

[0007] UBR stands for ubiquitin-protein ligase E3 component N-recognition protein, and is an N-recognition protein that recognizes N-terminal degradation signals of proteins. At least seven types of UBRs, UBR1 to UBR7, are known to exist in mammals. The UBR box domain, a zinc finger motif with approximately 70 residues, is a highly conserved substrate-binding domain shared by all UBRs [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].

[0008] That is, UBR is an N-recognition protein associated with the N-end rule pathway, which is a proteolytic pathway, and the UBR box domain in the UBR is a substrate binding domain. In particular, among UBR1 to UBR7, UBR1, UBR2, UBR3, and UBR5 are known to act as ubiquitin-protein ligase E3s and have a RING domain or a HECT domain. N-end rule substrates bound to the UBR are degraded through 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 via the RING domain or the HECT domain, thereby degrading the substrate via the proteasome pathway. For example, when misfolded proteins remain in the cell for a long time, the proteins may aggregate to block the proteasome or reduce other cellular functions, thereby being degraded via the ubiquitin proteasome pathway (Ji and Kwon, 2017).

[0009] Specifically, the UBR box domain plays an important role in intracellular proteolytic pathways by recognizing N-terminal degradation signals. Therefore, ligands binding to the UBR box domain may affect intracellular proteolytic pathways.

[0010] As stated above, the present description relates to compounds that are ligands that bind to UBR box domains that are associated with intracellular proteolytic pathways. Summary of the Invention

[0011] Technical issues

[0012] This specification provides a small molecule compound that binds to a UBR box domain. In this case, the UBR box domain includes the UBR box domains in UBR1 to UBR7. The small molecule compound can function as a ligand suitable for binding to the UBR box domain.

[0013] In one embodiment, the present disclosure provides a composition for inhibiting substrate binding of a UBR box domain, comprising a ligand compound that binds to the UBR box domain.

[0014] In a specific embodiment, the present specification provides a pharmaceutical composition for treating UBR-related diseases and its use, wherein the composition comprises a ligand compound that binds to the UBR box domain.

[0015] In a more specific embodiment, the present disclosure provides a pharmaceutical composition and its use for treating the following diseases, wherein the composition comprises a ligand compound that binds to the UBR box domain, wherein the diseases include: muscle loss caused by muscular dystrophy (Becker, Congennital, Duchenne, Distal, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, myotonic, ocuophargyngeal); muscle wasting diseases mediated by muscle loss or degeneration, including sarcopenia or cancer cachexia; diseases caused by excessive protein degradation, including liposarcoma, cystic fibrosis, Johanson-Blizzard syndrome, obstructive uropathy (urethral obstruction sequence); sequence)), autoimmune pancreatitis; or known diseases associated with the UBR box and UBR protein, including Usher syndrome.

[0016] Technical Solution

[0017] This specification provides a compound having a structure of Formula 1 or a salt thereof.

[0018] [Formula 1]

[0019]

[0020] wherein X1 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted by one or more R2;

[0021] Each R2 is independently selected from alkyl, alkoxy, amino, aminoalkyl, -NO2, =O, -NHC2H4OH, -C(=NH)NH2, -C(=O)NH2, -C(=O)NHCH3, -C(=O)OH, phenyl or heterocycloalkyl;

[0022] X4 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted by one or more R3;

[0023] Each R3 is independently selected from alkyl, alkoxy, amino, halogen, hydroxy, alkylamino, dialkylamino, -NO2, -CONR'R", -CO2R', -NHCOR', phenyl or heterocycloalkyl;

[0024] Each R' and R" is independently -H or alkyl;

[0025] X2 is SO2 or CR a R b ;

[0026] R a and R b are each independently H or CH3;

[0027] X3 is NH or CH2;

[0028] B1 is CH2 or NH;

[0029] A1 is CH2 or NH.

[0030] In this case, as an example, in Formula 1, -X2-B1-X3 is selected from the group consisting of -SO2-NH-NH, -SO2-NH-CH2, -SO2-CH2-NH and -CH2-NH-NH,

[0031] X1 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted by one or more R2;

[0032] Each R2 is independently selected from alkyl, alkoxy, amino, aminoalkyl, -NO2, =O, -NHC2H4OH, -C(=NH)NH2, -C(=O)NH2, -C(=O)NHCH3, -C(=O)OH, phenyl or heterocycloalkyl;

[0033] X4 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted by one or more R3;

[0034] Each R3 is independently selected from alkyl, alkoxy, amino, halogen, hydroxy, alkylamino, dialkylamino, -NO2, -CONR'R", -CO2R', -NHCOR', phenyl or heterocycloalkyl; wherein each R' and R" is independently -H or alkyl;

[0035] A1 is CH2 or NH,

[0036] I is an integer of 0 or 1.

[0037] As a specific example, each X1 and X4 is independently substituted or unsubstituted phenyl, cycloalkyl or heterocyclic; wherein each X1 and X4 can be independently selected from substituted or unsubstituted phenyl, cyclohexyl, cyclopentyl, furyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indole, 1H-indazolyl, isoindolinyl, indolin-2-one, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyridinyl.

[0038] In this case, as an example, each R2 can be independently selected from methyl, ethyl, amino, aminoalkyl, amino(hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH2, -C(=O)NH2, -C(=O)NHCH3, -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl and morpholinyl.

[0039] In this case, by way of example, each R3 is independently selected from hydroxy, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, alkylamino, dialkylamino, -NO2, -C(=O)NH2, -CO2R', -NHCOR', -CONR'R" and phenyl;

[0040] Each R' and R" is independently -H or alkyl.

[0041] As an example, this specification provides a compound or a salt thereof, wherein Formula 1 is Formula 1-1:

[0042] [Formula 1-1]

[0043]

[0044] Wherein, A1 is CH2 or NH,

[0045] I is an integer of 0 or 1.

[0046] In this case, as an example, the present specification provides a compound or a salt thereof, wherein Formula 1 is Formula 1-2:

[0047] [Formula 1-2]

[0048]

[0049] In this case, as an example, the present specification provides a compound or a salt thereof, wherein Formula 1 is Formula 1-3:

[0050] [Formula 1-3]

[0051]

[0052] In this case, as an example, the present specification provides a compound or a salt thereof, wherein Formula 1 is Formula 1-4:

[0053] [Formula 1-4]

[0054]

[0055] In this case, in Formula 1-1, Formula 1-2, Formula 1-3 and Formula 1-4, as an example, wherein X1 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted with one or more R2, wherein each R2 is independently selected from alkyl, alkoxy, amino, aminoalkyl, -NO2, =O, -NHC2H4OH, -C(=NH)NH2, -C(=O)NH2, -C(=O)NHCH3, -C(=O)OH, phenyl or heterocycloalkyl;

[0056] X4 is phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted by one or more R3; wherein each R3 is independently selected from alkyl, alkoxy, amino, halogen, hydroxy, alkylamino, dialkylamino, -NO2, -CONR'R", -CO2R', -NHCOR', phenyl or heterocycloalkyl; each R' and R" is independently -H or alkyl.

[0057] As a specific example, each X1 and X4 is independently substituted or unsubstituted phenyl, cycloalkyl or heterocyclyl; wherein each X1 and X4 can be independently selected from substituted or unsubstituted phenyl, cyclohexyl, cyclopentyl, furyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indole, 1H-indazolyl, isoindolinyl, indolin-2-one, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyridinyl.

[0058] In this case, as an example, each R2 can be independently selected from methyl, ethyl, amino, aminoalkyl, amino(hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH2, -C(=O)NH2, -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl and morpholinyl.

[0059] In this case, as a specific example, the present specification provides a compound or a salt thereof, wherein R2 is an amino group.

[0060] In this case, as an example, the present specification provides a compound or a salt thereof, wherein X1 is

[0061] In this case, by way of example, each R3 is independently selected from hydroxy, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, alkylamino, dialkylamino, -NO2, -C(=O)NH2, -CO2R', -NHCOR', -CONR'R" and phenyl;

[0062] Each R' and R" is independently -H or alkyl.

[0063] In this case, as a specific example, the present specification provides a compound or a salt thereof, wherein R3 is a hydroxyl group.

[0064] In this case, as an example, the present specification provides a compound or a salt thereof, wherein X4 is

[0065] In this case, as an example, the compound may be selected from:

[0066] N'-(4-hydroxybenzoyl)-4-methylbenzenesulfonylhydrazide;

[0067] 4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide;

[0068] 4-amino-N'-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonylhydrazide;

[0069] N'-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonylhydrazide;

[0070] N'-(4-hydroxybenzoyl)indoline-5-sulfonylhydrazide;

[0071] N'-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonylhydrazide;

[0072] N'-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonylhydrazide;

[0073] 3-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide;

[0074] 4-(1-aminoethyl)-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide;

[0075] 3,5-Diamino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide;

[0076] N'-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonylhydrazide;

[0077] N'-(4-hydroxybenzoyl)-4-methoxybenzenesulfonylhydrazide;

[0078] 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)benzamidine;

[0079] 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)benzamide;

[0080] 6-amino-N'-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonylhydrazide;

[0081] 4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide;

[0082] 4-amino-N'-(1H-indole-3-carbonyl)benzenesulfonylhydrazide;

[0083] 4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide;

[0084] N'-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide;

[0085] 4-amino-N'-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonylhydrazide;

[0086] N'-(4-hydroxybenzoyl)-1H-pyrazole-4-sulfonylhydrazide;

[0087] N'-(4-hydroxybenzoyl)indoline-4-sulfonylhydrazide;

[0088] N'-(4-hydroxybenzoyl)-1H-indole-4-sulfonylhydrazide;

[0089] 2-((4-aminophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide;

[0090] 4-amino-N'-(1H-indole-4-carbonyl)-3-morpholinobenzenesulfonylhydrazide;

[0091] 4-amino-N'-(indoline-4-carbonyl)benzenesulfonylhydrazide;

[0092] 4-amino-N'-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonylhydrazide;

[0093] 4-amino-N'-(2,3-dihydro-1H-indene-2-carbonyl)benzenesulfonylhydrazide;

[0094] 4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonylhydrazide;

[0095] N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonylhydrazide;

[0096] 4-amino-N'-(2-phenylacetyl)benzenesulfonylhydrazide;

[0097] N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonylhydrazide;

[0098] 4-amino-N'-(indoline-6-carbonyl)benzenesulfonylhydrazide;

[0099] 4-amino-N'-(indoline-3-carbonyl)benzenesulfonylhydrazide;

[0100] N'-(4-hydroxybenzoyl)piperidine-4-sulfonylhydrazide;

[0101] 4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonylhydrazide;

[0102] 4-amino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide;

[0103] 4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide;

[0104] N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonylhydrazide;

[0105] (1S,4S)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide;

[0106] (1R,4R)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide;

[0107] 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)-5-methylfuran-2-carboxylic acid;

[0108] N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonylhydrazide;

[0109] N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonylhydrazide;

[0110] 2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide;

[0111] 2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide;

[0112] N'-(4-aminobenzyl)-4-hydroxybenzohydrazide;

[0113] 4-Hydroxy-N'-(4-methoxybenzyl)benzohydrazide;

[0114] N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide;

[0115] 4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide;

[0116] 4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide;

[0117] 3,5-Diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide;

[0118] N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide;

[0119] 4-Hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide;

[0120] N-(((4-Aminophenyl)sulfonyl)methyl)-[1,1'-biphenyl]-4-carboxamide.

[0121] In another aspect, the present disclosure provides a pharmaceutical composition for treating a UBR-related disease, and a method for treating a UBR-related disease by using the compound, wherein the pharmaceutical composition comprises the compound or a pharmaceutically acceptable salt thereof.

[0122] In this case, as an example, the UBR-associated disease can be selected from the following diseases: muscle loss caused by muscular dystrophy (Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dauer muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy); muscle wasting diseases mediated by muscle loss or degeneration, including sarcopenia or cancer cachexia; diseases caused by excessive protein degradation, including liposarcoma, cystic fibrosis, Johanson-Blizzard syndrome, obstructive uropathy (urethral obstructive sequence), autoimmune pancreatitis; or Usher syndrome.

[0123] Beneficial effects

[0124] The invention disclosed herein provides ligand compounds with high binding strength relative to the UBR box domain.

[0125] UBR box domain ligand compounds can inhibit UBR box domain substrate binding and provide various applications utilizing this property. For example, UBR-related diseases (e.g., muscle loss, etc.) can be treated with UBR box domain ligand compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 Describes the results of an experiment using immunoblotting to confirm whether actin is a substrate of the Arg / N-degron pathway.

[0127] Figure 2The results of experiments using an in vitro transcription / translation method to confirm whether degradation of R-nsp4, which should be degraded by allowing compounds (Compound 2, Compound 3, Compound 7, Compound 12, Compound 14, and Compound 16) to bind to UBR1, is inhibited are described.

[0128] Figure 3 The results of an experiment using immunoblotting to confirm whether the degradation of RGS4, which is a substrate of the UBR protein in embryonic kidney cells and should be degraded by allowing compounds (Compound 2 and Compound 3) to bind to UBR1, is inhibited are described.

[0129] Figure 4 The present invention describes the results of an experiment using immunoblotting to confirm whether compounds (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, and Compound 16) inhibit the degradation of actin, which is a substrate of UBR protein in muscle cells.

[0130] Figure 5 The results of an experiment using immunoblotting to confirm whether compounds (Compound 35, Compound 36, Compound 37, Compound 38, Compound 39, Compound 43, Compound 44, Compound 45, Compound 46, and Compound 47) inhibit actin degradation in muscle cells are described.

[0131] Figure 6 The results of an experiment using immunoblotting to confirm whether compounds (Compound 8, Compound 9, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, and Compound 24) inhibit actin degradation in muscle cells are described.

[0132] Figure 7 The results of an experiment using immunoblotting to confirm whether compounds (Compound 25, Compound 26, Compound 27, Compound 28, Compound 32, Compound 33, and Compound 34) inhibit actin degradation in muscle cells are described.

[0133] Figure 8 The results of an experiment using immunoblotting to confirm whether compounds (Compound 41 and Compound 42) inhibit actin degradation in muscle cells are shown.

[0134] Figure 9 The results of an experiment using immunoblotting to confirm whether compounds (Compound 12, Compound 13, Compound 14, Compound 15, Compound 17, Compound 29, Compound 30, and Compound 31) inhibit actin degradation in muscle cells are described.

[0135] Figure 10 and Figure 11 Described are the results of experiments using immunoblotting to confirm the efficacy of a compound (Compound 2) in binding to UBR1, UBR2, UBR3, and UBR5 in cells.

[0136] Figures 12 to 19 The results of microscale thermophoresis (MST) experiments to confirm whether compounds (Compound 1, Compound 2, Compound 5, Compound 8, Compound 9, Compound 11, Compound 12, and Compound 13) bind to UBR1 are described. DETAILED DESCRIPTION

[0137] Hereinafter, the content of the present invention will be described in more detail by specific exemplary embodiments and examples with reference to the accompanying drawings. It should be noted that the accompanying drawings include some exemplary embodiments of the present invention, but not all exemplary embodiments. The invention disclosed in this specification can be implemented in many ways and is not limited to the specific exemplary embodiments described herein. A person of ordinary skill in the art to which the invention disclosed herein belongs will be able to think of many modifications and other exemplary embodiments of the invention disclosed herein. Therefore, it should be understood that the invention disclosed herein is not limited to the specific exemplary embodiments described herein, and modifications and other exemplary embodiments thereof are also within the scope of the claims.

[0138] Definition of terms

[0139] The following are definitions of the main terms used in this article.

