Compound as a PU.1 inhibitor

Abstract

The present application discloses compounds of formula (I) as PU.1 inhibitors. The present application also provides methods for preparing these compounds. [Formula 1] JPEG2023549962000076.jpg16128

Description

Cross - reference to related applications

[0001] This application claims priority to PCT International Application No. PCT / CN2020 / 130512, filed on November 20, 2020, the disclosure of which is hereby incorporated by reference in its entirety into this application.

Technical Field

[0002] The present disclosure relates to novel inhibitors of the transcription factor PU.1, their chemical synthesis, and their use in the treatment of disorders such as leukemia and fibrosis.

Background Art

[0003] T-cell acute lymphoblastic leukemia (T-ALL) is a type of hematopoietic cancer caused by the abnormal proliferation of T-cell progenitor cells. It accounts for 15% of pediatric patients and 25% of adult patients. T-ALL is a heterogeneous disease at both the biological process and genetic levels (Belver, L. & Ferrando, A. Nat. Rev. Cancer 16, 494-507, doi:10.1038 / nrc.2016.63 (2016)). Although T-ALL has heterogeneous characteristics, major genetic lesions include chromosomal translocations that affect the expression of specific cancer genes, mutations and deletions of specific genes related to signaling pathways and the cell cycle (Teachey, E. A. R. a. D. T. Hematology, 8 (2016)). Abnormal activation of NOTCH1 accounts for approximately 60% of T-ALL patients (Tosello, V. & Ferrando, A. A. Therapeutic advances in hematology 4, 199-210, doi:10.1177 / 2040620712471368 (2013)), and it has been reported that deletions or mutations of the well-known tumor suppressor gene PTEN account for approximately 20% of T-ALL patients (Guan, W., Jing, Y. & Yu, L. Zhongguo shi yan xue ye xue za zhi 25, 587-591, doi:10.7534 / j.issn.1009-2137.2017.02.050 (2017)).Intensified high-dose chemotherapy improves the outcome of T-ALL patients, but some patients who receive chemotherapy again after relapse die from this disease (Pui, C. H., Sailan, S., Relling, M. V., Masera, G. & Evans, W. E. Leukemia 15, 707-715, doi:10.1038 / sj.leu.2402111 (2001); Nguyen, K. et al. Leukemia 22, 2142-2150, doi:10.1038 / leu.2008.251 (2008); Reismueller, B. et al. Journal of Pediatric Hematology Oncology 35, E200-E204, doi:10.1097 / MPH.0b013e318290c3d6 (2013)). The most important factor in drug resistance is the presence of leukemia-initiating cells (LICs). LICs have the ability to self-renew and differentiate into leukemic blasts. Previous studies have reported that leukemic blasts, rather than LICs, can be eliminated by targeted activation pathways. LICs are a troublesome population in T-ALL targeted therapy.

[0004] To study the onset and mechanism of leukemia, a Pten-null T-ALL model was constructed. In this model, Pten was deficient by 40% in the fetal liver hematopoietic stem cells of mice, and then the PI3K-AKT pathway was activated, the c-Myc oncogene was overexpressed, and the hematopoietic system was destroyed. Within about 2 months after birth, the mice developed aggressive T-ALL (Guo, W. et al., Nature 453, 529-533, doi:10.1038 / nature06933(2008)). Using c-kit, a marker similar to the state of stem cells, T-ALL cells can be separated into blasts and LICs. In the laboratory, it was confirmed that TIM-3 is an important surface marker, highly expressed on the membrane of LICs but not expressed in blasts and normal cells. PU.1 is an ETS family transcription factor that binds to the TIM-3 promoter, regulates the expression of TIM-3, and can maintain the "stemness" of LICs. There was a high correlation between the expression levels of TIM-3 and PU.1 in LICs. A series of LIC signature genes are potential PU.1 targets (Zhu, H. et al., eLife 7, doi:10.7554 / eLife.38314(2018)).

[0005] PU.1 is a transcription factor belonging to the ETS family and plays an important role in the hematopoietic process. Its expression levels vary among different hematopoietic progenitor cells and their descendants. In long-term HSCs (LT-HSCs), the expression level of PU.1 is low, but it is highly expressed when differentiating into progenitor cells such as CMPs and CLPs. PU.1 also has different expressions in various mature lineages, with higher expression in macrophages than in B cells, and lower expression levels in T cells, erythrocytes, and megakaryocytes. In the GMP population, the expression of PU.1 in its neutrophil and monocyte descendants is very necessary. The role of PU.1 in myelopoiesis has been demonstrated by several mouse models. The absence of PU.1 causes a lack of CMPs and mature macrophages. Furthermore, since PU.1 can regulate the expression of several myeloid-specific genes including GM-CSFRa, G-CSFR, M-CSFR, and IL-7R, it is important for committed myeloid cells. In addition to being a major regulator of the myeloid lineage, PU.1 also plays an important role in the regulation of lymphoid lineage differentiation and the processes of B and T lineage production and lineage selection. Studies using mice with GFP reporter genes have confirmed that the PU.1 expression level increases as B cells mature but is silenced in mature T cells. PU.1-null CLPs can produce B cells. Similar to B cells, PU.1 is required at the stage of T progenitor cells but decreases in mature T cells. Overexpression of PU.1 in mature T cells may cause the cells to exhibit a stem cell-like state, with growth arrest and potential inhibition of maturation (Mak, K. S. et al., International Journal of Cell Biology 2011, 808524, doi:10.1155 / 2011 / 808524(2011)). Recently, it has been shown that PU.1 can control fibroblast polarization and tissue fibrosis, and PU.1 inhibition may be a promising treatment for a wide range of fibrotic diseases (Wohlfahrt, T. et al., Nature 566, 344-349, doi:10.1038 / s41586-019-0896-x(2019)).Furthermore, PU.1 inhibitors can reduce the cell proliferation and clonogenic ability of acute myeloid leukemia (AML) cells and increase apoptosis of AML cells, and PU.1 inhibition has the potential as a treatment strategy for AML (Antony-Debre, I. et al., J Clin Invest 127, 4297-4313, doi:10.1172 / JCI92504(2017)).

[0006] Fibrosis is a reparative or reactive process characterized by the formation and deposition of excessive fibrous connective tissue and extracellular matrix, causing progressive structural remodeling and further dysfunction of almost all tissues and organs such as the lung, skin, liver, kidney, and heart (Rockey, D. C. et al., N Engl JMed 373, 96, doi:10.1056 / NEJMc1504848(2015)). Therefore, fibrosis is a serious factor inducing morbidity and mortality, and it is estimated to cause more than 45% of deaths in the United States (Wynn, T. A., Nat Rev Immunol 4, 583-594, doi:10.1038 / nri1412(2004)). In response to stimuli (e.g., wound healing and inflammatory responses), fibroblasts differentiate into a phenotype that produces matrix, promoting the accumulation of extracellular matrix, which serves as the initiating switch for fibrotic diseases (Palumbo-Zerr, K. et al., NatMed 21, 150-158, doi:10.1038 / nm.3777(2015); Ramming, A. et al., Pharmacol Res 100, 93-100, doi:10.1016 / j.phrs.2015.06.012(2015); Chakraborty, D. et al., Nat Commun 8, 1130, doi:10.1038 / s41467-017-01236-6(2017)). The accompanying inflammatory response activates immune cells (mainly tissue macrophages) and is involved in fibrosis-mediated homeostasis regulation. Currently, there are relatively few methods for treating organ fibrosis, and the therapeutic effects are limited.

[0007] Non-alcoholic fatty liver disease (NAFLD) develops when abnormal and large amounts of fat accumulate (steatosis) in the liver without excessive alcohol consumption, and then progresses to steatohepatitis (non-alcoholic steatohepatitis, NASH), fibrosis with inflammatory reactions and collagen deposition, and may progress to cirrhosis and cancer (Adams, L. A. et al., J Hepatol 62, 1002-1004, doi:10.1016 / j.jhep.2015.02.005(2015); Ratziu, V., Lancet 385, 922-924, doi:10.1016 / S0140-6736(14)62010-9(2015)). In developed countries, more than one-third of people suffer from hepatic steatosis, not only showing a tendency to become younger, but also liver failure via NASH being a major problem in liver transplantation (Cohen, J. C. et al., Science 332, 1519-1523, doi:10.1126 / science.1204265(2011); Stine, J. G. et al., Liver Transpl 21, 1016-1021, doi:10.1002 / lt.24134(2015)). Unfortunately, drug intervention for NASH is poor, and only the PPAR α / γ agonist Saroglitazar has obtained approval from the Indian Drugs Controller General. Therefore, it is urgent to understand how fibrosis occurs and progresses in order to discover new targets for drug development and identify potential treatments for NASH and organ fibrosis.

[0008] In previous studies, the ETS family transcription factor PU.1 is a major regulator of LIC signature genes, and it has been shown that PU.1 is extremely important for the "stemness" of LICs and the development of T-ALL (Zhu, H. et al., eLife 7, doi:10.7554 / eLife.38314 (2018)). Furthermore, PU.1 is highly expressed in fibrotic fibroblasts but is reported to be silenced in matrix-degrading fibroblasts, and treatment with the PU.1 inhibitor DB1976 can alleviate skin, liver, and lung fibrosis (Wohlfahrt, T. et al., Nature 566, 344-349, doi:10.1038 / s41586-019-0896-x (2019)). Our co-researchers and I also confirmed that PU.1 inhibition via the application of DB1976 or shRNA showed beneficial effects on the progression of NASH, including the reduction of hepatic steatosis, inflammation, fibrosis, and the improvement of glucose homeostasis in vivo (Liu, Q. et al. J Hepatol 73, 361-370, doi:10.1016 / j.jhep.2020.02.025 (2020)). These efforts indicate that PU.1 is a potentially effective target for drug research and development for leukemia, liver diseases, and multi-organ fibrosis. However, existing PU.1 inhibitors such as DB1976 have limited efficacy and have inhibitory activity against other ETS family members, posing potential risks for further drug development.

[0009] To treat hematological T-ALL and other pathologies associated with PU.1 dysfunction such as NASH and organ fibrosis, improved methods of using new potent and selective PU.1 inhibitors are needed, where the PU.1 inhibitors are scientifically important and have potential medicinal efficacy. The present disclosure addresses such a need.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present disclosure provides compounds that can block the interaction between the ETS family transcription factor PU.1 and target DNA, downregulate TIM-3 expression, effectively kill leukemia cells, and alleviate organ fibrosis. These compounds can be widely used in the treatment of disorders such as leukemia and fibrosis.

Means for Solving the Problems

[0011] In one aspect, there is provided a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0012]

Chemical formula

[0013] (Wherein X, X’, x, x’, y, y’, R1, R2, R3, R4, A, Z, B, C, and n are as disclosed in the present application.)

[0014] In another aspect, there is provided a method for producing a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the method comprising the step of converting a compound of formula (II), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof into a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0015]

Chemical formula

[0016] (Wherein X, X’, x, x’, y, y’, R1, R2, R3, R4, A, Z, B, C, and n are as disclosed in the present application.)

[0017] In another aspect, provided is a method for treating a PU.1-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the present application. In some embodiments, provided is the use of a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the present application in the treatment of a PU.1-mediated disease. In some embodiments, provided is a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the present application in the manufacture of a medicament for treating a PU.1-mediated disease. In some embodiments, the PU.1-mediated disease is leukemia or fibrosis. In some embodiments, the PU.1-mediated disease or disorder is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), dermal fibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, or cardiac fibrosis. In some embodiments, the PU.1-mediated disease is NASH.

[0018] In another aspect, provided is a composition, such as a pharmaceutical composition, comprising a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the present application and a pharmaceutically acceptable carrier or excipient. Also provided is a kit comprising a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

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Mode for Carrying Out the Invention

[0020] The following description shows exemplary embodiments of the present disclosure. However, it should be recognized that such description is not intended to limit the scope of the present disclosure and is provided as an explanation of the exemplary embodiments.

[0021] Definitions As used herein, the following words, phrases, and symbols are intended to have the meanings as described below, unless otherwise specified in the context in which they are used.

[0022] The term "about" means a variation of ±1%, ±3%, ±5%, or ±10% of a specified value. For example, in some embodiments, "about 50" can include the range from 45 to 55. In the case of an integer range, the term "about" may include one or two integers greater than and / or less than the integers listed at each end of the range. Unless otherwise indicated herein, the term "about" is intended to include values close to the recited range, such as weight percentages, which are equivalent in terms of the functionality of the individual components, compositions, or embodiments. When a value or parameter "about" is recited herein, embodiments (and descriptions) with respect to that value or parameter itself are included. For example, the recitation "about X" includes the recitation "X".

[0023] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, when "the compound" is recited, it includes such plural compounds and references to one or more compounds and equivalents thereof known to those skilled in the art.

[0024] "Alkyl" means a straight or branched chain saturated hydrocarbon chain. As used herein, alkyl has from 1 to 10 carbon atoms (i.e., C 1~10 alkyl, or C1-C 10 alkyl), from 1 to 8 carbon atoms (i.e., C 1~8 alkyl, or C1-C8 alkyl), from 1 to 6 carbon atoms (i.e., C 1~6 alkyl, or C1-C6 alkyl), or from 1 to 4 carbon atoms (i.e., C 1~4It has an alkyl group, or a C1-C4 alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, secondary butyl, isobutyl, t-butyl, pentyl, 2-pentyl, isoamyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbon atoms is named by a chemical name or determined by a molecular formula, it can include all positional isomers having that number of carbon atoms. Thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), secondary butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and t-butyl (i.e., -C(CH3)3), and "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2). Note that the term "alkyl" also considers divalent moieties.

[0025] "Haloalkyl" means that one or more of the hydrogen atoms in the linear or branched alkyl defined above are substituted with a halogen. For example, when a residue is substituted with one or more halogens, it can be described using a prefix corresponding to the number of attached halogen moieties. Dihaloalkyl and trihaloalkyl refer to an alkyl substituted with two ("2") or three ("3") halogen groups, and these may be the same halogen but not necessarily. Examples of haloalkyl include difluoromethyl (-CHF2) and trifluoromethyl (-CF3).

[0026] "Alkoxy" means "-O-alkyl". Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, secondary butoxy, n-pentoxy, n-hexyl, and 1,2-dimethylbutoxy.

[0027] "Aryl" means an aromatic carbocyclic group having a monocyclic (e.g., monocyclic ring) or polycyclic (e.g., bicyclic or tricyclic) ring system including fused rings. As used herein, aryl has 6 to 20 cyclic carbon atoms (i.e., C 6~20 aryl, or C6-C 20 aryl), 6 to 12 carbocyclic atoms (i.e., C 6~12 aryl, or C6-C 12 aryl) or 6 to 10 carbocyclic atoms (i.e., C 6~10 aryl, or C6-C 10 aryl). Examples of aryl include, but are not limited to, phenyl, naphthyl, fluorenyl and anthracenyl. However, aryl does not include heteroaryl as defined below or overlap with these heteroaryls in any way. When one or more aryls are fused to heteroaryl, the resulting ring system is heteroaryl. When one or more aryls are fused to heterocyclyl, the resulting ring system is heterocyclyl. It should be understood that the term "aryl" also contemplates divalent moieties.

[0028] "Cycloalkyl" means a saturated or partially unsaturated cyclic alkyl having a monocyclic or polycyclic (including fused rings, bridged rings and spiro ring systems) ring system. The term "cycloalkyl" includes cycloalkenyl (i.e., a cyclic group having at least one double bond) and a carbocyclic fused ring system having at least one sp 3 carbon atom (i.e., at least one non-aromatic ring). As used herein, cycloalkyl has 3 to 20 cyclic carbon atoms (i.e., C 3~20 cycloalkyl, or C3-C 20 cycloalkyl), 3 to 12 cyclic carbon atoms (i.e., C 3~12 cycloalkyl, or C3-C 12 cycloalkyl), 3 to 10 cyclic carbon atoms (i.e., C 3~10 cycloalkyl, or C3-C 10 cycloalkyl), 3 to 8 cyclic carbon atoms (i.e., C 3~8cycloalkyl, or C3-C8 cycloalkyl), or 3 to 6 cyclic carbon atoms (i.e., C 3~6 has cycloalkyl, or C3-C6 cycloalkyl). Monocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Further, the term "cycloalkyl" is intended to encompass non-aromatic rings that can be fused to an aryl ring, regardless of the bond to the remainder of the molecule. Further, cycloalkyl includes "spirocycloalkyl" when there are two substitution positions on the same carbon atom. It should be understood that the term "cycloalkyl" also contemplates divalent moieties.

