Compounds as pu.1 inhibitors

Abstract

Disclosed herein are compounds of Formula (I) as PU.1 inhibitors. Also provided herein are methods of making these compounds.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT International Application No. PCT / CN2020 / 130512, filed on November 20, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to novel inhibitors of transcription factor PU.1, their chemical synthesis, and their use in the treatment of conditions such as leukemia and fibrosis. Background Technology

[0004] T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic cancer caused by the abnormal proliferation of T-cell progenitor cells. It accounts for 15% of pediatric cases and 25% of adult cases. T-ALL is a heterogeneous disease at both the biological and genetic levels. Belver, L. & Ferrando, A. Nat. Rev. Cancer 16, 494-507, doi:10.1038 / nrc.2016.63 (2016). Despite the heterogeneity of T-ALL, the major genetic abnormalities include chromosomal translocations affecting the expression of certain oncogenes and mutations or deletions in certain genes related to signal transduction pathways or the cell cycle. Teachey, EARaDT Hematology, 8 (2016). Aberrant activation of NOTCH1 accounts for approximately 60% of T-ALL cases (Tosello, V. & Ferrando, AA Therapeutic advances in hematology 4, 199-210, doi:10.1177 / 2040620712471368 (2013)). It has been reported that deletion or mutation of the well-known tumor suppressor gene PTEN accounts 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)). Intensive and high-dose chemotherapy can improve outcomes in T-ALL patients, but some patients who relapse and receive chemotherapy again still die from the disease. Pui, CH, Sailan, S., Relling, MV, Masera, G. & Evans, WE 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 leukemia blasts. Previous studies have reported that leukemia blasts, rather than LIC, can be eliminated by targeting activated pathways. LIC is a challenging group for targeted therapy in T-ALL.

[0005] To investigate the development and mechanisms of leukemia, a Pten-null T-ALL model was established. In this model, Pten is 40% absent in mouse fetal liver hematopoietic stem cells, subsequently activating the PI3K-AKT pathway, overexpressing the c-Myc oncogene, and disrupting the hematopoietic system. Within approximately two months of birth, mice develop aggressive T-ALL. (Guo, W. et al., Nature 453, 529-533, doi:10.1038 / nature06933 (2008)). Using c-kit, a marker similar to stem cell status, we were able to separate T-ALL cells into blast cells and LICs. Subsequent work in our laboratory has identified TIM-3 as an important surface marker, highly expressed in the LIC membrane but not in blast cells or normal cells. PU.1, an ETS family transcription factor, binds to the TIM-3 promoter and regulates TIM-3 expression, as well as maintaining the "steminess" of LICs. In LICs, the expression levels of TIM-3 and PU.1 are highly correlated. A series of LIC signature genes are potential PU.1 targets. Zhu, H. et al., eLife 7, doi:10.7554 / eLife.38314 (2018).

[0006] PU.1, a transcription factor belonging to the ETS family, plays a crucial role in hematopoiesis. Its expression levels vary among different hematopoietic progenitor cells and their progeny. In long-term hematopoietic stem cells (LT-HSCs), PU.1 expression is low, but it is highly expressed when differentiating into progenitor cells such as CMPs and CLPs. PU.1 expression also differs across maturation lineages, with higher expression in macrophages than in B cells, and lower expression in T cells, erythroid cells, and megakaryocytes. In the GMP population, PU.1 expression is highly desired in its progeny neutrophils and monocytes. Several mouse models have demonstrated the role of PU.1 in myelogenesis. PU.1 deficiency leads to a lack of CMPs and mature macrophages. Furthermore, PU.1 is important for targeted myelocyte development because it regulates the expression of several bone marrow-specific genes, including GM-CSFRa, G-CSFR, M-CSFR, and IL-7R. Besides being a major regulator of bone marrow lineage, PU.1 plays a crucial role in regulating lymphoid lineage differentiation and the generation and selection of B and T lineages. Studies using mice with GFP reporter genes have established that PU.1 expression levels increase with B cell maturation but are silenced in mature T cells. PU.1-null CLP can generate B cells. Similar to B cells, PU.1 is essential in the T progenitor stage but decreases in mature T cells. If PU.1 is overexpressed in mature T cells, the cells may exhibit a stem cell-like state and growth arrest, as well as maturation arrest. (Mak, KSetal., International Journal of Cell Biology 2011, 808524, doi:10.1155 / 2011 / 808524 (2011)). More recently, it has been shown that PU.1 can control fibroblast polarization and tissue fibrosis, and PU.1 inhibition may represent a promising therapeutic approach for treating 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 growth and clonogenic capacity of acute myeloid leukemia (AML) cells, leading to increased apoptosis in AML cells. PU.1 inhibition has the potential to be a therapeutic strategy for AML. Antony-Debre, I. et al., J Clin Invest 127, 4297-4313, doi:10.1172 / JCI92504 (2017).

[0007] Fibrosis is a restorative or reactive process characterized by the excessive formation and deposition of fibrous connective tissue and extracellular matrix, leading to progressive structural remodeling and further failure of almost all tissues and organs, such as the lungs, skin, liver, kidneys, and heart. Rockey, DC et al., N Engl J Med 373, 96, doi:10.1056 / NEJMc1504848 (2015). Therefore, fibrosis is a serious factor contributing to morbidity and mortality, estimated to account for over 45% of deaths in the United States. Wynn, TA, Nat Rev Immunol 4, 583-594, doi:10.1038 / nri1412 (2004). Under stimuli such as wound healing or inflammatory responses, fibroblasts differentiate into a matrix-producing phenotype and promote extracellular matrix accumulation, which is the trigger for fibrotic diseases. Palumbo-Zerr, K. et al., Nat Med 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 then leads to the activation of immune cells (mainly tissue macrophages) and their participation in the regulation of fibrosis-mediated homeostasis. Currently, there are few methods for treating organ fibrosis, and their efficacy is limited.

[0008] Nonalcoholic fatty liver disease (NAFLD) is caused by abnormal and excessive fat accumulation (steatodegeneration) in the liver without excessive alcohol consumption, which then develops into steatohepatitis (nonalcoholic steatohepatitis, NASH) and fibrosis with inflammation and collagen deposition, which may progress to cirrhosis and cancer. Adams, LA 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, not only does more than one-third of the population suffer from hepatic steatosis, which is increasingly affecting younger people, but NASH-mediated liver failure is also 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, with only the PPARα / γ agonist saroglitazar receiving approval from the Drugs and Medicinal Products Control Authority of India. Therefore, there is an urgent need to understand how fibrosis occurs and develops in order to discover new targets for drug development and identify potential treatments for NASH and organ fibrosis.

[0009] Previous studies have shown that the ETS family transcription factor PU.1 is a major regulator of the LIC signature gene and is crucial for the “stem” development of LIC and T-ALL. (Zhu, H. et al., eLife 7, doi:10.7554 / eLife.38314 (2018)). Furthermore, PU.1 has been reported to be highly expressed in fibrotic fibroblasts but silenced in matrix-degrading fibroblasts, and treatment with the PU.1 inhibitor DB1976 has been shown to alleviate skin, liver, and lung fibrosis. Wohlfahrt, T. et al., Nature 566, 344-349, doi:10.1038 / s41586-019-0896-x (2019). We and our collaborators also identified PU.1 inhibition mediated by DB1976 or shRNA application, showing beneficial effects on NASH progression, including reducing hepatic steatosis, inflammation, fibrosis, and improving glucose homeostasis. Liu, Q. et al. J Hepatol 73, 361-370, doi:10.1016 / j.jhep.2020.02.025 (2020). These works suggest that PU.1 is a potentially effective target for drug development and research in leukemia, liver disease, and multi-organ fibrosis. However, existing PU.1 inhibitors, such as DB1976, have limited potency and inhibitory activity against other ETS family members, posing potential risks to further drug development.

[0010] There is a need for improved approaches to treat hematologic T-ALL and other conditions associated with PU.1 dysfunction, such as NASH and organ fibrosis, using novel, potent, and selective PU.1 inhibitors, where such PU.1 inhibitors have scientific significance and potential pharmaceutical value. This disclosure addresses this need. Invention Overview

[0012] This 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 have broad applications in the treatment of conditions such as leukemia and fibrosis.

[0013] On the one hand, compounds of formula (I) or their stereoisomers or their pharmaceutically acceptable salts are provided.

[0014]

[0015] Where X, X', x, x', y, y', R1, R2, R3, R4, A, Z, B, C and n are as disclosed in this document.

[0016] On the other hand, a method for preparing a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof is provided, comprising taking a compound of formula (II) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0017]

[0018] The compounds are converted into compounds of formula (I) or their stereoisomers or pharmaceutically acceptable salts thereof, wherein X, X', x, x', y, y', R1, R2, R3, R4, A, Z, B, C and n are as disclosed herein.

[0019] On the other hand, a method for treating PU.1-mediated diseases in individuals in need is provided, comprising administering to said individual an effective amount of a compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, a compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided for treating PU.1-mediated diseases. In some embodiments, the use of a compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided in the preparation of a medicament for treating PU.1-mediated diseases. In some embodiments, the PU.1-mediated disease is leukemia or fibrosis. In some embodiments, the PU.1-mediated disease or condition is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), skin fibrosis, pulmonary fibrosis, renal fibrosis, liver fibrosis, or cardiac fibrosis. In some embodiments, the PU.1-mediated disease is NASH.

[0020] On the other hand, a composition, such as a pharmaceutical composition, is provided comprising the compound described herein or its stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. A kit is also provided comprising the compound as described herein or its stereoisomer or a pharmaceutically acceptable salt thereof.

[0021] Brief description of the attached figures

[0022] Figure 1 This study demonstrates the rational design of novel small-molecule PU.1 inhibitors that eliminate PU.1 binding to DNA and the biological evaluation of their efficacy. (a) Distinguishing between immediately disclosed PU.1 inhibitors such as compound I-1 and DB2115 (using rigid or AT-selective adapters instead of flexible adapters), and (b) q-PCR analysis (24h treatment) of compounds inhibiting the activity of the signature gene TIM-3 of LIC in Blast-PU.1 cells.

[0023] Figure 2The effects of compound I-1 and rapamycin combined treatment on leukemia burden in Pten-null T-ALL mice are shown. (a) High proportion of Blast and Tim-3 LIC in bone marrow of mice after treatment with compound I-1 and rapamycin or in combination. (b) Hematoxylin-eosin (H&E) staining of organs from mice treated with compound I-1 and rapamycin or in combination. Scale bar, 300 μm. (c) Immunohistochemical (IHC) analysis of mouse spleen from mice B220 (CD45R) treated with compound I-1 and rapamycin or in combination. Scale bar, 100 μm. (d) Survival curves of Pten-null T-ALL mice treated with compound I-1 (left) or DB1976 (right) and rapamycin, and in combination.

[0024] Figure 3 The effects of compound I-1 on the prevention and treatment of dermatofibrosis are shown (compound I-1, 5 mpk; DB1976, 5 mpk; medium, saline). (ae) Compound I-1 prevents bleomycin-induced dermatofibrosis (n=6). (fj) Compound I-1 alleviates and reverses bleomycin-induced dermatofibrosis (n=6). (a and f) Experimental design of the bleomycin-induced dermatofibrosis prevention and treatment model. (b and g) Pathological sections and staining of skin from different groups. Top, H&E staining; middle, Sirius red staining; bottom, Masson staining. Scale bar, 500 μm. (c and h) Quantified epidermal skin thickness. (de and ij) Relative mRNA levels of Col1a1 and Col1a2, normalized by GAPDH. Data are shown as mean ± sem of each of the n biologically independent samples. P-values ​​were determined by one-way ANOVA with Tukey multiple comparison post-hoc test. Compared with the bleomycin / carrier group, *P<0.05, **P<0.01 and ***P<0.001.

[0025] Figure 4The effects of compound I-1 on the prevention and treatment of pulmonary fibrosis are shown (compound I-1, 5 mpk; DB1976, 5 mpk; mediator, physiological saline). (ag) Compound I-1 prevents bleomycin-induced pulmonary fibrosis (n=5). (hn) Compound I-1 alleviates and reverses bleomycin-induced pulmonary fibrosis (n=5). (a and h) Experimental design of the bleomycin-induced pulmonary fibrosis prevention and treatment model. (b and i) Lung photographs after the above treatments. (c and j) H&E staining of lungs from different groups. Left panel, lower magnification, scale bar, 500 μm; right panel, higher magnification, 100 μm. (d and k) Ashcroft scores. (e and l) Sirius red staining of lungs from different groups. Left panel, lower magnification, scale bar, 500 μm; right panel, higher magnification, 100 μm. Relative mRNA levels of (fg and mn) Col1a1 and Col1a2 were normalized by GAPDH. Data are presented as mean ± sem of each of the n biologically independent samples. P-values ​​were determined by one-way ANOVA with post-hoc tests of Tukey multiple comparisons. *P<0.05, **P<0.01 and ***P<0.001 compared to the bleomycin / carrier group.

[0026] Figure 5 The effects of compound I-1 on hepatic lipid accumulation and NASH treatment are shown. (a) Experimental design of the NASH diet-induced model and compound treatment arrangement, n=8. (bc) Body weight and liver / body weight radio values ​​of different groups at the final time point. (d) H&E staining of liver tissue after tissue collection. Scale bar, 250 μm. (e) Oil Red O staining of liver tissue after tissue collection. Top, lower magnification, scale bar, 250 μm; bottom, higher magnification, scale bar, 50 μm. (f) NAFLD activity score conforms to criteria. (gi) Serum ALT, LDL-C, and total cholesterol levels in different groups. (jk) Inflammation-related genes, IL-6, and IL-1β mRNA levels. (lm) Fibrosis-related genes, Col1a1 and Col1a2 mRNA levels. Normalized by GAPDH. Data are presented as mean ± sem of each of the n biologically independent samples. P-values ​​were determined by one-way ANOVA and Tukey's post-hoc test for multiple comparisons. Compared with the carrier / NASH diet group, *P<0.05, **P<0.01 and ***P<0.001.

[0027] Figure 6The effects of compound I-1 on HFD / CCL4-induced NASH and liver fibrosis in mice are shown. (a) Experimental design of the HFD / CCL4-induced NASH and liver fibrosis model and compound treatment arrangements (n = 6–8). (b) Body weight of different groups at the final time point. (cd) Weight percentage of white adipose tissue (inguinal white adipose tissue, iWAT; gonadal white adipose tissue, gWAT) in different groups. (ef) Mediated levels of triglycerides (TG) and total cholesterol (TC) in fasting serum of different groups. (g) H&E and Sirius red staining of liver tissue after tissue collection. For H&E staining, top, lower magnification, scale bar, 250 μm; bottom, higher magnification, scale bar, 50 μm. For Sirius red staining, scale bar, 500 μm. (hi) Hepatic steatosis and inflammation scores based on H&E staining. (jk) Levels of inflammation-related genes, IL-6, and IL-1β mRNA in the liver. Normalized by GAPDH. (l) Quantitative data of Sirius red-positive regions based on Sirius red staining. (mn) mRNA levels of fibrosis-related genes, Col1a1, and Col1a2 in the liver. Normalized by GAPDH. (o) Fasting serum ALT levels in different groups. Data are presented as mean ± sem of n biologically independent samples. P-values ​​were determined by one-way ANOVA and Tukey multiple comparison post-hoc test. Compared with the mediator (saline) / HFD+CCL4 group, *P<0.05, **P<0.01, and ***P<0.001.