[0140] Ubiquitin-protein ligase E3 component n-recognition protein (UBR)

[0141] The term UBR as used herein refers to the abbreviation of the ubiquitin protein ligase E3 component n-recognition protein. UBR is an N-recognition protein that recognizes the N-terminal residue of a protein, and at least seven types, UBR1 to UBR7, are known to exist in mammals. UBR is an N-recognition protein and is associated with the N-end rule pathway, which is a proteolytic pathway in vivo. Specifically, UBR recognizes the N-terminal degradation signal (N-degron) of a protein and participates in the process of degrading substrate proteins via the ubiquitin proteasome pathway.

[0142] UBR box domain

[0143] As used herein, the term "UBR box domain" refers to a domain present in UBR proteins and is a zinc finger motif. UBR proteins include UBR1 to UBR7 proteins. The UBR box domain is known as a substrate protein binding domain. Compounds disclosed herein as UBR box domain ligands can inhibit UBR box domain substrate binding by binding to the UBR box domain. Furthermore, compounds disclosed herein as UBR box domain ligands can affect intracellular proteolytic pathways.

[0144] RING domain

[0145] The term "RING domain," as used herein, is known to be present in UBR1, UBR2, and UBR3 proteins. A RING domain can also be interchanged with a RING ubiquitination domain. A RING domain is a zinc finger motif and is present in proteins. The RING domain plays an important role in the transfer of ubiquitin present in E2 to substrate proteins, and serves to allow the transfer of ubiquitin to substrate proteins to occur in a single step.

[0146] HECT domain

[0147] The term HECT domain, as used herein, is known to be present in the UBR5 protein. The HECT domain can also be interchanged with the HECT ubiquitination domain. The HECT domain plays an important role in the transfer of ubiquitin present in E2 to substrate proteins. Ubiquitin present in E2 is delivered to the HECT domain and then transferred to the substrate protein. In other words, the HECT domain allows the transfer of ubiquitin to the substrate protein in two steps.

[0148] zinc finger motif

[0149] As used herein, the term zinc finger motif 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 the RING domain of the present specification are zinc finger motifs.

[0150] ligand

[0151] As used herein, the term ligand refers to a material that specifically binds to a protein. Proteins include enzymes or receptors. When the protein is an enzyme, the ligand may refer to a substrate that binds to the enzyme, or when the protein is a receptor, the ligand may refer to a hormone that binds to the receptor.

[0152] The compounds provided herein as UBR box domain ligands are compounds that bind to the UBR box domain. By way of example, the compounds are compounds that bind to the UBR box domain in a UBR protein. As a specific example, the compounds are compounds that bind to the UBR box domain present in one or more proteins of UBR1 to UBR7. However, the compounds are not limited thereto.

[0153] The compounds provided herein as UBR box domain ligands can act competitively with the substrate of the UBR box domain. That is, the compounds can inhibit substrate binding of the UBR box domain. In addition, the compounds can inhibit the degradation of the substrate by inhibiting the binding of the substrate.

[0154] aminoalkyl

[0155] As used herein, the term aminoalkyl refers to an alkyl moiety substituted with an amino group. Aminoalkyl groups include -CH(NH2)CH3 and -CH2(NH2).

[0156] Cycloalkyl and heterocycloalkyl

[0157] As used herein, the term cycloalkyl refers to a carbocyclic group containing one or more saturated ring structures, and includes bicyclic groups. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0158] Heterocycloalkyl refers to a ring structure containing one or more heteroatoms selected from P, N, O and S in addition to the ring-carbon atoms of the cycloalkyl group.

[0159] Heterocyclic group

[0160] As used herein, the term heterocyclyl refers to an unsaturated, saturated or partially unsaturated monocyclic, bicyclic or tricyclic group having 12 to 14 ring carbon atoms and containing, in addition to the ring-carbon atoms, one or more heteroatoms selected from P, N, O and S. Heterocyclyl includes heterocycloalkyl. In various exemplary embodiments, the heterocyclyl group is attached to another moiety through a carbon or heteroatom and is optionally substituted on the carbon or heteroatom. Examples of heterocyclic groups include azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolyl, isoindolyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolyl, tetrazolopyridinyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepine benzophenone, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothiophenyl, and the like.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0162] Hereinafter, the specific contents of the present invention will be disclosed.

[0163] I. UBR box domain

[0164] 1. Overview

[0165] The compounds provided herein as UBR box domain ligands bind to the UBR box domain. The UBR box domain is known as a domain that binds to an N-terminal residue sequence or an N-terminal degradation signal. This domain is involved in the process by which proteins are degraded via the N-end rule pathway. Thus, the compounds can affect proteolysis via the N-end rule pathway.

[0166] 2. N-end rule pathway

[0167] Cells regulate the amount of protein by proteolysis. In this case, known protein degradation processes are carried out by the process of identifying degron as protein degradation signal. Specifically, proteolysis is regulated according to the N-terminal residue sequence of protein, and the proteolytic signal present in the N-terminal is collectively referred to as N-degron. N-degron is included in those N-degrons with positively charged residues (for example, arginine, lysine and histidine) or large hydrophobic residues (phenylalanine, leucine, tryptophan, isoleucine and tyrosine) at the N-terminal. As mentioned above, based on the association that the half-life of protein is determined by the amino acid residues present in the N-terminal of protein, the term N-end rule has been used.

[0168] 3. UBR box domain

[0169] In the N-end rule pathway, N-degrons are recognized by N-recognition proteins, and the ubiquitin-protein ligase E3 component N-recognition protein (UBR) has been discovered as an N-recognition protein. It is known that the UBR recognizes the N-terminal residue sequence or N-terminal degradation signal via the UBR box domain. Specifically, the UBR recognizes the protein degradation signal via the UBR box domain, and the protein degradation process is carried out by recognizing the protein degradation signal.

[0170] The protein degradation process via UBR can include the following: The UBR box domain recognizes substrates with N-terminal degradation signals, ubiquitin binds to the substrate, and the ubiquitin-bound substrate can be degraded by the proteasome. In other words, substrates with N-terminal degradation signals can be degraded by the ubiquitin proteasome system (UPS).

[0171] II. UBR box domain ligands

[0172] 1. Overview

[0173] 1) The compounds of this specification reflect the structure of the UBR box domain and the properties of binding to N-terminal pathway substrates.

[0174] The compounds disclosed herein as UBR box domain ligands are designed taking into account the structure of the UBR box domain and the binding pattern of the UBR box domain to N-terminal pathway substrates.

[0175] The various amino acids present in the UBR box domain interact and bind to amino acids in the N-terminal pathway substrate through ionic interactions, hydrogen bonds, hydrophobic interactions, and the like. By analyzing these binding modes, small molecule compounds capable of forming suitable binding modes with the UBR box domain were synthesized and provided herein. Further, compounds according to Formulas 1 to 55 are provided below.

[0176] 2) The compound of this specification has a core structure that enhances binding to the UBR box domain.

[0177] In this specification, the binding pattern of the UBR box domain to the amino acid of the N-terminal pathway substrate was analyzed as described above, and the core structure of the compound was deduced. The compound provided herein may have the following structure [Formula 1] derived based on the core structure of the compound. [Formula 1] is as follows:

[0178] [Formula 1]

[0179]

[0180] In this specification, various compounds are designed and provided based on [Formula 1]. In this case, considering the binding mode to the UBR box domain, candidates X1, X2, X3, B1, A1, and X4 were derived. A more detailed description of the various compounds based on [Formula 1] is provided below.

[0181] 2. Formula 1

[0182]

[0183] [Formula 1]

[0184] 1) X2, B1, X3, and A1

[0185] ①X2

[0186] In Formula 1, X2 can be a structure that causes a kink structure in the compound disclosed herein. The kink structure helps to smoothly maintain the charge-charge interaction or hydrogen bond or hydrophobic interaction between the compound X1 disclosed herein and the UBR box domain and enhance the binding strength. Therefore, as an example, X2 can be one of the various structures that can cause a kink structure. As a specific example, X2 can be SO2 or CR a R b In this case, R a and R bEach of X2 and X2 can be independently selected from H or CH2. In addition, X2 can be CH2, CH(CH3) or C(CH3)2. As another specific example, X2 can be SO2.

[0187] ②B1, X3 and A1

[0188] In Formula 1, as an example, A1 may be CH2 or NH.

[0189] In Formula 1, as an example, B1 may be CH2 or NH.

[0190] In Formula 1, as an example, X3 may be CH2 or NH.

[0191] In this case, as an example, when B1 in Formula 1 is CH2, X3 may not be CH2.

[0192] However, the compound is not limited thereto.

[0193] ③ Examples of X2, B1, X3, and A1

[0194] Formula 1 may have a structure selected from the following structures:

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] [Formula 1-45]

[0201]

[0202] As a specific example, in Formula 1, -X2-B1-X3 is selected from the group consisting of -SO2-NH-NH, -SO2-NH-CH2, -SO2-CH2-NH and -CH2-NH-NH,

[0203] A1 is CH2 or NH, and

[0204] I is an integer of 0 or 1.

[0205] Formula 1 may have a structure selected from the structures described below as specific examples:

[0206] [Formula 1-1]

[0207]

[0208] [Formula 1-2]

[0209]

[0210] [Formula 1-3]

[0211]

[0212] [Formula 1-4]

[0213]

[0214] 2) X1

[0215] As a result of structural analysis of the complex of the UBR box of UBR1 and UBR2 with N-degron and molecular docking studies on a compound having a core structure of Formula 1, in Formula 1, X1 corresponds to the side chain of the first residue (N1) of N-degron, and X1 is expected to bind to the negatively charged surrounding region. Therefore, as an example, in Formula 1, X1 may have a ring structure containing a charge or including a portion that forms a hydrogen bond. As a specific example, X1 may be a ring structure with a charge or a ring structure having a planar structure including a portion that forms a hydrogen bond. In addition, when the compound is used in combination with another material, X1 in Formula 1 may include a structure that can bind to a linker.

[0216] As an example, X1 can be phenyl, cycloalkyl or heterocyclyl, which are optionally substituted or unsubstituted with one or more R2. As a specific example, X1 can be selected from phenyl, cyclohexyl, cyclopentyl, furyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indole, 1H-indazolyl, isoindolinyl, indolin-2-one, 2,3-dihydro-1H-indenyl and 1H-pyrrolopyridinyl. In this case, each R2 can be independently selected from alkyl, alkoxy, amino, aminoalkyl, -NO2, =O, -NHC2H4OH, -C(=NH)NH2, -C(=O)NH2, -C(=O)NHCH3, -C(=O)OH, phenyl or heterocyclyl. As an example, each R can be independently selected from methyl, ethyl, amino, aminoalkyl, amino(hydroxyalkyl), methoxy, ethoxy, -C(=NH)NH, -C(=O)NH, -C(=O)OH, phenyl, pyrrolidinyl, piperazinyl, piperidinyl and morpholinyl. As a specific example, R can be amino.

[0217] As a more specific example, X1 can be selected from the following structures:

[0218]

[0219]

[0220]

[0221] As a further more specific example, X1 can be selected from the following structures:

[0222]

[0223] 3) X4

[0224] In Formula 1, X4 corresponds to the side chain of the second residue of the N-degron and may have a cyclic or chain structure to fill the binding space when bound to the UBR box. In this case, as an example, the cyclic or chain structure can enhance binding strength by introducing a moiety that has a charge or forms a hydrogen bond. In addition, when the compound of this specification is subsequently used in combination with another material, X4 may include a structure that can function as a linker.

[0225] As an example, X4 can be phenyl, cycloalkyl or heterocyclyl, which is optionally substituted or unsubstituted with one or more R3. As a specific example, X4 can be selected from phenyl, cyclohexyl, cyclopentyl, furyl, thiazolyl, 1H-pyrazolyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolinyl, 1H-indolinyl, 1H-indole, 1H-indazolyl, isoindolinyl, indolin-2-one, 2,3-dioxo-1H-indenyl and 1H-pyrrolopyridinyl. In this case, each R3 can be independently selected from alkyl, alkoxy, amino, halogen, hydroxy, alkylamino, dialkylamino, -NO2, CONR'R", -C02R', -NHCOR', phenyl or heterocycloalkyl. As an example, each R3 can be independently selected from hydroxy, fluoro, chloro, bromo, amino, methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, alkylamino, dialkylamino, -NO2, -C(=O)NH2, -C02R', -NHCOR', -CONR'R", and phenyl. As a specific example, R3 can be hydroxy. In this case, each R' and R" can independently be -H or alkyl.

[0226] As a more specific example, X4 can be selected from the following structures:

[0227]

[0228]

[0229] As a further more specific example, X4 can be selected from the structures described below:

[0230]

[0231] The compounds disclosed herein may exist in the form of stereoisomers or salts thereof, and the isomers or salt forms of such compounds are included within the scope of the present specification.

[0232] III. Specific Examples of Compounds as UBR Box Domain Ligands

[0233] 1. Specific Examples of Compounds

[0234] The following are specific examples of the compounds disclosed herein. The following specific examples are provided to facilitate understanding of the exemplary structures of the compounds disclosed herein, and the scope of the compounds disclosed herein is not limited to the following examples.

[0235] As a specific example, the compound disclosed herein may have the structure of [Formula 1-1]:

[0236]

[0237] In this case, in the compound, A1 is CH2 or NH, and I is an integer of 0 or 1.

[0238] The application of X1 and X4 is the same as that described in 2) X1 and 3) X4 of the content II. UBR box domain ligand.

[0239] As a further more specific example, specific exemplary compounds of [Formula 1-1] can be selected from the compounds described below.

[0240] [Table 1]

[0241] Specific exemplary compounds of formula [1-1]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] In another specific example, the compound of the present specification may have the structure of [Formula 1-2]:

[0251] [Formula 1-2]

[0252]

[0253] In this case, compounds X1 and X4 are used in the same manner as described in Section II. UBR-box domain ligands 2) X1 and 3) X4.

[0254] Specific exemplary compounds of [Formula 1-2] may be selected from the following:

[0255] [Table 2]

[0256] Specific exemplary compounds of formula [1-2]

[0257]

[0258]

[0259] In another specific example, the compound of the present specification may have the structure of [Formula 1-3]:

[0260] [Formula 1-3]

[0261]

[0262] In this case, compounds X1 and X4 are used in the same manner as described in Section II. UBR-box domain ligands 2) X1 and 3) X4.

[0263] Specific exemplary compounds of [Formula 1-3] may be selected from the following:

[0264] [Table 3]

[0265] Specific exemplary compounds of [Formula 1-3]

[0266]

[0267]

[0268] In another specific example, the compound of the present specification may have the structure of [Formula 1-4]:

[0269] [Formula 1-4]

[0270]

[0271] Specific exemplary compounds of [Formula 1-4] may be selected from the following:

[0272] [Table 4]

[0273] Specific exemplary compounds of [Formula 1-4]

[0274]

[0275]

[0276] In this case, the compound may be considered in the form of its possible isomers or in the form of a mixture thereof. For example, all stereoisomers, including enantiomers and diastereomers or mixtures thereof (eg, racemic mixtures) may be considered.

[0277] 2. Salts of compounds

[0278] As compounds disclosed herein, the form of their salts can be considered. In this case, the salts include pharmaceutically acceptable salts. Salts disclosed herein include acid addition salts or base addition salts. Exemplary acids that form salts include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, glutaric acid, etc., and exemplary bases that form salts include lithium, sodium, potassium, calcium, magnesium, methylamine, trimethylamine, etc. However, acids and bases are not limited thereto and can be easily selected by those skilled in the art.