[0029] "Heteroaryl" means an aromatic group having a monocyclic, polycyclic or polycondensed ring having one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl has 1 to 20 cyclic carbon atoms (i.e., C 1~20 heteroaryl), 3 to 12 cyclic carbon atoms (i.e., C 3~12 heteroaryl) or 3 to 8 carbocyclic atoms (i.e., C 3~8(heteroaryl), and 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur, are included. In some cases, heteroaryl, each independently, has 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur, and includes a 5- to 12-membered ring system, a 5- to 10-membered ring system, a 5- to 7-membered ring system, or a 5- to 6-membered ring system. Any aromatic ring having a single or multiple fused rings and containing at least one heteroatom is considered to be heteroaryl regardless of its connection to the rest of the molecule (i.e., via any fused ring). Heteroaryl does not include or overlap with the aryl defined above. Examples of heteroaryl include, but are not limited to, pyridyl, pyrimidyl, thienyl, furyl, thiazolyl, oxazolyl, isoxazolyl, thienyl, pyrrolyl, pyrazolyl, 1,3,4-oxadiazolyl, imidazolyl, isothiazolyl, triazolyl, 1,3,4-thiadiazolyl, tetrazolyl, benzofuryl, benzothienyl, pyrazolopyridyl, indazolyl, benzothiazolyl, benzoxazolyl, and benzimidazolyl. It should be understood that the term "heteroaryl" also contemplates divalent moieties.

[0030] "Heterocyclyl" means a saturated or partially unsaturated cycloalkyl having one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. The term "heterocyclyl" includes heterocycloalkenyl (i.e., heterocyclyl having at least one double bond), bridged heterocyclyl, fused heterocyclyl, and spiroheterocyclyl. Heterocyclyl may be monocyclic or polycyclic, and polycyclic may be fused, bridged, or spiro rings, and may have one or more (e.g., 1 to 3) oxo (=O) or N-oxide (N + -O -may also contain an (oxide) moiety. Any non-aromatic ring containing at least one heteroatom is considered a heterocyclyl regardless of its linking method (i.e., it can be linked via a carbon atom or a heteroatom). Further, the term heterocyclyl is intended to include any non-aromatic ring containing at least one heteroatom, and this ring can be fused to an aryl or heteroaryl ring regardless of its linkage to the rest of the molecule. As used herein, heterocyclyl has 2 to 20 ring carbon atoms (i.e., C 2~20 or C2-C 20 heterocyclyl), 2 to 12 ring carbon atoms (i.e., C 2~12、 or C2-C 12 heterocyclyl), 2 to 10 ring carbon atoms (i.e., C 2~10 or C2-C 10 heterocyclyl), 2 to 8 ring carbon atoms (i.e., C 2~8 or C2-C8 heterocyclyl), 3 to 12 ring carbon atoms (i.e., C 3~12、 or C3-C 12 heterocyclyl), 3 to 8 ring carbon atoms (i.e., C 3~8 or C3-C8 heterocyclyl), or 3 to 6 ring carbon atoms (C 3~6or a C3-C6 heterocyclyl), and 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, sulfur or oxygen, and has. In some cases, the heterocyclyl has 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen and sulfur, respectively, and includes a 3- to 12-membered ring system, a 5- to 10-membered ring system, a 5- to 7-membered ring system or a 5- to 6-membered ring system. When there are two substitution positions on the same carbon atom, the term "heterocyclyl" also includes "spiroheterocyclyl". Examples of heterocyclyl include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, etc. It should be understood that the term "heterocyclyl" also takes into account divalent moieties.

[0031] "Oxo" means =O.

[0032] "Halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

[0033] The term "optional" or "optionally" means that the event or situation described later may or may not occur.

[0034] As used herein, "substituted" means that one or more (e.g., 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, or 3-4) hydrogen atoms of a group are replaced by the substituents listed for that group, and the substituents may be the same or different. "Optionally substituted" means that the group is either unsubstituted or may be substituted by one or more (e.g., 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, or 3-4) of the substituents listed for that group, and the substituents may be the same or different.

[0035] Also provided are stereoisomers, mixtures of stereoisomers, tautomers, hydrates, solvates, isotopically enriched analogs, and pharmaceutically acceptable salts of the compounds described herein.

[0036] The compounds or pharmaceutically acceptable salts thereof disclosed herein may contain asymmetric centers and, accordingly, can give rise to enantiomers, diastereomers, and other stereoisomers that can be defined in terms of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- with respect to amino acids. This disclosure is intended to cover all possible such isomers, and their racemic and optically pure forms. The optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using a chiral synthon or chiral reagents, or using conventional techniques such as chromatography and fractional crystallization resolution. Conventional techniques for preparing / separating a single enantiomer include chiral synthesis from an appropriate optically pure precursor, or resolution by use such as chiral high performance liquid chromatography (HPLC) of a racemate (or a racemate of a salt or derivative). When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, unless otherwise specified, the compounds are intended to include E- and Z- geometric isomers.

[0037] "Stereoisomers" refer to compounds in which the same atoms are linked by the same bonds but have different three-dimensional structures and are non-interconvertible. This disclosure covers various stereoisomers and mixtures thereof, including "enantiomers", which means that the molecules of two stereoisomers are mirror images that do not overlap with each other, and "diastereomers", which means stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Therefore, all stereoisomers (e.g., geometric isomers, optical isomers, etc.) of a compound (including salts, solvates, and hydrates of the compound) that may exist due to chiral carbons on various substituents are considered, and these include enantiomeric forms (which may exist even if there are no chiral carbons), rotational isomeric forms, atropisomeric forms, and diastereomeric forms.

[0038] Diastereomer mixtures can be separated into their individual diastereomers by methods well known to those skilled in the art, such as chromatography and / or fractional crystallization, based on their physicochemical differences. Enantiomers can be separated by reacting them with a suitable optically active compound (e.g., a chiral alcohol or a chiral auxiliary such as Mosher's acid chloride) to convert the enantiomer mixture into a diastereomer mixture, separating the diastereomers, and converting the individual diastereomers into the corresponding pure enantiomers (e.g., by hydrolysis). Additionally, some of the compounds disclosed herein may be atropisomers and are considered to be part of this specification. Stereoisomers can also be separated using chiral HPLC.

[0039] Some compounds exist in tautomeric forms. The tautomers are in equilibrium with each other. For example, amide-containing compounds can exist in equilibrium with imidic acid tautomers. Regardless of the types of tautomers shown and regardless of the nature of the equilibrium between tautomers, it will be understood by those skilled in the art that the compounds include amide and imidic acid tautomers. Thus, amide-containing compounds are understood as including the imidic acid tautomers thereof. Similarly, imidic acid-containing compounds are understood as including the amide tautomers thereof.

[0040] Any compound or structure described herein is intended to represent not only the isotopically labeled form but also the unlabeled form of the compound. Compounds of these forms are also referred to as "isotope-enriched analogs". Isotopically labeled compounds have the structures described herein except that one or more atoms are replaced with atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include, for example, the respective isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I are included. The various isotopically labeled compounds of the present disclosure are, for example 3 H and 14It is a compound incorporated with a radioisotope such as C. Such isotope-labeled compounds are useful in metabolic studies, reaction kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution measurement or radiation therapy. Such compounds can exhibit increased resistance to metabolism and are thus useful for increasing the half-life of any compound in administration to mammals, particularly humans. Such compounds are synthesized by methods well known to those skilled in the art, such as using starting materials in which one or more hydrogens are replaced with deuterium.

[0041] The term "inhibit / inhibiting / inhibition" means to slow, stop, or reverse the growth or progression of a disease, infection, condition, or cell population. Inhibition of, for example, about 20%, 40%, 60%, 80%, 90%, 95%, or 99% or more is possible compared to growth or progression that occurs without treatment or contact.

[0042] As used herein, "individual" is a mammal including a human. In some embodiments, the individual includes a pig, cow, feline, canine, primate, rodent, or human. In some embodiments, the individual is a human.

[0043] As used herein, "treatment" is a method for obtaining a beneficial or desirable result, including clinical results. For the purposes of the present disclosure, beneficial or desirable results include reducing one or more symptoms caused by a disease or disorder, reducing the degree of the disease or disorder, stabilizing the disease or disorder (e.g., preventing or delaying the worsening of the disease or disorder), delaying the onset or recurrence of the disease or disorder, delaying or retarding the progression of the disease or disorder, improving the condition of the disease or disorder, providing remission (partial or complete) of the disease or disorder, reducing the dosage of one or more other drugs required for the treatment of the disease or disorder, enhancing the action of other drugs used in the treatment of the disease or disorder, delaying the progression of the disease or disorder, improving the quality of life, and / or extending the survival period of the patient, but these "treatments" also include pathological results that reduce the disease or condition. The methods of the present disclosure take into account any one or more of these therapeutic aspects.

[0044] As used herein, the term "effective amount" means an amount sufficient to treat a particular disorder, condition or disease, e.g., a compound or composition that improves, alleviates, reduces and / or delays one or more symptoms. In some embodiments, the effective amount is an amount sufficient to delay growth. In some embodiments, the effective amount is an amount sufficient to delay onset and / or prevent recurrence. The effective amount can be administered in one or more administrations.

[0045] As used herein, the term "carrier" means a non-toxic compound or reagent that facilitates the incorporation of a compound into a cell or tissue.

[0046] As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, e.g., the material can be incorporated into a pharmaceutical composition administered to a patient without causing significant undesirable biological effects or interacting harmfully with other components of the composition. Pharmaceutically acceptable carriers or excipients preferably meet the requirements criteria of toxicity tests and manufacturing tests and / or are included in the inactive ingredient guidelines defined by the U.S. Food and Drug Administration.

[0047] "Pharmaceutically acceptable salts" are salts that retain at least a portion of the biological activity of the free (non-salt) compound and can be administered to an individual as a drug or medicament. These salts include, for example, (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; or acid addition salts formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid; (2) salts formed when acidic protons present in the parent compound are replaced by metal ions (e.g., alkali metal ions, alkaline earth metal ions, aluminum ions); or those that coordinate with organic bases. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc. Other examples of pharmaceutically acceptable salts include those described in Berge et al., Pharmaceutical Salts, J. Pharm. Sci. 1977 Jan;66(1):1-19. Pharmaceutically acceptable salts can be prepared in situ during the manufacturing process or by reacting the purified compounds of the present disclosure in the form of the free acid or base with the appropriate organic or inorganic base or acid, respectively, and isolating the resulting salt during a subsequent purification process.

[0048] As used herein, the term "excipient" means an inert or inactive substance useful in the manufacture of a drug or medicament, such as a tablet, containing a compound of the present disclosure as an active ingredient. The term excipient can encompass a variety of substances including, but not limited to, binders, disintegrants, coatings, compression / capsulation aids, creams or lotions, lubricants, solutions for parenteral administration, materials for chewable tablets, sweeteners or flavoring agents, suspending / gelling agents, or wet granulating agents. Examples of binders include, for example, carbomers, povidone, xanthan gum, and the like. Examples of coatings include, for example, cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, enteric coatings, and the like. Examples of compression / capsulation aids include, for example, calcium carbonate, glucose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrous or monohydrate, optionally in combination with aspartame, cellulose or microcrystalline cellulose), starch dc, sucrose, and the like. Examples of disintegrants include, for example, croscarmellose sodium, gellan gum, sodium starch glycolate, and the like. Examples of creams or lotions include, for example, maltodextrin, carrageenan, and the like. Examples of lubricants include, for example, magnesium stearate, stearic acid, sodium stearoyl fumarate, and the like. Examples of materials for chewable tablets include, for example, glucose, fructose dc, lactose (monohydrate, optionally in combination with aspartame or cellulose), and the like. Examples of suspending / gelling agents include, for example, carrageenan, sodium starch glycolate, xanthan gum, and the like. Examples of sweeteners include, for example, aspartame, glucose, fructose dc, sorbitol, sucrose dc, and the like. Examples of wet granulating agents include, for example, calcium carbonate, maltodextrin, microcrystalline cellulose, and the like.

[0049] Compound In one aspect, there is provided a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0050] [Chemistry]

[0051] (wherein, x and x' are each independently 0, 1, 2, 3 or 4; R1 and R2 are each independently -R a , -N(R a )2, -OR a , -C(O)OR a , -OC(O)R a , -NHC(O)R a , -C(O)N(R a )2, -OC(O)N(R a )2, -NHC(O)N(R a )2, -S(O)2R a , -S(O)2N(R a )2, -C(O)R a , -NHS(O)2R a , -NHS(O)2N(R a )2, nitro, cyano, or halogen, wherein R a is each independently hydrogen, C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, and wherein any two of R1 or any two of R2, together with the atoms to which they are attached, can form C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, wherein C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl and 5- to 12-membered heteroaryl are each independently optionally substituted with R9; y and y' are each independently 0, 1, 2, 3 or 4; R3 is

[0052] [Chemistry]

[0053] and wherein, R5 is O, S or NH, and R6 and R7 are each independently hydrogen, C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, 5- to 12-membered heteroaryl, -C(O)OR d or -S(O)2R d wherein R is each independently hydrogen, C 1~12 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, and wherein R6 and R7, together with the nitrogen atom to which they are attached, can form a 3- to 12-membered heterocyclyl or a 5- to 12-membered heteroaryl, or when y is 2, 3 or 4, two R3s, together with the atoms to which they are attached, can form a C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, wherein C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, and 5- to 12-membered heteroaryl are each independently optionally substituted with R9; R4 is

[0054]

Chemical formula

[0055] wherein, R'5 is O, S or NH, and R'6 and R'7 are each independently hydrogen, C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, 5- to 12-membered heteroaryl, -C(O)OR d or -S(O)2R d wherein R dis, independently of one another, hydrogen, C 1~12 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, and where R’6 and R’7, together with the nitrogen atom to which they are attached, can form a 3- to 12-membered heterocyclyl, or a 5- to 12-membered heteroaryl, or when y’ is 2, 3 or 4, two R4s, together with the atoms to which they are attached, can form a C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, where C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, and 5- to 12-membered heteroaryl are each independently optionally substituted with R9; X is O, S, NH or NR8, and X’ is O, S, NH or NR’8, where R8 and R’8 are each independently C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl; A and B are each independently -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)2NH-, or -NHS(O)2-; C is a chemical bond or -NH-, provided that when B is -C(O)-, or -S(O)2-, C is -NH-, and when B is -C(O)NH-, -NHC(O)-, -S(O)2NH-, or -NHS(O)2-, C is a chemical bond; n is an integer selected from 1 to 6; Z is, independently of one another, C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, each independently optionally R cis replaced, where R c is, independently of one another, C 1~6 alkyl, C 1~6 alkoxy, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, 5- to 12-membered heteroaryl, amino, hydroxy, carboxy, nitro, cyano, or halogen, provided that at least one Z is, independently of one another, optionally substituted with R c and is C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl; and R9 is, independently of one another, -R b , -N(R b )2, -OR b , -C(O)OR b , -OC(O)R b , -NHC(O)R b , -C(O)N(R b )2, -OC(O)N(R b )2, -NHC(O)N(R b )2, -S(O)2R b , -S(O)2N(R b )2, -C(O)R b , -NHS(O)2R b , -NHS(O)2N(R b )2, nitro, cyano, or halogen, where R b is, independently of one another, hydrogen, C 1~6 alkyl, C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to at 12-membered heteroaryl.)

[0056] In some embodiments of any compound related to formula (I) or the formula, x is 0, 1, 2, or 3. In some embodiments, x is 0, 1, or 2. In some embodiments, x is 0 or 1. In some embodiments, x is 1, 2, or 3. In some embodiments, x is 1 or 2. In some embodiments, x is 2 or 3. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4.

[0057] In some embodiments of any compound related to formula (I) or the formula, x' is 0, 1, 2, or 3. In some embodiments, x' is 0, 1, or 2. In some embodiments, x' is 0 or 1. In some embodiments, x' is 1, 2, or 3. In some embodiments, x' is 1 or 2. In some embodiments, x' is 2 or 3. In some embodiments, x' is 0. In some embodiments, x' is 1. In some embodiments, x' is 2. In some embodiments, x' is 3. In some embodiments, x' is 4.

[0058] In some embodiments of any compound related to formula (I) or the formula, x is equal to x'. In some embodiments, x is equal to x' and is 0. In some embodiments, x is equal to x' and is 1. In some embodiments, x is equal to x' and is 2. In some embodiments, x is equal to x' and is 3. In some embodiments, x is equal to x' and is 4. In some embodiments, x and x' are each independently 2 or 3. In some embodiments, x is equal to x' and is 2 or 3.