[0028] Figure 7 The effects of compound I-1 on CCL-induced liver fibrosis are shown. (a) Experimental design of the CCL4-induced liver fibrosis model and compound treatment arrangement (n=6). (bc) Sirius red staining and quantitative data of Sirius red-positive areas in liver tissue after tissue collection. Scale bar, 500 μm. (de) Fibrosis-related genes, Col1a1, Col1a2 mRNA levels. Normalized by GAPDH. (f) H&E staining of liver tissue after tissue collection. Top, lower magnification, scale bar, 250 μm; bottom, higher magnification, scale bar, 50 μm. (gh) Inflammation-related genes, IL-6, and IL-1β mRNA levels. Normalized by GAPDH. (i) Serum AST levels from different groups. Data are shown as mean ± sem for each of the n biologically independent samples. P-values ​​were determined by one-way ANOVA with Tukey multiple comparison post-hoc test. Compared with CCL4 / medium, *P<0.05, **P<0.01 and ***P<0.001. Invention Details

[0030] The following description illustrates exemplary embodiments of this disclosure. However, it should be understood that such description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0031] definition

[0032] As used in this specification, the following words, phrases and symbols are generally intended to have the meanings set forth below, unless otherwise stated in the context in which they are used.

[0033] The term "about" refers to a variation of ±1%, ±3%, ±5%, or ±10% of a specified value. For example, in some embodiments, "about 50" may include a range from 45 to 55. For integer ranges, 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 stated herein, the term "about" is intended to include values ​​close to the listed range, such as weight percentages, that are equivalent in function of a single ingredient, composition, or embodiment. References to "about" values ​​or parameters herein include (and describe) embodiments for that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0034] Unless the context clearly specifies otherwise, the singular forms “a” and “the” include plural references. Thus, for example, reference to “the compound” includes multiple such compounds and includes reference to one or more compounds and their equivalents known to those skilled in the art.

[0035] "Alkyl" refers to a straight-chain or branched saturated hydrocarbon chain. As used herein, alkyl groups have 1 to 10 carbon atoms (i.e., C64-C ... 1-10 Alkyl or C1-C 10 Alkyl groups, 1 to 8 carbon atoms (i.e., C64) 1-8 Alkyl or C1-C8 alkyl), 1 to 6 carbon atoms (i.e., C 1-6 Alkyl or C1-C6 alkyl), or 1 to 4 carbon atoms (i.e., C6 alkyl). 1-4Alkyl or C1-C4 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbons is named by its chemical name or determined by its molecular formula, it may include all positional isomers having that number of carbons; thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3); "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2). It should be understood that the term "alkyl" also considers the divalent portion.

[0036] "Haloalkyl" refers to a straight-chain or branched alkyl group as defined above, in which one or more hydrogen atoms are substituted with a halogen. For example, when a residue is substituted with more than one halogen, it can be referred to by using a prefix corresponding to the number of halogen moieties attached. Dihaloalkyl and trihaloalkyl refer to alkyl groups substituted with two ("di") or three ("tri") halogen groups, which may but are not necessarily the same halogen. Examples of haloalkyl include difluoromethyl (-CHF2) and trifluoromethyl (-CF3).

[0037] "Alkoxy" refers to the "-O-alkyl" group. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, and 1,2-dimethylbutoxy.

[0038] "Aryl" refers to an aromatic carbocyclic group having a monocyclic (e.g., monocyclic) or polycyclic (e.g., bicyclic or tricyclic) structure, including fused systems. As used herein, aryl groups have 6 to 20 cyclic carbon atoms (i.e., C64-C ... 6-20 Aryl or C6-C 20 aryl), 6 to 12 carbon ring atoms (i.e., C 6-12 Aryl or C6-C 12 aryl group) or 6 to 10 carbon ring atoms (i.e., C46, ​​C56, C6 ... 6-10 Aryl or C6-C 10 Aryl. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, and anthracene. However, aryl groups do not include or overlap in any way with heteroaryl groups as defined below. If one or more aryl groups are fused with a heteroaryl group, the resulting ring system is heteroaryl. If one or more aryl groups are fused with a heterocyclic group, the resulting ring system is heterocyclic. It should be understood that the term "aryl" also considers the divalent portion.

[0039] "Cycloalkyl" refers to a saturated or partially unsaturated cyclic alkyl group having a monocyclic or polycyclic structure (including fused rings, bridged rings, and spirocyclic systems). The term "cycloalkyl" includes cycloalkenyl groups (i.e., cyclic groups having at least one double bond) and cycloalkenyl groups having at least one sp... 3 Carbon-cyclic fused ring systems (i.e., at least one non-aromatic ring). As used herein, cycloalkyl groups have 3 to 20 ring carbon atoms (i.e., C164-C2 ... 3-20 cycloalkyl or C3-C 20 cycloalkyl groups), 3 to 12 cyclic carbon atoms (i.e., C12) 3-12 cycloalkyl or C3-C 12 cycloalkyl groups), 3 to 10 cyclic carbon atoms (i.e., C145-C ... 3-10 cycloalkyl or C3-C 10 cycloalkyl groups), 3 to 8 cyclic carbon atoms (i.e., C14 and C24) 3-8 Cycloalkyl or C3-C8 cycloalkyl) or 3 to 6 cyclic carbon atoms (i.e., C 3-6 Cycloalkyl (or C3-C6 cycloalkyl). Monocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Furthermore, the term cycloalkyl is intended to cover any non-aromatic ring that can fused with an aryl ring, regardless of its connection to the rest of the molecule. Further still, cycloalkyl also includes "spirocycloalkyl" when there are two substitution positions on the same carbon atom. It should be understood that the term "cycloalkyl" also considers the divalent moiety.

[0040] "Heteroaryl" refers to an aromatic group having a monocyclic, polycyclic, or polyfused ring structure, comprising one or more cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryls include 1 to 20 cyclic carbon atoms (i.e., C atoms). 1-20 heteroaryl), 3 to 12 cyclic carbon atoms (i.e., C 3-12 (heteroaryl) or 3 to 8 carbon ring atoms (i.e., C) 3-8A heteroaryl group comprises 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 heteroatom of a ring, wherein the ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. In some cases, a heteroaryl group comprises a 5-12 membered ring system, a 5-10 membered ring system, a 5-7 membered ring system, or a 5-6 membered ring system, each independently having 1 to 4, 1 to 3, 1 to 2, or 1 heteroatom of a ring, wherein the ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. Any aromatic ring having one or more fused rings and containing at least one heteroatom is considered a heteroaryl group, regardless of its connection to the rest of the molecule (i.e., through any fused ring). A heteroaryl group does not include or overlaps with an aryl group as defined above. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrimidinyl, thiopheneyl, furanyl, thiazolyl, oxazolyl, isoxazolyl, thiopheneyl, pyrroleyl, pyrazolyl, 1,3,4-oxadiazolyl, imidazolyl, isothiazolyl, triazolyl, 1,3,4-thiadiazolyl, tetrazolyl, benzofuranyl, benzothiopheneyl, pyrazolopyridyl, indazoleyl, benzothiazolyl, benzoxazolyl, and benzimidazolyl. It should be understood that the term "heteroaryl" also considers the divalent portion.

[0041] "Heterocyclic group" refers to a saturated or partially unsaturated cycloalkyl group having one or more cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. The term "heterocyclic group" includes heterocyclic alkenyl groups (i.e., heterocyclic groups having at least one double bond), bridged heterocyclic groups, fused heterocyclic groups, and spirocyclic groups. Heterocyclic groups can be monocyclic or polycyclic, wherein the polycyclic group can be fused, bridged, or spirocyclic, and can contain one or more (e.g., 1 to 3) oxo (=O) or N-oxide (N... + -O - The term "heterocyclic group" is used to describe any non-aromatic ring containing at least one heteroatom, regardless of its connection method (i.e., whether it can be bonded by carbon atoms or heteroatoms). Furthermore, the term "heterocyclic group" is intended to include any non-aromatic ring containing at least one heteroatom that can be fused to an aryl or heteroaryl ring, regardless of its connection to the rest of the molecule. As used herein, heterocyclic groups have 2 to 20 ring carbon atoms (i.e., C atoms). 2-20 Or C2-C 20 Heterocyclic groups), 2 to 12 ring carbon atoms (i.e., C 2-12 Or C2-C 12 Heterocyclic group), 2 to 10 ring carbon atoms (i.e., C 2-10 Or C2-C 10 Heterocyclic group), 2 to 8 ring carbon atoms (i.e., C 2-8 (or C2-C8 heterocyclic group), 3 to 12 ring carbon atoms (i.e., C 3-12 Or C3-C 12 Heterocyclic group), 3 to 8 ring carbon atoms (i.e., C 3-8Or C3-C8 heterocyclic group), or 3 to 6 ring carbon atoms (i.e., C 3-6 (or C3-C6 heterocyclic group); having 1 to 5 cyclic heteroatoms, 1 to 4 cyclic heteroatoms, 1 to 3 cyclic heteroatoms, 1 to 2 cyclic heteroatoms, or 1 cyclic heteroatomum, wherein the cyclic heteroatoms are independently selected from nitrogen, sulfur, or oxygen. In some cases, heterocyclic groups include 3-12 membered ring systems, 5-10 membered ring systems, 5-7 membered ring systems, or 5-6 membered ring systems, each independently having 1-4 cyclic heteroatoms, 1-3 cyclic heteroatoms, 1 to 2 cyclic heteroatoms, or 1 cyclic heteroatomum, wherein the cyclic heteroatoms are independently selected from nitrogen, oxygen, and sulfur. When there are two substitution positions on the same carbon atom, the term "heterocyclic group" also includes "spirocyclic group". Examples of heterocyclic groups include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazineyl, pyrrolylyl, thiazolinyl, thiazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, etc. It should be understood that the term "heterocyclic group" also considers a divalent portion.

[0042] "Oxytochemical" means =O.

[0043] "Halogen" or "halogenated" includes fluorine, chlorine, bromine, and iodine.

[0044] The terms “optional” or “optionally” indicate that the event or situation described below may or may not occur.

[0045] As used herein, “substituted” means that one or more hydrogen atoms of a group (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) are substituted with substituents listed for that group, and the substituents may be the same or different. “Optionally substituted” means that a group may be unsubstituted or substituted with one or more substituents listed for that group (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), wherein the substituents may be the same or different.

[0046] Stereoisomers, mixtures of stereoisomers, tautomers, hydrates, solvates, isotopically enriched analogs, and pharmaceutically acceptable salts of the compounds described herein are also provided.

[0047] The compounds disclosed herein, or their pharmaceutically acceptable salts, may contain asymmetric centers, thus yielding enantiomers, diastereomers, and other stereoisomers, which may be defined by absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- for amino acids. This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, the compound is intended to include E- and Z-geometric isomers unless otherwise specifically stated.

[0048] "Stereoisomers" are compounds composed of identical atoms linked by identical bonds, but with different three-dimensional structures and are not interchangeable. This disclosure covers a variety of stereoisomers and mixtures thereof, including "enantiomers" and "diastereomers." An "enantiomer" is a stereoisomer whose molecules are non-overlapping mirror images of each other, while a "diastereomer" is a stereoisomer having at least two asymmetric atoms but not being mirror images of each other. Therefore, all stereoisomers (e.g., geometric isomers, optical isomers, etc.) of compounds (including salts, solvates, and hydrates of compounds) are considered, such as those that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even if the asymmetric carbon is absent), rotational isomeric forms, transasterized isomers, and diastereomeric forms.

[0049] A mixture of diastereomers can be separated into their individual diastereomers based on their physicochemical differences using methods well known to those skilled in the art, such as chromatography and / or fractional crystallization. Enantiomers can be separated by reacting an enantiomer mixture with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), converting the diastereomers, and converting (e.g., hydrolyzing) individual diastereomers to their respective pure enantiomers. Furthermore, some of the compounds disclosed herein can be transisomers and are considered part of this disclosure. Stereoisomers can also be separated using chiral HPLC.

[0050] Some compounds exist as tautomers. These tautomers are in equilibrium with each other. For example, amide-containing compounds can exist in equilibrium with imine tautomers. Regardless of the type of tautomer shown and the nature of the equilibrium between the tautomers, those skilled in the art will understand that compounds include both amide and imine tautomers. Therefore, amide-containing compounds are understood to include their imine tautomers. Similarly, imine-containing compounds are understood to include their amide tautomers.

[0051] Any compound or structure presented herein is intended to represent both unlabeled and isotopically labeled forms of the compound. These forms of the compound may also be referred to as “isotopically enriched analogs.” Isotopically labeled compounds have the structures described herein, except that one or more atoms are substituted with atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as… 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. Various isotope-labeled compounds disclosed herein, such as those doped with radioactive isotopes, such as... 3 H and 14 Compounds labeled with C. These isotope-labeled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays or radiotherapy in patients. Such compounds can exhibit increased resistance to metabolism and are therefore used to increase the half-life of any compound when administered to mammals, particularly humans. These compounds are synthesized using methods well known in the art, for example, by using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0052] The term "inhibit / inhibiting / inhibition" refers to slowing down, stopping, or reversing the growth or progression of a disease, infection, condition, or cell population. Inhibition can be greater than, for example, about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to growth or progression that occurs without treatment or exposure.

[0053] As used herein, "individual" refers to a mammal, including humans. In some embodiments, an individual includes pigs, cattle, felines, canines, primates, rodents, or humans. In some embodiments, an individual is a human.

[0054] As used herein, “treatment” is a method of achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this disclosure, beneficial or desired outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by a disease or condition; reduction of the severity of a disease or condition; stabilization of a disease or condition (e.g., prevention or delay of its worsening); delay of the onset or recurrence of a disease or condition; delay or slow the progression of a disease or condition; improvement of the state of a disease or condition; provision of remission of a disease or condition (whether partial or complete); reduction of the dosage of one or more other medications required to treat a disease or condition; enhancement of the effect of another medication used to treat a disease or condition; delay of the progression of a disease or condition; improvement of quality of life; and / or prolongation of patient survival. “Treatment” also encompasses the reduction of pathological outcomes of a disease or condition. The methods of this disclosure consider any one or more of these treatment aspects.

[0055] As used herein, the term "effective amount" refers to an amount of compound or composition sufficient to treat a particular condition, ailment, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. In some embodiments, the effective amount is an amount sufficient to delay development. In some embodiments, the effective amount is an amount sufficient to delay onset and / or prevent recurrence. The effective amount may be administered in one or more applications.

[0056] As used herein, the term "carrier" refers to a relatively non-toxic compound or reagent that facilitates the incorporation of a compound into cells or tissues.