[0279] IV. Uses of the Compounds

[0280] 1. Inhibition of UBR box domain substrate binding

[0281] Compositions for inhibiting substrate binding of UBR box domains

[0282] The compounds disclosed herein can be used to prepare compositions for inhibiting substrate binding to a UBR box domain. As an example, a composition comprising a compound disclosed herein can be used to inhibit substrate binding to a UBR box domain by binding to the UBR box domain. As another example, a composition comprising the compound can be used to prevent degradation of a substrate that binds to and is degraded by the UBR box domain. As a specific example, a composition comprising the compound can be used to prevent degradation of a substrate that binds to the UBR box domain by the ubiquitin-proteasome pathway.

[0283] As a specific example, a composition comprising a compound disclosed herein can be used to inhibit the binding of a substrate having an N-terminal residue that binds to a UBR box domain. As a specific example, a composition comprising a compound disclosed herein can be used to inhibit the binding of a substrate having an N-terminal residue (e.g., arginine (Arg), lysine (Lys), histidine (His), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), and isoleucine (Ile)). However, the use is not limited thereto, and the composition can be used to inhibit the binding of a material known in the art as a UBR box domain substrate.

[0284] With reference to the Examples, it can be confirmed that the compounds disclosed herein inhibit the degradation of substrates by binding to UBR (see Figures 1 to 19 ).

[0285] 2. Treatment of UBR-related diseases

[0286] The compounds or salts thereof disclosed herein have the property of binding to the UBR box domain. Specifically, the compounds disclosed herein function as ligands that bind to the UBR box domain. Therefore, these compounds can be used to inhibit the degradation of proteins that are degraded by binding to the UBR box domain in vivo. This mechanism can be used to treat UBR-related diseases.

[0287] 1) Pharmaceutical composition

[0288] The compounds disclosed herein can be used to prepare a pharmaceutical composition for treating a subject in need thereof.

[0289] In this case, treatment includes the effect of improving the symptoms of a specific medical condition or delaying the progression of the disease. In this case, the subject includes humans and non-human animals. In this case, the pharmaceutical composition may include a pharmaceutically acceptable carrier, excipient and / or additive and the above-mentioned compound. Pharmaceutically acceptable carriers, excipients and additives include water, saline, ethylene glycol, glycerol, animal and vegetable fats, oils, starch, etc., but are not limited thereto, and also include all pharmaceutically acceptable carriers, excipients and / or additives known in the art.

[0290] 2) Treatment methods

[0291] This specification provides a method of treatment comprising administering a compound disclosed herein, or a pharmaceutically acceptable salt thereof, to a subject in need thereof. In this case, administration of the compound or a pharmaceutically acceptable salt thereof may have the effect of alleviating the symptoms of a particular medical condition or slowing the progression of the disease, compared to a subject not administered the compound or salt thereof. In this case, the subject includes humans and non-human animals.

[0292] -UBR-related diseases

[0293] As an example, this specification provides a treatment method comprising administering a compound or a pharmaceutically acceptable salt thereof to a subject suffering from a UBR-related disease. That is, the compounds disclosed herein or their pharmaceutically acceptable salts can be used to treat UBR-related diseases. As a specific example, the compounds or their pharmaceutically acceptable salts can be used to treat a specific disease by inhibiting the degradation of a protein, wherein the protein is degraded by binding to the UBR box domain.

[0294] Specific diseases include: muscle loss caused by muscular dystrophy (Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dauer muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy); muscle wasting diseases mediated by muscle loss or degeneration, including sarcopenia or cancer cachexia; diseases caused by excessive protein degradation, including liposarcoma, cystic fibrosis, Johanson-Blizzard syndrome, obstructive uropathy (urethral obstructive sequence), autoimmune pancreatitis; or diseases known to be associated with the UBR box and UBR protein, including Usher syndrome.

[0295] As an example, the compound or a pharmaceutically acceptable salt thereof can be used to treat muscle loss mediated by UBR. For example, the rapid loss of muscle mass associated with disease conditions (such as cancer, sepsis, and hyperthyroidism) is associated with increased degradation of intramuscular protein, which is known to be associated with activation of the ubiquitin proteasome system. In this case, it is known that ubiquitin binding is increased, particularly by activating the N-end rule pathway, leading to the occurrence of muscle loss [ALFRED L. GOLDBERG et al., 1998, 1999]. Therefore, by binding to the UBR box domain to prevent the activation of the muscle loss pathway, the compound disclosed herein or a pharmaceutically acceptable salt thereof can be used to treat the disease. However, the present invention is not limited thereto, and specific diseases include all diseases known in the art that are associated with UBR.

[0296] V. Examples

[0297] Example 1. Synthesis of compounds

[0298] [Table 5]

[0299] Compound List

[0300]

[0301]

[0302]

[0303] Recorded on a Bruker Avance III 400 MHz and a Bruker Fourier 300 MHz 1H NMR spectra were performed using TMS as an internal standard. LCMS was performed on an Agilent 1260 HPLC and 6120 MSD quadrupole mass spectrometer (column: C18 (50×4.6 mm, 5 μm)) operating in ES (+) or (-) ionization mode; T = 30°C; flow rate = 1.5 mL / min; detection wavelengths: 220 nm, 254 nm.

[0304] Experimental Example 1-1. Preparation of Compound 1 (N'-(4-hydroxybenzoyl)-4-methylbenzenesulfonylhydrazide)

[0305]

[0306] Step 1) Synthesis of A2

[0307] 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 hours. The solvent was concentrated under reduced pressure to give a crude product, which was purified by flash column (DCM / MeOH=50 / 1 to 1 / 1) to give A2 (4-hydroxybenzoic acid hydrazide, 2.0 g, 60% yield) as a white solid.

[0308]

[0309] Step 2) Synthesis of Compound 1

[0310] A mixture of A2 (4-hydroxybenzoyl hydrazide, 0.3 g, 1.97 mmol, 1.0 eq) and 4-methylbenzenesulfonyl chloride (0.3 g, 1.57 mmol, 0.8 eq) in pyridine (5 mL) was stirred at 80° C. for 16 hours. 1N HCl was then added to the mixture until pH=3 and extracted with EA (20 mL×3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated to give a crude product (400 mg). Approximately 130 mg of the crude product was purified by preparative HPLC. The collected fractions were concentrated to remove most of the CH 3 CN. The remainder was freeze-dried to give compound 1 (N '-(4-hydroxybenzoyl)-4-methylbenzenesulfonyl hydrazide, 50 mg, 24.8% yield) as a white solid.

[0311]

[0312] Experimental Example 1-2. Preparation of Compound 2 (4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide)

[0313]

[0314] Step 1) Synthesis of A3

[0315] A mixture of A2 (4-hydroxybenzoylhydrazide, 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) was stirred at 80° C. for 16 hours. 1N HCl was then added to the mixture until pH = 3, and the mixture was extracted with EA (20 mL×3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated to give crude A3 (N′-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 370 mg) as a yellow solid.

[0316]

[0317] Step 2) Synthesis of Compound 2

[0318] A mixture of A3 (N'-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 150 mg, 0.45 mmol, 1.0 eq) and 5% Pd / C (200 mg, 50% in water) in EtOH (10 mL) was stirred at 10°C for 4 hours using a H2 balloon. The mixture was then filtered and the filtrate was concentrated to give the crude product, which was purified by preparative HPLC. The collected fractions were concentrated to remove most of the CH3CN. The remaining portion was freeze-dried to give compound 2 (4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide), 50 mg, 36.6% yield) as a white solid.

[0319]

[0320] Experimental Example 1-3. Preparation of Compound 3 (4-amino-N'-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonylhydrazide)

[0321]

[0322] Step 1) Synthesis of A4

[0323] To a mixture of A2 (4-hydroxybenzoylhydrazide, 500 mg, 3.29 mmol, 1.0 eq) in pyridine (5 mL) was added dropwise 3-fluoro-4-nitrobenzene-1-sulfonyl chloride (790 mg, 3.29 mmol, 1.0 eq) in pyridine (3 mL). 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×3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated to give a crude product, which was purified by flash column chromatography (DCM / MeOH = 50 / 1 to 30 / 1) to give A4 (3-fluoro-N′-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 500 mg, 42.8%) as a white solid.

[0324]

[0325] Step 2) Synthesis of A5

[0326] To a mixture of A4 (3-fluoro-N'-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 500 mg, 1.41 mmol, 1.0 eq) and morpholine (184 mg, 2.11 mmol, 1.5 eq) in DMF (10 mL) was added KCO (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 Na2SO4, and concentrated to give a crude product, which was purified by flash column (DCM / MeOH = 50 / 1 to 30 / 1) to give A5 (N'-(4-hydroxybenzoyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 180 mg, 30.3%) as a white solid.

[0327]

[0328] Step 3) Synthesis of Compound 3

[0329] To a mixture of A5 (N'-(4-hydroxybenzoyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 180 mg, 0.427 mmol, 1.0 eq) in EtOH (10 mL) was added Pd / C (200 mg) at 25°C. The mixture was then stirred under a balloon of H2 at 25°C for 4 hours. The solution was filtered, and the filtrate was concentrated and purified by preparative HPLC to give compound 3 (4-amino-N'-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonylhydrazide, 40 mg, 23.9%) as a white solid. (TLC: N / A)

[0330]

[0331] Experimental Example 1-4. Preparation of Compound 4 (N'-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonylhydrazide)

[0332]

[0333] Step 1) Synthesis of A7

[0334] 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 hours and then at 68 ° C for 1 hour. The mixture was cooled and carefully poured into water. The precipitate formed was collected by filtration, washed with water, and dried under vacuum to obtain A7 (2-oxoindoline-5-sulfonyl chloride, 0.7 g, crude product) as a pink solid.

[0335]

[0336] Step 2) Synthesis of Compound 4

[0337] A mixture of A2 (4-hydroxybenzoyl hydrazide, 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) was stirred at 30° C. for 5 hours. The mixture was poured into water. The formed precipitate was collected by filtration, washed with water, and dried under vacuum. The solid was stirred at 30° C. for 30 minutes in DCM. The mixture was filtered and the filter cake was dried to give compound 4 (N'-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonyl hydrazide, 50 mg, 11%) as a pink solid.

[0338]

[0339] Experimental Example 1-5. Preparation of Compound 5 (N'-(4-hydroxybenzoyl)indoline-5-sulfonylhydrazide)

[0340]

[0341] Step 1) Synthesis of A8

[0342] A mixture of A2 (4-hydroxybenzoyl hydrazide, 293 mg, 1.93 mmol, 1.0 eq) and 1-acetyl indoline-5-sulfonyl chloride (500 mg, 1.93 mmol, 1.0 eq) in pyridine (10 mL) was stirred at 30° C. for 5 hours. After completion, the reaction mixture was diluted with H O (20 mL) and extracted with EA (20 mL×3). The combined organic layers were washed with brine, dried over Na SO, and concentrated to give A8 (1-acetyl-N′-(4-hydroxybenzoyl) indoline-5-sulfonyl hydrazide, 500 mg, crude product) as a yellow solid.

[0343] Step 2) Synthesis of Compound 5

[0344] A mixture of A8 (1-acetyl-N'-(4-hydroxybenzoyl)indoline-5-sulfonylhydrazide, 200 mg, 0.53 mmol, 1.0 eq) and 2N HCl (6 mL) in THF (10 mL) was stirred at 50° C. for 5 hours. After completion, the reaction mixture was diluted with H2O (20 mL) and extracted with EA (20 mL×3). The combined organic layers were washed with brine, dried over Na2SO4, and then concentrated. The crude product was purified by preparative HPLC and lyophilized to give compound 5 (N'-(4-hydroxybenzoyl)indoline-5-sulfonylhydrazide, 50 mg, 28.2%) as a white solid.

[0345]

[0346] Experimental Example 1-6. Preparation of Compound 6 (N'-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonylhydrazide)

[0347]

[0348] Step 1) Synthesis of A10

[0349] To a solution of bromobenzene (400 mg, 2.5 mmol) in dioxane was added methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (A9, 801 mg, 3.0 mmol), K3PO4 (541 mg, 7.5 mmol) and Pd(dppf)Cl2-CH2Cl2 (208 mg, 0.25 mmol) 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 MgSO4 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.

[0350]

[0351] Step 2) Synthesis of A11

[0352] 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 hours. After the reaction was completed, the reaction mixture was cooled and then concentrated under reduced pressure, and then purified by flash column chromatography (DCM / MeOH=15 / 1) to give the crude product A11 ([1,1'-biphenyl]-4-carbohydrazide, 152 mg, theoretical yield: 100%) as a white solid.

[0353]

[0354] Step 3) Synthesis of A12

[0355] 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). 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 give the crude product A12 (N'-([1,1'-biphenyl])-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 60 mg, yield: 21%) as a yellow solid.

[0356]

[0357] Step 4) Synthesis of Compound 6

[0358] To a solution of A12 (N'-([1,1'-biphenyl]-4-carbonyl)-4-nitrobenzenesulfonyl hydrazide, 60 mg, 0.15 mmol) in THF:MeOH=3:1 (12 mL) was added Zn (99 mg, 1.5 mmol) and NH4Cl (81 mg, 1.5 mmol). The mixture was stirred at room temperature for 12 hours. After the reaction was complete, 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 6 (N'-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonyl hydrazide, 10 mg, yield: 20%) as a white solid.

[0359]

[0360] Experimental Example 1-7. Preparation of Compound 7 (N'-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonylhydrazide)

[0361]

[0362] Step 1) Synthesis of A14

[0363] To a solution of bromobenzene (400 mg, 2.5 mmol) in dioxane was added methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (A13, 801 mg, 3.0 mmol), K3PO4 (541 mg, 7.5 mmol) and Pd(dppf)Cl2-CH2Cl2 (208 mg, 0.25 mmol) 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 MgSO4 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.

[0364]

[0365] Step 2) Synthesis of A15

[0366] 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 hours. After the reaction was completed, the reaction mixture was cooled and then concentrated under reduced pressure, and then purified by flash column chromatography (DCM / MeOH=15 / 1) to give the crude product A15 ([1,1'-biphenyl]-3-carbohydrazide, 200 mg, yield: 100%) as a yellow solid.

[0367]

[0368] Step 3) Synthesis of A16

[0369] 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 give the crude product A16 (N'-([1,1'-biphenyl]-3-carbonyl)-4-nitrobenzenesulfonylhydrazide, 102 mg, yield: 53%) as an ivory solid.

[0370]

[0371] Step 4) Synthesis of Compound 7

[0372] To a solution of A16 (N'-([1,1'-biphenyl]-3-carbonyl)-4-nitrobenzenesulfonylhydrazide, 102 mg, 0.25 mmol) in THF:MeOH=3:1 (10 mL) were added Zn (168 mg, 2.5 mmol) and NHCl (137 mg, 2.5 mmol). The mixture was stirred at room temperature for 12 hours. After the reaction was completed, 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 7 (N'-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonylhydrazide, 14 mg, yield: 14%, purity: 96.8%) as a white solid.

[0373]

[0374] Experimental Example 1-8. Preparation of Compound 8 (3-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide)

[0375]

[0376] Step 1) Synthesis of A17

[0377] 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 completion of the reaction, the resulting mixture was cooled and evaporated to remove pyridine, and then purified by flash column chromatography (DCM / MeOH=15 / 1) to give the crude product A17 (N'-(4-hydroxybenzoyl)-3-nitrobenzenesulfonylhydrazide), 129 mg, yield: 43%), as an ivory solid.

[0378]

[0379] Step 2) Synthesis of Compound 8

[0380] To a solution of A17 (N'-(4-hydroxybenzoyl)-3-nitrobenzenesulfonylhydrazide, 129 mg, 0.38 mmol) in THF:MeOH=3:1 (10 mL) was added Zn (250 mg, 3.8 mmol) and NHCl (204 mg, 3.8 mmol). The mixture was stirred at room temperature for 12 hours. After the reaction was complete, 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 8 (3-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 25 mg, yield: 21%, purity: 98.0%) as a white solid.