[0059] In some embodiments of any compound related to formula (I) or the formula, R1 are each independently -R a 、-OR aor a halogen. In some embodiments, each R1 is independently -R a , -OR a or a halogen, where each R1 is independently hydrogen or C 1~6 alkyl. In some embodiments, each R1 is independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C 1~6 alkyl. In some embodiments, R1 is methyl. In some embodiments, R1 is -O-C 1~6 alkyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is a halogen. In some embodiments, R1 is fluorine. In some embodiments, two R1s, together with the atom to which they are attached, form C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, each optionally substituted with R9. In some embodiments, two R1s, together with the atom to which they are attached, form C 3~8 cycloalkyl. In some embodiments, two R1s, together with the atom to which they are attached, form 3- to 12-membered heterocyclyl optionally substituted with R9 and optionally substituted with R9. In some embodiments, two R1s, together with the atom to which they are attached, form C 6~12 aryl optionally substituted with R9. In some embodiments, two R1s, together with the atom to which they are attached, form 5- to 12-membered heteroaryl optionally substituted with R9.

[0060] In some embodiments of any compound related to formula (I) or the formula, each R2 is independently -R a , -OR a or a halogen. In some embodiments, each R2 is independently -R a , -OR a or a halogen, where R a is independently hydrogen or C 1~6It is alkyl. In some embodiments, each R2 is independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, R2 is hydrogen. In some embodiments, R2 is C 1~6 alkyl. In some embodiments, R2 is methyl. In some embodiments, R2 is -O-C 1~6 alkyl. In some embodiments, R2 is methoxy. In some embodiments, R2 is halogen. In some embodiments, R2 is fluorine. In some embodiments, two R2s, together with the atoms to which they are attached, may be substituted with R9 and form a C 3~8 cycloalkyl. In some embodiments, two R2s, together with the atoms to which they are attached, may be substituted with R9 and form a 3- to 12-membered heterocyclyl. In some embodiments, two R2s, together with the atoms to which they are attached, may be substituted with R9 and form a C 6~12 aryl. In some embodiments, two R2s, together with the atoms to which they are attached, may be substituted with R9 and form a 5- to 12-membered heteroaryl.

[0061] In some embodiments of any compound related to formula (I) or the formula, R1 and R2 are each independently -R a , -OR a or halogen. In some embodiments, R1 and R2 are each independently -R a , -OR a or halogen, where R a is each independently hydrogen or C 1~6 alkyl. In some embodiments, R1 and R2 are each independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, both R1 and R2 are hydrogen. In some embodiments, two R1s and / or two R2s, together with the atoms to which they are attached, may each independently be substituted with R9 and form a C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl.

[0062] In some embodiments of any compound related to formula (I) or the formula, y is 0, 1, 2, or 3. In some embodiments, y is 0, 1, or 2. In some embodiments, y is 0 or 1. In some embodiments, y is 1, 2, or 3. In some embodiments, y is 1 or 2. In some embodiments, y is 2 or 3. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4.

[0063] In some embodiments of any compound related to formula (I) or the formula, y' is 0, 1, 2, or 3. In some embodiments, y' is 0, 1, or 2. In some embodiments, y' is 0 or 1. In some embodiments, y' is 1, 2, or 3. In some embodiments, y' is 1 or 2. In some embodiments, y' is 2 or 3. In some embodiments, y' is 0. In some embodiments, y' is 1. In some embodiments, y' is 2. In some embodiments, y' is 3. In some embodiments, y' is 4.

[0064] In some embodiments of any compound related to formula (I) or the formula, y is equal to y'. In some embodiments, y is equal to y' and is 0. In some embodiments, y is equal to y' and is 1. In some embodiments, y is equal to y' and is 2. In some embodiments, y is equal to y' and is 3. In some embodiments, y is equal to y' and is 4. In some embodiments, y and y' are each independently 1 or 2. In some embodiments, y is equal to y' and is 1 or 2. In some embodiments, x + y and x' + y' are equal to 4.

[0065] In some embodiments of any compound related to formula (I) or the formula, R5 is O. In some embodiments, R5 is S. In some embodiments, R5 is NH. In some embodiments, R5 is O or NH.

[0066] In some embodiments of any compound related to formula (I) or the formula, R6 and R7 are each independently hydrogen or -C(O)OR d wherein. In some embodiments, R6 and R7 are each independently hydrogen or -C(O)OR d wherein, R d is C 1~12 alkyl. In some embodiments, both R6 and R7 are hydrogen. In some embodiments, R6 and R7, together with the nitrogen atom to which they are attached, form a 3- to 12-membered heterocyclyl or a 5- to 12-membered heteroaryl.

[0067] In some embodiments of any compound related to formula (I) or the formula, R5 is O, and R6 and R7 are each independently hydrogen or -C(O)OR d wherein, R d is C 1~12 alkyl. In some embodiments, R5 is S, and R6 and R7 are each independently hydrogen or -C(O)OR d wherein, R d is C 1~12 alkyl. In some embodiments, R5 is NH, and R6 and R7 are each independently hydrogen or -C(O)OR d wherein, R d is C 1~12 alkyl. In some embodiments, R5 is O or NH, and R6 and R7 are each independently hydrogen or -C(O)OR d wherein, R d is C 1~12It is alkyl. In some embodiments, R5 is O, and both R6 and R7 are hydrogen. In some embodiments, R5 is NH, and both R6 and R7 are hydrogen. In some embodiments, R5 is S, and both R6 and R7 are hydrogen.

[0068] In some embodiments of any compound related to formula (I) or the formula, R'5 is O. In some embodiments, R'5 is S. In some embodiments, R'5 is NH. In some embodiments, R'5 is O or NH.

[0069] In some embodiments of any compound related to formula (I) or the formula, R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein. In some embodiments, R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein R d is C 1~12 alkyl. In some embodiments, both R'6 and R'7 are hydrogen. In some embodiments, R'6 and R'7, together with the nitrogen atom to which they are attached, can form a 3- to 12-membered heterocyclyl or a 5- to 12-membered heteroaryl.

[0070] In some embodiments of any compound related to formula (I) or the formula, R'5 is O, and R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein R d is C 1~12 alkyl. In some embodiments, R'5 is S, and R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein R d is C 1~12 alkyl. In some embodiments, R'5 is NH, and R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein R d is C 1~12It is alkyl. In some embodiments, R’5 is O or NH, and R’6 and R’7 are each independently hydrogen or -C(O)OR d wherein R d is C 1~12 alkyl. In some embodiments, R’5 is O, and both R’6 and R’7 are hydrogen. In some embodiments, R’5 is NH, and both R’6 and R’7 are hydrogen. In some embodiments, R’5 is S, and both R’6 and R’7 are hydrogen.

[0071] In some embodiments of any compound related to formula (I) or the formula, two R3s, together with the atoms to which they are attached, form C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, each independently optionally substituted with R9. In some embodiments, two R3s, together with the atoms to which they are attached, form optionally substituted C 3~8 cycloalkyl with R9. In some embodiments, two R3s, together with the atoms to which they are attached, form optionally substituted 3- to 12-membered heterocyclyl with R9. In some embodiments, two R3s, together with the atoms to which they are attached, form optionally substituted C 6~12 aryl with R9. In some embodiments, two R3s, together with the atoms to which they are attached, form optionally substituted 5- to 12-membered heteroaryl with R9. In some embodiments, two R3s, together with the atoms to which they are attached, form optionally substituted 5- or 6-membered heteroaryl with R9. In some embodiments, two R3s, together with the atoms to which they are attached,

[0072]

Chemical formula

[0073] form. In some embodiments, two R3s, together with the atoms to which they are attached,

[0074] [Chem.]

[0075] to form.

[0076] In some embodiments of any compound related to formula (I) or the formula, two R4, together with the atoms to which they are attached, are C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, each independently optionally substituted with R9. In some embodiments, two R4, together with the atoms to which they are attached, are C 3~8 cycloalkyl optionally substituted with R9. In some embodiments, two R4, together with the atoms to which they are attached, form 3- to 12-membered heterocyclyl optionally substituted with R9. In some embodiments, two R4, together with the atoms to which they are attached, are C 6~12 aryl. In some embodiments, two R4, together with the atoms to which they are attached, form 5- to 12-membered heteroaryl optionally substituted with R9. In some embodiments, two R4, together with the atoms to which they are attached, form 5- or 6-membered heteroaryl optionally substituted with R9. In some embodiments, two R4, together with the atoms to which they are attached,

[0077] [Chem.]

[0078] to form. In some embodiments, two R4, together with the atoms to which they are attached,

[0079] [Chem.]

[0080] to form.

[0081] In some embodiments of any compound related to formula (I) or the formula, two R3 and / or two R4, together with the atoms to which they are attached, are C 3~8 cycloalkyl, 3- to 12-membered heterocyclyl, C 6~12 aryl, or 5- to 12-membered heteroaryl, each independently optionally substituted with R9. In some embodiments, two R3 and / or two R4, together with the atoms to which they are attached, form a 5- to 12-membered heteroaryl optionally substituted with R9. In some embodiments, two R3 and / or two R4, together with the atoms to which they are attached, form a 5- or 6-membered heteroaryl each independently optionally substituted with R9. In some embodiments, two R3 and / or two R4, together with the atoms to which they are attached,

[0082]

Chemical formula

[0083] form. In some embodiments, two R3 and / or two R4, together with the atoms to which they are attached,

[0084]

Chemical formula

[0085] form.

[0086] In some embodiments of any compound related to formula (I) or the formula, X is O. In some embodiments, X is S. In some embodiments, X is NH. In some embodiments, X is NR8. In some embodiments, X is NH or NR8. In some embodiments, R8 is C 1~6 alkyl. In some embodiments, R8 is methyl.

[0087] In some embodiments of any compound related to formula (I) or the formula, X' is O. In some embodiments, X' is S. In some embodiments, X' is NH. In some embodiments, X' is NR'8. In some embodiments, X' is NH or NR'8. In some embodiments, R'8 is C 1~6 alkyl. In some embodiments, R'8 is methyl.

[0088] In some embodiments of any compound related to formula (I) or the formula, X is NH or NR8, and X' is NH or NR'8, where R8 and R'8 are each independently C 1~6 alkyl. In some embodiments, X is NH or NR8, and X' is NH or NR'8, where both R8 and R'8 are methyl. In some embodiments, both X and X' are NH.

[0089] In some embodiments of any compound related to formula (I) or the formula, A is -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)2NH-, or -NHS(O)2-. In some embodiments, A is -C(O)-, -C(O)NH-, or -NHC(O)-. In some embodiments, A is -C(O)-. In some embodiments, A is -C(O)NH-. In some embodiments, A is -NHC(O)-. In some embodiments, A is -S(O)2-. In some embodiments, A is -S(O)2NH-. In some embodiments, A is -NHS(O)2-.

[0090] In some embodiments of any compound related to formula (I) or the formula, B is -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)2NH-, or -NHS(O)2-. In some embodiments, B is -C(O)-, -C(O)NH-, or -NHC(O)-. In some embodiments, B is -C(O)-. In some embodiments, B is -C(O)NH-. In some embodiments, B is -NHC(O)-. In some embodiments, B is -S(O)2-. In some embodiments, B is -S(O)2NH-. In some embodiments, B is -NHS(O)2-.

[0091] In some embodiments of any compound related to formula (I) or the formula, A and B are each independently -C(O)-, -C(O)NH-, or -NHC(O)-. In some embodiments, each A and B is -C(O)-.

[0092] In some embodiments of any compound related to formula (I) or the formula, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is from 2 to 6. In some embodiments, n is from 2 to 5. In some embodiments, n is from 2 to 4. In some embodiments, n is from 2 to 3. In some embodiments, n is from 3 to 6. In some embodiments, n is from 3 to 5. In some embodiments, n is from 3 to 4. In some embodiments, n is from 4 to 6. In some embodiments, n is from 4 to 5.

[0093] In some embodiments of any compound related to formula (I) or the formula, Z is each independently C 1~6 alkyl, 3- to 12-membered heterocyclyl, or 5- to 12-membered heteroaryl, each independently optionally substituted with R c In some embodiments, Z is each independently optionally substituted with R cis a 3- to 12-membered heterocyclyl which is replaced by. In some embodiments, Z is, independently of each other, C optionally substituted with Rc 3~8 is cycloalkyl. In some embodiments, Z is, independently of each other, optionally R c substituted C 6~12 is aryl. In some embodiments, Z is, independently of each other, optionally R c substituted 5- to 12-membered heteroaryl. In some embodiments, Z is, independently of each other, -CH2-, -CH2CH2-,

[0094]

Chemical formula

[0095] and is, independently of each other, optionally substituted with R c Note that each wavy line represents a connection point with the rest of the molecule, and the connection point may be on any atom permitted by the valence. For example,

[0096]

Chemical formula

[0097] Examples include, but are not limited to,

[0098]

Chemical formula

[0099] are contemplated. In some embodiments, Z is, independently of each other, methyl,

[0100]

Chemical formula

[0101] and is, independently of each other, optionally substituted with R c In some embodiments, Z is, independently of each other, optionally Rc is replaced by

[0102]

Chem.

[0103] is. In some embodiments, each Z is

[0104]

Chem.

[0105] is. In some embodiments, each Z is, independently of one another, optionally R c is replaced by

[0106]

Chem.

[0107] is. In some embodiments, each Z is, independently of one another,

[0108]

Chem.

[0109] is.

[0110] It should be understood that the specific values described herein are those of the compounds of formula (I) or, alternatively, those of the compounds of any related formula (e.g., formula (II)). Two or more values can be combined. Thus, any variable of a compound of formula (I) or a related formula may be combined with any other variable of a compound of formula (I) or a related formula, and it should be understood that each combination of variables is equivalent to being specifically and individually listed. For example, in some embodiments, there is provided a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein x and x' are each 2 or 3, R1 and R2 are each hydrogen, y and y' are each independently 1 or 2, where two R3 and / or two R4, together with the atoms to which they are attached,

[0111] [Chemical Formula]

[0112] can form, R5 is O or NH, R6 and R7 are each independently hydrogen or -C(O)OR d wherein R'5 is O or NH, R'6 and R'7 are each independently hydrogen or -C(O)OR d wherein X is NH or NR8 and X' is NH or NR'8, where R8 and R'8 are each independently C 1~6 alkyl, A and B are each independently -C(O)-, -C(O)NH-, or -NHC(O)-, n is 2, and Z is each independently C 1~6 alkyl, a 3- to 12-membered heterocyclyl, or a 5- to 12-membered heteroaryl, each independently optionally substituted with R c .

[0113] Exemplary compounds according to the present disclosure include, but are not limited to, the compounds shown in Table 1 or their stereoisomers, tautomers, hydrates, solvates, isotopically labeled forms, or pharmaceutically acceptable salts. In some embodiments, there is provided a compound shown in Table 1, its stereoisomer, or its pharmaceutically acceptable salt. In some embodiments, there is provided a compound shown in Table 1 or its pharmaceutically acceptable salt.

[0114]

Table 1-1

[0115]

Table 1-2

[0116] Treatment methods In another aspect, there is provided a method of treating a PU.1-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound, its stereoisomer, or its pharmaceutically acceptable salt described herein. There is provided a compound, its stereoisomer, or its pharmaceutically acceptable salt described herein for treating a PU.1-mediated disease. In some embodiments, there is provided the use of a compound, its stereoisomer, or its pharmaceutically acceptable salt described herein in the manufacture of a medicament for treating a PU.1-mediated disease. In some embodiments, the PU.1-mediated disease is leukemia or fibrosis. In some embodiments, the PU.1-mediated disease is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), dermal fibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, or cardiac fibrosis. In some embodiments, the PU.1-mediated disease is NASH.

[0117] In some embodiments, there is provided a method of inhibiting PU.1 comprising contacting a cell with an effective amount of a compound, its stereoisomer, or its pharmaceutically acceptable salt disclosed herein.

[0118] Compositions In another aspect, there is provided a composition, such as a pharmaceutical composition, comprising a compound described in the present application, its stereoisomer or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to the present application can be in a form suitable for oral, buccal, parenteral (e.g., intravenous, intramuscular, infusion or subcutaneous injection), nasal, topical or rectal administration, or a form suitable for administration by inhalation.

[0119] In some embodiments, the compound described in the present application may be in a purified form. In some embodiments, a composition comprising a compound described in the present application, its stereoisomer or its pharmaceutically acceptable salt is in a substantially pure form. Unless otherwise specified, "substantially pure" refers to a composition containing no more than 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5% or 0.1% of impurities, where the impurities are compounds different from the desired compound or its pharmaceutically acceptable salts.