[0057] As used herein, "pharmaceuticalally acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, for example, that can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a harmful manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet toxicological and manufacturing testing requirements and / or are included in the inactive ingredient guidelines established by the U.S. Food and Drug Administration.

[0058] “Pharmaceutically acceptable salts” are those salts that retain at least some of the biological activity of the free (non-salt) compound and can be administered to an individual as a drug or medicine. 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, etc.; or with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, etc.; (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion); or salts coordinated with an organic base. 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 listed in Berge et al., Pharmaceutical Salts, J. Pharm. Sci. 1977 Jan; 66(1): 1-19. Pharmaceutically acceptable salts can be prepared in situ during manufacturing, or by reacting the purified compound of the present disclosure in free acid or base form with a suitable organic or inorganic base or acid, and then separating the resulting salt during a subsequent purification process.

[0059] As used herein, the term "excipient" refers to an inert or inactive substance that can be used in the production of a medicine or pharmaceutical preparation, such as a tablet containing a compound of the present disclosure as an active ingredient. The term excipient may cover a wide range of substances, including but not limited to any substance used as a binder, disintegrant, coating, compression / encapsulation aid, cream or emulsion, lubricant, parenteral solution, chewable tablet material, sweetener or flavoring agent, suspending / gelling agent, or wet granulation agent. Binders include, for example, carbomer, povidone, xanthan gum, etc.; coatings include, for example, cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, enteric coating, etc.; compression / encapsulation aids include, for example, calcium carbonate, glucose, fructose DC (DC = "directly compressible"), honey DC, lactose (anhydrous or monohydrate; optionally combined with aspartame, cellulose or microcrystalline cellulose), starch DC, sucrose, etc.; disintegrants include, for example, sodium croscarmellose, gellan gum, sodium glycolate starch, etc.; creams or Emulsions include, for example, maltodextrin and carrageenan; lubricants include, for example, magnesium stearate, stearic acid, and sodium stearoyl fumarate; chewable tablet materials include, for example, glucose, fructose DC, and lactose (monohydrate, optionally combined with aspartame or cellulose); suspending / gelling agents include, for example, carrageenan, sodium glycolate starch, and xanthan gum; sweeteners include, for example, aspartame, glucose, fructose DC, sorbitol, and sucrose DC; wet granulation agents include, for example, calcium carbonate, maltodextrin, and microcrystalline cellulose.

[0060] compound

[0061] On the one hand, it provides a compound of formula (I) or its stereoisomer or its pharmaceutically acceptable salt.

[0062]

[0063] in:

[0064] x and x' are each independently 0, 1, 2, 3 or 4;

[0065] Each R1 and R2 is 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 each R a Independently hydrogen, C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, wherein any two of R1 or any two of R2 can form C with the atoms they are attached to. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 aryl or 5-12 heteroaryl, wherein each C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl and 5-12 heteroaryl groups are independently and optionally replaced by R9;

[0066] y and y' are each independently 0, 1, 2, 3 or 4;

[0067] R3 is in

[0068] R5 is O, S, or NH, and

[0069] R6 and R7 are independently hydrogen and C, respectively. 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl, 5-12 heteroaryl, -C(O)OR d or -S(O)2R d Each R is independently hydrogen, C 1-12 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12-membered heteroaryl, wherein R6 and R7 can form 3-12-membered heterocyclic groups or 5-12-membered heteroaryl groups together with the nitrogen atoms they are attached to, or

[0070] When y is 2, 3, or 4, the two R3 atoms can form C together with the atoms they are attached to. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 aryl or 5-12 heteroaryl, wherein each C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl and 5-12 heteroaryl groups are independently and optionally replaced by R9;

[0071] R4 is in

[0072] R'5 is O, S, or NH, and

[0073] R'6 and R'7 are independently hydrogen and C, respectively. 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl, 5-12 heteroaryl, -C(O)OR d or -S(O)2R d , where each R d Independently hydrogen, C 1-12 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12-membered heteroaryl, wherein R'6 and R'7 can form 3-12-membered heterocyclic groups or 5-12-membered heteroaryl groups together with the nitrogen atoms to which they are attached, or

[0074] When y' is 2, 3, or 4, the two R4 atoms can form C together with the atoms they are attached to. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 aryl or 5-12 heteroaryl, wherein each C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl and 5-12 heteroaryl groups are independently and optionally replaced by R9;

[0075] X is O, S, NH, or NR8, and X' is O, S, NH, or NR'8, where

[0076] R8 and R'8 are each independently C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups;

[0077] A and B are each independently -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)2NH- or -NHS(O)2-;

[0078] C is a chemical bond or -NH-, provided that...

[0079] When B is -C(O)- or -S(O)2-, then C is -NH-, and

[0080] When B is -C(O)NH-, -NHC(O)-, -S(O)2NH- or -NHS(O)2-, then C is a chemical bond;

[0081] n is an integer selected from 1 to 6;

[0082] Each Z is independently C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12Aryl or 5-12 heteroaryl groups, each independently and optionally R c Replace, where each R c Independently for C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl, 5-12 heteroaryl, amino, hydroxyl, carboxyl, nitro, cyano, or halogen,

[0083] The premise is that at least one Z is C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, each independently and optionally R c Replace; and

[0084] Each R9 is independently -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, wherein each R b Independently hydrogen, C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups.

[0085] In some embodiments of the compound of formula (I) or any related 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.

[0086] In some embodiments of the compound of formula (I) or any related 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.

[0087] In some embodiments of the compound of formula (I) or any related formula, x equals x'. In some embodiments, x equals x' and is 0. In some embodiments, x equals x' and is 1. In some embodiments, x equals x' and is 2. In some embodiments, x equals x' and is 3. In some embodiments, x equals x' and is 4. In some embodiments, x and x' are each independently 2 or 3. In some embodiments, x equals x' and is 2 or 3.

[0088] In some embodiments of compounds of formula (I) or any related formula, each R1 is independently -R a -OR a Or halogen. In some implementations, each R1 is independently -R a -OR a Or halogen, wherein each R1 is independently hydrogen or C. 1-6 Alkyl group. In some embodiments, each R1 is independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C2. 1-6 Alkyl group. In some embodiments, R1 is methyl. In some embodiments, R1 is -OC. 1-6 Alkyl group. In some embodiments, R1 is a methoxy group. In some embodiments, R1 is a halogen. In some embodiments, R1 is fluorine. In some embodiments, the two R1s together with the atoms they are bonded to form a C group. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, each optionally and independently substituted with R9. In some embodiments, the two R1 groups, together with the atoms they are attached to, form C. 3-8 Cycloalkyl groups, optionally substituted with R9. In some embodiments, the two R1 groups, together with the atoms they are attached to, form 3-12 membered heterocyclic groups, optionally substituted with R9. In some embodiments, the two R1 groups, together with the atoms they are attached to, form C... 6-12The aryl group is optionally substituted with R9. In some embodiments, the two R1 groups together with the atoms they are attached to form a 5-12 membered heteroaryl group, which is optionally substituted with R9.

[0089] In some embodiments of the compound of formula (I) or any related formula, each R2 is independently -R a -OR a Or halogen. In some implementations, each R2 is independently -R a -OR a Or halogen, where each R a Independently hydrogen or C 1-6 Alkyl group. In some embodiments, each R2 is independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, R2 is hydrogen. In some embodiments, R2 is C2. 1-6 Alkyl group. In some embodiments, R2 is methyl. In some embodiments, R2 is -OC. 1-6 Alkyl group. In some embodiments, R2 is a methoxy group. In some embodiments, R2 is a halogen. In some embodiments, R2 is fluorine. In some embodiments, the two R2 groups together with the atoms they are bonded to form a C group. 3-8 Cycloalkyl groups, optionally substituted with R9. In some embodiments, the two R2 groups, together with the atoms they are attached to, form 3-12 membered heterocyclic groups, optionally substituted with R9. In some embodiments, the two R2 groups, together with the atoms they are attached to, form C... 6-12 The aryl group is optionally substituted with R9. In some embodiments, two R2 groups together with the atoms they are attached to form a 5-12 membered heteroaryl group, which is optionally substituted with R9.

[0090] In some embodiments of compounds of formula (I) or any related formula, each R1 and R2 is independently -R a -OR a Or halogen. In some implementations, each R1 and R2 is independently -R a -OR a Or halogen, where each R a Independently hydrogen or C 1-6 Alkyl group. In some embodiments, each R1 and R2 is independently hydrogen, methyl, methoxy, or fluorine. In some embodiments, both R1 and R2 are hydrogen. In some embodiments, the two R1s and / or the two R2s together with the atoms they are attached to form a C group. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, each of which is independently and optionally replaced by R9.

[0091] In some embodiments of the compound of formula (I) or any related 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.

[0092] In some embodiments of the compound of formula (I) or any related 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.

[0093] In some embodiments of the compound of formula (I) or any related formula, y equals y'. In some embodiments, y equals y' and is 0. In some embodiments, y equals y' and is 1. In some embodiments, y equals y' and is 2. In some embodiments, y equals y' and is 3. In some embodiments, y equals y' and is 4. In some embodiments, y and y' are each independently 1 or 2. In some embodiments, y equals y' and is 1 or 2. In some embodiments, x+y and x'+y' equal 4.

[0094] In some embodiments of the compound of formula (I) or any related formula, R5 is O. In some embodiments, R5 is S. In some embodiments, R5 is NH. In some embodiments, R5 is O or NH.

[0095] In some embodiments of compounds of formula (I) or any related formula, R6 and R7 are each independently hydrogen or -C(O)OR d In some implementations, R6 and R7 are each independently hydrogen or -C(O)OR d , where R d C 1-12 Alkyl group. In some embodiments, both R6 and R7 are hydrogen atoms. In some embodiments, R6 and R7 may form 3-12 membered heterocyclic groups or 5-12 membered heteroaryl groups together with the nitrogen atoms to which they are attached.

[0096] In some embodiments of compounds of formula (I) or any related formula, R5 is O; R6 and R7 are each independently hydrogen or -C(O)OR d , where R d C 1-12 Alkyl group. In some embodiments, R5 is S; R6 and R7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R5 is NH; R6 and R7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R5 is O or NH; R6 and R7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R5 is O; R6 and R7 are both hydrogen. In some embodiments, R5 is NH; R6 and R7 are both hydrogen. In some embodiments, R5 is S; R6 and R7 are both hydrogen.

[0097] In some embodiments of the compound of formula (I) or any related 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.

[0098] In some embodiments of compounds of formula (I) or any related formula, R'6 and R'7 are each independently hydrogen or -C(O)OR d In some implementations, R'6 and R'7 are each independently hydrogen or -C(O)OR d , where R d C 1-12 Alkyl group. In some embodiments, both R'6 and R'7 are hydrogen atoms. In some embodiments, R'6 and R'7 may form a 3-12 membered heterocyclic group or a 5-12 membered heteroaryl group together with the nitrogen atom to which they are attached.

[0099] In some embodiments of compounds of formula (I) or any related formula, R'5 is O; R'6 and R'7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R'5 is S; R'6 and R'7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12Alkyl group. In some embodiments, R'5 is NH; R'6 and R'7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R'5 is O or NH; R'6 and R'7 are each independently hydrogen or -C(O)OR. d , where R d C 1-12 Alkyl group. In some embodiments, R'5 is O; R'6 and R'7 are both hydrogen. In some embodiments, R'5 is NH; R'6 and R'7 are both hydrogen. In some embodiments, R'5 is S; R'6 and R'7 are both hydrogen.

[0100] In some embodiments of compounds of formula (I) or any related formula, the two R3 atoms together with the atoms they are attached to form C. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, each optionally and independently substituted with R9. In some embodiments, the two R3 groups, together with the atoms they are attached to, form C. 3-8 Cycloalkyl groups, optionally substituted with R9. In some embodiments, the two R3 groups, together with the atoms they are attached to, form 3-12 membered heterocyclic groups, optionally substituted with R9. In some embodiments, the two R3 groups, together with the atoms they are attached to, form C... 6-12 An aryl group, optionally substituted with R9. In some embodiments, the two R3 groups together with the atoms they are attached to form a 5-12 membered heteroaryl group, optionally substituted with R9. In some embodiments, the two R3 groups together with the atoms they are attached to form a 5 or 6 membered heteroaryl group, optionally substituted with R9. In some embodiments, the two R3 groups together with the atoms they are attached to form In some implementations, the two R3 atoms are formed together with the atoms they are attached to.

[0101] In some embodiments of compounds of formula (I) or any related formula, the two R4 atoms together with the atoms they are attached to form C. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl or 5-12 heteroaryl groups, each optionally and independently substituted with R9. In some embodiments, the two R4 groups, together with the atoms they are attached to, form C. 3-8 Cycloalkyl groups, optionally substituted with R9. In some embodiments, the two R4 groups, together with the atoms they are attached to, form 3-12 membered heterocyclic groups, optionally substituted with R9. In some embodiments, the two R4 groups, together with the atoms they are attached to, form C... 6-12An aryl group, optionally substituted with R9. In some embodiments, two R4 groups together with the atoms they are attached to form a 5-12 membered heteroaryl group, optionally substituted with R9. In some embodiments, two R4 groups together with the atoms they are attached to form a 5 or 6 membered heteroaryl group, optionally substituted with R9. In some embodiments, two R4 groups together with the atoms they are attached to form In some implementations, two R4 atoms are formed together with the atoms they are attached to.

[0102] In some embodiments of compounds of formula (I) or any related formula, the two R3s and / or the two R4s together with the atoms to which they are attached form C. 3-8 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 An aryl or 5-12-membered heteroaryl group, each optionally substituted with R9 independently. In some embodiments, two R3s and / or two R4s together with the atoms they are attached to form a 5-12-membered heteroaryl group, optionally substituted with R9. In some embodiments, two R3s and / or two R4s together with the atoms they are attached to form a 5 or 6-membered heteroaryl group, each optionally substituted with R9. In some embodiments, two R3s and / or two R4s together with the atoms they are attached to form... In some implementations, two R3s and / or two R4s are formed together with the atoms they are attached to.

[0103] In some embodiments of compounds of formula (I) or any related 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 group. In some embodiments, R8 is methyl.

[0104] In some embodiments of compounds of formula (I) or any related 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 group. In some embodiments, R'8 is methyl.

[0105] In some embodiments of compounds of formula (I) or any related formula, X is NH or NR8, and X' is NH or NR'8, wherein R8 and R'8 are each independently C 1-6Alkyl group. In some embodiments, X is NH or NR8, and X' is NH or NR'8, wherein both R8 and R'8 are methyl groups. In some embodiments, both X and X' are NH.

[0106] In some embodiments of the compound of formula (I) or any related 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-.

[0107] In some embodiments of the compound of formula (I) or any related 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-.

[0108] In some embodiments of the compound of formula (I) or any related 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)-.

[0109] In some embodiments of the compound of formula (I) or any related 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 2-6. In some embodiments, n is 2-5. In some embodiments, n is 2-4. In some embodiments, n is 2-3. In some embodiments, n is 3-6. In some embodiments, n is 3-5. In some embodiments, n is 3-4. In some embodiments, n is 4-6. In some embodiments, n is 4-5.