[0381] Experimental Example 1-9. Preparation of Compound 9 (4-(1-aminoethyl)-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide)

[0382]

[0383] Step 1) Synthesis of A18

[0384] A mixture of A2 (4-hydroxybenzoyl hydrazide, 500 mg, 3.29 mmol, 1.0 eq) and 4-acetophenone-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×2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated to give A18 (4-acetyl-N′-(4-hydroxybenzoyl)benzenesulfonyl hydrazide, 0.5 g, crude product) as a brown solid.

[0385]

[0386] Step 2) Synthesis of Compound 9

[0387] A18 (4-acetyl-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 200 mg, 0.60 mmol, 1.0 eq), NH4OAc (461 mg, 5.98 mmol, 10 eq) and sodium cyanoborohydride (188 mg, 2.99 mmol, 5 eq) in MeOH (5 mL) was stirred at 60° C. for 16 hours. The mixture was concentrated and purified by preparative HPLC and lyophilized to give compound 9 (4-(1-aminoethyl)-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 55 mg, 24%) as a white solid.

[0388]

[0389] Experimental Example 1-10. Preparation of Compound 10 (3,5-diamino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide)

[0390]

[0391] Step 1) Synthesis of A20

[0392] To a mixture of 3,5-dinitroaniline (A19, 500 mg, 2.73 mmol, 1.0 eq) in concentrated HCl (10 mL) at 0°C was added NaNO₂ (226 mg, 3.28 mmol, 1.2 eq) in H₂O (2 mL). 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 H₂O (10 mL) at 0°C was added SOCl₂ (1.30 g, 10.9 mmol, 4.0 eq). The diazonium salt solution was then added dropwise at 0°C. The mixture was stirred at 0°C for 3 h and then poured into water. The resulting precipitate was collected by filtration and dried under vacuum to afford A20 (3,5-dinitrobenzenesulfonyl chloride, 0.5 g, crude) as a yellow solid.

[0393] Step 2) Synthesis of A21

[0394] A mixture of A20 (3,5-dinitrobenzenesulfonyl chloride, 175 mg, 0.66 mmol, 1.0 eq) and A2 (4-hydroxybenzoylhydrazide, 100 mg, 0.66 mmol, 1.0 eq) in pyridine (5 mL) 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 Na2SO4, and concentrated to afford A21 (N'-(4-hydroxybenzoyl)-3,5-dinitrobenzenesulfonylhydrazide, 0.1 g, crude) as a pink solid.

[0395] Step 3) Synthesis of Compound 10 (3,5-diamino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide)

[0396] A mixture of A21 (N'-(4-hydroxybenzoyl)-3,5-dinitrobenzenesulfonylhydrazide, 0.1 g, 0.26 mmol, 1.0 eq) and 10% Pd / C (0.1 g) in EtOH (5 mL) was stirred under a H2 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, which was stirred in MeOH (5 mL) for 10 minutes. The mixture was filtered and the filter cake was dried in vacuo to give compound 10 (3,5-diamino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 27 mg, 32%) as a yellow solid.

[0397]

[0398] Experimental Example 1-11. Preparation of Compound 11 (N'-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonylhydrazide)

[0399]

[0400] A mixture of compound 2 (4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 500 mg, 1.63 mmol, 1.0 eq) and ethylene oxide (72 mg, 1.63 mmol, 1.0 eq) in AcOH / H2O=1:1 (4 mL) was stirred at 25°C for 4 hours. NaHCO3 was then added to the mixture until pH=8, and extracted with EA (50 mL×3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give a crude product, which was purified by preparative TLC to give compound 11 (N'-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonylhydrazide, 50 mg, 8.8% yield) as a white solid.

[0401]

[0402] Experimental Example 1-12. Preparation of Compound 12 (4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide)

[0403]

[0404] Step 1) Synthesis of A23

[0405] 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 (2.83 g, 17.7 mmol, 1.0 eq) 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-bromoethan-1-one, 2.5 g, 46.3%) as a white solid.

[0406] Step 2) Synthesis of A24

[0407] A mixture of 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 eq) in DCM (20 mL) was stirred at 10 °C for 2 hours. The mixture was then filtered and the filter cake was dissolved in EtOH (15 mL) and HCl (5 mL). The mixture was stirred at 85 °C for 2 hours. The mixture was filtered and the filter cake was dried to give A24 (2-amino-1-(4-(benzyloxy)phenyl)ethan-1-one hydrochloride, 2.0 g, crude) as a white solid.

[0408]

[0409] Step 3) Synthesis of A25

[0410] A mixture of A24 (2-amino-1-(4-(benzyloxy)phenyl)ethan-1-one hydrochloride, 2.00 g, 7.20 mmol, 1.0 eq), 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) in DCM (20 mL) was stirred at 20° C. for 1 hour. The mixture was poured into water and extracted with DCM. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated to give the crude product, which was stirred in PE for 30 minutes. The mixture was filtered, and the filter cake was dried to give A25 (N-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-4-nitrobenzenesulfonamide, 1.0 g, 35.8% over 2 steps) as a white solid.

[0411]

[0412] Step 4) Synthesis of Compound 12

[0413] A mixture of A25 (N-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-4-nitrobenzenesulfonamide, 200 mg, 0.47 mmol, 1.0 eq) and Pd / C (150 mg, 50% in water) in EtOH (10 mL) was stirred under a balloon of H at 25° C. for 4 h. The mixture was then filtered and concentrated, then purified by preparative HPLC and lyophilized to afford compound 12 (4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide, 69 mg, 48.0% yield) as a white solid.

[0414]

[0415] Experimental Example 1-13. Preparation of Compound 13 (N'-(4-hydroxybenzoyl)-4-methoxybenzenesulfonylhydrazide)

[0416]

[0417] To a solution of A2 (4-hydroxybenzoyl hydrazide, 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). The mixture was allowed to stand at room temperature for 12 hours. After completion of the reaction, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, 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-methoxybenzenesulfonyl hydrazide, 25 mg, yield: 12%, purity: 96.9%) as a white solid.

[0418]

[0419] Experimental Example 1-14. Preparation of Compound 14 (4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)benzamidine

[0420]

[0421] Step 1) Synthesis of A26

[0422] To a mixture of A2 (4-hydroxybenzoylhydrazide, 1.00 g, 6.57 mmol, 1.0 eq) in pyridine (10 mL) was added dropwise 4-cyanobenzene-1-sulfonyl chloride (1.33 g, 6.57 mmol, 1.0 eq) in pyridine (3 mL). 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×3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated to give a crude product, which was purified by flash column chromatography (PE / EA=1 / 1) to give A26 (4-cyano-N′-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 1.40 g, 67.1%) as a white solid.

[0423]

[0424] Step 2) Synthesis of compound 14

[0425] A mixture of A26 (4-cyano-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 200 mg, 0.63 mmol, 1.0 eq) in HCl / EtOH (5 mL, 6 mol / L) was stirred at 25 ° C for 3 hours. The above solution was concentrated and added to MeOH. The mixture was concentrated again. The residue was added to MeOH (10 mL), and then NH4OAc (485 mg, 6.30 mmol, 10 eq) was added. The mixture was stirred at 25 ° C for 16 hours. The mixture was concentrated and then purified by preparative HPLC and lyophilized to give compound 14 (4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)benzamidine, 26 mg, 10.8%) as a white solid.

[0426]

[0427] Experimental Example 1-15. Preparation of Compound 15 (4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)benzamide)

[0428]

[0429] To a cooled (0°C) solution of A26 (4-cyano-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide, 210 mg, 0.66 mmol) in DMSO was added hydrogen peroxide 35% w / w aqueous solution (0.42 mL, 4.8 mmol) and potassium carbonate (30 mg, 0.20 mmol). The reaction was allowed to warm to room temperature and stirred for 12 hours. After completion of the reaction, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM / MeOH=5 / 1) to give compound 15 (4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)benzamide, 20 mg, yield: 10%, purity: 96.5%) as a white solid.

[0430]

[0431] Experimental Example 1-16. Preparation of Compound 16 (N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide)

[0432]

[0433] Step 1) Synthesis of A28

[0434] To a mixture of A27 (4-hydroxybenzamide, 4.0 g, 29.2 mmol, 1.0 eq) in DMF (40 mL) was added KCO (6.05 g, 43.8 mmol, 1.5 eq) and BnBr (4.99 g, 29.2 mmol, 1.0 eq). 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 NaSO, and concentrated to afford A28 (4-(benzyloxy)benzamide, 6.0 g, 90.5%) as a white solid.

[0435] Step 2) Synthesis of A29

[0436] A mixture of A28 (4-(benzyloxy)benzamide, 4.0 g, 17.6 mmol, 1.0 eq), KCO (0.24 g, 1.76 mmol, 0.1 eq) and HCOH (1.43 g, 17.6 mmol, 37% in water, 1.0 eq) in THF / H2O (40 mL, v / v = 1 / 1) was stirred at 65°C for 16 hours. The mixture was then concentrated and filtered. The filter cake was dried to afford A29 (4-(benzyloxy)-N-(hydroxymethyl)benzamide, 4.0 g, crude) as a white solid.

[0437] Step 3) Synthesis of A30

[0438] A mixture of 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 the pH was adjusted to 8 with aqueous NaHCO 3 solution. The mixture was extracted with EA. The organic layer was washed with brine, dried over Na 2 SO 4 , and concentrated to afford A30 (4-(benzyloxy)-N-(((4-nitrophenyl)thio)methyl)benzamide, 2.0 g, 32.6% over 2 steps) as a white solid.

[0439] Step 4) Synthesis of A31

[0440] A mixture of A30 (4-(benzyloxy)-N-(((4-nitrophenyl)thio)methyl)benzamide, 500 mg, 1.27 mmol, 1.0 eq) and m-CPBA (656 g, 3.80 mmol, 3.0 eq) in DCM (20 mL) was stirred at 20° C. for 16 hours. The mixture was poured into aqueous Na2O3S2 and extracted. The organic layer was washed with aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated to afford A31 (4-(benzyloxy)-N-(((4-nitrophenyl)sulfonyl)methyl)benzamide, 300 mg, crude) as a white solid.

[0441]

[0442] Step 5) Synthesis of Compound 16

[0443] A mixture of A31 (4-(benzyloxy)-N-(((4-nitrophenyl)sulfonyl)methyl)benzamide, 300 mg, 0.47 mmol, 1.0 eq) and Pd / C (150 mg, 50% in water) in EtOH (10 mL) was stirred under a balloon of H at 25° C. for 2 h. The mixture was then filtered, concentrated, purified by preparative HPLC, and lyophilized to afford compound 16 (N-(((4-aminophenyl)sulfonyl)methyl)-4-hydroxybenzamide, 35 mg, 9.0% yield over 2 steps) as a white solid.

[0444]

[0445] Experimental Example 1-17. Preparation of Compound 17 (6-amino-N'-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonylhydrazide)

[0446]

[0447] Step 1) Synthesis of A33

[0448] A mixture of 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)Cl2 (337 mg, 0.09 mmol, 0.1 eq), and AcOK (1.14 g, 13.8 mmol, 3.0 eq) in toluene (10 mL) was stirred at 90°C under N2 for 16 hours. 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 Na2SO4, and concentrated. The crude product was purified by flash column chromatography (PE / EA = 30 / 1 to 10 / 1) to give A33 (6-nitro-[1,1'-biphenyl]-3-amine, 1 g, crude) as a yellow solid.

[0449] Step 2) Synthesis of A34

[0450] To a mixture of A33 (6-nitro-[1,1'-biphenyl]-3-amine, 500 mg, 2.33 mmol, 1.0 eq) in AcOH (10 mL) and concentrated HCl (5 mL) was added dropwise NaNO (193 mg, 2.8 mmol, 1.2 eq) in H2O (2 mL) 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 H2O (10 mL) at 0°C was added SOCl (1.39 g, 11.6 mmol, 5.0 eq). The diazonium salt solution was then added dropwise at 0°C. The mixture was stirred at 0°C for 3 h and then poured into water. The precipitate that formed was collected by filtration and dried under vacuum to afford A34 (6-nitro-[1,1'-biphenyl]-3-sulfonyl chloride, 400 mg, 57.6%) as a yellow solid.

[0451]

[0452] Step 3) Synthesis of A35

[0453] A mixture of A34 (6-nitro-[1,1'-biphenyl]-3-sulfonyl chloride, 200 mg, 0.67 mmol, 1.0 eq) and 4-hydroxybenzoylhydrazide (122 mg, 0.81 mmol, 1.2 eq) in pyridine (5 mL) was stirred at 30°C for 0.5 h. The mixture was 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 NaSO, and concentrated to afford A35 (N'-(4-hydroxybenzoyl)-6-nitro-[1,1'-biphenyl]-3-sulfonylhydrazide, 200 mg, crude) as a brown solid.

[0454] Step 4) Synthesis of Compound 17

[0455] A mixture of A35 (N'-(4-hydroxybenzoyl)-6-nitro-[1,1'-biphenyl]-3-sulfonylhydrazide, 200 mg, 0.48 mmol, 1.0 eq) and 10% Pd / C (200 mg) in EtOH (10 mL) was stirred at 30 ° C. under a H2 balloon for 3 hours. The reaction mixture was filtered, and the filter cake was washed with EA (50 mL×2). The combined filtrates were concentrated to give a crude product, which was purified by preparative HPLC and lyophilized to give compound 17 (6-amino-N'-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonylhydrazide, 45 mg, 17.5% over 2 steps) as a white solid.

[0456]

[0457] Experimental Example 1-18. Preparation of Compound 18 (4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide)

[0458]

[0459] Step 1) Synthesis of A37

[0460] To a cooling (0 ℃) solution of 4-(chlorocarbonyl)benzoic acid methyl ester (A36, 1.5 g, 7.5 mmol) in DCM, add ammonia solution (25-30%) (1.8 mL, 15 mmol). Allow the reaction to warm to room temperature and stir for 4 hours. After the reaction is complete, the reaction mixture is evaporated, and water is then added. The precipitate formed is collected by filtration. The filter cake is washed with water and dried to give A37 (methyl 4-carbamoylbenzoate, 1.0 g, 74%) as a white solid.

[0461]

[0462] Step 2) Synthesis of A38

[0463] 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 hours. 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.

[0464]

[0465] Step 3) Synthesis of A39

[0466] 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). The mixture was kept at room temperature for 12 hours. After completion of the reaction, the reaction mixture was evaporated and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4, 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%) as a white solid. LCMS Calcd m / z for C 14 H 12 N4O6 S[M+H] + 365.33Found 365.

[0467] Step 4) Synthesis of Compound 18

[0468] To 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) was added Zn (657 mg, 10 mmol) and NHCl (537 mg, 10 mmol) and allowed to stand at 60°C for 5 hours. After completion of the reaction, the reaction mixture was cooled and 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%) as a white solid.

[0469]

[0470] Experimental Example 1-19. Preparation of Compound 19 (N'-(4-aminobenzyl)-4-hydroxybenzohydrazide)

[0471]

[0472] Step 1) Synthesis of A40

[0473] 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). The mixture was allowed to stand 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 salt water, dried over anhydrous MgSO4, 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.

[0474]

[0475] Step 2) Synthesis of Compound 19

[0476] To a solution of A40 (4-hydroxy-N'-(4-nitrobenzyl)benzohydrazide, 410 mg, 1.4 mmol) in THF:MeOH=3:1 (12 mL) was added Zn (933 mg, 14 mmol) and NH4Cl (764 mg, 14 mmol). The mixture was stirred at room temperature for 12 hours. After the reaction was complete, ethyl acetate was added. The mixture was filtered through diatomaceous earth. 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%) as a white solid.