[0120] Kit The present application also provides a kit comprising a compound disclosed in the present application, its stereoisomer or its pharmaceutically acceptable salt or a composition disclosed in the present application. In some embodiments, the kit comprises unit doses of the compound or composition described in the present application and / or instructions for administering them.

[0121] Manufacturing method In another aspect, there is provided a method for manufacturing a compound disclosed in the present application, its stereoisomer or its pharmaceutically acceptable salt, the method comprising the step of converting a compound of formula (II), its stereoisomer or its pharmaceutically acceptable salt into a compound of formula (I), its stereoisomer or its pharmaceutically acceptable salt.

[0122]

Chemical formula

[0123] (Wherein X, X', x, x', y, y', R1, R2, R3, R4, A, Z, B, C, and n are as disclosed in the present application.)

[0124] In some embodiments, the compound of formula (II) is a compound of formula (13′), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[0125]

Chemical formula

[0126] The method comprises (a) reacting a compound of formula (11′), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof with a compound of formula (5′), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[0127]

Chemical formula

[0128] (b) converting a compound of formula (6), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof into a compound of formula (11′), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and / or

[0129]

Chemical formula

[0130] (c) further comprising converting a compound of formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof into a compound of formula (5′), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0131]

Chemical formula

[0132] In some embodiments, the compound of formula (II) is a compound of formula (50), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[0133]

Chemical formula

[0134] The method comprises (a) reacting a compound of formula (45), a stereoisomer thereof or a pharmaceutically acceptable salt thereof with a compound of formula (41), a stereoisomer thereof or a pharmaceutically acceptable salt thereof;

[0135]

Chemical formula

[0136] (b) converting a compound of formula (42), a stereoisomer thereof or a pharmaceutically acceptable salt thereof into a compound of formula (45), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and / or

[0137]

Chemical formula

[0138] (c) further comprising converting a compound of formula (6), a stereoisomer thereof or a pharmaceutically acceptable salt thereof into a compound of formula (41), a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0139]

Chemical formula

[0140] In some embodiments, the compound of formula (II) is a compound of formula (54), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0141]

Chemical formula

[0142] The method comprises (a) Converting the compound of formula (53), its stereoisomer or its pharmaceutically acceptable salt to the compound of formula (54), its stereoisomer or its pharmaceutically acceptable salt;

[0143] [Chemical formula]

[0144] (b) Converting the compound of formula (47), its stereoisomer or its pharmaceutically acceptable salt to the compound of formula (53), its stereoisomer or its pharmaceutically acceptable salt, and / or

[0145] [Chemical formula]

[0146] (c) Further comprising converting the compound of formula (45), its stereoisomer or its pharmaceutically acceptable salt to the compound of formula (47), its stereoisomer or its pharmaceutically acceptable salt.

[0147] [Chemical formula]

[0148] In some embodiments, one or more steps of the manufacturing methods disclosed in the present application include acylation, condensation, reduction, protection and / or deprotection.

[0149] The following provides representative schemes for manufacturing the compounds disclosed in the present application.

[0150] Scheme 1

[0151] [Chemical formula]

[0152] Scheme 2

[0153] [Chemistry]

[0154] Scheme 3

[0155] [Chemistry]

[0156] Scheme 4

[0157] [Chemistry]

[0158] Scheme 5

[0159] [Chemistry]

[0160] The compounds of formula (I) or any related formula of the present application can be synthesized by standard synthetic techniques known to those skilled in the art. The compounds of the present disclosure can be synthesized according to the general synthetic procedures described in the schemes provided above and the examples provided below.

[0161] If it is desired to obtain a specific enantiomer of a compound, the corresponding enantiomer can be obtained from a mixture of enantiomers using any suitable conventional procedure for separating or resolving the enantiomers. Thus, for example, diastereomeric derivatives can be prepared by reacting a mixture of enantiomers (e.g., a racemate and a suitable chiral compound). The diastereomers can then be separated by any convenient method such as crystallization, and the desired enantiomer can be recovered. In another resolution process, the racemate can be separated using chiral high performance liquid chromatography. Alternatively, if desired, a specific enantiomer can be obtained using a suitable chiral intermediate in one of the described procedures. Examples

[0162] Synthesis Examples The compounds according to the present disclosure can be produced by commercially available raw materials and the production methods described in this specification. The following examples are used to illustrate the compounds and their production methods disclosed in this specification. These examples and production steps described below should not be regarded as limiting the scope of the present disclosure. The structure of the compounds according to the present disclosure was 1 confirmed by 1H NMR. Unless otherwise stated, all compounds or intermediates in the synthesis steps are purified by column chromatography or preparative reverse-phase HPLC. The reaction process can be measured by thin-layer chromatography, and the elution systems commonly used in the purification stage are petroleum ether / ethyl acetate and dichloromethane / methanol.

[0163] Example S1: Synthesis of (S)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-1)

[0164] [Chemical formula]

[0165] Step 1: Synthesis of 4-formylbenzoyl chloride (Compound 2) 4-Formylbenzoic acid 1 (4 g, 26.64 mmol) was suspended in a mixture of toluene (64 mL) and SOCl2 (8 mL), and the mixture was refluxed at 110 °C overnight. The obtained clear solution was cooled to room temperature and concentrated under vacuum. By co-evaporation with toluene, excess SOCl2 was removed and dried under vacuum to obtain the target product 2 (4.40 g, 98%) as a white solid. 11H NMR (400 MHz, CDCl3) δ 10.15 (s, 1H), 8.29 (d, J = 8.3 Hz, 2H), 8.03 (d, J = 8.6 Hz, 2H).

[0166] Step 2: Synthesis of (4-Formylbenzoyl)-L-proline t-butyl ester (Compound 4) At 0 °C, a solution of Compound 2 (1.98 g, 11.74 mmol) in DCM (18 mL) was slowly added to a solution of L-proline t-butyl ester 3 (2.01 g, 11.74 mmol) in DCM (18 mL) and TEA (2 mL). Then, the mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was washed with aqueous HCl solution (1 M, 3 × 60 mL). The combined aqueous portions were extracted with DCM, and the combined organic portions were further washed with saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give a pale yellow oil 4 (1.87 g), which was used in the next step without further purification.

[0167] Step 3: Synthesis of (4-Formylbenzoyl)-L-proline (Compound 5) A solution of Compound 4 (1.87 g, 6.16 mmol) in DCM (18 mL) and TFA (18 mL) was stirred at room temperature for 12 h. After completion of the reaction, the solvent was removed. The residue was dissolved in saturated aqueous NaHCO3 and washed with EtOAc. The organic portion was extracted with saturated aqueous NaHCO3. The aqueous portion was acidified to pH = 2 by adding 2 M HCl, and then the combined aqueous portions were extracted with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (2% MeOH in DCM) to give the desired product 5 as a white solid (850 mg, 29% over two steps). 11H NMR (400 MHz, CDCl3) δ 10.07 (s, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.1 Hz, 2H), 4.78 (dd, J = 8.3, 5.0 Hz, 1H), 3.58 - 3.51 (m, 2H), 3.34 (s, 1H), 2.41 - 2.35 (m, 1H), 2.31 - 2.25 (m, 1H), 2.12 - 2.02 (m, 1H), 2.00 - 1.90 (m, 1H).

[0168] Step 4: Synthesis of 2-(4-Nitrophenyl)-1,3-dithiolane (Compound 8) To a solution of 4-Nitrobenzaldehyde 6 (6.92 g, 45.79 mmol) in DCM (180 mL) was added ethan-1,2-dithiol 7 (20 mL, 0.24 mol), and then boron trifluoride diethyl etherate (1.2 mL) was added. After stirring at room temperature for 6 h, the solution was washed with 10% NaOH, water, and brine. The obtained bright yellow solution was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give the desired product 8 (9.78 g, 94%) as a yellow solid. 1 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 5.65 (s, 1H), 3.56 - 3.48 (m, 2H), 3.45 - 3.37 (m, 2H).

[0169] Step 5: Synthesis of 4-(1,3-Dithiolan-2-yl)aniline (Compound 9) A solution of compound 8 (5.0 g, 22.00 mmol) and tin(II) dichloride dihydrate (24.82 g, 0.11 mol) in anhydrous EtOH (44 mL) was heated at 70 °C for 0.5 h. After cooling to room temperature, the orange solution was poured onto ice in a large beaker and then treated with saturated aqueous NaHCO3 until the pH value reached 7 - 8. About 200 mL of EtOAc was added and the mixture was vacuum filtered through a glass funnel. The filtrate was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give the desired product 9 (3.52 g, 81%) as a light yellow solid. 1 1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 7.8 Hz, 2H), 6.62 (d, J = 7.7 Hz, 2H), 5.61 (s, 1H), 3.69 (s, 2H), 3.53 - 3.45 (m, 2H), 3.37 - 3.29 (m, 2H).

[0170] Step 6: Synthesis of (9H-fluoren-9-yl)methyl (S)-2-((4-(1,3-dithiolan-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (compound 10) To a solution of freshly prepared compound 9 (3.08 g, 15.61 mmol) and Fmoc-L-proline (5.26 g, 15.59 mmol) in DMF (15 mL) were added a solution of HOBT in DMF (1 M, 15 mL) and a solution of DCC in DCM (1 M, 15 mL), and the reaction mixture was stirred at room temperature for 24 h. Then 75 mL of EtOAc was added and the mixture was filtered through a glass funnel. After removing the solvent, the residue was diluted with CHCl3 / i-PrOH (3:1) and then washed with water, 0.1 M HCl, saturated aqueous NaHCO3 and brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (0 - 0.5% MeOH in DCM) to give the desired product 10 (5.89 g, 73%) as a pale yellow solid. 11H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.82 - 7.28 (m, 12H), 5.62 (s, 1H), 4.56 - 4.40 (m, 3H), 4.26 (s, 1H), 3.57 - 3.30 (m, 6H), 2.56 (s, 1H), 1.96 (s, 3H).

[0171] Step 7: Synthesis of (S)-N-(4-(1,3-dithiolan-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound 11) To a solution of Compound 10 (3.16 g, 6.12 mmol) in DMF (24 mL) was added piperidine (6 mL), and the reaction mixture was stirred at room temperature for 1 h. After removing the solvent, the residue was dissolved in EtOAc and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (0 - 2% MeOH in DCM) to give the desired product 11 (1.37 g, 76%) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 7.5 Hz, 2H), 5.63 (s, 1H), 3.85 (dd, J = 9.0, 5.2 Hz, 1H), 3.53 - 3.46 (m, 2H), 3.38 - 3.31 (m, 2H), 3.11 - 3.05 (m, 1H), 3.00 - 2.94 (m, 1H), 2.26 - 2.16 (m, 1H), 2.07 - 1.99 (m, 1H), 1.79 - 1.70 (m, 2H).

[0172] Step 8: Synthesis of (S)-N-(4-(1,3-dithiolan-2-yl)phenyl)-1-((4-formylbenzoyl)-L-prolyl)pyrrolidine-2-carboxamide (Compound 12) To a solution of compound 11 (880 mg, 2.99 mmol) and compound 5 (740 mg, 2.99 mmol) in DCM (20 mL) was added EDCI (688 mg, 3.59 mmol), and the reaction mixture was stirred at room temperature for 16 h. Then, the solution was concentrated in vacuo to give white solid 12 (900 mg), which was used in the next step without further purification.

[0173] Step 9: Synthesis of (S)-1-((4-formylbenzoyl)-L-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (Compound 13) To a solution of compound 12 (900 mg, 1.72 mmol) in AcOH (35 mL) was added SeO2 (954 mg, 8.60 mmol), and the reaction mixture was stirred at room temperature for 36 h. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was dissolved in DCM, washed with saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (pure EtOAc) to give the desired product 13 (710.1 mg, 53% over 2 steps) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 10.07 (d, J = 8.0 Hz, 1H), 9.93 (dd, J = 26.6, 22.2 Hz, 2H), 8.13 (d, J = 8.6 Hz, 0.5H), 7.95 (dd, J = 13.0, 8.0 Hz, 2H), 7.85 (d, J = 8.7 Hz, 0.5H), 7.79 - 7.67 (m, 5H), 4.87 - 4.81 (m, 1.5H), 4.55 (dd, J = 17.1, 7.7 Hz, 0.5H), 3.97 (dd, J = 16.8, 9.1 Hz, 1H), 3.76 - 3.62 (m, 2H), 3.57 - 3.52 (m, 1H), 2.49 - 1.95 (m, 8H).

[0174] Step 10: Synthesis of (S)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-1) A solution of compound 13 (143.4 mg, 0.32 mmol), 3,4-diaminobenzamidine hydrochloride 14 (120 mg, 0.64 mmol), and p-benzoquinone (70.0 mg, 0.64 mmol) in anhydrous EtOH (13 mL) was heated under reflux for 12 h. The reaction mixture was cooled to room temperature and stirred in acetone (80 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (30 mL) and EtOH (30 mL), filtered, the volume was reduced to 20 mL, and acidified with saturated HCl-EtOH (2 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-1 (101.7 mg, 37%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.27 - 8.23 (m, 4H), 8.15 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.8 Hz, 4H), 7.90 - 7.82 (m, 4H), 4.77 - 4.72 (m, 1H), 4.06 - 3.99 (m, 1H), 3.86 - 3.60 (m, 4H), 2.59 - 2.50 (m, 1H), 2.45 - 2.36 (m, 1H), 2.24 - 1.99 (m, 6H).

[0175] Example S2: Synthesis of (R)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-2)

[0176]

Chem.

[0177] Step 1: Synthesis of (4-formylbenzoyl)-D-proline tert-butyl ester (Compound 16) At 0 °C, a solution of Compound 2 (984 mg, 11.74 mmol) in DCM (10 mL) was slowly added to a solution of D-proline tert-butyl ester 15 (1 g, 5.84 mmol) in DCM (10 mL) and TEA (1 mL). Then, the mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was washed with aqueous HCl solution (1 M, 3 × 30 mL). The combined aqueous portions were extracted with DCM, and the combined organic portions were further washed with saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, concentrated in vacuo to give a pale yellow oil 16 (798.1 mg), which was used in the next step without further purification.

[0178] Step 2: Synthesis of (4-formylbenzoyl)-D-proline (Compound 17) A solution of Compound 16 (798.1 mg, 2.63 mmol) in DCM (9 mL) and TFA (9 mL) was stirred at room temperature for 12 h. After completion of the reaction, the solvent was removed. The residue was dissolved in saturated aqueous NaHCO3 and washed with EtOAc. The organic portion was extracted with saturated aqueous NaHCO3. The aqueous portion was acidified to pH = 2 by adding 2 M HCl and then extracted with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (2% MeOH in DCM) to give the desired product 17 (444 mg, 31% over 2 steps) as a white solid. 11H NMR (400 MHz, CDCl3) δ 10.07 (s, 1H), 8.58 (s, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 4.76 (t, J = 6.7 Hz, 1H), 3.62 - 3.50 (m, 2H), 2.32 (dd, J = 13.5, 6.7 Hz, 2H), 2.12 - 2.02 (m, 1H), 2.00 - 1.90 (m, 1H).

[0179] Step 3: Synthesis of (9H-fluoren-9-yl)methyl (R)-2-((4-(1,3-dithiolan-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 18) A solution of freshly prepared compound 9 (1.90 g, 9.63 mmol) and Fmoc-D-proline (3.25 g, 9.63 mmol) in DMF (9.5 mL) was added to a solution of HOBT in DMF (1 M, 9.6 mL) and a solution of DCC in DCM (1 M, 9.6 mL). The reaction mixture was stirred at room temperature for 24 h. Then, 50 mL of EtOAc was added and the mixture was filtered through a glass funnel. After removing the solvent, the residue was diluted with CHCl3 / i-PrOH (3:1) and then washed with water, 0.1 M HCl, saturated aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (0 - 0.5% MeOH in DCM) to give the desired product 18 (3.13 g, 63%) as a pale yellow solid. 1 1H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.81 - 7.30 (m, 12H), 5.62 (s, 1H), 4.56 - 4.41 (m, 3H), 4.26 (s, 1H), 3.56 - 3.31 (m, 6H), 2.57 (s, 1H), 1.98 (s, 3H).