[0110] In some embodiments of compounds of formula (I) or any related formula, each Z is independently C. 1-6 Alkyl, 3-12-membered heterocyclic or 5-12-membered heteroaryl, each independently and optionally R c Replacement. In some embodiments, each Z is independently a 3-12 membered heterocyclic group, which is optionally replaced by R. c Replacement. In some implementations, each Z is independently C. 3-8 Cycloalkyl, optionally substituted with Rc. In some embodiments, each Z is independently C. 6-12 Aryl groups, which are optionally R c Replacement. In some embodiments, each Z is independently a 5-12 membered heteroaryl group, which is optionally replaced by R. c Replacement. In some implementations, each Z is independently -CH2-, -CH2CH2-, Each of them independently and arbitrarily subject to R c Substitution. It should be understood that each wavy line represents a point of connection with the rest of the molecule, and these points can be on any atom allowed by the valence. For example, Consider but not limited to In some implementations, each Z is independently methyl, Each of them independently and arbitrarily subject to R c Replacement. In some implementations, each Z is independently... Its optional use by R c Replacement. In some implementations, each Z is In some implementations, each Z is independently for Its optional use by R c Replacement. In some implementations, each Z is independently...

[0111] It should be understood that the specific values ​​described herein are values ​​of compounds of formula (I) or any applicable related formula (e.g., formula (II)). Two or more values ​​may be combined. Therefore, it should be understood that any variable of a compound of formula (I) or any related formula may be combined with any other variable of a compound of formula (I) or any related formula, as if each combination of variables were specifically and individually listed. For example, in some embodiments, a compound of formula (I) or its stereoisomers or pharmaceutically acceptable salts thereof are provided, wherein x and x' are each 2 or 3; R1 and R2 are both hydrogen; y and y' are each independently 1 or 2, wherein the two R3s and / or the two R4s may form together with the atoms to which they are attached. R5 is O or NH; R6 and R7 are each independently hydrogen or -C(O)OR d R'5 is O or NH; R'6 and R'7 are each independently hydrogen or -C(O)OR d X is NH or NR8, X' is NH or NR'8, where R8 and R'8 are each independently C. 1-6 Alkyl group; A and B are each independently -C(O)-, -C(O)NH-, or -NHC(O)-; n is 2; each Z is independently C 1-6 Alkyl, 3-12-membered heterocyclic or 5-12-membered heteroaryl, each independently and optionally R c replace.

[0112] The exemplary compounds provided in this 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 thereof. In some embodiments, compounds shown in Table 1 or their stereoisomers or pharmaceutically acceptable salts thereof are provided.

[0113] Table 1

[0114]

[0115]

[0116]

[0117] Treatment

[0118] On the other hand, a method for treating PU.1-mediated diseases in individuals in need is provided, comprising administering to the individual an effective amount of a compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. A compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is also provided for the treatment of PU.1-mediated diseases. In some embodiments, the use of a compound as described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided in the preparation of a medicament for treating PU.1-mediated diseases. 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), skin fibrosis, pulmonary fibrosis, renal fibrosis, liver fibrosis, or cardiac fibrosis. In some embodiments, the PU.1-mediated disease is NASH.

[0119] In some embodiments, a method for inhibiting PU.1 is provided, which includes contacting cells with an effective amount of the compound disclosed herein or its stereoisomer or its pharmaceutically acceptable salt.

[0120] Composition

[0121] On the other hand, a composition, such as a pharmaceutical composition, is provided, comprising the compound described herein or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions provided herein may be in forms suitable for oral, oral, parenteral (e.g., intravenous, intramuscular, infusion, or subcutaneous), nasal, topical, or rectal administration, or in forms suitable for inhalation administration.

[0122] In some embodiments, the compounds described herein may be in purified form. In some embodiments, compositions comprising the compounds described herein, their stereoisomers, or pharmaceutically acceptable salts thereof are in substantially pure form. Unless otherwise stated, “substantially pure” means a composition containing no more than 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% impurities, wherein the impurities represent compounds different from the desired compound or pharmaceutically acceptable salts thereof.

[0123] Reagent test kit

[0124] This document also provides kits containing the compounds disclosed herein, their stereoisomers, or pharmaceutically acceptable salts thereof, or the compositions disclosed herein. In some embodiments, the kit contains a unit dose of the compounds or compositions described herein and / or instructions for administration thereof.

[0125] Preparation method

[0126] On the other hand, a method for preparing the compounds disclosed herein, or their stereoisomers or pharmaceutically acceptable salts thereof, is provided, comprising taking a compound of formula (II), or its stereoisomers or pharmaceutically acceptable salts thereof,

[0127]

[0128] To convert into a compound of formula (I) or its stereoisomer or its pharmaceutically acceptable salt.

[0129]

[0130] Where X, X', x, x', y, y', R1, R2, R3, R4, A, Z, B, C and n are as disclosed in this document.

[0131] In some embodiments, the compound of formula (II) is a compound of formula (13') or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0132]

[0133] The method also includes:

[0134] (a) to make a compound of formula (11') or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0135]

[0136] Reaction with compounds of formula (5') or their stereoisomers or their pharmaceutically acceptable salts;

[0137]

[0138] (b) The compound of formula (6) or its stereoisomer or its pharmaceutically acceptable salt.

[0139]

[0140] Converted to a compound of formula (11') or its stereoisomer or its pharmaceutically acceptable salt; and / or

[0141] (c) The compound of formula (1) or its stereoisomer or its pharmaceutically acceptable salt.

[0142]

[0143] The compound is converted into formula (5') or its stereoisomer or its pharmaceutically acceptable salt.

[0144] In some embodiments, the compound of formula (II) is a compound of formula (50) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0145]

[0146] The method also includes:

[0147] (a) to make a compound of formula (45) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0148]

[0149] Reaction with compounds of formula (41) or their stereoisomers or their pharmaceutically acceptable salts;

[0150]

[0151] (b) a compound of formula (42) or its stereoisomer or its pharmaceutically acceptable salt.

[0152]

[0153] The compounds converted to formula (45) or their stereoisomers or pharmaceutically acceptable salts thereof; and / or

[0154] (c) The compound of formula (6) or its stereoisomer or its pharmaceutically acceptable salt.

[0155]

[0156] The compound is converted into a compound of formula (41) or its stereoisomer or its pharmaceutically acceptable salt.

[0157] In some embodiments, the compound of formula (II) is a compound of formula (54) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0158]

[0159] The method also includes:

[0160] (a) A compound of formula (53) or its stereoisomer or its pharmaceutically acceptable salt.

[0161]

[0162] The compound of formula (54) or its stereoisomer or its pharmaceutically acceptable salt is converted into it;

[0163] (b) A compound of formula (47) or its stereoisomer or its pharmaceutically acceptable salt.

[0164]

[0165] The compounds converted to formula (53) or their stereoisomers or pharmaceutically acceptable salts thereof; and / or

[0166] (c) A compound of formula (45) or its stereoisomer or its pharmaceutically acceptable salt.

[0167]

[0168] The compound is converted into a compound of formula (47) or its stereoisomer or its pharmaceutically acceptable salt.

[0169] In some embodiments, one or more steps of the preparation methods disclosed herein include acylation, condensation, reduction, protection, and / or deprotection.

[0170] The following provides representative schemes for preparing the compounds disclosed herein.

[0171] Option 1

[0172]

[0173] Option 2

[0174]

[0175] Option 3

[0176]

[0177] Option 4

[0178]

[0179] Option 5

[0180]

[0181] Compounds of formula (I) or any related formula described herein can be synthesized using standard synthetic techniques known to those skilled in the art. The compounds of this disclosure can be synthesized using the general synthetic procedures illustrated in the schemes provided above and the examples provided below.

[0182] When it is desired to obtain a specific enantiomer of a compound, this can be achieved from the corresponding mixture of enantiomers using any suitable conventional procedure for separating or resolving enantiomers. Thus, for example, diastereomeric derivatives can be produced by reacting a mixture of enantiomers (e.g., a racemic mixture and a suitable chiral compound). The diastereomers can then be separated by any convenient method, such as crystallization, and the desired enantiomers can be recovered. In another resolution process, the racemic mixture can be separated using chiral high-performance liquid chromatography. Alternatively, if desired, in one of the described processes, the specific enantiomer can be obtained by using a suitable chiral intermediate. Example

[0183] Synthesis Examples

[0184] The compounds disclosed herein can be prepared from commercially available raw materials and using the preparation methods described herein. The following examples illustrate the compounds disclosed herein and their preparation methods. These examples and preparation processes described below should not be considered as limiting the scope of this disclosure.

[0185] The structure of the compound disclosed herein is determined by 1Confirmed by ¹H NMR. Unless otherwise stated, all compounds or intermediates in the synthesis steps were purified by column chromatography or preparative reversed-phase HPLC. The reaction process can be detected by thin-layer chromatography, and the commonly used elution systems during the purification stage are petroleum ether / ethyl acetate and dichloromethane / methanol.

[0186] Example S1: Synthesis of (S)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-1)

[0187]

[0188] 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 overnight at 110 °C. The resulting clear solution was cooled to room temperature and concentrated under vacuum. Excess SOCl2 was removed by co-evaporation with toluene, followed by drying under vacuum to give the desired product 2 as a white solid (4.40 g, 98%).

[0189] 1 H NMR (400MHz, CDCl3) δ10.15 (s, 1H), 8.29 (d, J = 8.3 Hz, 2H), 8.03 (d, J = 8.6 Hz, 2H).

[0190] Step 2: Synthesis of (4-formylbenzoyl)-L-proline tert-butyl ester (compound 4). At 0 °C, a solution of 2 (1.98 g, 11.74 mmol) in DCM (18 mL) was slowly added to a solution of L-proline tert-butyl ester 3 (2.01 g, 11.74 mmol) in DCM (18 mL) and TEA (2 mL). The mixture was then heated to room temperature and stirred for another 3 h. The reaction mixture was washed with aqueous HCl solution (1 M, 3 × 60 mL). The combined aqueous fraction was extracted with DCM, and the combined organic fraction was further washed with saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain a pale yellow oil 4 (1.87 g), which was used for the next step without further purification.

[0191] Step 3: Synthesis of (4-formylbenzoyl)-L-proline (compound 5). A solution of 4 (1.87 g, 6.16 mmol) in DCM (18 mL) and TFA (18 mL) was stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed. The residue was dissolved in a saturated aqueous NaHCO3 solution and washed with EtOAc. The organic fraction was extracted with a saturated aqueous NaHCO3 solution. The aqueous fraction was acidified by adding 2 M HCl until pH = 2, and then the combined aqueous fractions were extracted with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. 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% for both steps).

[0192] 1 H NMR (400MHz, CDCl3) δ10.07(s,1H),7.96(d,J=8.2Hz,2H),7.72(d,J=8.1Hz,2H),4.78(dd,J=8.3,5.0Hz,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).

[0193] Step 4: Synthesis of 2-(4-nitrophenyl)-1,3-dithiopentane (compound 8). Ethane-1,2-dithiol 7 (20 mL, 0.24 mol) was added to a solution of 4-nitrobenzaldehyde 6 (6.92 g, 45.79 mmol) in DCM (180 mL), followed by the addition of boron trifluoride diethyl ether (1.2 mL). After stirring at room temperature for 6 h, the solution was washed with 10% NaOH, water, and brine. The resulting bright yellow solution was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the desired product 8 as a yellow solid (9.78 g, 94%).

[0194] 1 H NMR (400MHz, CDCl3) δ8.17(d,J=8.4Hz,2H),7.67(d,J=8.3Hz,2H),5.65(s,1H),3.56–3.48(m,2H),3.45–3.37(m,2H).

[0195] Step 5: Synthesis of 4-(1,3-dithiopentane-2-yl)aniline (compound 9). A solution of compound 8 (5.0 g, 22.00 mmol) and tin 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 a saturated aqueous solution of NaHCO3 until the pH reached 7-8. Approximately 200 mL of EtOAc was added and the mixture was filtered under vacuum through a glass funnel. The filtrate was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give the desired product 9 as a bright yellow solid (3.52 g, 81%).

[0196] 1 H NMR (400MHz, CDCl3) δ7.32(d,J=7.8Hz,2H),6.62(d,J=7.7Hz,2H),5.61(s,1H),3.69(s,2H),3.53–3.45(m,2H),3.37–3.29(m,2H).

[0197] Step 6: Synthesis of (S)-2-((4-(1,3-dithiopentane-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylic acid (9H-fluorene-9-yl)methyl ester (compound 10). A solution of HOBT in DMF (1M, 15 mL) and DCC in DCM (1M, 15 mL) was added to a freshly prepared solution of 9 (3.08 g, 15.61 mmol) and Fmoc-L-proline (5.26 g, 15.59 mmol), 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 solvent removal, the residue was diluted with CHCl3 / i-PrOH (3:1) and washed with water, 0.1 M HCl, saturated NaHCO3 aqueous solution, and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (0-0.5% MeOH in DCM) to obtain the desired product 10, which was a light yellow solid (5.89 g, 73%).

[0198] 1 H NMR (400MHz, CDCl3) δ9.18(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).

[0199] Step 7: Synthesis of (S)-N-(4-(1,3-dithiopentane-2-yl)phenyl)pyrrolidine-2-carboxamide (compound 11).

[0200] To a solution of 10 (3.16 g, 6.12 mmol) in DMF (24 mL), piperidine (6 mL) was added, 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 under vacuum. The crude product was purified by silica gel chromatography (0–2% MeOH in DCM) to give the desired product 11 as a white solid (1.37 g, 76%).

[0201] 1 H NMR (400MHz, CDCl3) δ9.75(s,1H),7.55(d,J=7.6Hz,2H),7.48(d,J=7.5Hz,2H),5.63(s,1H),3.85(dd,J=9.0,5.2Hz,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).

[0202] Step 8: Synthesis of (S)-N-(4-(1,3-dithiopentane-2-yl)phenyl)-1-((4-formylbenzoyl)-L-prolyl)pyrrolidine-2-carboxamide (compound 12). EDCI (688 mg, 3.59 mmol) was added to a solution of 11 (880 mg, 2.99 mmol) and 5 (740 mg, 2.99 mmol) in DCM (20 mL), and the reaction mixture was stirred at room temperature for 16 h. The solution was then concentrated under vacuum to obtain a white solid 12 (900 mg), which was used in the next step without further purification.

[0203] Step 9: Synthesis of (S)-1-((4-formylbenzoyl)-L-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (compound 13). SeO2 (954 mg, 8.60 mmol) was added to a solution of 12 (900 mg, 1.72 mmol) in AcOH (35 mL), 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 a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (pure EtOAc) to give the desired product 13 as a white solid (710.1 mg, 53% from step 2).

[0204] 1 H NMR (400MHz, CDCl3) δ10.07 (d, J=8.0Hz, 1H), 9.93 (dd, J=26.6, 22.2Hz, 2H), 8. 13(d,J=8.6Hz,0.5H),7.95(dd,J=13.0,8.0Hz,2H),7.85(d,J=8.7Hz,0.5H),7 .79–7.67(m,5H),4.87–4.81(m,1.5H),4.55(dd,J=17.1,7.7Hz,0.5H),3.97(d d,J=16.8,9.1Hz,1H),3.76–3.62(m,2H),3.57–3.52(m,1H),2.49–1.95(m,8H).