[0477]

[0478] Experimental Example 1-20. Synthesis of Compound 20 (4-amino-N'-(1H-indole-3-carbonyl)benzenesulfonylhydrazide)

[0479]

[0480] Step 1) Synthesis of A42

[0481] To a mixture of A41 (1H-indole-3-carbohydrazide, 500 mg, 2.85 mmol, 1.0 eq) in pyridine (5 mL) was added dropwise 4-nitrobenzene-1-sulfonyl chloride (632 mg, 2.85 mmol, 1.0 eq) in pyridine (5 mL). 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 Na2SO4, and concentrated to give the crude product, which was purified by flash column chromatography (DCM / MeOH = 50 / 1 to 30 / 1) to give A42 (N'-(1H-indole-3-carbonyl)-4-nitrobenzenesulfonylhydrazide, 0.9 g, 87.5% yield) as a yellow solid. (TLC: DCM / MeOH = 20 / 1, Rf = 0.5)

[0482] Step 2) Synthesis of Compound 20

[0483] To a mixture of A42 (N'-(1H-indole-3-carbonyl)-4-nitrobenzenesulfonylhydrazide, 300 mg, 0.83 mmol, 1.0 eq) in MeOH (10 mL) was added Pd / C (100 mg). The mixture was then stirred under a balloon of H2 at 10°C for 2 hours. The solution was filtered, and the filtrate was purified by preparative HPLC and lyophilized to afford compound 20 (4-amino-N'-(1H-indole-3-carbonyl)benzenesulfonylhydrazide, 30 mg, 10.9% yield) as a yellow solid.

[0484]

[0485] Experimental Examples 1-21 and 1-22. Preparation of Compound 21 (4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide) and Compound 22 (N'-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide)

[0486]

[0487] Step 1) Synthesis of Compound 22

[0488] To a mixture of A4 (3-fluoro-N'-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 400 mg, 1.13 mmol, 1.0 eq) and KCO (389 mg, 2.81 mmol, 2.5 eq) in DMF (10 mL) 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 Na2SO4, and concentrated to give a crude product, which was purified by flash column (DCM / MeOH=50 / 1~30 / 1) to give compound 22 (N'-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide, 176 mg, yield 38.5%) as a yellow solid.

[0489]

[0490] Step 2) Synthesis of Compound 21

[0491] To a mixture of compound 22 (N'-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide, 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 balloon of H2 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 minutes. The mixture was filtered and the filter cake was washed with MeOH and dried to give compound 21 (4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide, 30 mg, 32.4% yield) as an off-white solid.

[0492]

[0493] Experimental Example 1-23. Synthesis of Compound 23 (4-amino-N'-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonylhydrazide)

[0494]

[0495] Step 1) Synthesis of A43

[0496] To a mixture of A4 (3-fluoro-N'-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 400 mg, 1.13 mmol, 1.0 eq) and KCO (389 mg, 2.81 mmol, 2.5 eq) in DMF (10 mL) at 10°C was added piperidine (115 mg, 1.35 mmol, 1.2 eq). 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 NaSO, and concentrated to give a crude product, which was purified by flash column chromatography (DCM / MeOH = 50 / 1 to 30 / 1) to give A43 (N'-(4-hydroxybenzoyl)-4-nitro-3-(piperidin-1-yl)benzenesulfonylhydrazide, 240 mg, 50.7% yield) as a yellow solid.

[0497]

[0498] Step 2) Synthesis of Compound 23

[0499] To a mixture of A43 (N'-(4-hydroxybenzoyl)-4-nitro-3-(piperidin-1-yl)benzenesulfonylhydrazide, 100 mg, 0.24 mmol, 1.0 eq) in MeOH (5 mL) was added Pd / C (30 mg). The mixture was then stirred under a balloon of H2 at 10°C for 16 hours. The solution was filtered, and the filtrate was purified by preparative HPLC and lyophilized to afford compound 23 (4-amino-N'-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonylhydrazide, 25 mg, 26.9% yield) as a yellow solid.

[0500]

[0501] Experimental Example 1-24. Synthesis of Compound 24 (N'-(4-hydroxybenzoyl)-1H-pyrazole-4-sulfonylhydrazide)

[0502]

[0503] Step 1) Synthesis of A44

[0504] A solution of 1H-pyrazole (1 g, 14.7 mmol, 1.0 eq) in chlorosulfonic acid (5 mL) was stirred overnight at 100° C. The solution was poured into water (30 mL) and extracted with EA (30 mL×3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated to afford A44 (1H-pyrazole-4-sulfonyl chloride, 340 mg, 14% yield) as an off-white solid.

[0505]

[0506] Step 2) Synthesis of Compound 24

[0507] A mixture of A44 (1H-pyrazole-4-sulfonyl chloride, 100 mg, 0.6 mmol, 1.0 eq) and A2 (4-hydroxybenzoyl hydrazide, 109 mg, 0.72 mmol, 1.2 eq) in pyridine (20 mL) was stirred at 80° C. overnight. The solution was poured into water (30 mL) and extracted with EA (30 mL×3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated to give the crude product. The residue was purified by preparative HPLC to give compound 24 (N '-(4-hydroxybenzoyl)-1H-pyrazole-4-sulfonyl hydrazide, 60 mg) as an off-white solid.

[0508]

[0509] Experimental Examples 1-25 and 1-26. Preparation of Compound 25 (N'-(4-hydroxybenzoyl)indoline-4-sulfonylhydrazide) and Compound 26 (N'-(4-hydroxybenzoyl)-1H-indole-4-sulfonylhydrazide)

[0510]

[0511] Step 1) Synthesis of A46

[0512] To a solution of A45 (4-bromo-1H-indole, 1.0 g, 5.10 mmol, 1.0 eq) in THF (10 mL) and Et2O (10 mL) at 0°C was added NaH (204 mg, 5.10 mmol, 60% in mineral oil, 1.0 eq). After stirring for 15 minutes, 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 slowly added. After 30 minutes, SO2 (gas, 1 L) was slowly added at -78°C. The mixture was then warmed to room temperature and stirred overnight. A mixture of acetic acid (307 mg, 5.10 mmol, 1.0 eq) in Et2O (15 mL) was added at 0°C. The mixture was stirred at 0°C for 30 minutes and then filtered. The filter cake was quickly washed with Et2O. The solid is suspended in Et2O (15mL), cooled to 0 ° C, and NCS (682g, 5.10mmol, 1.0eq) is carefully added. The resulting suspension is rapidly stirred for 30 minutes and then filtered. The filter cake is washed with Et2O. The combined filtrate is concentrated to provide A46 (1H-indole-4-sulfonyl chloride, 420mg, crude product) as a brown solid.

[0513] Step 2) Synthesis of Compound 26

[0514] A mixture of A46 (1H-indole-4-sulfonyl chloride, 420 mg, 1.95 mmol, 1.0 eq) and 4-hydroxybenzoylhydrazide (A2, 297 mg, 1.95 mmol, 1.0 eq) in pyridine (30 mL) was stirred at 25° C. for 30 minutes. The solution was poured into water (30 mL). The formed solid was collected by filtration and the filter cake was washed with water. The crude product was purified by preparative HPLC and lyophilized to give compound 26 (N′-(4-hydroxybenzoyl)-1H-indole-4-sulfonylhydrazide, 200 mg, 31.0% yield) as a white solid.

[0515]

[0516] Step 3) Synthesis of Compound 25

[0517] To a mixture of compound 26 (N'-(4-hydroxybenzoyl)-1H-indole-4-sulfonylhydrazide, 150 mg, 0.48 mmol, 1.0 eq) in TFA (5 mL) and DCM (5 mL) was added NaBH3CN (89 mg, 1.43 mmol, 3.0 eq) at 0°C. The mixture was stirred at 10°C for 30 minutes. The above solution was poured into water (30 mL) and extracted with DCM (30 mL×3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give a crude product, which was purified by preparative HPLC and lyophilized to give compound 25 (N'-(4-hydroxybenzoyl)indoline-4-sulfonylhydrazide, 30 mg, 20.0% yield) as a gray solid.

[0518]

[0519] Experimental Example 1-27. Preparation of Compound 27 (2-((4-aminophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide)

[0520]

[0521] Step 1) Synthesis of A47

[0522] 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) dropwise. The mixture was then stirred at 10° C. for 2 hours. The above solution was poured into water (100 mL) and the solution was 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, 69.7% yield) as a yellow solid.

[0523]

[0524] Step 2) Synthesis of A48

[0525] 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 MeOH / HCl (30 mL, 6 mol / L). The mixture was then stirred at 10°C for 2 hours. The solution was concentrated to afford A48 (4-nitrobenzenesulfonylhydrazine hydrochloride, 2.0 g, 83.4% yield) as a yellow solid.

[0526]

[0527] Step 3) Synthesis of A49

[0528] To a mixture of A48 (4-nitrobenzenesulfonylhydrazine 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 phenyl isocyanate (235 mg, 1.97 mmol, 1.0 eq) at 10°C. The mixture was then stirred at 10°C for 2 hours. The above solution was poured into water (80 mL). The formed solid was filtered and the filter cake was stirred in EA (20 mL) for 30 minutes. The mixture was then filtered again and the filter cake was dried to give A49 (2-((4-nitrophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide, 340 mg, 51.3% yield) as a white solid.

[0529]

[0530] Step 4) Synthesis of Compound 27

[0531] A mixture of 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) was stirred under a balloon of H at 10° C. for 15 h. 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, 16.2% yield) as a white solid.

[0532]

[0533] Experimental Example 1-28. Preparation of Compound 28 (4-amino-N'-(1H-indole-4-carbonyl)-3-morpholinobenzenesulfonylhydrazide)

[0534]

[0535] Step 1) Synthesis of A51

[0536] A mixture of A50 (1H-indole-4-carboxylic acid methyl ester, 1.00 g, 5.71 mmol, 1.0 eq) in N₂H₄H₂O (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 Na₂SO₄ and concentrated to afford A51 (1H-indole-4-carbohydrazide, 500 mg, crude) as a yellow solid.

[0537] Step 2) Synthesis of A52

[0538] To a mixture of A51 (1H-indole-4-carbohydrazide, 100 mg, 0.57 mmol, 1.0 eq) in pyridine (2 mL) was added dropwise 3-fluoro-4-nitrobenzene-1-sulfonyl chloride (175 mg, 0.57 mmol, 1.0 eq) in pyridine (2 mL). 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 Na2SO4, and concentrated to afford A52 (3-fluoro-N'-(1H-indole-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 200 mg, crude) as a yellow solid.

[0539] Step 3) Synthesis of A53

[0540] To a mixture of A52 (3-fluoro-N'-(1H-indole-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 200 mg, 0.52 mmol, 1.0 eq) and KCO (183 mg, 1.30 mmol, 2.5 eq) in DMF (5 mL) at 10°C was added morpholine (54 mg, 0.62 mmol, 1.2 eq). 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 NaSO, and concentrated to give A53 (N'-(1H-indole-4-carbonyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 100 mg, crude) as a yellow solid.

[0541] Step 4) Synthesis of Compound 28

[0542] To a mixture of A53 (N'-(1H-indole-4-carbonyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 100 mg, 0.22 mmol, 1.0 eq) in EtOH (5 mL) was added Fe (61.6 mg, 1.10 mmol, 5.0 eq) and saturated aqueous NH4Cl (3 mL). The mixture was then stirred at 85°C for 3 hours. The solution was filtered and the filtrate was purified by preparative HPLC and lyophilized to give compound 28 (4-amino-N'-(1H-indole-4-carbonyl)-3-morpholinobenzenesulfonylhydrazide, 20 mg, 21.5% yield) as a white solid.

[0543]

[0544] Experimental Example 1-29. Preparation of Compound 29 (4-amino-N'-(indoline-4-carbonyl)benzenesulfonylhydrazide)

[0545]

[0546] Step 1) Synthesis of A54

[0547] To a mixture of A51 (1H-indole-4-carbohydrazide, 400 mg, 2.29 mmol, 1.0 eq) in pyridine (5 mL) was added dropwise 4-nitrobenzene-1-sulfonyl chloride (505 mg, 2.29 mmol, 1.0 eq) in pyridine (5 mL). 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 Na2SO4, and concentrated to afford A54 (N'-(1H-indole-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 300 mg, crude) as a yellow solid.

[0548] Step 2) Synthesis of A55

[0549] To a mixture of A54 (N'-(1H-indole-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 300 mg, 0.83 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1.5 mL) and NaBH3CN (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 adjusted to pH = 7 with saturated aqueous NaHCO3. The mixture was filtered, the filter cake was washed with MTBE, and dried in vacuo to afford A55 (N'-(indoline-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 150 mg, crude) as a yellow solid.

[0550] Step 3) Synthesis of Compound 29

[0551] A mixture of A55 (N'-(indoline-4-carbonyl)-4-nitrobenzenesulfonylhydrazide, 150 mg, 0.41 mmol, 1.0 eq) and Pd / C (100 mg) in MeOH (5 mL) was stirred under a balloon of H2 at 10°C for 2 hours. The solution was filtered, and the filtrate was concentrated to give the crude product, which was stirred in MeOH (10 mL) for 30 minutes. The mixture was filtered, and the filter cake was dried in vacuo to give compound 29 (4-amino-N'-(indoline-4-carbonyl)benzenesulfonylhydrazide, 40 mg, 29.1% yield) as a white solid.

[0552]

[0553] Experimental Example 1-30. Preparation of Compound 30 (4-amino-N'-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonylhydrazide)

[0554]

[0555] Step 1) Synthesis of A56

[0556] To a mixture of A4 (3-fluoro-N'-(4-hydroxybenzoyl)-4-nitrobenzenesulfonylhydrazide, 500 mg, 1.41 mmol, 1.0 eq) and KCO (290 mg, 2.10 mmol, 1.5 eq) in DMF (5 mL) at 10° C. was added tert-butyl piperazine-1-carboxylate (315 mg, 1.69 mmol, 1.2 eq). 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×3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give a crude product, which was purified by flash column (DCM / MeOH=50 / 1~30 / 1) to give A56 (tert-butyl 4-(5-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)-2-nitrophenyl)piperazine-1-carboxylate, 310 mg, crude) as a yellow solid.

[0557] Step 2) Synthesis of A57

[0558] To a mixture of A56 (tert-butyl 4-(5-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)-2-nitrophenyl)piperazine-1-carboxylate, 250 mg, 0.48 mmol, 1.0 eq) in EtOH (3 mL) was added saturated aqueous NH4Cl (3 mL) and Fe (135 mg, 2.41 mmol, 5.0 eq). 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 Na2SO4, and concentrated to give A57 (tert-butyl 4-(2-amino-5-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)phenyl)piperazine-1-carboxylate, 210 mg, crude) as a yellow solid.

[0559] Step 3) Synthesis of Compound 30

[0560] To a mixture of A57 (tert-butyl 4-(2-amino-5-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)phenyl)piperazine-1-carboxylate, 270 mg, 0.55 mmol, 1.0 eq) in DCM (5 mL) 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 give a crude product, which was purified by preparative HPLC and lyophilized to give the crude product. The crude product was stirred in MeOH (2.5 mL) and CHCN (2.5 mL) for 5 minutes. The mixture was filtered, the filter cake was washed with MeOH, and dried to give compound 30 (4-amino-N'-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonylhydrazide, 20 mg, 9.30% yield) as a white solid.