[0180] Step 4: Synthesis of (R)-N-(4-(1,3-dithiolan-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound 19) To a solution of compound 18 (1.51 g, 2.92 mmol) in DMF (10 mL) was added piperidine (2.6 mL), and the reaction mixture was stirred at room temperature for 1 h. After removing the solvent, the residue was dissolved in EtOAc and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (0 - 2% MeOH in DCM) to give the desired product 19 (790 mg, 92%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.74 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 5.63 (s, 1H), 3.85 (dd, J = 9.3, 5.2 Hz, 1H), 3.54 - 3.46 (m, 2H), 3.38 - 3.31 (m, 2H), 3.10 - 3.04 (m, 1H), 3.00 - 2.94 (m, 1H), 2.25 - 2.17 (m, 1H), 2.07 - 1.99 (m, 1H), 1.79 -1.71 (m, 2H).

[0181] Step 5: Synthesis of (R)-N-(4-(1,3-dithiolan-2-yl)phenyl)-1-((4-formylbenzoyl)-D-prolyl)pyrrolidine-2-carboxamide (Compound 20) To a solution of compound 19 (210 mg, 0.71 mmol) and compound 17 (176 mg, 0.71 mmol) in DCM (5 mL) was added EDCI (164 mg, 0.85 mmol), and the reaction mixture was stirred at room temperature for 16 h. Then, the solution was concentrated in vacuo to give white solid 20 (319.3 mg), which was used in the next step without further purification.

[0182] Step 6: Synthesis of (R)-1-((4-formylbenzoyl)-D-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (Compound 21) A solution of compound 20 (319.3 mg, 0.61 mmol) in AcOH (12 mL) was added with SeO2 (338 mg, 3.05 mmol), and the reaction mixture was stirred at room temperature for 36 h. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was dissolved in DCM, washed with saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (pure EtOAc) to afford the desired product 21 (200.6 mg, 63% in 2 steps) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 10.07 (d, J = 9.4 Hz, 1H), 9.93 (dd, J = 32.9, 28.7 Hz, 2H), 8.13 (d, J = 8.6 Hz, 0.5H), 7.94 (dd, J = 15.5, 8.1 Hz, 2H), 7.85 (d, J = 8.7 Hz, 0.5H), 7.79 - 7.65 (m, 5H), 4.86 - 4.80 (m, 1.5H), 4.55 (dd, J = 17.1, 7.7 Hz, 0.5H), 3.96 (dd, J = 16.7, 9.0 Hz, 1H), 3.76 - 3.63 (m, 2H), 3.57 - 3.51 (m, 1H), 2.45 - 1.90 (m, 8H).

[0183] Step 7: Synthesis of (R)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-2) A solution of compound 21 (87.6 mg, 0.20 mmol), 3,4-diaminobenzamidine hydrochloride 14 (73 mg, 0.39 mmol), and p-benzoquinone (42.6 mg, 0.39 mmol) in anhydrous EtOH (8 mL) was heated under reflux for 8 h. The reaction mixture was cooled to room temperature and stirred in acetone (50 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Subsequently, the solid was dissolved in a 1:1 mixture of hot MeOH (18 mL) and EtOH (18 mL), filtered, the volume was reduced to 12 mL, and it was acidified with saturated HCl-EtOH (1.2 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried under vacuum. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-2 (40.4 mg, 24%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.27 - 8.23 (m, 4H), 8.16 (d, J = 8.8 Hz, 2H), 7.97 - 7.93 (m, 4H), 7.91 - 7.82 (m, 4H), 4.75 - 4.70 (m, 1H), 4.06 - 3.98 (m, 1H), 3.87 - 3.59 (m, 4H), 2.58 - 2.50 (m, 1H), 2.44 - 2.36 (m, 1H), 2.26 - 1.95 (m, 6H).

[0184] Example S3: Synthesis of (S)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-3)

[0185]

Chemical formula

[0186] Step 1: Synthesis of (S)-N-(4-(1,3-dithiolan-2-yl)phenyl)-1-((4-formylbenzoyl)-D-prolyl)pyrrolidine-2-carboxamide (Compound 22) To a solution of Compound 11 (213 mg, 0.72 mmol) and Compound 17 (179 mg, 0.72 mmol) in DCM (5 mL) was added EDCI (166 mg, 0.86 mmol), and the reaction mixture was stirred at room temperature for 20 h. Then, the solution was concentrated in vacuo to afford white solid 22 (255 mg), which was used in the next step without further purification.

[0187] Step 2: Synthesis of (S)-1-((4-formylbenzoyl)-D-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (Compound 23) To a solution of Compound 22 (255 mg, 0.49 mmol) in AcOH (10 mL) was added SeO2 (270 mg, 2.43 mmol), and the reaction mixture was stirred at room temperature for 36 h. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was dissolved in DCM, washed with saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (pure EtOAc) to afford the desired product 23 (210.5 mg, 65% over 2 steps) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 10.08 (s, 1H), 9.84 (s, 1H), 9.13 (s, 1H), 7.95 (d, J = 7.8 Hz, 4H), 7.69 (dd, J = 10.5, 8.4 Hz, 4H), 4.82 - 4.76 (m, 2H), 4.25 - 4.19 (m, 1H), 3.73 - 3.59 (m, 3H), 2.51 - 2.46 (m, 1H), 2.32 - 2.10 (m, 6H), 2.01 - 1.94 (m, 1H).

[0188] Step 3: Synthesis of (S)-1-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-3) A solution of Compound 23 (75 mg, 0.17 mmol), 3,4-diaminobenzamidine hydrochloride 14 (62.5 mg, 0.33 mmol) and p-benzoquinone (36.5 mg, 0.33 mmol) in anhydrous EtOH (8 mL) was heated under reflux for 8 h. The reaction mixture was cooled to room temperature and stirred in acetone (50 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether and dried to give a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (18 mL) and EtOH (18 mL), filtered, the volume was reduced to 12 mL, and acidified with saturated HCl-EtOH (1.2 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to give a precipitate, washed with ethyl ether and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-3 (44.6 mg, 31%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.41 - 8.29 (m, 4H), 8.27 - 8.24 (m, 2H), 8.11 (s, 4H), 8.01 - 7.93 (m, 4H), 5.00 (t, J = 7.1 Hz, 1H), 4.73 - 4.66 (m, 1H), 4.25 - 4.18 (m, 1H), 3.88 - 3.81 (m, 1H), 3.78 - 3.68 (m, 2H), 2.53 - 2.45 (m, 1H), 2.42 - 2.34 (m, 1H), 2.31 - 2.24 (m, 1H), 2.20 - 2.00 (m, 5H).

[0189] Example S4: Synthesis of 2-(4-((S)-1-((4-(6-carbamoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamido) phenyl)-1H-benzo[d]imidazole-6-carboxamide (Compound I-4)

[0190]

Chem.

[0191] Step 1: Synthesis of 4-amino-3-nitrobenzamide (Compound 25) To a suspension of 4-amino-3-nitrobenzoic acid 24 (1 g, 5.49 mmol), HOBT (816 mg, 6.04 mmol), and EDCI (1.16 g, 6.05 mmol) in THF (50 mL) under stirring was added DIPEA (1 mL, 6.06 mmol), and the reaction mixture was stirred at room temperature for 10 min. Then, (NH4)2CO3 (1.58 g, 16.44 mmol) was added all at once, and the resulting suspension was stirred for an additional 24 h. The reaction mixture was concentrated in vacuo, and then a 1:1 NaHCO3 / H2O mixture (40 mL) was added and stirred for a further 2 h. The suspension was filtered, and the solid was dried in vacuo (40 °C, 24 h) to obtain the desired product 25 (878.7 mg, 88%) as a brown solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 7.94 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.76 (s, 2H), 7.25 (s, 1H), 7.01 (d, J = 8.9 Hz, 1H).

[0192] Step 2: Synthesis of 3,4-diaminobenzamide (Compound 26) To a solution of compound 25 (400 mg, 2.21 mmol) in DMF (2 mL) and EtOH (3 mL) was added Pd / C (78 mg, 10%). The flask was then evacuated and purged three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with EtOH. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (0.5% MeOH in DCM) to afford the desired product 26 (289.2 mg, 87%) as a brown solid. 1 1H NMR (400 MHz, DMSO-d6) δ 7.39 (s, 1H), 7.05 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.71 (s, 1H), 6.45 (d, J = 8.0 Hz, 1H), 4.94 (s, 2H), 4.50 (s, 2H).

[0193] Step 3: Synthesis of 2-(4-((S)-1-((4-(6-carbamoyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamido)phenyl)-1H-benzo[d]imidazole-6-carboxamide (Compound I-4) A solution of compound 13 (99.8 mg, 0.22 mmol), 3,4-diaminobenzamide 26 (67.7 mg, 0.45 mmol), and p-benzoquinone (48.6 mg, 0.45 mmol) in anhydrous EtOH (9 mL) was heated to reflux for 8 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to give a brown solid. The solid was then dissolved in a hot 1:1 mixture of MeOH (20 mL) and EtOH (20 mL), filtered, the volume reduced to 13.5 mL, and acidified with saturated HCl-EtOH (3 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to give a precipitate, which was washed with ethyl ether and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to afford the desired product I-4 (67 mg, 35%) as a dark green solid. 11H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.72 (s, 0.5H), 8.52 - 8.21 (m, 14H), 8.08 - 7.76 (m, 12H), 7.66 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 14.2 Hz, 3H), 4.83 (dd, J = 8.3, 4.5 Hz, 1H), 4.75 (dd, J = 8.4, 3.5 Hz, 0.5H), 4.60 (dd, J = 8.3, 4.8 Hz, 1H), 4.17 (dd, J = 8.1, 4.5 Hz, 0.5H), 3.87 - 3.79 (m, 1H), 3.71 - 3.48 (m, 4H), 3.40 - 3.32 (m, 0.5H), 3.08 - 3.00 (m, 0.5H), 2.39 - 2.33 (m, 1H), 2.30 - 2.21 (m, 1H), 2.10 - 1.76 (m, 9H), 1.74 - 1.63 (m, 1H).

[0194] Example S5: Synthesis of (S)-1-((4-(1,7-Dihydroimidazo[4,5-f]indazol-6-yl)benzoyl)-L-prolyl)-N-(4-(1,7-dihydroimidazo[4,5-f]indazol-6-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-5)

[0195]

Chemical Structure

[0196] Step 1: Synthesis of 5,6-Dinitro-1H-indazole (Compound 28) A mixture of 6-nitro-1H-indazole 27 (1 g, 6.13 mmol) in concentrated H2SO4 (14 mL) was cooled to 0 °C and slowly added to a solution of concentrated HNO3 (0.42 mL) in concentrated H2SO4 (6 mL) while stirring at 0 °C. The reaction mixture was stirred at room temperature for 16 h and then poured onto ice. The solid was collected by filtration, washed with water, and dissolved in CHCl3 / i-PrOH (3:1). Then, the mixture was washed with brine and saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the desired product 28 (595 mg, 47%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 14.35 (s, 1H), 8.85 (s, 1H), 8.54 (s, 1H), 8.45 (s, 1H).

[0197] Step 2: Synthesis of 1H-indazole-5,6-diamine (Compound 29) Ammonium formate (900 mg, 14.27 mmol) was added to a mixture of compound 28 (300 mg, 1.44 mmol) and Pd / C (30 mg, 10%) in MeOH (9 mL), and the mixture was refluxed for 4 h. Then, it was filtered through a pad of diatomaceous earth to remove the catalyst and washed with MeOH. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (2 - 10% MeOH in DCM) to afford the desired product 29 (121.4 mg, 57%) as a brown solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 7.52 (s, 1H), 6.71 (s, 1H), 6.56 (s, 1H), 4.80 (s, 2H), 4.29 (s, 2H).

[0198] Step 3: Synthesis of (S)-1-((4-(1,7-dihydroimidazo[4,5-f]indazol-6-yl)benzoyl)-L-prolyl)-N-(4-(1,7-dihydroimidazo[4,5-f]indazol-6-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-5) A solution of compound 13 (77.4 mg, 0.17 mmol), 1H-indazole-5,6-diamine 29 (51.2 mg, 0.34 mmol) and p-benzoquinone (37.7 mg, 0.34 mmol) in anhydrous EtOH (7 mL) was heated under reflux for 8 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to give a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (15 mL) and EtOH (15 mL), filtered, the volume was reduced to 10.5 mL, and acidified with saturated HCl-EtOH (2.1 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to give a precipitate, washed with ethyl ether and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-5 (16.8 mg, 12%) as a brown solid. 1 H NMR (600 MHz, DMSO-d6) δ 10.85 (s, 1H), 10.76 (s, 0.5H), 8.55 - 8.08 (m, 16H), 7.97 (d, J = 8.6 Hz, 2H), 7.93 - 7.80 (m, 8H), 7.70 (d, J = 8.1 Hz, 1H), 4.84 (dd, J = 8.3, 4.5 Hz, 1H), 4.77 (dd, J = 8.0, 3.7 Hz, 0.5H), 4.59 (dd, J = 8.4, 4.7 Hz, 1H), 4.16 (dd, J = 8.2, 4.3 Hz, 0.5H), 3.86 - 3.81 (m, 1H), 3.70 - 3.57 (m, 4H), 3.40 - 3.36 (m, 0.5H), 3.10 - 3.05 (m, 0.5H), 2.40 - 2.34 (m, 1H), 2.30 - 2.23 (m, 1H), 2.11 - 2.05 (m, 1H), 2.04 - 1.99 (m, 1H), 1.98 - 1.84 (m, 7H), 1.74 - 1.66 (m, 1H).

[0199] Example S6: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-6)

[0200]

Chem.

[0201] Step 1: Synthesis of N-(4-cyano-3-methylphenyl)acetamide (Compound 31) To a solution of 4-amino-2-methylbenzonitrile 30 (4.68 g, 35.41 mmol) in DCM (145 mL) was added dropwise Ac2O (4.32 mL, 42.49 mmol), and the reaction mixture was stirred at room temperature for 18 h. After completion of the reaction, the solvent was removed under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (pure DCM) to give the desired product 31 (5.98 g, 97%) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 2.52 (s, 3H), 2.21 (s, 3H).

[0202] Step 2: Synthesis of N-(4-cyano-5-methyl-2-nitrophenyl)acetamide (Compound 32) At 0 °C, compound 31 (2.6 g, 14.92 mmol) was added to a solution of KNO3 (3 g, 29.67 mmol) in concentrated H2SO4 (50 mL). The reaction mixture was stirred at 0 °C for 3 h and then poured onto ice. The resulting precipitate was recrystallized from MeOH to give the desired product 32 (2.39 g, 73%) as a yellow solid. 11H NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 8.86 (s, 1H), 8.48 (s, 1H), 2.61 (s, 3H), 2.32 (s, 3H).

[0203] Step 3: Synthesis of 4-amino-2-methyl-5-nitrobenzonitrile (Compound 33) A mixture of Compound 32 (1.17 g, 5.34 mmol) in H2SO4 (70 mL, 10%) was heated under reflux for 3 h. After cooling to room temperature, the mixture was extracted with CHCl3 / i-PrOH (3:1), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (pure DCM) to give the desired product 33 (920 mg, 97%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.98 (s, 2H), 6.93 (s, 1H), 2.35 (s, 3H).

[0204] Step 4: Synthesis of ethyl 4-amino-2-methyl-5-nitrobenzimidate hydrochloride (Compound 34) Dry HCl gas was passed through a stirred suspension of Compound 33 (354 mg, 2.00 mmol) in EtOH (20 mL) cooled in an ice-salt bath. After saturating the reaction mixture with HCl, the mixture was stirred at room temperature for 4 d. The reaction mixture was then concentrated under reduced pressure to produce a yellow mixture of 33 and 34 (482.9 mg), which was used in the next step without further purification.

[0205] Step 5: Synthesis of 4-amino-2-methyl-5-nitrobenzimidamide hydrochloride (Compound 35) To a mixture of Compound 33 and Compound 34 (482.9 mg, 1.86 mmol) in EtOH (4 mL) was added NH3 (7 M in MeOH, 6 mL), and the reaction mixture was refluxed overnight. Then, the mixture was concentrated in vacuo to obtain a crude product, which was purified by silica gel chromatography (10 - 20% MeOH in DCM) to remove unreacted Compound 33, and an orange residue (306.3 mg, 66% over 2 steps) containing Compound 35 was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 2H), 9.04 (s, 1H), 8.14 (s, 1H), 7.85 (s, 2H), 6.94 (s, 1H), 2.31 (s, 3H).