[0205] Step 10: Synthesis of (S)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-1). A solution of 13 (143.4 mg, 0.32 mmol), 14 (120 mg, 0.64 mmol) of 3,4-diaminobenzoamide hydrochloride, and p-benzoquinone (70.0 mg, 0.64 mmol) in anhydrous EtOH (13 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (30 mL) and EtOH (30 mL), filtered, and the volume was reduced to 20 mL and acidified with HCl-saturated EtOH (2 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-1 as a brown solid (101.7 mg, 37%).

[0206] 1 H NMR (400MHz, methanol-d4) δ8.27–8.23(m,4H),8.15(d,J=8.6Hz,2H),7.94(d,J=8.8Hz,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).

[0207] Example S2: Synthesis of (R)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-2)

[0208]

[0209] Step 1: Synthesis of (4-formylbenzoyl)-D-proline tert-butyl ester (compound 16). At 0 °C, a solution of 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 (1 mL) and TEA (1 mL). The mixture was then heated to room temperature and stirred for another 3 h. The reaction mixture was washed with aqueous HCl solution (1 M, 3 × 30 mL). The combined aqueous fractions were extracted with DCM, and the combined organic fractions were further washed with saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a pale yellow oil 16 (798.1 mg), which was used in the next step without further purification.

[0210] Step 2: Synthesis of (4-formylbenzoyl)-D-proline (compound 17). A solution of 16 (798.1 mg, 2.63 mmol) in DCM (9 mL) and TFA (9 mL) was stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed. The residue was dissolved in a saturated aqueous NaHCO3 solution and washed with EtOAc. The organic fraction was extracted with a saturated aqueous NaHCO3 solution. The aqueous fraction was acidified by adding 2 M HCl until pH = 2, and then extracted with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (2% MeOH in DCM) to give the desired product 17 as a white solid (444 mg, 31% of 2 steps).

[0211] 1 H NMR (400MHz, CDCl3) δ10.07(s,1H),8.58(s,1H),7.96(d,J=7.9Hz,2H),7.72(d,J=7.6Hz,2H),4.76(t ,J=6.7Hz,1H),3.62–3.50(m,2H),2.32(dd,J=13.5,6.7Hz,2H),2.12–2.02(m,1H),2.00–1.90(m,1H).

[0212] Step 3: Synthesis of (R)-2-((4-(1,3-dithiopentane-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylic acid (9H-fluorene-9-yl)methyl ester (compound 18). A solution of HOBT in DMF (1M, 9.6mL) and DCC in DCM (1M, 9.6mL) was added to a freshly prepared solution of 9 (1.90 g, 9.63 mmol) and Fmoc-D-proline (3.25 g, 9.63 mmol) in DMF (9.5 mL), and 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 solvent removal, the residue was diluted with CHCl3 / i-PrOH (3:1) and washed with water, 0.1 M HCl, saturated NaHCO3 aqueous solution, and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (0-0.5% MeOH in DCM) to obtain the desired product 18, which was a pale yellow solid (3.13 g, 63%).

[0213] 1 H NMR (400MHz, CDCl3) δ9.18(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).

[0214] Step 4: Synthesis of (R)-N-(4-(1,3-dithiopentane-2-yl)phenyl)pyrrolidine-2-carboxamide (compound 19). Piperidine (2.6 mL) was added to a solution of 18 (1.51 g, 2.92 mmol) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 1 h. After solvent removal, the residue was dissolved in EtOAc and washed with brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (0–2% MeOH in DCM) to give the desired product 19 as a white solid (790 mg, 92%).

[0215] 1H NMR (400MHz, CDCl3) δ9.74(s,1H),7.55(d,J=8.6Hz,2H),7.48(d,J=8.6Hz,2H),5.63(s,1H),3.85(dd,J=9.3,5.2Hz,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).

[0216] Step 5: Synthesis of (R)-N-(4-(1,3-dithiopentane-2-yl)phenyl)-1-((4-formylbenzoyl)-D-prolyl)pyrrolidine-2-carboxamide (compound 20). EDCI (164 mg, 0.85 mmol) was added to a solution of 19 (210 mg, 0.71 mmol) and 17 (176 mg, 0.71 mmol) in DCM (5 mL), and the reaction mixture was stirred at room temperature for 16 h. The solution was then concentrated under vacuum to obtain a white solid 20 (319.3 mg), which was used in the next step without further purification.

[0217] Step 6: Synthesis of (R)-1-((4-formylbenzoyl)-D-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (compound 21). SeO2 (338 mg, 3.05 mmol) was added to a solution of 20 (319.3 mg, 0.61 mmol) in AcOH (12 mL), 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 a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (pure EtOAc) to give the desired product 21 as a white solid (200.6 mg, 63% of step 2).

[0218] 1H NMR (400MHz, CDCl3) δ10.07 (d, J=9.4Hz, 1H), 9.93 (dd, J=32.9, 28.7Hz, 2H), 8. 13(d,J=8.6Hz,0.5H),7.94(dd,J=15.5,8.1Hz,2H),7.85(d,J=8.7Hz,0.5H),7 .79–7.65(m,5H),4.86–4.80(m,1.5H),4.55(dd,J=17.1,7.7Hz,0.5H),3.96(d d,J=16.7,9.0Hz,1H),3.76–3.63(m,2H),3.57–3.51(m,1H),2.45–1.90(m,8H).

[0219] Step 7: Synthesis of (R)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-2). A solution of 21 (87.6 mg, 0.20 mmol), 14 (73 mg, 0.39 mmol) of 3,4-diaminobenzoamidin hydrochloride, and p-benzoquinone (42.6 mg, 0.39 mmol) in anhydrous EtOH (8 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (18 mL) and EtOH (18 mL), filtered, and the volume was reduced to 12 mL and acidified with HCl-saturated EtOH (1.2 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-2 as a brown solid (40.4 mg, 24%).

[0220] 1 H NMR (400MHz, methanol-d4) δ8.27–8.23(m,4H),8.16(d,J=8.8Hz,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).

[0221] Example S3: Synthesis of (S)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-3)

[0222]

[0223] Step 1: Synthesis of (S)-N-(4-(1,3-dithiopentane-2-yl)phenyl)-1-((4-formylbenzoyl)-D-prolyl)pyrrolidine-2-carboxamide (compound 22). EDCI (166 mg, 0.86 mmol) was added to a solution of 11 (213 mg, 0.72 mmol) and 17 (179 mg, 0.72 mmol) in DCM (5 mL), and the reaction mixture was stirred at room temperature for 20 h. The solution was then concentrated under vacuum to obtain a white solid 22 (255 mg), which was used in the next step without further purification.

[0224] Step 2: Synthesis of (S)-1-((4-formylbenzoyl)-D-prolyl)-N-(4-formylphenyl)pyrrolidine-2-carboxamide (compound 23). SeO2 (270 mg, 2.43 mmol) was added to a solution of 22 (255 mg, 0.49 mmol) in AcOH (10 mL), 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 a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (pure EtOAc) to give the desired product 23 as a white solid (210.5 mg, 65% of step 2).

[0225] 1 H NMR (400MHz, CDCl3) δ10.08(s,1H),9.84(s,1H),9.13(s,1H),7.95(d,J=7.8Hz,4H),7.69(dd,J=10.5,8.4Hz,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).

[0226] Step 3: Synthesis of (S)-1-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoyl)-D-prolyl)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-3). A solution of 23 (75 mg, 0.17 mmol), 14 (62.5 mg, 0.33 mmol) of 3,4-diaminobenzoamidinium hydrochloride, and p-benzoquinone (36.5 mg, 0.33 mmol) in anhydrous EtOH (8 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (18 mL) and EtOH (18 mL), filtered, and the volume was reduced to 12 mL and acidified with HCl-saturated EtOH (1.2 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-3 as a brown solid (44.6 mg, 31%).

[0227] 1 H NMR (400MHz, methanol-d4) δ8.41–8.29(m,4H),8.27–8.24(m,2H),8.11(s,4H),8.01–7.93(m,4H),5.00(t,J=7.1Hz,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).

[0228] 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]imidazol-6-carboxamide (compound I-4)

[0229]

[0230] Step 1: Synthesis of 4-amino-3-nitrobenzamide (compound 25). DIPEA (1 mL, 6.06 mmol) was added to a suspension of 4-amino-3-nitrobenzic 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, and the reaction mixture was stirred at room temperature for 10 min. Then, (NH4)2CO3 (1.58 g, 16.44 mmol) was added in a single batch, and the resulting suspension was stirred for an additional 24 h. The reaction mixture was concentrated under vacuum, and then a 1:1 NaHCO3 / H2O mixture (40 mL) was added, with stirring continued for 2 h. The suspension was filtered, and the solid was dried under vacuum (40 °C, 24 h) to give the desired product 25 as a brown solid (878.7 mg, 88%).

[0231] 1 H NMR (400MHz, DMSO-d6) δ8.57(s,1H),7.94(s,1H),7.86(d,J=8.8Hz,1H),7.76(s,2H),7.25(s,1H),7.01(d,J=8.9Hz,1H).

[0232] Step 2: Synthesis of 3,4-Diaminobenzamide (compound 26). Pd / C (78 mg, 10%) was added to a solution of 25 (400 mg, 2.21 mmol) in DMF (2 mL) and EtOH (3 mL). The flask was then evacuated, washed three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with EtOH. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography (0.5% MeOH in DCM) to give the desired product 26 as a brown solid (289.2 mg, 87%).

[0233] 1 H NMR (400MHz, DMSO-d6) δ7.39 (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).

[0234] 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]imidazol-6-carboxamide (compound I-4). A solution of 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 refluxed and heated for 8 h. The reaction mixture was cooled to room temperature and concentrated under vacuum to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (20 mL) and EtOH (20 mL), filtered, reduced to a volume of 13.5 mL, and acidified with HCl-saturated EtOH (3 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to obtain the desired product I-4 as a dark green solid (67 mg, 35%).

[0235] 1 H NMR(500MHz,DMSO-d6)δ10.82(s,1H),10.72(s,0.5H),8.52–8.21(m,14H),8.08–7.76(m,12H),7.66(d, J=8.0Hz,1H),7.52(d,J=14.2Hz,3H),4.83(dd,J=8.3,4.5Hz,1H),4.75(dd,J=8.4,3.5Hz,0.5H),4.60(d d,J=8.3,4.8Hz,1H),4.17(dd,J=8.1,4.5Hz,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).

[0236] Example S5: Synthesis of (S)-1-((4-(1,7-dihydroimidazo[4,5-f]indazole-6-yl)benzoyl)-L-prolyl)-N-(4-(1,7-dihydroimidazo[4,5-f]indazole-6-yl)phenyl)pyrrolidine-2-carboxamide (compound I-5)

[0237]

[0238] 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 at 0 °C to a stirred solution of concentrated HNO₃ (0.42 mL) in concentrated H₂SO₄ (6 mL). The reaction mixture was stirred at room temperature for 16 h and then poured onto ice. The solid was filtered off, washed with water, and dissolved in CHCl₃ / i-PrOH (3:1). The mixture was then washed with brine and a saturated aqueous solution of NaHCO₃, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give the desired product 28 as a yellow solid (595 mg, 47%).

[0239] 1 H NMR (400MHz, DMSO-d6) δ14.35(s,1H),8.85(s,1H),8.54(s,1H),8.45(s,1H).

[0240] Step 2: Synthesis of 1H-indazole-5,6-diamine (compound 29). Ammonium formate (900 mg, 14.27 mmol) was added to a mixture of 28 (300 mg, 1.44 mmol) and Pd / C (30 mg, 10%) in MeOH (9 mL), and the mixture was refluxed for 4 h. The catalyst was then removed by filtration through a diatomaceous earth pad and washed with MeOH. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography (2-10% MeOH in DCM) to give the desired product 29 as a brown solid (121.4 mg, 57%).

[0241] 1 H NMR (400MHz, DMSO-d6) δ12.02(s,1H),7.52(s,1H),6.71(s,1H),6.56(s,1H),4.80(s,2H),4.29(s,2H).

[0242] Step 3: Synthesis of (S)-1-((4-(1,7-dihydroimidazo[4,5-f]indazole-6-yl)benzoyl)-L-prolyl)-N-(4-(1,7-dihydroimidazo[4,5-f]indazole-6-yl)phenyl)pyrrolidine-2-carboxamide (compound I-5). A solution of 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 refluxed and heated for 8 h. The reaction mixture was cooled to room temperature and concentrated under vacuum to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (15 mL) and EtOH (15 mL), filtered, and the volume was reduced to 10.5 mL. The mixture was then acidified with HCl-saturated EtOH (2.1 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H₂O containing 0.05% HCl) to give the desired product I-5 as a brown solid (16.8 mg, 12%).

[0243] 1 H NMR (600MHz, DMSO-d6) δ10.85(s,1H),10.76(s,0.5H),8.55–8.08(m,16H),7.97(d,J=8.6Hz,2H),7.93–7.80(m,8 H),7.70(d,J=8.1Hz,1H),4.84(dd,J=8.3,4.5Hz,1H),4.77(dd,J=8.0,3.7Hz,0.5H),4.59(dd,J=8.4,4.7Hz,1H) ,4.16(dd,J=8.2,4.3Hz,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).

[0244] Example S6: Synthesis of (S)-1-((4-(6-formamidinyl-5-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-6)

[0245]

[0246] Step 1: Synthesis of N-(4-cyano-3-methylphenyl)acetamide (compound 31). Ac₂O (4.32 mL, 42.49 mmol) was added dropwise to a solution of 4-amino-2-methylbenzonitrile 30 (4.68 g, 35.41 mmol) in DCM (145 mL), and the reaction mixture was stirred at room temperature for 18 h. After the reaction was complete, the solvent was removed under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (pure DCM) to obtain the desired product 31 as a white solid (5.98 g, 97%).

[0247] 1 H NMR (400MHz, CDCl3) δ7.56 (s, 1H), 7.54 (d, J = 8.5Hz, 1H), 7.40 (d, J = 8.4Hz, 1H), 7.32 (s, 1H), 2.52 (s, 3H), 2.21 (s, 3H).

[0248] Step 2: Synthesis of N-(4-cyano-5-methyl-2-nitrophenyl)acetamide (compound 32). 31 (2.6 g, 14.92 mmol) was added to a solution of KNO3 (3 g, 29.67 mmol) in concentrated H2SO4 (50 mL) at 0 °C. 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 as a yellow solid (2.39 g, 73%).

[0249] 1 H NMR (400MHz, CDCl3) δ10.53(s,1H),8.86(s,1H),8.48(s,1H),2.61(s,3H),2.32(s,3H).

[0250] Step 3: Synthesis of 4-amino-2-methyl-5-nitrobenzenenitrile (compound 33). A mixture of 32 (1.17 g, 5.34 mmol) in H₂SO₄ (70 mL, 10%) was refluxed and heated 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 under vacuum. The crude product was purified by silica gel chromatography (pure DCM) to give the desired product 33 as a yellow solid (920 mg, 97%).