[0561]

[0562] Experimental Example 1-31. Preparation of Compound 31 (4-amino-N'-(2,3-dihydro-1H-indene-2-carbonyl)benzenesulfonylhydrazide)

[0563]

[0564] Step 1) Synthesis of A59

[0565] To a mixture of A58 (2,3-dihydro-1H-indene-2-carboxylic acid, 5.0 g, 30.8 mmol, 1.0 eq) in MeOH (50 mL) was added dropwise HSO (5 mL). 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 NaSO, and concentrated to afford A59 (methyl 2,3-dihydro-1H-indene-2-carboxylate, 5.3 g, 97.5% yield) as a yellow oil.

[0566] Step 2) Synthesis of A60

[0567] To a mixture of A59 (methyl 2,3-dihydro-1H-indene-2-carboxylate, 3.0 g, 17.1 mmol, 1.0 eq) in MeOH (30 mL) was added N2H4.H2O (8.56 g, 171 mmol, 10 eq) dropwise. The mixture was then stirred at 80°C for 12 hours. The solution was concentrated to afford A60 (2,3-dihydro-1H-indene-2-carbohydrazide, 2.0 g, 87.5% yield) as a yellow solid.

[0568]

[0569] Step 3) Synthesis of A61

[0570] 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) 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 Na2SO4, and concentrated to afford A61 (N'-(2,3-dihydro-1H-indene-2-carbonyl)-4-nitrobenzenesulfonylhydrazide, 1.4 g, 34.1% yield) as a yellow solid. (TLC: DCM / MeOH = 10 / 1, Rf = 0.5)

[0571] Step 4) Synthesis of compound 31

[0572] To a mixture of A61 (N'-(2,3-dihydro-1H-indene-2-carbonyl)-4-nitrobenzenesulfonylhydrazide, 200 mg, 0.83 mmol, 1.0 eq) in MeOH (10 mL) was added Pd / C (100 mg). The mixture was then stirred under a balloon of H2 at 10°C for 12 hours. The solution was filtered, and the filtrate was purified by preparative HPLC and lyophilized to afford compound 31 (4-amino-N'-(2,3-dihydro-1H-indene-2-carbonyl)benzenesulfonylhydrazide, 80 mg, 43.6% yield) as a white solid.

[0573]

[0574] Experimental Example 1-32. Preparation of Compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonylhydrazide)

[0575]

[0576] Step 1) Synthesis of A63

[0577] To a mixture of A62 (isoindoline, 1.00 g, 8.4 mmol, 1.0 eq) in dichloromethane (10 mL) were added triethylamine (2 mL) and 4-nitrophenyl chloroformate (1.68 g, 8.4 mmol). The mixture was stirred at room temperature for 16 hours. The above solution was poured into water (30 mL) and extracted with dichloromethane (30 mL×3). The combined organic layers were dried over Na2SO4 and concentrated. The product was added to tetrahydrofuran (10 mL) and N2H4H2O (2 mL). The mixture was stirred at 60°C for 16 hours. The above solution was poured into water (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over sodium sulfate (Na2SO4) and concentrated to give A63 (isoindoline-2-carbohydrazide, 600 mg, crude) as a yellow solid. (TLC: DCM / MeOH=10 / 1, Rf=0.6)

[0578]

[0579] Step 2) Synthesis of A64

[0580] To a mixture of A63 (isoindoline-2-carbohydrazide, 600 mg, 3.39 mmol, 1.0 eq) in pyridine (10 mL) was added dropwise 4-nitrobenzene-1-sulfonyl chloride (750 mg, 3.39 mmol, 1.0 eq) in pyridine (5 mL). 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 Na2SO4, and concentrated to afford A64 (N'-(isoindoline-2-carbonyl)-4-nitrobenzenesulfonylhydrazide, 200 mg, crude) as a yellow solid. (TLC: DCM / MeOH = 20 / 1, Rf = 0.5)

[0581]

[0582] Step 3) Synthesis of Compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonylhydrazide)

[0583] 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 saturated aqueous NH4Cl (6 mL). The mixture was then stirred at 85°C for 3 hours. The solution was filtered and the filtrate was concentrated to give a crude product. The crude product was purified by preparative HPLC and lyophilized to give compound 32 (4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonylhydrazide, 20 mg, 11.0% yield) as a white solid.

[0584]

[0585] Experimental Example 1-33. Preparation of Compound 33 (N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonylhydrazide)

[0586]

[0587] Step 1) Synthesis of A66

[0588] 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, 2.5 M in hexane, 10.0 mmol, 1.1 eq) at -70 ° C. After 1 hour, SO2 (gas, 1 L) was slowly added at -70 ° C. The reaction mixture was then warmed to 10 ° C over a period of 2 hours. The 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 × 20 mL) and brine (2 × 20 mL). The organic phase 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, 34.4% yield) as a brown oil.

[0589]

[0590] Step 2) Synthesis of A67

[0591] To a stirred solution of 4-hydroxybenzoic acid hydrazide (481 mg, 3.17 mmol, 1.0 eq) in pyridine (10 mL) was added a solution of A66 (tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate, 1.0 g, 3.17 mmol, 1.0 eq) in pyridine (5 mL) 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 give A67 (tert-butyl 2-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)-1H-indole-1-carboxylate, 500 mg, 36.5% yield) as a white solid.

[0592] Step 3) Synthesis of compound 33

[0593] 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 preparative HPLC, and lyophilized to afford compound 33 (N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonylhydrazide, 50 mg, 13% yield) as an off-white solid.

[0594]

[0595] Experimental Example 1-34. Preparation of Compound 34 (4-amino-N'-(2-phenylacetyl)benzenesulfonylhydrazide)

[0596]

[0597] Step 1) Synthesis of A69

[0598] 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, 1.0 eq) in pyridine (5 mL) 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×3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated to give a crude product, which was stirred in DCM (30 mL) for 30 minutes. The mixture was filtered, and the filter cake was dried to give A69 (4-nitro-N′-(2-phenylacetyl)benzenesulfonylhydrazide, 1 g, 89.6% yield) as a yellow solid.

[0599]

[0600] Step 1) Synthesis of compound 34

[0601] 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 at 10° C. under a H2 balloon for 2 hours. The solution was filtered, and the filtrate was concentrated. The crude product was crystallized three times from MeOH (10 mL) to afford compound 34 (4-amino-N′-(2-phenylacetyl)benzenesulfonylhydrazide, 20 mg, 10.9% yield) as a gray solid. (TLC: DCM / MeOH=10 / 1, Rf=0.3)

[0602]

[0603] Experimental Example 1-35. Preparation of Compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonylhydrazide)

[0604]

[0605] Step 1) Synthesis of A70

[0606] To a stirred solution of 1H-indazole-3-amine (1.00 g, 7.5 mmol, 1.0 eq) in acetic acid (16 mL), concentrated hydrochloric acid (1.6 mL) and formic acid (1.6 mL) was added NaNO2 (0.62 g, 9.0 mmol, 1.2 eq) at 0°C. The mixture was stirred for 1 hour. SO2 (gas, 1 L) and CuCl2 (0.38 g, 2.3 mmol, 0.3 eq) were slowly added 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, 30.8% yield) as a brown solid.

[0607]

[0608] Step 2) Synthesis of Compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonylhydrazide)

[0609] To a stirred solution of 4-hydroxybenzoylhydrazide (225 mg, 1.48 mmol, 0.8 eq) in pyridine (5 mL) was added a solution of A70 (1H-indazole-3-sulfonyl chloride, 0.40 g, 1.85 mmol, 1.0 eq) in pyridine (5 mL) at 0°C. The mixture was stirred at room temperature for 5 hours. The mixture was concentrated. The residue was purified by preparative HPLC and lyophilized to give compound 35 (N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonylhydrazide, 60 mg, 9.77% yield) as a white solid.

[0610]

[0611] Experimental Example 1-36. Preparation of Compound 36 (4-amino-N'-(indoline-6-carbonyl)benzenesulfonylhydrazide)

[0612]

[0613] Step 1) Synthesis of A72

[0614] A mixture of hydrazine monohydrate (10 mL) and A71 (1H-indole-6-carboxylic acid methyl ester, 500 mg, 2.86 mmol, 1.0 eq) was stirred at 100° C. for 3 hours. The mixture was then cooled to 0° C. and filtered. The filter cake was washed with ice water and dried in vacuo to afford A72 (1H-indole-6-carbohydrazide, 300 mg, 60.0% yield) as a white solid.

[0615]

[0616] Step 2) Synthesis of A73

[0617] To a mixture of A72 (300 mg, 1.71 mmol, 1.0 eq) in pyridine (5 mL) at 0°C was added 4-nitrobenzenesulfonyl chloride (380 mg, 1.71 mmol, 1.0 eq). 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 Na2SO4, and concentrated to give the crude product, which was purified by flash column chromatography (DCM / MeOH = 50 / 1 to 30 / 1) to give A73 (N'-(1H-indole-6-carbonyl)-4-nitrobenzenesulfonylhydrazide, 500 mg, 81.0% yield) as a yellow solid.

[0618]

[0619] Step 3) Synthesis of A74

[0620] To a mixture of A73 (400 mg, 1.11 mmol, 1.0 eq) in DCM (15 mL) and TFA (5 mL) was added NaBH3CN (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 the pH was adjusted to 7-8 with a saturated aqueous solution of NaHCO3. The solution was filtered, and the filter cake was washed with PE and dried to afford A74 (N'-(indoline-6-carbonyl)-4-nitrobenzenesulfonylhydrazide, 200 mg crude) as a yellow solid.

[0621]

[0622] Step 3) Synthesis of Compound 36

[0623] A mixture of A74 (200 mg, 0.55 mmol, 1.0 eq), NH4Cl (146 mg, 2.75 mmol, 5.0 eq), and Fe (154 mg, 2.75 mmol, 5.0 eq) in H2O (10 mL) and EtOH (20 mL) was stirred at 85°C for 2 hours. 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 Na2SO4, and concentrated. The crude product was recrystallized from methanol, and the solid was lyophilized to give compound 36 (4-amino-N'-(indoline-6-carbonyl)benzenesulfonylhydrazide, 60 mg, 32.7%) as a white solid.

[0624]

[0625] Experimental Example 1-37. Preparation of Compound 37 (4-amino-N'-(indoline-3-carbonyl)benzenesulfonylhydrazide)

[0626]

[0627] Step 1) Synthesis of A76

[0628] 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) was added dropwise BocO (1.37 g, 6.28 mmol, 1.1 eq) 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 NaSO, and concentrated to afford A76 (1-(tert-butyl) 3-methyl 1H-indole-1,3-dicarboxylate, 1.20 g, crude) as a yellow solid.

[0629] Step 2) Synthesis of A77

[0630] 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 under H2 (50 psi) for 12 hours. 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 to 15:1) to give A77 (1-(tert-butyl) 3-methylindoline-1,3-dicarboxylate, 0.80 g, 66.2% yield) as a white solid.

[0631] Step 3) Synthesis of A78

[0632] To a mixture of A77 (800 mg, 2.89 mmol, 1.0 eq) in MeOH (20 mL) was added N H .H O (1.6 mL). The mixture was then stirred at 80° C. for 4 hours. The reaction mixture was concentrated to afford A78 (tert-butyl 3-(hydrazinecarbonyl)indoline-1-carboxylate 700 mg, 87.5% yield) as a white solid.

[0633] Step 3) Synthesis of A79

[0634] 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) 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×3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated to afford A79 (tert-butyl 3-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)indoline-1-carboxylate, 200 mg, 39.7% yield) as a yellow solid.

[0635] Step 4) Synthesis of A80

[0636] In the mixture of A79 (200 mg, 0.43 mmol, 1.0 eq) in EA (10 mL), Pd / C (50 mg) was added. The mixture was then stirred at 10° C. for 12 hours under a H balloon. The above solution was filtered, and the filtrate was concentrated to give A80 (tert-butyl 3-(2-((4-aminophenyl) sulfonyl) hydrazine-1-carbonyl) indoline-1-carboxylate, 200 mg, yield: 100%) as a yellow solid.

[0637] Step 5) Synthesis of Compound 37 (4-amino-N'-(indoline-3-carbonyl)benzenesulfonylhydrazide)

[0638] 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 above solution was concentrated, H O (5 mL) was added to the residue, and the pH was adjusted to 9-10 with K CO aqueous solution. The solution was extracted with EA (20 mL×3). The combined organic layers were washed with brine, dried over Na SO and concentrated to give a crude product. The crude product was purified by preparative HPLC and lyophilized to give compound 37 (4-amino-N′-(indoline-3-carbonyl)benzenesulfonylhydrazide, 30.0 mg, 19.6% yield) as a pale solid.

[0639]

[0640] Experimental Example 1-38. Preparation of Compound 38 (N'-(4-hydroxybenzoyl)piperidine-4-sulfonylhydrazide)

[0641]

[0642] Step 1) Synthesis of A82

[0643] To a mixture of 4-hydroxybenzoylhydrazide (A2, 268 mg, 1.77 mmol, 1.0 eq) in pyridine (2 mL) was added dropwise a solution of A81 (tert-butyl 4-(chlorosulfonyl)piperidine-1-carboxylate, 500 mg, 1.77 mmol, 1.0 eq) in pyridine (1 mL) at 0 ° C. The mixture was stirred at room temperature for 5 hours. The above solution was filtered. The filtrate was concentrated and purified by column chromatography (DCM / MeOH=100: 1-10: 1) to give A82 (tert-butyl 4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)piperidine-1-carboxylate, 300 mg, 42% yield) as a yellow solid.

[0644]

[0645] Step 2) Synthesis of Compound 38 (N'-(4-hydroxybenzoyl)piperidine-4-sulfonylhydrazide)

[0646] 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 and purified by preparative HPLC and lyophilized to afford compound 38 (N′-(4-hydroxybenzoyl)piperidine-4-sulfonylhydrazide, 50 mg, 22% yield) as a yellow solid.

[0647]

[0648] Experimental Example 1-39. Preparation of Compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonylhydrazide)

[0649]

[0650] Step 1) Synthesis of A83

[0651] To a mixture of A72 (1H-indole-6-carbohydrazide, 500 mg, 2.86 mmol, 1.0 eq) in pyridine (5 mL) was added dropwise 3-fluoro-4-nitrobenzene-1-sulfonyl chloride (686 mg, 2.86 mmol, 1.0 eq) in pyridine (2 mL) 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-nitrobenzenesulfonylhydrazide, 700 mg, yield: 64.8%) as a yellow solid.

[0652]

[0653] Step 2) Synthesis of A84

[0654] To a mixture of A83 (700 mg, 1.85 mmol, 1.0 eq) and KCO (640 mg, 4.64 mmol, 2.5 eq) in DMF (7 mL) was added morpholine (193 mg, 2.22 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 NaSO, and concentrated to afford A84 (N'-(1H-indole-6-carbonyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 600 mg, 72.8% yield) as a yellow solid.

[0655]

[0656] Step 3) Synthesis of A85

[0657] To a mixture of A84 (600 mg, 1.35 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1 mL) and NaBH CN (251 mg, 4.04 mmol, 3.0 eq) at 10° C. The mixture was then stirred at 25° C. for 4 hours. The above solution was poured into water (30 mL) and extracted with EA (30 mL×3). The combined organic layers were washed with brine, dried over Na SO and concentrated to give A85 (N′-(indoline-6-carbonyl)-3-morpholino-4-nitrobenzenesulfonylhydrazide, 450 mg, crude product) as a yellow solid.

[0658]

[0659] Step 4) Synthesis of Compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonylhydrazide)

[0660] To the mixture of A85 (450mg, 1.01mmol, 1.0eq) in EtOH (5mL), add Fe (282mg, 5.05mmol, 5.0eq) and NH4Cl saturated aqueous solution (1mL).Then this mixture was stirred 3 hours at 85 ℃.Then the above solution was concentrated, DMSO (5mL) was added, and filtered.The filtrate was purified by preparative HPLC, and lyophilized to obtain compound 39 (4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonylhydrazide, 30mg, 7.0% productive rate), which was a pale solid.