[0206] Step 6: Synthesis of 4,5-diamino-2-methylbenzimidamide hydrochloride (Compound 36) To a solution of Compound 35 (304 mg, 1.32 mmol) in EtOH (30 mL) was added Pd / C (60.8 mg, 10%). Then, the flask was evacuated and purged with H2 three times, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with MeOH. The filtrate was concentrated under reduced pressure to obtain yellow solid 36 (280 mg, quantitative). 1 H NMR (400 MHz, methanol-d4) δ 6.81 (s, 1H), 6.60 (s, 1H), 2.29 (s, 3H).

[0207] Step 7: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-carboxamide (Compound I-6) A solution of compound 13 (61.4 mg, 0.14 mmol), 4,5-diamino-2-methylbenzimidamide hydrochloride 36 (55.1 mg, 0.27 mmol), and p-benzoquinone (29.9 mg, 0.27 mmol) in anhydrous EtOH (6 mL) was heated under reflux for 12 h. The reaction mixture was cooled to room temperature and stirred in acetone (50 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to give a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (13 mL) and EtOH (13 mL), filtered, the volume was reduced to 9 mL, and acidified with saturated HCl-EtOH (0.9 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-6 (26.0 mg, 21%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 9.53 (d, J = 8.0 Hz, 1H), 9.21 (d, J = 8.5 Hz, 1H), 8.30 - 8.27 (m, 2H), 8.20 - 8.16 (m, 2H), 8.07 - 8.00 (m, 3H), 7.97 - 7.84 (m, 3H), 4.97 (dd, J = 8.2, 5.6 Hz, 1H), 4.69 (dd, J = 8.2, 4.8 Hz, 1H), 4.05 - 3.98 (m, 1H), 3.86 - 3.77 (m, 1H), 3.71 - 3.56 (m, 2H), 2.67 (d, J = 4.6 Hz, 6H), 2.56 - 2.47 (m, 1H), 2.44 - 2.36 (m, 1H), 2.28 - 1.94 (m, 6H).

[0208] Example S7: Synthesis of hexyl ((2-(4-((S)-1-((4-(6-(N-((hexyloxy)carbonyl)carbamimidoyl)-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamide)phenyl)-1H-benzo[d]imidazol-6-yl)(imino)methyl)carbamate (Compound I-7)

[0209]

Chem.

[0210] Step 1: Synthesis of hexyl ((3,4-diaminophenyl)(imino)methyl)carbamate (Compound 38) A solution of Compound 14 (1.25 g, 6.70 mmol) in acetone (5 mL) was cooled to 0 °C in an ice / water bath, and then NaOH solution (5 mL, 16 wt%) and Compound 37 (1.1 mL, 6.70 mmol) were slowly added. The reaction mixture was stirred at 0 °C for an additional 1 h. After cooling to room temperature, the mixture was concentrated under reduced pressure, diluted with CHCl3 / i-PrOH (3:1), and then washed with water. The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (2% MeOH in DCM) to give the desired product 38 (926.3 mg, 50%) as a pale yellow solid. 1 1H NMR (400 MHz, methanol-d4) δ 7.23 (d, J = 2.1 Hz, 1H), 7.19 (dd, J = 8.2, 2.1 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 4.10 (t, J = 6.7 Hz, 2H), 1.72 - 1.65 (m, 2H), 1.47 - 1.39 (m, 2H), 1.37 - 1.32 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).

[0211] Step 2: Synthesis of hexyl ((2-(4-((S)-1-((4-(6-(N-((hexyloxy)carbonyl)carbamimidoyl)-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamide)phenyl)-1H-benzo[d]imidazol-6-yl)(imino)methyl)carbamate (Compound I-7) A solution of Compound 13 (37.8 mg, 0.08 mmol), hexyl ((3,4-diaminophenyl)(imino)methyl)carbamate 38 (47.0 mg, 0.16 mmol) and p-benzoquinone (18.4 mg, 0.16 mmol) in anhydrous EtOH (10 mL) was heated under reflux for 12 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to give a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (7.5 mL) and EtOH (7.5 mL), filtered, the volume was reduced to 5 mL, and acidified with saturated HCl-EtOH (1 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to give a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-7 (19.3 mg, 24%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.29 - 8.13 (m, 6H), 7.98 - 7.76 (m, 8H), 4.99 - 4.94 (m, 1H), 4.68 (dd, J = 8.3, 4.7 Hz, 1H), 4.41 (td, J = 6.7, 2.5 Hz, 4H), 4.07 - 3.98 (m, 1H), 3.86 - 3.77 (m, 1H), 3.74 - 3.59 (m, 2H), 2.55 - 2.33 (m, 2H), 2.27 - 1.93 (m, 6H), 1.81 (p, J = 6.8 Hz, 4H), 1.47 (p, J = 6.8 Hz, 4H), 1.38 (h, J = 3.5 Hz, 8H), 0.96 - 0.91 (t, J = 6.9 Hz, 6H).

[0212] Example S8: Synthesis of 4-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzamide)-N-((4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-1-methyl-1H-pyrrole-2-carboxamide (Compound I-8)

[0213]

Chemical formula

[0214] Step 1: Synthesis of 2-(4-nitrophenyl)-1,3-dioxolane (Compound 40) Ethane-1,2-diol 39 (6.6 mL, 0.12 mol) was added to a solution of 4-nitrobenzaldehyde 6 (3.46 g, 22.90 mmol) in DCM (90 mL), and then boron trifluoride diethyl etherate (0.6 mL) was added. After stirring at room temperature for 9 h, the solution was washed with 10% NaOH, water, and brine. The resulting bright yellow solution was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain the desired product 40 (4.14 g, 93%) as a yellow solid. 1 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 5.90 (s, 1H), 4.14 - 4.05 (m, 4H).

[0215] Step 2: Synthesis of 4-(1,3-dioxolan-2-yl)aniline (Compound 41) A solution of 40 (2.45 g, 12.55 mmol) in anhydrous EtOH (150 mL) was added to a mixture of PtO2 (565 mg, 2.49 mmol) and NaHCO3 (1.05 g, 12.50 mmol). Then, the flask was evacuated, purged three times with H2, filled with H2, and stirred at room temperature for 2 h. Subsequently, the reaction mixture was filtered through a diatomaceous earth pad and washed with MeOH. The filtrate was concentrated under reduced pressure to obtain the crude product, which was dissolved in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the desired product 41 (2.03 g, 98%) was obtained as a pale yellow oil. 1 1H NMR (400 MHz, CDCl3) δ 7.28 -7.26 (m, 2H), 6.70 - 6.66 (m, 2H), 5.70 (s, 1H), 4.15 - 4.10 (m, 2H), 4.03 - 3.98 (m, 2H), 3.72 (s, 2H).

[0216] Step 3: Synthesis of 2,2,2-trichloro-1-(1-methyl-1H-pyrrol-2-yl)ethan-1-one (Compound 43) A solution of 1-methyl-1H-pyrrole 42 (7.34 g, 90.48 mmol) in anhydrous ethyl ether (25 mL) was added dropwise to a solution of 2,2,2-trichloroacetyl chloride (16.45 g, 90.47 mmol) in anhydrous ethyl ether (25 mL). The reaction mixture was stirred at room temperature for 1.5 h. Subsequently, a K2CO3 solution (20 mL, 20 mmol) was added dropwise to the mixture to quench it, and it was extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, concentrated in vacuo to obtain the crude product, which was washed with hexane and dried under vacuum to obtain the desired product 43 (13.24 g, 65%) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ 7.51 (dd, J = 4.4, 1.5 Hz, 1H), 6.97 (s, 1H), 6.23 (dd, J = 4.4, 2.4 Hz, 1H), 3.98 (s, 3H).

[0217] Step 4: Synthesis of 2,2,2-trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethan-1-one (Compound 44) Fuming nitric acid (4 mL) was added dropwise to a stirred solution of Compound 43 (10.67 g, 47.11 mmol) in Ac2O (50 mL), and the solution was maintained at -5 °C in an ice / NaCl bath. After the addition was complete, the temperature was gradually raised to room temperature and stirring was continued for an additional 3 h. The reaction mixture was then poured into ice water (200 mL) and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (10 - 50% EtOAc in petroleum ether) to give the desired product 44 as a pale yellow solid (9.46 g, 74%). 1 H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 1.7 Hz, 1H), 7.75 (d, J = 1.3 Hz, 1H), 4.05 (s, 3H).

[0218] Step 5: Synthesis of 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid (Compound 45) Compound 44 (3.10 g, 11.42 mmol) was added to a solution of NaOH (1.37 g, 34.25 mmol) in water (60 mL), and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction mixture was extracted with EtOAc, and the aqueous layer was acidified to pH = 3 with 2M HCl. The resulting solid was filtered and dried under vacuum to give the desired product 45 as a white solid (1.60 g, 82%). 1 H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 4.01 (s, 3H).

[0219] Step 6: Synthesis of N-(4-(1,3-dioxolan-2-yl)phenyl)-1-methyl-4-nitro-1H-pyrrole-2-carboxamide (Compound 46) To a solution of compound 45 (761 mg, 4.47 mmol) and HBTU (2.04 g, 5.38 mmol) in DMF (20 mL) under stirring was added DIPEA (1.5 mL, 9.08 mmol). After stirring at room temperature for 10 min, compound 41 (739 mg, 4.47 mmol) was added and the mixture was stirred for an additional 18 h. After removing the solvent, the residue was dissolved in CHCl3 / i-PrOH (3:1) and washed with water. Then, the organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give yellow solid 46 (1.42 g), which was used in the next step without further purification.

[0220] Step 7: Synthesis of N-(4-(1,3-dioxolan-2-yl)phenyl)-4-amino-1-methyl-1H-pyrrole-2-carboxamide (compound 47) To a solution of compound 46 (1.42 g, 4.47 mmol) in DMF (50 mL) was added Pd / C (1.42 g, 10%). Then, the flask was evacuated and purged with H2 three times, filled with H2 and stirred at room temperature for 18 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with MeOH. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (0.5 - 1% MeOH in DCM) to afford the desired product 47 (741.3 mg, 58% over 2 steps) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.56 (dd, J = 5.2, 3.2 Hz, 3H), 7.44 (d, J = 8.5 Hz, 2H), 6.34 (d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 5.78 (s, 1H), 4.14 - 4.09 (m, 2H), 4.05 - 4.00 (m, 2H), 3.85 (s, 3H), 2.93 (s, 2H).

[0221] Step 8: Synthesis of N-(4-(1,3-dioxolan-2-yl)phenyl)-1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamide (compound 48) To a stirred solution of compound 45 (207 mg, 1.22 mmol) and HBTU (555 mg, 1.46 mmol) in DMF (15 mL) was added DIPEA (0.5 mL, 3.03 mmol). After stirring for 10 min at room temperature, compound 47 (350 mg, 1.22 mmol) was added and the mixture was stirred for an additional 18 h. After removing the solvent, the residue was dissolved in CHCl3 / i-PrOH (3:1) and washed with water. Then, the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give yellow solid 48 (530 mg), which was used in the next step without further purification.

[0222] Step 9: Synthesis of N-(4-(1,3-dioxolan-2-yl)phenyl)-4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamide (Compound 49) To a solution of compound 48 (530 mg, 1.21 mmol) in DMF (50 mL) was added Pd / C (800 mg, 10%). The flask was then evacuated, purged with H2 three times, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with MeOH. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (2 - 5% MeOH in DCM) to give the desired product 49 (298.2 mg, 60% over 2 steps) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J = 1.7 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 6.18 (d, J = 2.0 Hz, 1H), 5.80 (s, 1H), 4.15 - 4.12 (m, 2H), 4.07 - 4.01 (m, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 2.97 (s, 2H).

[0223] Step 10: Synthesis of 4-(4-Formylbenzamide)-N-(5-((4-Formylphenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-1-methyl-1H-pyrrole-2-carboxamide (Compound 50) At 0 °C, a solution of Compound 2 (41 mg, 0.24 mmol) in DCM (6 mL) was slowly added to a solution of Compound 49 (100 mg, 0.24 mmol) in DCM (6 mL) and TEA (60 μL). Then, the mixture was warmed to room temperature and stirred for 12 h. After removing the solvent, the residue was dissolved in EtOAc and washed with aqueous HCl solution (1 M, 3 × 20 mL) and saturated aqueous NaHCO3. Then, the organic part was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (2 - 5% MeOH in DCM) to obtain the target product 50 (72.8 mg, 60% in 2 steps) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 10.27 (s, 1H), 10.11 (s, 1H), 10.08 (s, 1H), 9.89 (s, 1H), 8.13 (d, J = 8.2 Hz, 2H), 8.05 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.39 - 7.36 (m, 2H), 7.27 (d, J = 1.5 Hz, 1H), 7.15 (d, J = 1.5 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H).

[0224] Step 11: Synthesis of 4-(4-(6-Carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzamide)-N-(5-((4-(6-Carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-1-methyl-1H-pyrrole-2-carboxamide (Compound I-8) A solution of compound 50 (33.4 mg, 0.067 mmol), 3,4-diaminobenzimidamide hydrochloride 14 (25 mg, 0.13 mmol), and p-benzoquinone (14.6 mg, 0.13 mmol) in anhydrous EtOH (6 mL) was heated under reflux for 12 h. The reaction mixture was cooled to room temperature and stirred in acetone (30 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (5 mL) and EtOH (5 mL), filtered, the volume was reduced to 4 mL, and it was acidified with saturated HCl-EtOH (0.6 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-8 (21.9 mg, 36%) as a brown solid. 1 H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.38 (s, 1H), 10.11 (s, 1H), 9.63 (s, 2H), 9.54 (s, 2H), 9.35 (s, 2H), 9.28 (s, 2H), 8.56 (d, J = 8.0 Hz, 2H), 8.50 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 5.4 Hz, 2H), 8.24 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 7.99 - 7.89 (m, 3H), 7.84 (d, J = 8.4 Hz, 1H), 7.45 - 7.31 (m, 3H), 7.23 (d, J = 10.2 Hz, 1H), 3.90 (d, J = 4.5 Hz, 6H).

[0225] Example S9: Synthesis of 4-(3-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzamido)propionamido)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)-1-methyl-1H-pyrrole-2-carboxamide (Compound I-9)

[0226]

Chem.

[0227] Step 1: Synthesis of (9H-Fluoren-9-yl)methyl (3-((5-((4-(1,3-Dioxolan-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-3-oxopropyl)carbamate (Compound 52) DIPEA (0.2 mL, 1.21 mmol) was added to a stirred solution of Fmoc-β-alanine 51 (115 mg, 0.37 mmol) and HBTU (167 mg, 0.44 mmol) in DMF (20 mL). After stirring at room temperature for 10 minutes, compound 47 (105 mg, 0.37 mmol) was added and the mixture was stirred for an additional 14 h. After removing the solvent, the residue was dissolved in CHCl3 / i-PrOH (3:1) and washed with water. Then, the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give orange solid 52 (212 mg), which was used in the next step without further purification.

[0228] Step 2: Synthesis of N-(4-(1,3-Dioxolan-2-yl)phenyl)-4-(3-aminopropionamido)-1-methyl-1H-pyrrole-2-carboxamide (Compound 53) Piperidine (0.34 mL) was added to a solution of compound 52 (212 mg, 0.36 mmol) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 1 h. After removing the solvent, the crude residue 53 (130 mg) was used in the next step without further purification.

[0229] Step 3: Synthesis of 4-(3-(4-Formylbenzamido)propionamido)-N-(4-formylphenyl)-1-methyl-1H-pyrrole-2-carboxamide (Compound 54) At 0 °C, a solution of compound 2 (62 mg, 0.37 mmol) in DCM (9 mL) was slowly added to a solution of compound 53 (130 mg, 0.36 mmol) in DCM (9 mL) and TEA (90 μL). Then, the mixture was warmed to room temperature and stirred for an additional 24 h. After removing the solvent, the residue was dissolved in EtOAc and washed with aqueous HCl solution (1 M, 3 × 20 mL) and saturated aqueous NaHCO3. Then, the organic portion was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (0 - 2% MeOH in DCM) to afford the desired product 54 (54.8 mg, 34% over 3 steps) as a white solid. 1 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 10.07 (s, 1H), 10.00 (s, 1H), 9.88 (s, 1H), 8.83 (t, J = 5.4 Hz, 1H), 8.04 -7.95 (m, 6H), 7.86 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 1.5 Hz, 1H), 7.06 (d, J = 1.5 Hz, 1H), 3.85 (s, 3H), 3.57 (dd, J = 12.7, 6.8 Hz, 2H), 2.59 (t, J = 7.0 Hz, 2H).