[0251] 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),7.98(s,2H),6.93(s,1H),2.35(s,3H).

[0252] Step 4: Synthesis of ethyl 4-amino-2-methyl-5-nitrobenzylimine hydrochloride (compound 34). Dry HCl gas was passed through a stirred suspension of 33 (354 mg, 2.00 mmol) cooled in an ice-salt bath in EtOH (20 mL) until the reaction mixture was saturated with HCl, and the mixture was stirred at room temperature for 4 days. 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.

[0253] Step 5: Synthesis of 4-amino-2-methyl-5-nitrobenzamide hydrochloride (compound 35). NH3 (7M, 6 mL in MeOH) was added to a mixture of 33 and 34 (482.9 mg, 1.86 mmol) in EtOH (4 mL), and the reaction mixture was refluxed overnight. The mixture was then concentrated under vacuum to give a crude product, which was purified by silica gel chromatography (10-20% MeOH in DCM) to remove unreacted 33 and give an orange residue containing 35 (306.3 mg, 66% from step 2).

[0254] 1 H NMR (400MHz, DMSO-d6) δ9.18(s,2H),9.04(s,1H),8.14(s,1H),7.85(s,2H),6.94(s,1H),2.31(s,3H).

[0255] Step 6: Synthesis of 4,5-diamino-2-methylbenzamide hydrochloride (compound 36). Pd / C (60.8 mg, 10%) was added to a solution of 35 (304 mg, 1.32 mmol) in EtOH (30 mL). The flask was then evacuated, rinsed three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with MeOH. The filtrate was concentrated under reduced pressure to give a yellow solid 36 (280 mg, quantitative).

[0256] 1 ¹H NMR (400 MHz, methanol-d⁴) δ 6.81 (s, ¹H), 6.60 (s, ¹H), 2.29 (s, ³H).

[0257] Step 7: Synthesis of (S)-1-((4-(6-formamidinyl-5-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-formamide (compound I-6). A solution of 13 (61.4 mg, 0.14 mmol), 36 (55.1 mg, 0.27 mmol) of 4,5-diamino-2-methylbenzoamidinium hydrochloride, and p-benzoquinone (29.9 mg, 0.27 mmol) in anhydrous EtOH (6 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (13 mL) and EtOH (13 mL), filtered, and the volume was reduced to 9 mL and acidified with HCl-saturated EtOH (0.9 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-6 as a brown solid (26.0 mg, 21%).

[0258] 1 ¹H NMR (400MHz, methanol-d⁴) δ 9.53 (d, J = 8.0Hz, 1H), 9.21 (d, J = 8.5Hz, 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, 5.6Hz, 1H) H),4.69(dd,J=8.2,4.8Hz,1H),4.05–3.98(m,1H),3.86–3.77(m,1H),3.71–3.56(m, 2H), 2.67 (d, J = 4.6Hz, 6H), 2.56–2.47 (m, 1H), 2.44–2.36 (m, 1H), 2.28–1.94 (m, 6H).

[0259] Example S7: Synthesis of ((2-(4-((S)-1-((4-(6-(N-((hexyloxy)carbonyl)formamidinyl)-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamido)phenyl)-1H-benzo[d]imidazol-6-yl)(imino)methyl)hexyl carbamate (compound I-7)

[0260]

[0261] Step 1: Synthesis of ((3,4-diaminophenyl)(imino)methyl)carbamate (compound 38). A solution of 14 (1.25 g, 6.70 mmol) in acetone (5 mL) was cooled to 0 °C in an ice / water bath. Then, a solution of NaOH (5 mL, 16 wt%) and 37 (1.1 mL, 6.70 mmol) were slowly added, and the reaction mixture was stirred at 0 °C for another 1 h. After cooling to room temperature, the mixture was concentrated under reduced pressure, diluted with CHCl3 / i-PrOH (3:1), and washed with water. The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (2% MeOH in DCM) to give the desired product 38 as a pale yellow solid (926.3 mg, 50%).

[0262] 1 ¹H NMR (400MHz, 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).

[0263] Step 2: Synthesis of ((2-(4-((S)-1-((4-(6-(N-((hexyloxy)carbonyl)formamidinyl)-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)pyrrolidine-2-carboxamido)phenyl)-1H-benzo[d]imidazol-6-yl)(imino)methyl)hexyl carbamate (compound I-7). A solution of 13 (37.8 mg, 0.08 mmol), 38 (47.0 mg, 0.16 mmol) of ((3,4-diaminophenyl)(imino)methyl)hexyl carbamate, and p-benzoquinone (18.4 mg, 0.16 mmol) in anhydrous EtOH (10 mL) was refluxed and heated for 12 h. The reaction mixture was cooled to room temperature and concentrated under vacuum to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (7.5 mL) and EtOH (7.5 mL), filtered, and the volume was reduced to 5 mL and acidified with HCl-saturated EtOH (1 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-7 as a brown solid (19.3 mg, 24%).

[0264] 1¹H NMR (400MHz, 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, 4.7Hz, 1H), 4.41 (td, J = 6.7, 2.5Hz, 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).

[0265] Example S8: Synthesis of 4-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzoylamino)-N-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrole-3-yl)-1-methyl-1H-pyrrole-2-carboxamide (compound I-8)

[0266]

[0267] 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), followed by boron trifluoride diethyl ether (0.6 mL). 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 under vacuum to obtain the desired product 40 as a yellow solid (4.14 g, 93%).

[0268] 1 H NMR (400MHz, CDCl3) δ8.24 (d, J = 8.8Hz, 2H), 7.66 (d, J = 8.6Hz, 2H), 5.90 (s, 1H), 4.14–4.05 (m, 4H).

[0269] Step 2: Synthesis of 4-(1,3-dioxolane-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). The flask was then evacuated, rinsed three times with H2, filled with H2, and stirred at room temperature for 2 h. The reaction mixture was then filtered through a diatomaceous earth mat and washed with MeOH. The filtrate was concentrated under reduced pressure to obtain a crude product, which was dissolved in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give the desired product 41 as a pale yellow oil (2.03 g, 98%).

[0270] 1 H NMR (400MHz, CDCl3) δ7.28–7.26(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).

[0271] Step 3: Synthesis of 2,2,2-trichloro-1-(1-methyl-1H-pyrrolo-2-yl)ethyl-1-one (compound 43). A solution of 1-methyl-1H-pyrrole 42 (7.34 g, 90.48 mmol) in anhydrous diethyl ether (25 mL) was added dropwise to a solution of 2,2,2-trichloroacetyl chloride (16.45 g, 90.47 mmol) in anhydrous diethyl ether (25 mL). The reaction mixture was stirred at room temperature for 1.5 h. The mixture was then quenched dropwise with K2CO3 solution (20 mL, 20 mmol), extracted with EtOAc, and the combined organic layers were dried over anhydrous Na2SO4, filtered, concentrated under vacuum to give a crude product, washed with hexane, and dried under vacuum to give the desired product 43 as a white solid (13.24 g, 65%).

[0272] 1 H NMR (400MHz, CDCl3) δ7.51 (dd, J=4.4, 1.5Hz, 1H), 6.97 (s, 1H), 6.23 (dd, J=4.4, 2.4Hz, 1H), 3.98 (s, 3H).

[0273] Step 4: Synthesis of 2,2,2-trichloro-1-(1-methyl-4-nitro-1H-pyrrolo-2-yl)ethyl-1-one (compound 44). Fuming nitric acid (4 mL) was added dropwise to a stirred solution of 43 (10.67 g, 47.11 mmol) in Ac₂O (50 mL), which was maintained at -5 °C using an ice / NaCl bath. After the addition was complete, the temperature was gradually raised to room temperature and stirred 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 under vacuum. 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%).

[0274] 1 H NMR (400MHz, CDCl3) δ7.94 (d, J = 1.7 Hz, 1H), 7.75 (d, J = 1.3 Hz, 1H), 4.05 (s, 3H).

[0275] Step 5: Synthesis of 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid (compound 45). 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 2 M HCl. The resulting solid was filtered and dried under vacuum to give the desired product 45 as a white solid (1.60 g, 82%).

[0276] 1 H NMR (400MHz, CDCl3) δ7.65 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 4.01 (s, 3H).

[0277] Step 6: Synthesis of N-(4-(1,3-dioxolane-2-yl)phenyl)-1-methyl-4-nitro-1H-pyrrole-2-carboxamide (compound 46). DIPEA (1.5 mL, 9.08 mmol) was added to a stirred solution of 45 (761 mg, 4.47 mmol) and HBTU (2.04 g, 5.38 mmol) in DMF (20 mL). After stirring for 10 min at room temperature, 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. The organic layer was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain a yellow solid 46 (1.42 g), which was used in the next step without further purification.

[0278] Step 7: Synthesis of N-(4-(1,3-dioxolane-2-yl)phenyl)-4-amino-1-methyl-1H-pyrrole-2-carboxamide (compound 47). Pd / C (1.42 g, 10%) was added to a solution of 46 (1.42 g, 4.47 mmol) in DMF (50 mL). The flask was then evacuated, washed three times with H2, filled with H2, and stirred at room temperature for 18 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with MeOH. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography (0.5–1% MeOH in DCM) to give the desired product 47 as a pale yellow solid (741.3 mg, 58% of both steps).

[0279] 1 H NMR (400MHz, CDCl3) δ7.56 (dd, J=5.2, 3.2Hz, 3H), 7.44 (d, J=8.5Hz, 2H), 6.34 (d, J=2.0Hz, 1H), 6. 24(d,J=2.0Hz,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).

[0280] Step 8: Synthesis of N-(4-(1,3-dioxolane-2-yl)phenyl)-1-methyl-4-(1-methyl-4-nitro-1H-pyrrolo-2-carboxamido)-1H-pyrrolo-2-carboxamide (compound 48). DIPEA (0.5 mL, 3.03 mmol) was added to a stirred solution of 45 (207 mg, 1.22 mmol) and HBTU (555 mg, 1.46 mmol) in DMF (15 mL). After stirring for 10 min at room temperature, 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. The organic layer was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain a yellow solid 48 (530 mg), which was used in the next step without further purification.

[0281] Step 9: Synthesis of N-(4-(1,3-dioxolane-2-yl)phenyl)-4-(4-amino-1-methyl-1H-pyrrolo-2-carboxamido)-1-methyl-1H-pyrrolo-2-carboxamido (compound 49). Pd / C (800 mg, 10%) was added to a solution of 48 (530 mg, 1.21 mmol) in DMF (50 mL). The flask was then evacuated, washed three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a diatomaceous earth pad and washed with MeOH. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography (2-5% MeOH in DCM) to give the desired product 49 as a yellow solid (298.2 mg, 60% from two steps).

[0282] 1 H NMR (400MHz, CDCl3) δ7.62(s,1H),7.58(d,J=8.6Hz,2H),7.46(d,J=8.5Hz,2H),7.34(s,1H),7.14(d,J=1.7Hz,1H),6.76(d,J=1.8Hz,1H) ,6.35(d,J=2.0Hz,1H),6.18(d,J=2.0Hz,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).

[0283] Step 10: Synthesis of 4-(4-formylbenzamido)-N-(5-((4-formylphenyl)carbamoyl)-1-methyl-1H-pyrrolo-3-yl)-1-methyl-1H-pyrrolo-2-carboxamide (Compound 50). At 0 °C, 2 (41 mg, 0.24 mmol) of a solution in DCM (6 mL) was slowly added to a solution of 49 (100 mg, 0.24 mmol) in DCM (6 mL) and TEA (60 μL). The mixture was then heated to room temperature and stirred for another 12 h. After removing the solvent, the residue was dissolved in EtOAc and washed with aqueous HCl (1 M, 3 × 20 mL) and saturated NaHCO3 aqueous solution. The organic fraction was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (2-5% MeOH in DCM) to obtain the desired product 50, which was a pale yellow solid (72.8 mg, 60% in 2 steps).

[0284] 1H NMR (400MHz, DMSO-d6) δ10.60(s,1H),10.27(s,1H),10.11(s,1H),10.08(s,1H),9.89(s,1H),8.13(d,J=8.2Hz,2H),8.05(d,J=8.2Hz,2H ),7.99(d,J=8.6Hz,2H),7.87(d,J=8.6Hz,2H),7.39–7.36(m,2H),7.27(d,J=1.5Hz,1H),7.15(d,J=1.5Hz,1H),3.90(s,3H),3.88(s,3H).

[0285] Step 11: Synthesis of 4-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzamido)-N-(5-((4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrolo-3-yl)-1-methyl-1H-pyrrolo-2-carboxamide (compound I-8). A solution of 50 (33.4 mg, 0.067 mmol), 14 (25 mg, 0.13 mmol) of 3,4-diaminobenzoamide hydrochloride, and p-benzoquinone (14.6 mg, 0.13 mmol) in anhydrous EtOH (6 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (5 mL) and EtOH (5 mL), filtered, and the volume was reduced to 4 mL and acidified with HCl-saturated EtOH (0.6 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-8 as a brown solid (21.9 mg, 36%).

[0286] 1H NMR(500MHz,DMSO-d6)δ10.68(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=8.0Hz,2H),8.50(d,J=8.4Hz,2H),8.29 (d,J=5.4Hz,2H),8.24(d,J=8.0Hz,2H),8.10(d,J=8.4Hz,2H),7.99–7.89(m,3H),7. 84(d,J=8.4Hz,1H),7.45–7.31(m,3H),7.23(d,J=10.2Hz,1H),3.90(d,J=4.5Hz,6H).

[0287] Example S9: Synthesis of 4-(3-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzamido)propamido)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)-1-methyl-1H-pyrrole-2-carboxamide (compound I-9)

[0288]

[0289] Step 1: Synthesis of (3-((5-((4-(1,3-dioxolane-2-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrolo-3-yl)amino)3-oxopropyl)carbamate (9H-fluorene-9-yl)methyl ester (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 min, 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. The organic layer was then dried with anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain orange solid 52 (212 mg), which was used in the next step without further purification.

[0290] Step 2: Synthesis of N-(4-(1,3-dioxolane-2-yl)phenyl)-4-(3-aminopropionamido)-1-methyl-1H-pyrrole-2-carboxamide (compound 53). Piperidine (0.34 mL) was added to a solution of 52 (212 mg, 0.36 mmol) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 1 h. After solvent removal, the crude residue 53 (130 mg) was used for the next step without further purification.

[0291] Step 3: Synthesis of 4-(3-(4-formylbenzoylamino)propionylamino)-N-(4-formylphenyl)-1-methyl-1H-pyrrole-2-carboxamide (compound 54). At 0 °C, a solution of 2 (62 mg, 0.37 mmol) in DCM (9 mL) was slowly added to a solution of 53 (130 mg, 0.36 mmol) in DCM (90 μL) and TEA (90 μL). The mixture was then heated to room temperature and stirred for 24 h. After solvent removal, the residue was dissolved in EtOAc and washed with aqueous HCl (1 M, 3 × 20 mL) and saturated NaHCO3 aqueous solution. The organic fraction was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (0–2% MeOH in DCM) to give the desired product 54 as a white solid (54.8 mg, 34% in 3 steps).