[0661]

[0662] Experimental Example 1-40. Preparation of Compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide)

[0663]

[0664] Step 1) Synthesis of A87

[0665] 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) was added dropwise 4-nitrobenzene-1-sulfonyl chloride (906 mg, 4.09 mmol, 1.0 eq) in pyridine (5 mL) 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 Na2SO4, and concentrated to give the crude product. The crude product was washed with EA (5 mL), filtered, and the solid dried in vacuo to give A87 (tert-butyl 4-(2-((4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 800 mg, 45.5% yield) as a yellow solid.

[0666]

[0667] Step 2) Synthesis of A88

[0668] 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 at 10° C. for 16 hours under a H balloon. The above solution was filtered, and the filtrate was concentrated to afford A88 (tert-butyl 4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 350 mg, 94% yield) as a yellow solid.

[0669] Step 3) Synthesis of Compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide)

[0670] 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 above solution was concentrated to obtain a crude product. MeOH (5 mL) and K CO (62 mg, 0.45 mmol) were added to the crude product and stirred at room temperature for 1 hour. The solution was filtered and purified by preparative HPLC. Compound 40 (4-amino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide, 50 mg, 38.7% yield) was obtained by lyophilization as a white solid.

[0671]

[0672] Experimental Example 1-41. Preparation of Compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide)

[0673]

[0674] Step 1) Synthesis of A89

[0675] 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) 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×3). The combined organic layers were washed with 1N HCl (30 mL×2) and brine, dried over Na₂SO₄, and concentrated to give A89 (tert-butyl 4-(2-((3-fluoro-4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 1.5 g, crude) as a yellow solid.

[0676] Step 2) Synthesis of A90

[0677] To a mixture of A89 (1.50 g, 3.35 mmol, 1.0 eq) and KCO (1.16 g, 8.37 mmol, 2.5 eq) in DMF (15 mL) was added morpholine (350 mg, 4.02 mmol, 1.2 eq) at 10°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 NaSO, and then concentrated to give a crude product, which was purified by column chromatography (DCM / MeOH = 50 / 1-10 / 1) to give A90 (tert-butyl 4-(2-((3-morpholino-4-nitrophenyl)sulfonyl)hydrazine-1-carbonyl)piperazine-1-carboxylate, 700 mg, 40.6% yield) as a yellow solid.

[0678]

[0679] Step 3) Synthesis of A91

[0680] To be in the mixture of A90 (700mg, 1.36mmol, 1.0eq) in THF (10mL), add Pd / C (200mg).Then with this mixture at H 2 (50psi) lower, at room temperature stirred 15 minutes.Above-mentioned solution is filtered, and filtrate is concentrated, obtain A91 (4-(2-((4-amino-3-morpholinophenyl) sulfonyl) hydrazine-1-carbonyl) piperazine-1-carboxylic acid tert-butyl ester, 300mg, crude product), be yellow solid.

[0681] Step 4) Synthesis of Compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide)

[0682] To the 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 and purified by preparative HPLC, and lyophilized to give compound 41 (4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide, 20.0 mg, 12.6% yield) as a pink solid.

[0683]

[0684] Experimental Example 1-42. Preparation of Compound 42 (N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonylhydrazide)

[0685]

[0686] Step 1) Synthesis of A92

[0687] 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 crystallized upon cooling. The mixture was triturated with deionized water, filtered, washed with copious amounts of water, and recrystallized from ethanol to afford A92 (N-(2,2,2-trichloro-1-hydroxyethyl)acetamide, 15 g) as a white solid.

[0688]

[0689] Step 2) Synthesis of A93

[0690] Zinc powder (5 g, 78 mmol) was gradually added to a stirred suspension of A92 (8 g, 39 mmol) in glacial acetic acid (50 mL) over a 3-hour period. During the addition of zinc, the temperature of the reaction mixture was maintained below 40°C. The reaction mixture was then stirred at room temperature for 24 hours. The precipitated zinc salt was then filtered off and washed with glacial acetic acid. The 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.

[0691]

[0692] Step 3) Synthesis of A94

[0693] Benzyl mercaptan (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-propyl alcohol (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-propyl alcohol or ethanol to give A94 (N-(1-(benzylthio)-2,2-dichloroethyl) acetamide, 2.5 g) as a white solid.

[0694]

[0695] Step 4) Synthesis of A95

[0696] Lawesson's reagent (7.6g, 18.8mmol) is added into the stirred solution of A94 (5mmol) in toluene (30mL). The reaction mixture was refluxed for 8 hours, then desolvated under reduced pressure. Resistates was triturated with a 10% NaOH aqueous solution and adjusted to pH 9. The crude product was filtered, dried, and recrystallized from 2-propyl alcohol. The liquid product was extracted with dichloromethane to obtain A95 (4-(benzylthio)-2-methylthiazole, crude product, 2.5g), which was a yellow oil.

[0697]

[0698] Step 5) Synthesis of A96

[0699] 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 at room temperature overnight. The reaction mixture was triturated with a 10% NaHCO3 aqueous solution, adjusted to pH = 8, and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give a crude product. The residue was purified on silica gel to give A96 (2-methylthiazole-4-sulfonyl chloride, 100 mg) as a yellow oil.

[0700]

[0701] Step 6) Synthesis of Compound 42 (N'-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonylhydrazide)

[0702] A mixture of A96 (100 mg, 0.5 mmol) and A2 (4-hydroxybenzoyl hydrazide, 80 mg, 0.5 mmol) in pyridine (20 mL) was stirred at 80° C. overnight. The above solution was poured into water (30 mL) and extracted with EA (30 mL×3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated to give a crude product. The residue was purified by preparative HPLC to give compound 42 (N '-(4-hydroxybenzoyl)-2-methylthiazole-4-sulfonylhydrazide, 30 mg) as an off-white solid.

[0703]

[0704] Experimental Example 1-43. Preparation of Compound 43 ((1S,4S)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide)

[0705]

[0706] Step 1) Synthesis of A98

[0707] To a mixture of A97 (tert-butyl((1r,4r)-4-hydroxycyclohexyl)carbamate, 27 g, 126 mmol, 1.0 eq) in pyridine (100 mL) was added 4-methylbenzenesulfonyl chloride (28.6 g, 151 mmol, 1.2 eq) in portions, and the mixture was stirred at room temperature overnight. The pyridine was removed in vacuo, and the residue was purified by silica gel column chromatography to afford A98 ((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl 4-methylbenzenesulfonate, 40 g, 86.4%) as a white solid.

[0708] Step 2) Synthesis of A99

[0709] 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 hours. The reaction mixture was quenched with brine (200 mL) and extracted with EtOAc (100 mL×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-butyloxycarbonyl)amino)cyclohexyl)thioacetate, 3.0 g, 40.5%) as a white solid.

[0710] Step 3) Synthesis of A100

[0711] To 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 for 30 minutes at 0° C. The two layers were separated, and the DCM layer was washed with aqueous sodium thiosulfate and brine, dried over sodium sulfate, filtered, and concentrated to afford crude A100 (tert-butyl ((1s,4s)-4-(chlorosulfonyl)cyclohexyl)carbamate) as a brown solid.

[0712] Step 4) Synthesis of A101

[0713] To a solution of 4-hydroxybenzoylhydrazide (A2, 2.76 g, 18.2 mmol, 2.0 eq) in pyridine (10 mL) was added a solution of A100 (crude) in DCM (5 mL) dropwise, and the mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the crude product was purified by silica gel column chromatography to afford A101 (tert-butyl ((1s, 4s)-4-((2-(4-hydroxybenzoyl)hydrazinyl)sulfonyl)cyclohexyl)carbamate, 0.2 g, 5.3% over 2 steps) as a white solid.

[0714] Step 5) Synthesis of Compound 43 ((1s,4s)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide)

[0715] 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 hours. The mixture was concentrated in vacuo and dissolved in MeOH. NH3 / MeOH was added to pH = 9. The solvent was concentrated in vacuo and the residue was dissolved in MeOH. The product was purified by preparative HPLC and freeze-dried to give compound 43 ((1s, 4s)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide, 20 mg, 13.2%) as a yellow solid.

[0716]

[0717] Experimental Example 1-44. Preparation of Compound 44 ((1R,4R)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide)

[0718] Compound 44 was synthesized as a white solid (26.7% yield) in the same manner as Experimental Example 1-45 by using tert-butyl ((1s,4s)-4-hydroxycyclohexyl)carbamate as a starting material instead of A97.

[0719]

[0720] Experimental Example 1-45. Preparation of Compound 45 (4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)-5-methylfuran-2-carboxylic acid)

[0721]

[0722] Step 1) Synthesis of A102

[0723] A stirred mixture of 5-methylfuran-2-carboxylic acid (10 g, 80 mmol), chlorosulfonic acid (30 mL) was stirred at 50° C. for 3 hours and then quenched 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 afford compound A102 (14 g) as a yellow solid.

[0724]

[0725] Step 2) Synthesis of Compound 45 (4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)-5-methylfuran-2-carboxylic acid)

[0726] 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 above solution was poured into water (30 mL) and extracted with EA (30 mL × 3). The combined organic layer was washed with salt water, dried over Na SO and concentrated to give a crude product. Residue was purified by preparative HPLC to give compound 45 (4-((2-(4-hydroxybenzoyl)hydrazine)sulfonyl)-5-methylfuran-2-carboxylic acid, 1.3 g) as a yellow solid.

[0727]

[0728] Experimental Example 1-46. Preparation of Compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonylhydrazide)

[0729]

[0730] Step 1) Synthesis of A104

[0731] 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) at 0 ° C. The mixture was stirred at room temperature for 2 hours. The mixture was quenched with H2O (100 mL) and extracted with DCM (100 mL × 2). 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, 70.6% yield) as a yellow oil. (PE / EA=10:1, Rf=0.6)

[0732] Step 2) Synthesis of A105

[0733] 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 hours. The mixture was treated with H O (200 mL) and extracted with EA (200 mL×2). The combined organic layers were washed with H O (100 mL×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, 43.4% yield) as a brown solid.

[0734] Step 3) Synthesis of A106

[0735] To a stirred solution of A105 (2.00 g, 8.16 mmol, 1.0 eq) in acetic acid (AcOH 30 mL) and H2O (30 mL) was added N-chlorosuccinimide (5.45 g, 40.8 mmol, 5.0 eq). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated and purified by column chromatography (PE / EA = 50:1 to 1:1) to give A106 (1.0 g, 45.6% yield) as a yellow oil. (PE / EA = 3:1, Rf = 0.5)

[0736]

[0737] Step 4) Synthesis of A107

[0738] To a stirred solution of 4-hydroxybenzoic acid hydrazide (0.56 g, 3.71 mmol, 1.0 eq) in pyridine (30 mL) was added a solution of A106 (1.00 g, 3.71 mmol, 1.0 eq) in pyridine (10 mL) 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, 34.9% yield) as a yellow oil. (DCM / MeOH = 10: 1, Rf = 0.4)

[0739] Step 5) Synthesis of Compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonylhydrazide)

[0740] 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 and purified by preparative HPLC and lyophilized to afford compound 46 (N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonylhydrazide, 30 mg, 7.0% yield) as a light yellow solid. (TLC: N / A)

[0741]

[0742] Experimental Example 1-47. Preparation of Compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonylhydrazide)

[0743]

[0744] Step 1) Synthesis of A109

[0745] To a mixture of 1H-pyrrolo[2,3-b]pyridine (A108, 6.00 g, 50.8 mmol, 1.0 eq) in THF (60 mL) was added NaH (2.44 g (60% w / w), 60.9 mmol, 1.2 eq) at 0°C and stirred at 0°C for 1 hour. TsCl (9.65 g, 50.8 mmol, 1.0 eq) in THF (20 mL) was then added dropwise. The solution was stirred for 16 hours. The above solution was poured into water (200 mL) and extracted with EA (100 mL×3). The combined organic layers were dried over Na2SO4 and concentrated to give the crude product, which was washed with PE (30 mL) for 1 hour, filtered, and the solid was collected. The solid was dried in vacuo to give A109 (11.0 g, 79.5% yield) as a white solid. (TLC: N / A)

[0746]

[0747] Step 2) Synthesis of A110

[0748] 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) at -76 ° C. The mixture was stirred at -76 ° C for 1 hour. The mixture was then stirred at -76 ° C to 10 ° C under a SO2 balloon for 1 hour, and the solution was concentrated. At 20 ° C, NCS (1.58 g, 11.7 mmol, 1.6 eq) was added to the residue in DCM (30 mL), and the mixture was stirred at 20 ° C for 1 hour. The above solution was poured into water (50 mL) and extracted with DCM (50 mL × 3). The combined organic layers were dried over Na2SO4 and concentrated to give A110 (1.30 g, 47.8% yield) as a yellow solid.

[0749]

[0750] Step 3) Synthesis of A111

[0751] To a mixture of A110 (1.30 g, 3.51 mmol, 1.0 eq) in pyridine (10 mL) was added dropwise 4-hydroxybenzoic acid hydrazide (A2, 587 mg, 3.86 mmol, 1.1 eq) in pyridine (5 mL) at 10 ° C. The mixture was then stirred at 10 ° C for 3 hours. The above solution was poured into water (50 mL) and extracted with EA (50 mL×3). The combined organic layers were washed with 1NHCl (50 mL×2) and brine (50 mL), dried over Na2SO4, and concentrated to give a crude product. The crude product was washed with EA (5 mL), filtered, and the solid was dried in vacuo to give A111 (600 mg, 35.3% yield) as a yellow solid. (TLC: N / A)

[0752]

[0753] Step 4) Synthesis of Compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonylhydrazide)

[0754] To a mixture of A111 (350 mg, 0.71 mmol, 1.0 eq) in MeOH (6 mL) was added concentrated hydrochloric acid (2 mL). The mixture was then stirred at 60°C for 3 hours. The solution was concentrated, and the crude product was purified by preparative HPLC and lyophilized to afford compound 47 (N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonylhydrazine, 20 mg, 8.36% yield) as a white solid. (TLC: N / A)

[0755]

[0756] Experimental Example 1-48. Preparation of Compound 48 (4-hydroxy-N'-(4-methoxybenzyl)benzoylhydrazide)

[0757]

[0758] Using 1-(bromomethyl)-4-methoxybenzene instead of 1-(bromomethyl)-4-nitrobenzene, Compound 48 was obtained by the same preparation method as in Experimental Example 1-19.

[0759] Experimental Example 1-49. Preparation of Compound 49 (N'-(4-aminobenzyl)-2,3-dihydro-1H-indene-2-carbohydrazide)

[0760]

[0761] 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.

[0762] Experimental Example 1-50. Preparation of Compound 50 (4-amino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)-3-morpholinobenzenesulfonamide)

[0763]

[0764] Compound 50 was obtained by a preparation method similar to that of Experimental Example 1-12, using 3-fluoro-4-nitrobenzenesulfonyl chloride instead of 4-nitrobenzene-1-sulfonyl chloride in Step 3 of Experimental Example 1-12.

[0765] Experimental Example 1-51. Preparation of Compound 51 (3,5-diamino-N-(2-(4-hydroxyphenyl)-2-oxoethyl)benzenesulfonamide)

[0766]

[0767] Compound 51 was obtained by a preparation method similar to that of Experimental Example 1-12, using 3,5-dinitrobenzenesulfonyl chloride (A20) instead of 4-nitrobenzene-1-sulfonyl chloride in Step 3 of Experimental Example 1-12.

[0768] Experimental Example 1-52. Preparation of Compound 52 (2-((4-aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide)

[0769]

[0770] 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.