[0230] Step 4: Synthesis of 4-(3-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)benzamido)propionamido)-N-(4-(6-carbamimidoyl-1H-benzo[d]imidazol-2-yl)phenyl)-1-methyl-1H-pyrrole-2-carboxamide (Compound I-9) A solution of compound 54 (80 mg, 0.18 mmol), 3,4-diaminobenzimidamide hydrochloride 14 (67 mg, 0.36 mmol), and p-benzoquinone (39 mg, 0.36 mmol) in anhydrous EtOH (7 mL) was heated under reflux for 10 h. The reaction mixture was cooled to room temperature and stirred in acetone (40 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (15 mL) and EtOH (15 mL), filtered, the volume was reduced to 10 mL, and acidified with saturated HCl-EtOH (1 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-9 (68 mg, 44%) as a brown solid. 1 H NMR (500 MHz, DMSO-d6) δ 10.34 (s, 1H), 10.17 (s, 1H), 9.64 (s, 2H), 9.54 (s, 2H), 9.34 (s, 2H), 9.26 (s, 2H), 8.89 (t, J = 5.5 Hz, 1H), 8.46 (dd, J = 11.2, 8.4 Hz, 4H), 8.27 (s, 2H), 8.11 (d, J = 8.2 Hz, 2H), 8.07 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H), 7.16 (s, 1H), 3.86 (s, 3H), 3.60 (q, J = 6.7 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H).

[0231] Example S10: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-methoxy-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-10)

[0232] [Chemical formula]

[0233] Step 1: Synthesis of 4-amino-2-fluoro-5-nitrobenzonitrile (Compound 56) At 0 °C, NH4OH (6.5 mL) was added to a solution of 2,4-difluoro-5-nitrobenzonitrile 55 (2.2 g, 11.95 mmol) in EtOH (1.5 mL). The resulting mixture was stirred at room temperature for 6 h. Then, the resulting precipitate was filtered and dried under vacuum to obtain the desired product 56 (2.21 g, 98%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 7.0 Hz, 1H), 8.24 (s, 2H), 6.88 (d, J = 11.9 Hz, 1H).

[0234] Step 2: Synthesis of 4-amino-2-fluoro-5-nitroethyl benzimidate hydrochloride (Compound 57) Dry HCl gas was passed through a suspension of Compound 56 (1.45 g, 8.00 mmol) in EtOH (40 mL) with stirring. The reaction mixture was saturated with HCl and stirred at room temperature for 36 h. Then, the reaction mixture was diluted with anhydrous ethyl ether. The orange solid imidate precipitate was filtered, washed with ethyl ether, and dried under vacuum to obtain orange solid 57 (1.88 g), which was used in the next step without further purification.

[0235] Step 3: Synthesis of 4-amino-2-methoxy-5-nitrobenzimidamide hydrochloride (Compound 58) A suspension of compound 57 (278 mg, 1.05 mmol) in MeOH (3 mL) during stirring was added with NH3 (7 M in MeOH, 3 mL), and the reaction mixture was refluxed overnight. Then, the reaction mixture was concentrated under vacuum and diluted with ethyl ether. It was filtered to obtain a precipitate, which was washed with ethyl ether and dried under vacuum to obtain yellow solid 58 (306.9 mg), which was used in the next step without further purification.

[0236] Step 4: Synthesis of 4,5-diamino-2-methoxybenzimidamide hydrochloride (compound 59) Pd / C (20 mg, 10%) was added to a solution of compound 58 (170 mg, 1.32 mmol) in EtOH (10 mL). Then, the flask was evacuated and purged with H2 three times, filled with H2, and stirred at room temperature for 18 h. The reaction mixture was filtered through a diatomaceous earth pad and washed with MeOH. The filtrate was concentrated under reduced pressure to obtain the desired product 59 (130.2 mg, 92% over 3 steps) as a yellow solid. 1 H NMR (400 MHz, Methanol-d4) δ 7.02 (s, 1H), 6.48 (s, 1H), 3.87 (s, 3H).

[0237] Step 5: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-methoxy-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-10) A solution of compound 13 (52 mg, 0.12 mmol), 4,5-diamino-2-methoxybenzimidamide hydrochloride 59 (50 mg, 0.23 mmol), and p-benzoquinone (25 mg, 0.23 mmol) in anhydrous EtOH (5 mL) was heated under reflux for 12 h. The reaction mixture was cooled to room temperature and stirred in acetone (50 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (10 mL) and EtOH (10 mL), filtered, the volume was reduced to 7 mL, and it was acidified with saturated HCl-EtOH (0.7 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried under vacuum. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-10 (29.8 mg, 28%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.23 (d, J = 8.2 Hz, 2H), 8.15 (d, J = 8.9 Hz, 2H), 8.03 - 7.97 (m, 4H), 7.88 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 15.1 Hz, 2H), 4.96 (dd, J = 8.3, 5.8 Hz, 1H), 4.68 (dd, J = 8.3, 4.7 Hz, 1H), 4.06 (s, 3H), 4.05 (s, 3H), 4.04 - 3.98 (m, 1H), 3.86 - 3.76 (m, 1H), 3.72 - 3.58 (m, 2H), 2.56 - 2.45 (m, 1H), 2.44 - 2.35 (m, 1H), 2.27 - 1.92 (m, 6H).

[0238] Example S11: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-fluoro-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-11)

[0239]

Chem.

[0240] Step 1: Synthesis of Ethyl 4,5-diamino-2-fluorobenzimidate Hydrochloride (Compound 60) To a solution of compound 57 (350 mg, 1.33 mmol) in EtOH (30 mL) was added Pd / C (40 mg, 10%). Then, the flask was evacuated and purged with H2 three times, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with MeOH. The filtrate was concentrated under reduced pressure to give orange solid 60 (323.4 mg), which was used in the next step without further purification.

[0241] Step 2: Synthesis of 4,5-diamino-2-fluorobenzimidamide Hydrochloride (Compound 61) To a suspension of compound 60 (320 mg, 1.37 mmol) in MeOH (15 mL) was added NH3 (7 M in MeOH, 2 mL), and the reaction mixture was refluxed overnight. Then, the reaction mixture was concentrated in vacuo, diluted with ethyl ether. Filtration gave a precipitate, which was washed with ethyl ether and dried in vacuo to give the desired product 61 (248.1 mg, 91% over 2 steps) as a reddish-brown solid. 1 1H NMR (400 MHz, methanol-d4) δ 6.91 (d, J = 7.1 Hz, 1H), 6.50 (d, J = 13.5 Hz, 1H).

[0242] Step 3: Synthesis of (S)-1-((4-(6-carbamimidoyl-5-fluoro-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-11) A solution of compound 13 (67.1 mg, 0.15 mmol), 4,5-diamino-2-fluorobenzimidamide hydrochloride 61 (61.4 mg, 0.30 mmol), and p-benzoquinone (32.7 mg, 0.30 mmol) in anhydrous EtOH (12 mL) was heated under reflux for 16 h. The reaction mixture was cooled to room temperature and stirred in acetone (80 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether, and dried to obtain a brown solid. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (13.2 mL) and EtOH (13.2 mL), filtered, the volume was reduced to 9 mL, and it was acidified with saturated HCl-EtOH (1.8 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether, and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-11 (17.4 mg, 13%) as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.31 - 8.18 (m, 6H), 8.06 - 7.85 (m, 6H), 5.00 - 4.95 (m, 1H), 4.69 (dd, J = 8.3, 4.7 Hz, 1H), 4.05 - 3.98 (m, 1H), 3.87 - 3.76 (m, 1H), 3.71 - 3.56 (m, 2H), 2.57 - 2.47 (m, 1H), 2.46 - 2.35 (m, 1H), 2.29 - 1.93 (m, 6H).

[0243] Example S12: Synthesis of (S)-1-((4-(6-carbamimidoyl-1-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-12)

[0244]

Chemical Structure

[0245] Step 1: Synthesis of 4-(methylamino)-3-nitrobenzonitrile (Compound 63) To a suspension of 4-chloro-3-nitrobenzonitrile 62 (1.5 g, 8.22 mmol) in EtOH (6 mL) was added CH3NH2 (27 - 32% in EtOH, 1.5 mL). The reaction mixture was stirred at room temperature for 1 h and then refluxed overnight. The reaction mixture was cooled and concentrated in vacuo. The residue was suspended in ethyl ether, filtered to obtain the crude product, and purified by silica gel chromatography (pure DCM) to give the desired product 63 (936.3 mg, 64%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 6.8 Hz, 1H), 8.50 (d, J = 2.0 Hz, 1H), 7.84 (ddd, J = 9.0, 2.1, 0.8 Hz, 1H), 7.11 (d, J = 9.1 Hz, 1H), 3.00 (d, J = 5.0 Hz, 3H).

[0246] Step 2: Synthesis of 4-(methylamino)-3-nitroethylbenzimidate hydrochloride (Compound 64) Dry HCl gas was passed through a stirred suspension of compound 63 (710 mg, 8.00 mmol) in EtOH (20 mL) cooled in an ice-salt bath. The reaction mixture was saturated with HCl and stirred at room temperature for 48 h. Then the reaction mixture was diluted with anhydrous ethyl ether. The imidate precipitate as an orange solid was filtered, washed with ethyl ether, and dried in vacuo to give orange solid 64 (1.08 g), which was used in the next step without further purification.

[0247] Step 3: Synthesis of 4-(methylamino)-3-nitrobenzimidamide hydrochloride (Compound 65) A suspension of compound 64 (1.08 g, 4.16 mmol) in MeOH (20 mL) was added with NH3 (7 M in MeOH, 3 mL), and the reaction mixture was stirred at room temperature overnight. Then, the reaction mixture was concentrated in vacuo and diluted with ethyl ether. It was filtered to obtain a precipitate, which was washed with ethyl ether and dried in vacuo to give the desired product 65 (945.8 mg, quantitative in 2 steps) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 2H), 8.98 (s, 2H), 8.70 (d, J = 2.4 Hz, 1H), 8.68 (d, J = 5.2 Hz, 1H), 7.98 (dd, J = 9.2, 2.4 Hz, 1H), 7.17 (d, J = 9.3 Hz, 1H), 3.03 (d, J = 5.0 Hz, 3H).

[0248] Step 4: Synthesis of 3-amino-4-(methylamino)benzimidamide hydrochloride (Compound 66) To a solution of compound 65 (686.8 mg, 3.00 mmol) in EtOH (30 mL) was added Pd / C (70 mg, 10%). Then, the flask was evacuated and purged with H2 three times, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with MeOH. The filtrate was concentrated under reduced pressure to give the desired product 66 (556.2 mg, 93%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 2H), 8.42 (s, 2H), 7.12 (dd, J = 8.3, 2.3 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 6.47 (d, J = 8.4 Hz, 1H), 5.76 (d, J = 5.1 Hz, 1H), 4.88 (s, 2H), 2.80 (d, J = 4.7 Hz, 3H).

[0249] Step 5: Synthesis of (S)-1-((4-(6-carbamimidoyl-1-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-carbamimidoyl-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (Compound I-12) A solution of Compound 13 (135.4 mg, 0.30 mmol), 3-amino-4-(methylamino)benzimidamide hydrochloride 66 (121.4 mg, 0.60 mmol) and p-benzoquinone (65.9 mg, 0.60 mmol) in anhydrous EtOH (12 mL) was heated under reflux for 6 h. The reaction mixture was cooled to room temperature and stirred in acetone (100 mL) for 0.5 h. The mixture was filtered, washed with anhydrous ethyl ether and dried to obtain the hydrochloride salt. Then, the solid was dissolved in a hot 1:1 mixture of MeOH (26 mL) and EtOH (26 mL), filtered, the volume was reduced to 18 mL and acidified with saturated HCl-EtOH (1.8 mL). After stirring overnight at room temperature, the mixture was diluted with ethyl ether, filtered to obtain a precipitate, washed with ethyl ether and dried in vacuo. The crude product was purified by preparative reverse-phase HPLC (5 - 100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-12 (141.6 mg, 53%) as a pink solid. 1 H NMR (400 MHz, methanol-d4) δ 8.30 (dd, J = 6.9, 1.3 Hz, 2H), 8.14 (d, J = 8.8 Hz, 1H), 8.06 - 7.99 (m, 6H), 7.98 - 7.89 (m, 5H), 4.98 (dd, J = 8.2, 5.7 Hz, 1H), 4.70 (dd, J = 8.2, 4.8 Hz, 1H), 4.16 (s, 3H), 4.10 (s, 3H), 4.07 - 3.99 (m, 1H), 3.87 - 3.78 (m, 1H), 3.74 - 3.60 (m, 2H), 2.57 - 2.47 (m, 1H), 2.46 - 2.36 (m, 1H), 2.30 - 1.96 (m, 6H). Biological Examples

[0250] Example B1: Biological Evaluation of the Efficacy of a PU.1 Inhibitor Using an acute T-cell lymphoblastic leukemia (T-ALL) disease model, the effect of a novel synthetic compound on PU.1 was evaluated. Pten, a well-known tumor suppressor gene, is absent in 40% of mouse hematopoietic stem cells and their differentiated progeny, resulting in progressive T-ALL at approximately 2 months of age. The immune checkpoint T-cell immunoglobulin mucin 3 (TIM-3), a surface marker for isolating pure leukemia-initiating cells (LICs), is thought to be transcriptionally regulated by the transcription factor PU.1 in the Pten-null T-ALL model. Therefore, the expression level of TIM-3 was detected and quantified to characterize the inhibitory effect of the compound after treatment with gradient concentrations of the compound for 24 hours. The blasts were transfected with a PU.1-EGFP vector or an EGFP vector to generate stable cell lines, blast-PU.1 and blast-EGFP, respectively. These cell lines were used for in vitro compound testing. Compounds DB1976 and DB2115 were also tested for comparison. blast-PU.1 and blast-EGFP are ideal cell lines for in vitro test compounds because they are T-ALL blasts with low expression levels of TIM-3 and PU.1. In compound I-1, when the flexible alkyl linker of DB2115 was replaced with rigid L-proline, the efficacy was significantly improved. Compound I-1 downregulated the expression level of TIM-3 by 40% at 10 nM in the Blast-PU.1 cell line (Figure 1b), because the TIM-3 level was too low to be detected in the DMSO and compound-treated groups (data not shown). On the other hand, its D,D-proline analog, compound I-2, and D,L-proline analog, compound I-3, did not show significant inhibitory effects at 10 μM and were inferior to compound I-1 (Figure 1b). In short, compound I-1 showed the most excellent activity in PU.1-mediated TIM-3 inhibition, and it was concluded that compound I-1 would be useful for future studies on biological functions.

[0251] Example B2: Effect of delaying the progression of leukemia by the combined use of compound I-1 and rapamycin To generate a Pten-null T-ALL mouse model, Pten was depleted by 40% in mouse fetal liver hematopoietic stem cells (HSCs), followed by activation of the PI3K-AKT pathway, hematopoietic disorders, and the development of T-ALL. At the T-ALL onset stage (crisis stage), T-ALL blasts and LICs infiltrate the hematopoietic and non-hematopoietic organs of the mouse. T-ALL mice were treated with a combination of rapamycin (a well-studied PI3K-AKT pathway inhibitor that shows promising effects in targeting T-ALL blasts) and compound I-1. Treatment was initiated at the blast onset stage and terminated 62 days after birth, and the direct effects of the compound that inhibits blasts and high TIM-3 LICs were observed. In the 2-day treatment, the proportion of blasts and live lymphocytes did not decrease with compound I-1 alone, but rapamycin showed high efficiency in targeting blasts, and the blasts decreased from 96.4% to 13.2% (Figure 2a). The combination treatment more significantly decreased the proportion of blasts in the bone marrow (Figure 2a), spleen, and thymus. In the high TIM-3 LIC group, treatment with compound I-1 alone significantly decreased the LIC proportion from 33.5% to 6.15% compared with the rapamycin treatment group (22.2%). Importantly, compared with the other three groups, the combination treatment showed a significant effect on the decrease in blasts and LICs. Previously, in a T-ALL mouse model using DB1976 and a PI3K inhibitor, it has been shown that targeting both blasts and LICs can reduce tumor burden. To test whether compound I-1 can achieve this, T-ALL mice were treated with compound I-1 and / or rapamycin for 1 month during the blast onset period. After treatment, the morphology of the hematopoietic and non-hematopoietic organs of the mice was analyzed using hematoxylin-eosin (H&E) staining. In the group treated with compound I-1 alone, the organ morphology showed no significant change compared to the T-ALL group, and targeting only LICs did not reduce the tumor burden. In contrast, in the rapamycin group, an improved therapeutic effect was shown because blasts are the main population of leukemia cells. In the combination treatment group, the morphology of the thymus and spleen recovered, and the infiltration of leukemia cells into the lungs, kidneys, and liver decreased significantly (Figure 2b). Studies have revealed that after Pten deficiency, B cell development is inhibited at the pro-B stage. As shown in Figure 2c, a small number of B220-positive cells were observed in the spleens of T-ALL mice. However, in mouse spleen B220 immunohistochemical slide glasses, the B cell population was rescued after combined treatment with compound I-1 and rapamycin (Figure 2c). In B lineage cells, PU.1 is a major regulatory factor controlling lineage commitment, and the expression of PU.1 gradually increases from pro-B cells to B cells. The expression levels of PU. and / or its downstream genes in the combination treatment group recovered normally, suggesting that lymphoid lineage commitment and cell differentiation may proceed normally. Furthermore, the combination of compound I-1 and rapamycin prolonged mouse survival compared to treatment with compound I-1 alone, rapamycin alone, or the combination of DB1976 and rapamycin (Figure 2d).