[0292] 1 H NMR (400MHz, DMSO-d6) δ10.21(s,1H),10.07(s,1H),10.00(s,1H),9.88(s,1H),8.83(t,J=5.4Hz,1H),8.04–7.95(m,6H),7.86( d,J=8.7Hz,2H),7.29(d,J=1.5Hz,1H),7.06(d,J=1.5Hz,1H),3.85(s,3H),3.57(dd,J=12.7,6.8Hz,2H),2.59(t,J=7.0Hz,2H).

[0293] Step 4: Synthesis of 4-(3-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)benzamido)propionamide)-N-(4-(6-formamidinyl-1H-benzo[d]imidazol-2-yl)phenyl)-1-methyl-1H-pyrrole-2-carboxamide (compound I-9). A solution of 54 (80 mg, 0.18 mmol), 14 (67 mg, 0.36 mmol) of 3,4-diaminobenzoamidinium hydrochloride, and p-benzoquinone (39 mg, 0.36 mmol) in anhydrous EtOH (7 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (15 mL) and EtOH (15 mL), filtered, and the volume was reduced to 10 mL and acidified with HCl-saturated EtOH (1 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-9 as a brown solid (68 mg, 44%).

[0294] 1 H NMR(500MHz,DMSO-d6)δ10.34(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=5.5Hz,1H),8.46(dd,J=11.2,8.4Hz,4H),8.27(s,2H),8.11(d,J=8. 2Hz,2H),8.07(d,J=8.6Hz,2H),7.94(d,J=8.6Hz,1H),7.89(d,J=8.5Hz,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 = 6.7Hz, 2H), 2.65 (t, J = 7.2Hz, 2H).

[0295] Example S10: Synthesis of (S)-1-((4-(6-formamidinyl-5-methoxy-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-10)

[0296]

[0297] Step 1: Synthesis of 4-amino-2-fluoro-5-nitrobenzenenitrile (compound 56). At 0 °C, NH4OH (6.5 mL) was added to a solution of 2,4-difluoro-5-nitrobenzenenitrile 55 (2.2 g, 11.95 mmol) in EtOH (1.5 mL), and the resulting mixture was stirred at room temperature for 6 h. The resulting precipitate was then filtered and dried under vacuum to give the desired product 56 as a yellow solid (2.21 g, 98%).

[0298] 1 H NMR (400MHz, DMSO-d6) δ 8.60 (d, J = 7.0 Hz, 1H), 8.24 (s, 2H), 6.88 (d, J = 11.9 Hz, 1H).

[0299] Step 2: Synthesis of ethyl 4-amino-2-fluoro-5-nitrobenzyl imine hydrochloride (compound 57). Dry HCl gas was passed through a stirred suspension of 56 (1.45 g, 8.00 mmol) in EtOH (40 mL) until the reaction mixture was saturated with HCl, and the mixture was stirred at room temperature for 36 h. The reaction mixture was then diluted with anhydrous diethyl ether. The imine ester precipitated as an orange solid was filtered, washed with diethyl ether, and dried under vacuum to give orange solid 57 (1.88 g), which was used in the next step without further purification.

[0300] Step 3: Synthesis of 4-amino-2-methoxy-5-nitrobenzoamide hydrochloride (compound 58). NH3 (7M, 3 mL in MeOH) was added to a stirred suspension of 57 (278 mg, 1.05 mmol) in MeOH (3 mL), and the reaction mixture was refluxed overnight. The reaction mixture was then concentrated under vacuum and diluted with diethyl ether. The resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum to give a yellow solid 58 (306.9 mg), which was used in the next step without further purification.

[0301] Step 4: Synthesis of 4,5-diamino-2-methoxybenzamide hydrochloride (compound 59). Pd / C (20 mg, 10%) was added to a solution of 58 (170 mg, 1.32 mmol) in EtOH (10 mL). The flask was then evacuated, washed three times with H2, filled with H2, and stirred at room temperature for 18 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with MeOH. The filtrate was concentrated under reduced pressure to give the desired product 59 as a yellow solid (130.2 mg, 92% of the three steps).

[0302] 1 H NMR (400MHz, Methanol-d4) δ7.02(s,1H),6.48(s,1H),3.87(s,3H).

[0303] Step 5: Synthesis of (S)-1-((4-(6-formamidinyl-5-methoxy-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-10). A solution of 13 (52 mg, 0.12 mmol), 59 (50 mg, 0.23 mmol) of 4,5-diamino-2-methoxybenzoamide hydrochloride, and p-benzoquinone (25 mg, 0.23 mmol) in anhydrous EtOH (5 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (10 mL) and EtOH (10 mL), filtered, and the volume was reduced to 7 mL and acidified with HCl-saturated EtOH (0.7 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-10 as a brown solid (29.8 mg, 28%).

[0304] 1 ¹H NMR (400MHz, methanol-d⁴) δ 8.23 ​​(d, J = 8.2Hz, 2H), 8.15 (d, J = 8.9Hz, 2H), 8.03–7.97 (m, 4H), 7.88 (d, J = 8.2Hz, 2H), 7.49 (d, J = 15.1Hz, 2H), 4.96 (dd, J = 8.3, 5.8Hz, 1H), 4.6 8(dd,J=8.3,4.7Hz,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).

[0305] Example S11: Synthesis of (S)-1-((4-(6-formamidinyl-5-fluoro-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-11)

[0306]

[0307] Step 1: Synthesis of ethyl 4,5-diamino-2-fluorobenzoimino acid hydrochloride (compound 60). Pd / C (40 mg, 10%) was added to a solution of 57 (350 mg, 1.33 mmol) in EtOH (30 mL). The flask was then evacuated, washed three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a diatomaceous earth pad and washed with MeOH. The filtrate was concentrated under reduced pressure to obtain orange solid 60 (323.4 mg), which was used in the next step without further purification.

[0308] Step 2: Synthesis of 4,5-diamino-2-fluorobenzoamide hydrochloride (compound 61). NH3 (7M, 2 mL in MeOH) was added to a suspension of 60 (320 mg, 1.37 mmol) in MeOH (15 mL), and the reaction mixture was refluxed overnight. The reaction mixture was then concentrated under vacuum and diluted with diethyl ether. The resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum to give the desired product 61 as a reddish-brown solid (248.1 mg, 91% of step 2).

[0309] 1 ¹H NMR (400MHz, methanol-d⁴) δ 6.91 (d, J = 7.1 Hz, 1H), 6.50 (d, J = 13.5 Hz, 1H).

[0310] Step 3: Synthesis of (S)-1-((4-(6-formamidinyl-5-fluoro-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-11). A solution of 13 (67.1 mg, 0.15 mmol), 4,5-diamino-2-fluorobenzoamidinium hydrochloride 61 (61.4 mg, 0.30 mmol), and p-benzoquinone (32.7 mg, 0.30 mmol) in anhydrous EtOH (12 mL) was refluxed and heated 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 diethyl ether, and dried to give a brown solid. The solid was then dissolved in a 1:1 mixture of hot MeOH (13.2 mL) and EtOH (13.2 mL), filtered, and the volume was reduced to 9 mL and acidified with HCl-saturated EtOH (1.8 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-11 as a brown solid (17.4 mg, 13%).

[0311] 1H NMR (400MHz, methanol-d4) δ8.31–8.18(m,6H),8.06–7.85(m,6H),5.00–4.95(m,1H),4.69(dd,J=8.3,4.7Hz,1H),4.0 5–3.98(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).

[0312] Example S12: Synthesis of (S)-1-((4-(6-formamidinyl-1-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-12)

[0313]

[0314] Step 1: Synthesis of 4-(methylamino)-3-nitrobenzenenitrile (compound 63). CH3NH2 (27-32% in EtOH, 1.5 mL) was added to a suspension of 4-chloro-3-nitrobenzenenitrile 62 (1.5 g, 8.22 mmol) in EtOH (6 mL), and the reaction mixture was stirred at room temperature for 1 h, then refluxed overnight. The reaction mixture was cooled and concentrated under vacuum. The residue was suspended in diethyl ether and filtered to obtain a crude product, which was purified by silica gel chromatography (pure DCM) to give the desired product 63 as a yellow solid (936.3 mg, 64%).

[0315] 1 H NMR (400MHz, 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).

[0316] Step 2: Synthesis of ethyl 4-(methylamino)-3-nitrobenzoimino acid hydrochloride (compound 64). Dry HCl gas was passed through a stirred suspension of 63 (710 mg, 8.00 mmol) cooled in an ice-salt bath in EtOH (20 mL) until the reaction mixture was saturated with HCl, and the mixture was stirred at room temperature for 48 h. The reaction mixture was then diluted with anhydrous diethyl ether. The imino acid ester precipitated as an orange solid was filtered, washed with diethyl ether, and dried under vacuum to give orange solid 64 (1.08 g), which was used for the next step without further purification.

[0317] Step 3: Synthesis of 4-(methylamino)-3-nitrobenzidine hydrochloride (compound 65). NH3 (7M, 3 mL in MeOH) was added to a suspension of 64 (1.08 g, 4.16 mmol) in MeOH (20 mL), and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under vacuum and diluted with diethyl ether. The resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum to give the desired product 65 as a yellow solid (945.8 mg, quantified in both steps).

[0318] 1 H NMR (400MHz, DMSO-d6) δ9.30 (s, 2H), 8.98 (s, 2H), 8.70 (d, J = 2.4Hz, 1H), 8.68 (d, J = 5 .2Hz, 1H), 7.98 (dd, J = 9.2, 2.4Hz, 1H), 7.17 (d, J = 9.3Hz, 1H), 3.03 (d, J = 5.0Hz, 3H).

[0319] Step 4: Synthesis of 3-amino-4-(methylamino)benzamide hydrochloride (compound 66). Pd / C (70 mg, 10%) was added to a solution of 65 (686.8 mg, 3.00 mmol) in EtOH (30 mL). The flask was then evacuated, rinsed three times with H2, filled with H2, and stirred at room temperature for 24 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with MeOH. The filtrate was concentrated under reduced pressure to give the desired product 66 as a yellow solid (556.2 mg, 93%).

[0320] 1 H NMR (400MHz, DMSO-d6) δ8.74(s,2H),8.42(s,2H),7.12(dd,J=8.3,2.3Hz,1H),6.92(d,J=2 .3Hz, 1H), 6.47 (d, J = 8.4Hz, 1H), 5.76 (d, J = 5.1Hz, 1H), 4.88 (s, 2H), 2.80 (d, J = 4.7Hz, 3H).

[0321] Step 5: Synthesis of (S)-1-((4-(6-formamidinyl-1-methyl-1H-benzo[d]imidazol-2-yl)benzoyl)-L-prolyl)-N-(4-(6-formamidinyl-1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidine-2-carboxamide (compound I-12). A solution of 13 (135.4 mg, 0.30 mmol), 3-amino-4-(methylamino)benzoamide hydrochloride 66 (121.4 mg, 0.60 mmol), and p-benzoquinone (65.9 mg, 0.60 mmol) in anhydrous EtOH (12 mL) was refluxed and heated 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 diethyl ether, and dried to give the hydrochloride. The solid was then dissolved in a 1:1 mixture of hot MeOH (26 mL) and EtOH (26 mL), filtered, and the volume was reduced to 18 mL and acidified with HCl-saturated EtOH (1.8 mL). After stirring overnight at room temperature, the mixture was diluted with diethyl ether, the resulting precipitate was filtered, washed with diethyl ether, and dried under vacuum. The crude product was purified by preparative reversed-phase HPLC (5-100% acetonitrile in H2O containing 0.05% HCl) to give the desired product I-12 as a pink solid (141.6 mg, 53%).

[0322] 1 H NMR (400MHz, methanol-d4) δ8.30 (dd, J=6.9, 1.3Hz, 2H), 8.14 (d, J=8.8Hz, 1H), 8.0 6–7.99(m,6H),7.98–7.89(m,5H),4.98(dd,J=8.2,5.7Hz,1H),4.70(dd,J=8. 2,4.8Hz,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).

[0323] Biological Examples

[0324] Example B1: Biological evaluation of the efficacy of PU.1 inhibitors

[0325] An acute T-cell lymphoblastic leukemia (T-ALL) disease model was used to evaluate the effects of newly synthesized compounds on PU.1. Pten is a well-known tumor suppressor gene, and its deletion in 40% of mouse hematopoietic stem cells and their differentiated progeny ultimately leads to progressive T-ALL approximately two months after birth. Immune checkpoint T-cell immunoglobulin mucin 3 (TIM-3, a surface marker used to isolate pure leukemia initiating cells (LIC)) is believed to be transcriptionally regulated by the transcription factor PU.1 in the Pten-null T-ALL model. Therefore, TIM-3 expression levels were detected and quantified to characterize the inhibitory efficacy of the compounds after 24 hours of treatment with gradient concentrations of the compounds. Progenitor cells were transfected with either the PU.1-EGFP-vector or the EGFP-vector to generate stable cell lines blast-PU.1 and blast-EGFP, respectively. These cell lines were used for in vitro compound assays. Compounds DB1976 and DB2115 were also tested for comparison. blast-PU.1 and blast-EGFP are ideal cell lines for in vitro testing of compounds because they are T-ALL blast cells with low levels of TIM-3 and PU.1 expression.

[0326] Replacing the flexible alkyl linker of DB2115 with a rigid L-proline in compound I-1 resulted in a significant enhancement in potency, as compound I-1 downregulated TIM-3 expression levels by 40% at 10 nM in the Blast-PU.1 cell line. Figure 1 b) TIM-3 levels were too low to be detected in both the DMSO and compound-treated groups (data not shown). In contrast, its D,D-proline analogs I-2 and I-3 showed no significant inhibitory effect at 10 μM, but were not as good as compound I-1. Figure 1 (b) In summary, compound I-1 exhibited the best activity in PU.1-mediated TIM-3 inhibition, and we conclude that compound I-1 can be used for further biological function studies.

[0327] Example B2: The role of the combination of compound I-1 and rapamycin in slowing the progression of leukemia.

[0328] To generate the Pten-null T-ALL mouse model, Pten was 40% lost in mouse fetal liver hematopoietic stem cells (HSCs), followed by PI3K-AKT pathway activation, hematopoietic dysfunction, and T-ALL development. During the T-ALL crisis phase, T-ALL blasts and LICs infiltrate both hematopoietic and non-hematopoietic organs in mice.

[0329] T-ALL mice were treated with a combination of rapamycin (a well-studied PI3K-AKT pathway inhibitor that has shown promising efficacy in targeting T-ALL blast cells) and compound I-1. Treatment was initiated at the blast crisis stage and discontinued 62 days after birth to observe the direct effects of the compounds on inhibiting blast cells and TIM-3-induced high LIC.