[0771] Experimental Example 1-53. Preparation of Compound 53 (2-((4-amino-3-morpholinophenyl)sulfonyl)-N-phenylhydrazine-1-carboxamide)

[0772]

[0773] Compound 53 was obtained by a preparation method similar to that of Experimental Example 1-27 using 3-fluoro-4-nitrobenzenesulfonyl chloride as a starting material instead of 4-nitrobenzenesulfonyl chloride.

[0774] Experimental Example 1-54. Preparation of Compound 54 (4-hydroxy-N-(((4-methoxyphenyl)sulfonyl)methyl)benzamide)

[0775]

[0776] Using 4-methoxybenzenethiol instead of 4-nitrobenzenethiol, compound 54 was obtained by the same preparation method as in Experimental Example 1-16.

[0777] Experimental Example 1-55. Preparation of Compound 55 (N-(((4-aminophenyl)sulfonyl)methyl)-[1,1'-biphenyl]-4-carboxamide)

[0778]

[0779] Compound 55 was obtained by a preparation method similar to that of Experimental Example 1-16, using [1,1′-biphenyl]-4-carboxamide as a starting material instead of Intermediate A28 of Experimental Example 1-16.

[0780] Example 2. Binding assay experiment

[0781] Example 2-1 confirmed whether actin is a substrate of the Arg / N-degron pathway.

[0782] L6 cell lines were cultured in a 5% CO2 incubator using DMEM medium containing 10% FBS and 1% streptomycin / penicillin. After culture, the cells were aliquoted into 12-well plates. L6 cell lines are rat myogenic cells. The cells were cultured for an additional 24 hours to allow them to fully adhere to the surface of the plate. To confirm whether MG132 increased UBR1 binding, cells were collected after 24 hours of treatment with MG132 (10 μM). To extract protein from the collected 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, aprotinin) was injected into each sample to lyse the cells. According to the measured total protein concentration, sample buffer was added to each sample, and the mixture was reacted at 100°C for 5 minutes. After taking 5 μL of the completely reacted sample and aliquoting it into each well of the acrylamide gel, immunoblotting was performed. Figure 1 ] The experimental results are described in .

[0783] For immunoblotting, a representative experiment was summarized from more than three independent experiments.

[0784] Reference Figure 1MG132 was used to confirm that the levels of ACTA1, ACTC1, and ACTG2 were increased compared to the control. Furthermore, it was confirmed that the levels of ACTA1 and ACTG2 increased when the UBR protein was knocked down. This confirms that actin is a substrate of the Arg / N-degron pathway.

[0785] Example 2-2 Confirmation of Inhibition of R-nsP4 Degradation by In Vitro Transcription / Translation Method

[0786] use A quick coupled transcription / translation system kit was used to confirm the R-nsP4 expression of the compound. After preparing a premix using Transcend biotin-lysyl-tRNA, methionine, bestatin, TnT quick master mix, and DHFR-Ub-R-nsP4 plasmid, the premix was mixed with the compound (1 μM). After each sample was reacted at 30°C for 40 minutes, 5X SDS loading dye was added. After the resulting mixture was reacted at 95°C for 2 minutes, 5 μL aliquots were removed and placed into each well of an acrylamide gel, followed by immunoblotting. Figure 2 For in vitro transcription / translation methods, representative experiments were summarized from three or more independent experiments.

[0787] Reference Figure 2 It was confirmed that the level of R-nsP4 was increased compared to the control by Compound 2, Compound 3, Compound 7, Compound 12, Compound 14, and Compound 16. In other words, it was confirmed that when treated with the compounds of the present invention, the level of R-nsP4 was increased by binding to UBR1.

[0788] Example 2-3 Evaluation of Inhibition of Intracellular RGS4 Degradation by Transfection

[0789] L6 cell lines, which are rat myogenic cells, were cultured in DMEM medium containing 10% FBS and 1% streptomycin / penicillin in an incubator maintained at 5% carbon dioxide. To measure the UBR1 binding strength of a representative compound selected from the compounds of the present invention, cells were aliquoted into 6-well plates. The cells were cultured for an additional 24 hours to allow them to fully adhere to the plate surface. Opti-MEM, Lipofectamine, and RGS4 plasmids were reacted for transfection. After the reaction was complete, the cells were treated to allow the DNA to be expressed intracellularly. Cells were collected after 24 hours of treatment with the compound (5 μM) alone to confirm whether the compound increased UBR1 binding after 24 hours. To extract protein from the collected 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, aprotinin) was injected into each sample to lyse the cells. Sample buffer was added to each sample based on the measured total protein concentration, and the mixture was reacted at 100°C for 5 minutes. 5 μL of the fully reacted sample was removed and aliquoted into each well of an acrylamide gel, followed by immunoblotting. Figure 3 For immunoblotting, the representative experiment was summarized from three or more independent experiments.

[0790] Reference Figure 3 It was confirmed that the level of RGS4 was increased compared to the control by Compound 2 and Compound 3. That is, it was confirmed that when treated with the compounds of the present invention, the level of RGS4 was further increased by binding to UBR1.

[0791] Example 2-4 Evaluation of Inhibition of Myocyte Actin Degradation by Immunoblotting

[0792] To evaluate the actin degradation of compounds in myocytes, L6 cell lines, which are rat myogenic cells, were cultured in a DMEM medium containing 10% FBS and 1% streptomycin / penicillin in an incubator maintained at 5% carbon dioxide. To measure UBR1 binding strength based on the treatment of representative compounds selected from the compounds of the present invention, cells were aliquoted into 12-well plates. The cells were cultured for an additional 24 hours to allow them to fully adhere to the surface of the plate. Cells were collected after 24 hours of treatment with the compound (5 μM) alone to confirm whether the compound increased UBR1 binding. To extract protein from the collected 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, aprotinin) was injected into each sample to lyse the cells. According to the measured total protein concentration, sample buffer was added to each sample, and the mixture was reacted at 100°C for 5 minutes. After taking 5 μL of the completely reacted sample and aliquoting it into each well of the acrylamide gel, immunoblotting was performed. Figure 4 ]、[ Figure 5 ]、[ Figure 6 ]、[ Figure 7 ]、[ Figure 8 ]and[ Figure 9 For immunoblotting, the representative experiment was summarized from three or more independent experiments.

[0793] Reference Figure 4 and Figure 5 It was confirmed that the level of ACTA1 was further increased by 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 compared to the control. In other words, it was confirmed that the degradation of the intramuscular protein ACTA1 was suppressed by binding to UBR1 when treated with the compounds of the present invention.

[0794] Reference Figure 6 and Figure 7It was confirmed that the level of ACTA2 was further increased by Compound 8, Compound 9, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 32, Compound 33, and Compound 34 compared to the control. In other words, it was confirmed that the degradation of the intramuscular protein ACTA2 was suppressed by binding to UBR1 when treated with the compounds of the present invention.

[0795] Reference Figure 8 It was confirmed that the level of ACTC1 was further increased by Compound 41 and Compound 42 compared to the control. That is, it was confirmed that the degradation of the intramuscular protein ACTC1 was inhibited by binding to UBR1 when treated with the compounds of the present invention.

[0796] Reference Figure 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. In other words, it was confirmed that the degradation of the intramuscular protein ACTG2 was inhibited by binding to UBR1 when treated with the compounds of the present invention.

[0797] Example 2-5 Evaluation of UBR Box Domain Binding Strength by Immunoprecipitation Analysis

[0798] To assess the binding strength of compounds to UBR1, UBR2, UBR3, and UBR5 via the UBR box domain, L6 cell lines, a rat myogenic cell line, were cultured in DMEM medium containing 10% FBS and 1% streptomycin / penicillin in an incubator maintained at 5% carbon dioxide. To measure the UBR1 binding strength of representative compounds selected from the compounds of the present invention, cells were aliquoted into 100 μl plates. The cells were cultured for an additional 24 hours to allow complete attachment to the plate surface. To confirm whether the compounds increased UBR1 binding, cells were treated with the compound (5 μM), the proteasome inhibitor MG132 (10 μM), or a positive control (5 μM) alone for 24 hours, and then the cells were harvested. To extract protein from the collected 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, aprotinin) was injected into each sample to lyse the cells. Based on the measured total protein concentration, each sample was reacted with the UBR1 antibody for 16 hours and then with protein A / G beads for 3 hours. Sample buffer was added to the fully reacted sample, and the resulting mixture was reacted at 100°C for 5 minutes. 20 μL of the fully reacted sample was removed and aliquoted into each well of an acrylamide gel, and then immunoblotting was performed. Figure 10 ]and[ Figure 11 For immunoblotting, the representative experiment was summarized from three or more independent experiments.

[0799] Reference Figure 10 and Figure 11 The results showed that compound 2, a compound of the present invention, actually binds to the UBR box domain of the UBR protein, as shown by the decrease in the binding strength of ACTA1, UBR2 and ACTA1, UBR3 and ACTA1, and UBR5 and ACTA1, similar to the positive control, when cells were treated with the compound. However, when cells were treated with a DMSO control group and the proteasome inhibitor MG132 used as a negative control, the binding strength between UBR1 and the substrate ACTA1 was maintained.

[0800] That is, it was confirmed that when treated with the compound of the present invention, the degradation of the intramuscular protein ACTG2 is suppressed by binding to UBR1, UBR2, UBR3, or UBR5.

[0801] Example 2-6 Evaluation of UBR Box Domain Binding Strength by MST

[0802] 1) Preparation of UBR1 protein

[0803] The Gln97-Pro168 portion of the UBR cassette corresponding to human UBR1 (UniProt ID: Q8IWV7) was cloned into a modified expression vector and expressed in E. coli. Following affinity chromatography, the tag was removed by protease, and Gly-His-Met was added to the N-terminus. Following ion chromatography, the final UBR cassette protein of UBR1 was purified using gel filtration chromatography in a buffer composition of 10 mM NaCl, 20 mM Tris-HCl, and 2 mM beta-mercaptoethanol (pH 7.5).

[0804] 2) UBR1 UBR box protein labeling

[0805] The Monolith Protein Labeling Kit RED-NHS Second Generation (Cat#MO-L011) contains a dye with an NHS-ester group that forms a covalent bond with primary amines (lysine residues). This dye is optimized for use with Monolith instruments equipped with RED detectors. This kit was used to label purified UBR1 UBR box protein according to the protocol described.

[0806] 3) Use MST to measure the binding between UBR1 and ligand

[0807] Thermophoresis is a phenomenon in which particles migrate due to a temperature gradient. Particles in high-temperature regions have greater kinetic energy than those in low-temperature regions and collide more frequently with surrounding particles with greater energy. As a result, particles migrate from high-temperature regions to low-temperature regions.

[0808] The thermophoresis of proteins is generally different from that of protein-ligand complexes. This is because the binding of the ligand changes its size, charge, and solvation energy. Furthermore, even if the ligand binding does not significantly change the size and charge of the protein, MST can detect the change in the solvent entropy of the protein molecule caused by ligand binding. Therefore, the binding of the UBR1 UBR box protein and the ligand compound was measured using MST, confirming that the proposed ligand binds to the UBR1 UBR box (refer to Figures 12 to 19 ).

Claims

1. Use of a compound or a salt thereof in the preparation of a composition for binding to a UBR box domain, in, The compound or a salt thereof acts as a ligand that binds to the UBR box domain, Wherein, the compound is selected from: 4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide; 4-amino-N'-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-2-oxoindoline-5-sulfonylhydrazide; N'-(4-hydroxybenzoyl)indoline-5-sulfonylhydrazide; N'-([1,1'-biphenyl]-4-carbonyl)-4-aminobenzenesulfonylhydrazide; N'-([1,1'-biphenyl]-3-carbonyl)-4-aminobenzenesulfonylhydrazide; 3-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide; 4-(1-aminoethyl)-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide; 3,5-Diamino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-4-((2-hydroxyethyl)amino)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-4-methoxybenzenesulfonylhydrazide; 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)benzamidine; 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)benzamide; 6-amino-N'-(4-hydroxybenzoyl)-[1,1'-biphenyl]-3-sulfonylhydrazide; 4-(2-((4-aminophenyl)sulfonyl)hydrazine-1-carbonyl)benzamide; 4-amino-N'-(1H-indole-3-carbonyl)benzenesulfonylhydrazide; 4-amino-N'-(4-hydroxybenzoyl)-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-4-nitro-3-(pyrrolidin-1-yl)benzenesulfonylhydrazide; 4-amino-N'-(4-hydroxybenzoyl)-3-(piperidin-1-yl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-1H-pyrazole-4-sulfonylhydrazide; N'-(4-hydroxybenzoyl)indoline-4-sulfonylhydrazide; N'-(4-hydroxybenzoyl)-1H-indole-4-sulfonylhydrazide; 4-amino-N'-(1H-indole-4-carbonyl)-3-morpholinobenzenesulfonylhydrazide; 4-amino-N'-(indoline-4-carbonyl)benzenesulfonylhydrazide; 4-amino-N'-(4-hydroxybenzoyl)-3-(piperazin-1-yl)benzenesulfonylhydrazide; 4-amino-N'-(2,3-dihydro-1H-indene-2-carbonyl)benzenesulfonylhydrazide; 4-amino-N'-(isoindoline-2-carbonyl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-1H-indole-2-sulfonylhydrazide; 4-amino-N'-(2-phenylacetyl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)-1H-indazole-3-sulfonylhydrazide; 4-amino-N'-(indoline-6-carbonyl)benzenesulfonylhydrazide; 4-amino-N'-(indoline-3-carbonyl)benzenesulfonylhydrazide; N'-(4-hydroxybenzoyl)piperidine-4-sulfonylhydrazide; 4-amino-N'-(indoline-6-carbonyl)-3-morpholinobenzenesulfonylhydrazide; 4-amino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide; 4-amino-3-morpholino-N'-(piperazine-1-carbonyl)benzenesulfonylhydrazide; (1S,4S)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide; (1R,4R)-4-amino-N'-(4-hydroxybenzoyl)cyclohexane-1-sulfonylhydrazide; 4-((2-(4-hydroxybenzoyl)hydrazino)sulfonyl)-5-methylfuran-2-carboxylic acid; N'-(4-hydroxybenzoyl)pyrrolidine-3-sulfonylhydrazide; N'-(4-hydroxybenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-sulfonylhydrazide; and 2-((4-Aminophenyl)sulfonyl)-N-(3-hydroxyphenyl)hydrazine-1-carboxamide.

2. The use according to claim 1, wherein The compound is selected from: 4-amino-N'-(4-hydroxybenzoyl)benzenesulfonylhydrazide; 4-amino-N'-(4-hydroxybenzoyl)-3-morpholinobenzenesulfonylhydrazide; and 4-Amino-N'-(1H-indole-4-carbonyl)-3-morpholinobenzenesulfonylhydrazide.

3. The use according to claim 1, wherein The compound has a structure selected from Compound 2-11, Compound 13-15, Compound 17, Compound 18, Compound 20-26, Compound 28-41 and Compound 43-47: [Compound 2] [Compound 3] [Compound 4] [Compound 5] [Compound 6] [Compound 7] [Compound 8] [Compound 9] [Compound 10] [Compound 11] [Compound 13] [Compound 14] [Compound 15] [Compound 17] [Compound 18] [Compound 20] [Compound 21] [Compound 22] [Compound 23] [Compound 24] [Compound 25] [Compound 26] [Compound 28] [Compound 29] [Compound 30] [Compound 31] [Compound 32] [Compound 33] [Compound 34] [Compound 35] [Compound 36] [Compound 37] [Compound 38] [Compound 39] [Compound 40] [Compound 41] [Compound 43] [Compound 44] [Compound 45] [Compound 46] and [Compound 47]

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