[0252] Example B3: Preventive and Therapeutic Effects of Compound I-1 on Scleroderma Method: To evaluate the preventive and therapeutic effects of compound I-1 on scleroderma, two different drug-interfered scleroderma animal models (6 - 8 weeks old, C57BL / 6, male) were created using bleomycin. Every other day, in a limited skin zone on the upper back that had been depilated (about 1 cm 2) Bleomycin (0.5 mg / mL, 0.1 mL / animal) was locally injected to induce skin fibrosis. As a control, physiological saline was subcutaneously injected. (I) Prevention model of bleomycin-induced skin fibrosis: Compound I-1, positive control DB1976 or vehicle (physiological saline) was intraperitoneally injected simultaneously with bleomycin for 4 weeks (Figure 3a). (II) Treatment model of bleomycin-induced skin fibrosis: After bleomycin was precharged in mice for 3 weeks to induce skin fibrosis, the mice were further treated with Compound I-1, positive control DB1976 or vehicle (physiological saline) for another 3 weeks, and the total time was set to 6 weeks after the first bleomycin treatment (Figure 3f). After the last day of treatment in both models, the mice were fasted overnight and euthanized. After a part of the skin was completely flattened on the foil, it was fixed with paraformaldehyde, embedded in paraffin, sliced, and stained with H&E, Sirius red, and Masson to examine the pathological characteristics. The epidermal thickness of each sample was quantitatively calculated using image J. Other parts of the skin were collected, rapidly frozen in liquid nitrogen, stored at -80 °C, subjected to RNA isolation (Code.R6934, OMEGA, USA), first-strand cDNA reverse transcription (first cDNA reverse, Code.AT341, TransGen, China), and SYBR mixing (Code.AQ601, TransGen, China), and Q-PCR (LightCycler(R)96, Roche) was performed to verify the mRNA levels of several fibrosis-related genes (such as Col1a1 and Col1a2). As a result, in the bleomycin-induced scleroderma prevention model, compared with the normal saline / vehicle group, when bleomycin was treated for 4 weeks, pathological features of scleroderma including increased skin epidermal thickness, collagen deposition, and increased levels of Col1a1 and Col1a2 mRNA were significantly induced, suggesting that the bleomycin-induced scleroderma model was successfully constructed. Treatment with Compound I-1 or DB1976 significantly blocked the progression of scleroderma, reduced epidermal thickness and collagen deposition, and decreased the mRNA levels of Col1a1 and Col1a2 compared with the bleomycin / vehicle group (Figures 3a-3e). In the bleomycin-induced scleroderma treatment model, treatment with bleomycin for 6 weeks significantly induced pathological features of scleroderma, producing a thicker epidermis, more collagen deposition, and higher levels of Col1a1 and Col1a2 mRNA compared with the normal saline / vehicle group, further suggesting that the scleroderma model induced by bleomycin stimulation was successfully constructed. Treatment with Compound I-1 or DB1976 for 3 weeks significantly alleviated and recovered bleomycin-induced scleroderma (Figures 3f-3j). These data suggest that Compound I-1 has preventive and therapeutic effects against bleomycin-induced scleroderma.

[0253] Example B4: Preventive and Therapeutic Effects of Compound I-1 against Pulmonary Fibrosis Methods: To evaluate the preventive and therapeutic effects of Compound I-1 on pulmonary fibrosis, two different drug-interfered pulmonary fibrosis animal models (6-8 weeks old, C57BL / 6, male) were constructed using bleomycin. Bleomycin (0.025 U, Code.D11063, OKA, China) was injected by single intratracheal administration. An equal volume of sterile saline was used as a control. (I) Preventive model of bleomycin-induced pulmonary fibrosis: Immediately after a single injection of bleomycin, the animals were treated with Compound I-1, positive control DB1976, or vehicle (saline) for 4 weeks (intraperitoneal injection, i.p.) (Figure 4a). (II) Therapeutic model of bleomycin-induced pulmonary fibrosis: Mice were pre-charged with bleomycin for 11 days to induce pulmonary fibrosis, and then treated with Compound I-1, positive control DB1976, or vehicle (saline) for 17 days, with a total time of 4 weeks after bleomycin treatment (Figure 4h). After the last day of treatment in both models, the mice were fasted overnight and euthanized. A part of the lung was fixed with paraformaldehyde, embedded in paraffin, sliced, and subjected to H&E staining and Sirius red staining to examine pathological features and Ashcroft scores (Hubner, R. H. et al. Biotechniques 44, 507-511, 514-507, doi:10.2144 / 000112729 (2008)). The other part of the lung was collected, rapidly frozen in liquid nitrogen, stored at -80°C, and subjected to RNA isolation (Code.R6934, OMEGA, USA), first-strand cDNA reverse transcription (Code.AT341, TransGen, China), SYBR mixing (Code.AQ601, TransGen, China), and Q-PCR (LightCycler(R)96, Roche) to verify the mRNA levels of several fibrosis-related genes (such as Col1a1, Col1a2). As a result, as shown in Figure 4, compared with the saline / vehicle group, 4 weeks of treatment with bleomycin significantly induced pathological features of pulmonary fibrosis, including pulmonary deterioration, collagen deposition, alveolar wall thickening, and destruction of the alveolar structure, suggesting that the bleomycin-induced pulmonary fibrosis model was successfully established. Treatments with Compound I-1 and DB1976 significantly blocked the progression of pulmonary fibrosis compared with the bleomycin / vehicle group, as measured by pathological changes based on staining and Ashcroft score, collagen deposition shown by Sirius red staining, and levels of Col1a1 and Col1a2 mRNA (Figure 4a-4g). In the treatment model, treatment with Compound I-1 restored and blocked bleomycin-induced pulmonary fibrosis, including the above pathological features (Figure 4h-4n). These data suggest that Compound I-1 has preventive and therapeutic effects against bleomycin-induced pulmonary fibrosis.

[0254] Example B5: Effect of treatment with Compound I-1 on NASH and hepatic fibrosis Methods: To evaluate the potential therapeutic effect of Compound I-1 on liver diseases (including hepatic steatosis and accumulation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis), three types of mouse models (C57BL / 6, male, 8 weeks old) were used. (I) NASH diet-induced NASH model (Code.TD.160785, ENVIGO, USA): All mice were given a NASH diet for 10 weeks. Then, the mice were randomly divided into three groups, and each group was administered vehicle, Compound I-1 (2.5 mpk or 5 mpk, i.p.), or DB1976 (2.5 mpk, i.p., positive control) daily for 6 weeks while continuing the NASH diet. The group fed a normal diet was treated with vehicle as a healthy control. (II) NASH model induced by the combination of high-fat diet (HFD) and CCl4 (low dose): Mice were given a normal diet or HFD (60% kcal fat - D12492, research diet) for 10 weeks, and then the HFD mice were randomly divided into two groups according to the rule of minimum body weight difference. Each group was injected with CCl4 (25% v / v in olive oil, 0.5 mL / kg body weight) or pure olive oil (i.p.) twice a week for 4 weeks continuously. While continuing the continuous HFD, Compound I-1 or vehicle (saline) was injected once a day (i.p.) for 4 weeks, and CCl4 was also injected. (III) CCl4 (high dose)-induced liver fibrosis model: To induce liver fibrosis, CCl4 (20% v / v in olive oil, 10 mL / kg body weight) was injected twice a week for 6 weeks. Simultaneously with the CCl4 administration, Compound I-1 or vehicle was injected once a day (i.p.) for 6 weeks. The mice in the above three types of models were observed daily. After the last day of treating all models, the mice were fasted overnight and euthanized. After centrifuging the whole blood at 4°C, the serum was collected, and the biochemical parameters of the blood were detected using an automatic biochemical analyzer (BS-240VET, Mindray). Some liver tissues were fixed with paraformaldehyde, embedded in paraffin, sliced, and the pathological features and NAFLD score were examined by H&E staining or Sirius red staining (Kleiner, D. E. et al. Hepatology 41, 1313 - 1321, doi:10.1002 / hep.20701 (2005)).Fresh liver tissue sections were taken, embedded in OCT compound, and sliced. After fixation of the sections in a PBS solution of 4% paraformaldehyde, they were stained with 0.5% Oil Red O according to standard procedures. Other parts of the liver tissue were collected, snap-frozen in liquid nitrogen, and stored at -80 °C. RNA isolation (Code.R6934, OMEGA, USA), first-strand cDNA reverse transcription (Code.AT341, TransGen, China), and SYBR mixture (Code.AQ601, TransGen, China) were performed, and Q-PCR (LightCycler(R)96, Roche) was carried out to verify the mRNA levels of fibrosis-related genes such as Col1a1 and Col1a2, and inflammation-related genes such as IL-6 and IL-1β. Result 1: In the NASH diet-induced NASH model, as shown in Figure 5, a 16-week NASH diet significantly increased body weight and liver / body ratio (Figures 5b - 5c). A large amount of fat accumulation in the liver containing larger and more fat droplets was induced based on pathological staining (Figures 5d - 5f). Furthermore, the application of the NASH diet increased not only serum parameters such as ALT, LDL-C, and total cholesterol (TC) (Figures 5g - 5i), but also inflammation- and fibrosis-related genes such as IL-6, IL-1β, and Col1a1, Col1a2 (Figures 5j - 5m). Treatment with Compound I-1 (5 mpk) and DB1976 (2.5 mpk) for 6 weeks could alleviate the above-mentioned metabolic disorders caused by the NASH diet. Compound I-1 (2 mpk) showed a lower therapeutic effect compared with DB1976 (2.5 mpk), but also showed a tendency to alleviate metabolic disorders such as a decrease in fat accumulation in the liver and a decrease in the mRNA levels of IL-6, IL-1β, Col1a1, and Col1a2. These data suggest that Compound I-1 has the potential to treat liver fat accumulation, inflammation, and NASH. Result 2: In the NASH model induced by the combination of a high-fat diet (HFD) and CCl4 (low dose), as shown in Figure 6, after HFD pretreatment and 6 weeks of CCl4 treatment, the body weight, weights of gonadal white adipose tissue (gWAT) and inguinal white adipose tissue (iWAT) were significantly increased, but these were reversed by treatment with DB1976 and I-1 (Figure 6b - 6d). HFD / CCL4 treatment induced lipid abnormalities in mice, and the application of DB1976 and I-1 decreased serum triglyceride (TG) and total cholesterol (TC) (Figure 6e - 6f). Furthermore, in histological detection by H&E staining, I-1 was unable to effectively reduce fat accumulation (Figure 6g), which was consistent with the hepatic steatosis score (Figure 6h), but was shown to effectively reduce the inflammatory response, as demonstrated by the decrease in the levels of hepatic mRNA of inflammatory infiltration (Figure 6g), inflammation score (Figure 6i), IL-1β (Figure 6j), and IL-6 (Figure 6k). In addition, the administration of DB1976 and I-1 significantly alleviated hepatic fibrosis induced by the application of HFD / CCL4, as shown by collagen deposition in Sirius red staining (Figure 6g and 6l), and decreased the levels of hepatic mRNA of Col1a1 (Figure 6m) and Col1a2 (Figure 6n). On the other hand, I-1 treatment showed a tendency to decrease serum ALT levels (Figure 6o), indicating no hepatotoxicity at the effective concentration of I-1. These data suggest that compound I-1 exhibits anti-inflammatory and anti-fibrotic potential in HFD / CCL4-induced NASH and hepatic fibrosis mice. Result 3: In CCl4 (high dose)-induced liver fibrosis, as shown in Fig. 7, by Sirius red staining and H&E staining, 6-week treatment with CCl4 significantly induced robust CCl4-induced liver fibrosis parameters such as massive collagen deposition, a high degree of fibrosis (Fig. 7b - 7c and 7f), and inflammatory responses. The use of CCl4 also significantly increased inflammatory and fibrosis-related genes, IL-6, IL-1β (Fig. 7g - 7h) and Col1a1, Col1a2 (Fig. 7d - 7e), except for the AST level in serum (Fig. 7i). Treatment with Compound I-1 (5 mpk or 10 mpk) alleviated CCl4-induced liver fibrosis, significantly decreased collagen deposition and Sirius red positive area, and improved the levels of abnormal mRNAs and blood biochemistry induced by the use of CCl4. These data suggest that Compound I-1 has potential for prevention and treatment against liver fibrosis. All publications, including patents, patent applications, and scientific papers described herein, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including the patent, patent application, or scientific paper, were specifically and individually indicated to be incorporated by reference.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (Wherein, x and x' are each independently 0 or 1; R １ and R ２ are each independently, -R ａ wherein, R ａ are each independently hydrogen; y and y' are each independently 1; R ３ is [Chemical Formula 2] and wherein, R ５ is NH and R ６ and R ７ are each independently hydrogen or -C(O)OR ｄ wherein R ｄ is C １～１２ alkyl; R ４ is 【Chemical Formula 3】 and wherein, R' ５ is NH and R' ６ and R' ７ are each independently hydrogen or -C(O)OR ｄ wherein R ｄ is C １～１２ alkyl; X is NH and X' is NH; A is -C(O)-, the symbol C in formula (I) is -NH-, and [Chemical Formula 4] is, 【Chemical Formula 5】 or; or A is -NHC(O)-, the symbol C in formula (I) is a chemical bond, B is -NHC(O)-, n is 2, and Z is a 5- to 12-membered heteroaryl, substituted with R ｃ wherein R ｃ is C １～６ alkyl. )

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following group. 【Chemical Formula 6】

3. A method for producing a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, comprising converting a compound of formula (II) or a pharmaceutically acceptable salt thereof into the compound of formula (I) or a pharmaceutically acceptable salt thereof by condensation, wherein, The compound of formula (II) is a compound of formula (13') or a pharmaceutically acceptable salt thereof, [Chemical Formula 7] (a) reacting a compound of formula (11') or a pharmaceutically acceptable salt thereof with a compound of formula (5') or a pharmaceutically acceptable salt thereof, [Chemical Formula 8] (b) converting a compound of formula (6) or a pharmaceutically acceptable salt thereof into the compound of formula (11') or a pharmaceutically acceptable salt thereof by protection, reduction, condensation and deprotection, and / or 【Chemical Formula 9】 (c) further comprising converting a compound of formula (1) or a pharmaceutically acceptable salt thereof into the compound of formula (5') or a pharmaceutically acceptable salt thereof by acylation, condensation and deprotection, 【Chemical Formula 10】 or, The compound of formula (II) is a compound of formula (50) or a pharmaceutically acceptable salt thereof, 【Chemical 11】 (a) reacting a compound of formula (45) or a pharmaceutically acceptable salt thereof with a compound of formula (41) or a pharmaceutically acceptable salt thereof, 【Chemical 12】 (b) converting a compound of formula (42) or a pharmaceutically acceptable salt thereof into the compound of formula (45) or a pharmaceutically acceptable salt thereof by acylation, nitration and hydrolysis, and / or 【Chemical 13】 (c) further comprising converting a compound of formula (6) or a pharmaceutically acceptable salt thereof into the compound of formula (41) or a pharmaceutically acceptable salt thereof by protection and reduction, a production method. 【Chemical Formula 14】 (Each symbol in the formula has the same meaning as in claim 1.)

4. A pharmaceutical composition comprising a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

5. A compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, which is used in the treatment method of PU.1-mediated diseases, wherein the PU.1-mediated diseases are leukemia or fibrosis.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein the leukemia or fibrosis is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), dermal fibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, or cardiac fibrosis.

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