[0330] After two days of treatment, compound I-1 alone did not reduce the ratio of blast cells and viable lymphocytes, while rapamycin showed high efficiency in targeting blast cells, resulting in a reduction of blast cells from 96.4% to 13.2%. Figure 2 a). Combined treatment causes bone marrow ( Figure 2 a) The proportion of blast cells in the spleen and thymus was significantly reduced. In the TIM-3 high LIC population, treatment with compound I-1 alone significantly reduced the LIC proportion from 33.5% to 6.15% compared with the rapamycin-treated group (22.2%). Importantly, the combination treatment showed a significant effect in reducing blast cells and LIC compared with the other three groups.

[0331] Previously, it had been shown that co-targeting blastocytes and LIC in a T-ALL mouse model using DB1976 and a PI3K inhibitor could reduce tumor burden. To test whether compound I-1 could achieve this, T-ALL mice were treated with compound I-1 and / or rapamycin for one month during the blastocyte crisis phase. After treatment, the morphology of hematopoietic and non-hematopoietic organs in the mice was analyzed using hematoxylin and eosin (H&E) staining. The organ morphology of the group treated with compound I-1 alone did not show significant changes compared to the T-ALL group, indicating that targeting LIC alone did not reduce tumor burden, while the rapamycin group showed improved treatment efficacy because blastocytes are the main population of leukemia cells. The morphology of the thymus and spleen was restored in the combined treatment group, and the infiltration of leukemia cells into the lungs, kidneys, and liver was significantly reduced. Figure 2 b).

[0332] Studies have shown that Pten deficiency inhibits B cell development in the pre-pro-B phase. For example... Figure 2 As shown in c, a small number of B220-positive cells were observed in the spleen of T-ALL mice; however, in the immunohistochemical slides of mouse spleen B220 cells, the B cell population was rescued after treatment with a combination of compound I-1 and rapamycin. Figure 2c). In B lineage cells, PU.1 is a major regulator of lineage stereotype, and PU.1 expression gradually increases from pro-B cells to B cells. It is possible that the expression levels of PU.1 and / or its downstream genes in the co-treatment group returned to normal, and lymphoid lineage stereotype and cell differentiation proceeded normally. Furthermore, the combination of compound I-1 and rapamycin prolonged mouse survival compared to compound I-1 alone, rapamycin alone, or the combination of DB1976 and rapamycin. Figure 2 d).

[0333] Example B3: The preventive and therapeutic effects of compound I-1 on cutaneous fibrosis

[0334] Methods: To evaluate the preventive and therapeutic effects of compound I-1 on cutaneous fibrosis, two different drug interventions were used to establish animal models of cutaneous fibrosis (6-8 weeks old, C57BL / 6, males). Every other day, a pre-haired, defined area (approximately 1 cm) of skin on the upper back was treated. 2 Local injection of bleomycin (0.5 mg / mL, 0.1 ml / mouse) induced skin fibrosis. Subcutaneous injection of physiological saline served as a control. (I) Prevention model of bleomycin-induced skin fibrosis: Compound I-1, positive control DB1976, or a medium (physiological saline) were simultaneously injected intraperitoneally with bleomycin for 4 weeks. Figure 3 a). (II) Treatment model of bleomycin-induced skin fibrosis: Mice were pre-treated with bleomycin for 3 weeks to induce skin fibrosis, and then treated with compound I-1, positive control DB1976, or a mediator (physiological saline) for another 3 weeks, for a total duration of 6 weeks after the first bleomycin treatment. Figure 3 f). On the last day of both model treatments, mice were fasted overnight and euthanized. A portion of skin was completely flattened on foil, fixed with paraformaldehyde, embedded in paraffin, and then sectioned for H&E, Sirius red, and Masson staining to examine pathological features. Epidermal thickness for each sample was quantitatively calculated using ImageJ. Another portion of skin was collected, flash-frozen in liquid nitrogen, and stored at -80°C for RNA isolation (Code. R6934, OMEGA, USA), first-strand cDNA reversed (Code. AT341, TransGen, China), and SYBR mixed (Code. AQ601, TransGen, China) for Q-PCR. 96, Roche) to verify the mRNA levels of several fibrosis-related genes (such as Col1a1 and Col1a2).

[0335] Results: In the bleomycin-induced skin fibrosis prevention model, compared with the saline / carrier group, bleomycin treatment for 4 weeks significantly induced pathological features of skin fibrosis, including increased epidermal thickness, collagen deposition, and increased Col1a1 and Col1a2 mRNA levels, indicating that the bleomycin-induced skin fibrosis model was successfully established. Compared with the bleomycin / carrier group, treatment with compound I-1 or DB1976 significantly inhibited the progression of skin fibrosis, reduced epidermal thickness and collagen deposition, and decreased Col1a1 and Col1a2 mRNA levels. Figure 3 (a-3e). In a bleomycin-induced skin fibrosis treatment model, 6 weeks of bleomycin treatment also significantly induced skin fibrosis pathological features, producing thicker epidermis, more collagen deposition, and higher Col1a1 and Col1a2 mRNA levels compared to the saline / carrier group, further demonstrating the successful establishment of a skin fibrosis model via bleomycin stimulation. Treatment with compound I-1 or DB1976 for 3 weeks significantly alleviated and reversed bleomycin-induced skin fibrosis (a-3e). Figure 3 (f-3j). These data indicate that compound I-1 has preventive and therapeutic effects on bleomycin-induced cutaneous fibrosis.

[0336] Example B4: The preventive and therapeutic effects of compound I-1 on pulmonary fibrosis

[0337] Methods: To evaluate the preventive and therapeutic effects of compound I-1 on pulmonary fibrosis, two different animal models of pulmonary fibrosis (6-8 weeks old, C57BL / 6, males) were established using bleomycin. Bleomycin (0.025U, Code. D11063, OKA, China) was administered via a single intratracheal injection. An equal volume of sterile saline served as a control. (I) Bleomycin-induced preventive model of pulmonary fibrosis. Immediately after a single bleomycin injection, patients were treated with compound I-1, the positive control DB1976, or the medium (saline) (intraperitoneal injection, ip) for 4 weeks. Figure 4 a). (II) Bleomycin-induced pulmonary fibrosis model. Mice were pre-fed with bleomycin for 11 days to induce pulmonary fibrosis, and then treated with compound I-1, positive control DB1976, or a mediator (physiological saline) for 17 days, for a total duration of 4 weeks after bleomycin treatment. Figure 4h). On the last day of both model treatments, mice were fasted overnight and euthanized. Partial lung tissue was fixed with paraformaldehyde, embedded in paraffin, and then sectioned for H&E and Sirius red staining to examine pathological features and Ashcroft scores. Hubner, R. Hetal. Biotechniques 44, 507-511, 514-507, doi:10.2144 / 000112729 (2008). Another partial lung tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C for RNA isolation (Code. R6934, OMEGA, USA), first-strand cDNA reverse-engineering (Code. AT341, TransGen, China), and SYBR mixed (Code. AQ601, TransGen, China) for Q-PCR ( 96, Roche) to verify the mRNA levels of several fibrosis-related genes (such as Col1a1 and Col1a2).

[0338] Result: As Figure 4 As shown, compared with the saline / carrier group, bleomycin treatment for 4 weeks significantly induced pulmonary fibrosis pathological features, including lung deterioration, collagen deposition, alveolar wall thickening, and alveolar structural destruction, indicating the successful establishment of a bleomycin-induced pulmonary fibrosis model. Compared with the bleomycin / carrier group, treatment with compounds I-1 and DB1976 significantly inhibited the progression of pulmonary fibrosis, as measured by pathological changes based on staining and Ashcroft scores, collagen deposition indicated by Sirius red staining, and Col1a1 and Col1a2 mRNA levels. Figure 4 a-4g). In the treatment model, treatment with compound I-1 reversed and prevented bleomycin-induced pulmonary fibrosis, including the aforementioned pathological features (a-4g). Figure 4 These data indicate that compound I-1 has preventive and therapeutic effects against bleomycin-induced pulmonary fibrosis.

[0339] Example B5: Effect of compound I-1 on NASH and liver fibrosis

[0340] Methods: To evaluate the potential therapeutic effects of compound I-1 on liver diseases, including hepatic steatosis and accumulation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatotic hepatitis (NASH), and liver fibrosis, three mouse models (C57BL / 6, male, 8 weeks old) were used. (I) NASH model induced by NASH diet (Code.TD.160785, ENVIGO, USA): All mice were fed the NASH diet for 10 weeks. Mice were then randomly assigned to three groups, each receiving daily injections of the mediator, compound I-1 (2.5 mpk or 5 mpk, ip), or DB1976 (2.5 mpk, ip, as a positive control) for an additional 6 weeks, while continuing the NASH diet. Groups with a normal regular diet were treated with the mediator as healthy controls. (II) NASH model induced by high-fat diet (HFD) combined with low-dose CCl4. Mice were fed a normal diet or HFD (60% kcal fat - D12492, study diet) for 10 weeks, and then HFD mice were randomly divided into two groups according to the minimum body weight difference rule. Each group was assigned to be injected with CCl4 (25% v / v in olive oil, 0.5 mL / kg body weight) or pure olive oil (ip) twice a week for 4 weeks. Compound I-1 or the mediator (physiological saline) was injected once daily (ip) for 4 weeks under continuous HFD, concurrently with CCl4 injection. (III) CCl4 (high-dose) induced liver fibrosis model. Liver fibrosis was induced by twice-weekly CCl4 injections (20% v / v in olive oil, 10 mL / kg body weight) for 6 weeks, concurrently with CCl4 administration and once daily (ip) of compound I-1 or the mediator for 6 weeks. Mice in the above three models were observed daily. On the last day of all model treatments, mice were fasted overnight and euthanized. Serum was collected after centrifuging whole blood at 4 degrees Celsius, and blood biochemical parameters were measured using a fully automated biochemical analyzer (BS-240VET, Mindray). Partial liver tissue was fixed with paraformaldehyde, embedded in paraffin, and sections were stained with H&E or Sirius red to examine pathological features and NAFLD scores. (Kleiner, DE et al. Hepatology 41, 1313-1321, doi:10.1002 / hep.20701 (2005)). Fresh partial liver tissue was taken, embedded in OCT embedding medium, and sectioned. After fixation in PBS solution with 4% paraformaldehyde, the sections were stained with 0.5% Oil Red O according to standard procedures. Another portion of liver tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C for RNA isolation (Code. R6934, OMEGA, USA). First-strand cDNA was reverse-engineered (Code. AT341, TransGen, China) and mixed with SYBR (Code. AQ601, TransGen, China) for Q-PCR. 96, Roche) to validate the mRNA levels of genes such as fibrosis-related genes, Col1a1 and Col1a2; and inflammation-related genes, IL-6 and IL-1β.

[0341] Result 1: A NASH diet-induced NASH model. (e.g.) Figure 5 As shown, the 16-week NASH diet significantly increased body weight and liver / body weight ratio. Figure 5 b-5c); induces significant fat accumulation in the liver, including larger and more numerous lipid droplets based on pathological staining (b-5c); Figure 5 In addition, the application of the NASH diet not only improved serum parameters such as ALT, LDL-C, and total cholesterol (TC), but also... Figure 5 g-5i), and also increased inflammation and fibrosis-related genes, IL-6, IL-1β and Col1a1, Col1a2 (g-5i), and increased the levels of these genes. Figure 5 Treatment with compound I-1 (5 mpk) and DB1976 (2.5 mpk) for 6 weeks effectively alleviated the aforementioned metabolic disturbances induced by the NASH diet. Compared with DB1976 (2.5 mpk), compound I-1 (2.5 mpk) showed lower efficacy, but also showed a trend toward alleviating metabolic disturbances, as evidenced by reduced fat accumulation in the liver and lower mRNA levels of IL-6, IL-1β, and Col1a1, Col1a2. These data suggest that compound I-1 has therapeutic potential in treating hepatic fat accumulation, inflammation, and NASH.

[0342] Result 2: A NASH model induced by a high-fat diet (HFD) combined with low-dose CCl4. Figure 6 As shown, after HFD pretreatment and 6 weeks of CCL4 treatment, body weight, gonadal white adipose tissue (gWAT), and inguinal white adipose tissue (iWAT) weights significantly increased, and these increases were reversed by treatment with DB1976 and I-1. Figure 6 b-6d). HFD / CCL4 treatment induced dyslipidemia in mice; the application of DB1976 and I-1 reduced serum triglycerides (TG) and total cholesterol (TC). Figure 6 e-6f). Furthermore, histological examination with H&E staining showed that I-1 was not effective in reducing fat accumulation (e-6f). Figure 6 g), consistent with the hepatic steatosis score ( Figure 6 h), but effectively reduces the inflammatory response, manifested as inflammatory infiltration ( Figure 6 g), inflammation score Figure 6 i) and IL-1β ( Figure 6 j) and IL-6 Figure 6The liver mRNA levels of (k) were reduced. Notably, administration of DB1976 and I-1 significantly alleviated HFD / CCL4 application-induced liver fibrosis and reduced collagen deposition indicated by Sirius red staining. Figure 6 g and 6l) and Col1a1 ( Figure 6 m) and Col1a2 ( Figure 6 The liver mRNA level (n) was measured. Simultaneously, I-1 treatment showed a trend towards reducing serum ALT levels. Figure 6 This indicates that I-1 is not hepatotoxic at its effective concentrations. These data suggest that compound I-1 exhibits anti-inflammatory and anti-fibrotic potential in HFD / CCL4-induced NASH and liver fibrosis mice.

[0343] Result 3: CCl4 (high dose) induced liver fibrosis. (e.g.,) Figure 7 As shown, based on Sirius red and H&E staining, CCl4 treatment for 6 weeks significantly induced strong CCl4-induced liver fibrosis parameters, such as abundant collagen deposition and high degree of fibrosis. Figure 7 b-7c and 7f) and inflammatory responses. CCl4 application also significantly increased inflammation and fibrosis-related genes, IL-6, IL-1β (b-7c and 7f), and inflammatory responses. Figure 7 g-7h) and Col1a1, Col1a2 ( Figure 7 d-7e), except for serum AST levels ( Figure 7 i). Treatment with compound I-1 (5 mpk or 10 mpk) alleviated CCl4-induced liver fibrosis, significantly reduced collagen deposition and Sirius red positive areas, and improved abnormal mRNA levels and blood biochemistry induced by CCl4 application. These data suggest that compound I-1 has the potential to prevent and treat liver fibrosis.

[0344] All publications mentioned in this specification, including patents, patent applications and scientific articles, are incorporated herein by reference in their entirety for all purposes, to the same extent as each individual publication, including patents, patent applications or scientific articles, is specifically and individually indicated to be incorporated by reference.

Claims

1. A compound of formula (I-1) or a pharmaceutically acceptable salt thereof, (I-1)。 2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

3. A kit comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating PU.1-mediated diseases, comprising administering an effective amount of the compound or a pharmaceutically acceptable salt thereof to an individual.

5. The use according to claim 4, wherein the disease mediated by said PU.1 is leukemia or fibrosis.

6. The use according to claim 4, wherein the PU.1-mediated disease is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia (CML), skin fibrosis, pulmonary fibrosis, renal fibrosis, liver fibrosis, or cardiac fibrosis.

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