Indirubin derivatives with novel heterobicyclic residues and uses thereof

Abstract

The present invention relates to indirubin derivatives with novel heterobicyclic residues and their use as inhibitors of FLT3 (fms-like tyrosine kinase 3; FMS-like tyrosine kinase 3) and rearranged during transfection (RET) kinases. The compounds of the present invention are capable of effectively inhibiting the activity of FLT3 and RET kinases and are useful in the prevention or treatment of mutant FLT3 and mutant RET-associated diseases, in particular acute myeloid leukemia.

Description

Technical Field

[0001] This invention relates to indirubin derivatives having novel heterobicyclic residues and their use as inhibitors of FLT3 (fms-like tyrosine kinase 3) and rearranged during transfection (RET) kinases. Background Technology

[0002] Fms-like tyrosine kinase 3 (FLT3) is a protein belonging to the type III receptor tyrosine kinase family. FLT3 has been identified and expressed in normal CD34-positive human bone marrow progenitor cells and dendritic progenitor cells, playing an important role in the proliferation and differentiation of these cells (Brown P et al., European Journal of Cancer, 40th, pp.707-721, 2004). Furthermore, FLT3 ligand (FL) is expressed in bone marrow stromal cells and T cells, and is one of the cytokines that influences the production of most hematopoietic cells and stimulates the proliferation of stem cells, progenitor cells, dendritic cells, and natural killer cells through interactions with other growth factors.

[0003] FLT3 is known to be a target antigen of acute myeloid leukemia (AML), and it is overexpressed in AML patient blasts when compared with healthy cells, and is expressed in most cells of patients (Carow et al., Feb 2, 1996 Blood: 87(3); Birg et al., Nov 1992 Blood: 99-6 2022-02-14 80(10)). In particular, FLT3 is a gene that is frequently mutated in AML patients, and mutations are associated with poor prognosis (Abu-Duhier et al., British Journal of Haematology 2000 Oct;111(1):190-5, Yamamoto et al., April15, 2001; Blood:97(8)). In addition, recent reports have indicated that FLT3 may be an important biomarker related to the prognosis of pancreatic cancer (Ger et al., Anticancer Research 38: 5759-5765 (2018)) and that FLT3 ligand (FL) promotes resistance in esophageal squamous cell carcinoma (Zhu et al., Frontiers in Pharmacology, May 2021, Vol 12, Article 659735). Furthermore, the correlation between FLT3 kinase and pancreatic cancer and esophageal cancer has been confirmed.

[0004] RET kinases are expressed in the development of various tissues, including the peripheral nervous system, central nervous system, and kidneys. They are single-transmembrane receptors of tyrosine kinases essential for the development, maturation, and maintenance of several tissues and cell types (Mulligan, LM, Nature Reviews Cancer, 2014, 14, 173-186). RET proteins consist of three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic portion of a tyrosine kinase domain split by an insertion of 27 amino acids. Activating mutations of RET kinases have been found in patients with lung cancer and thyroid cancer, and are considered to be inducible mutations in these cancers (Kohno, T. et al., Nature Medicine, 2012, 18 (3), pp. 375-377, Matsubara, D. et al., Journal of Thoracic Oncology, 2012, 7 (12), pp. 1872-1876). In addition, reports have indicated the interdependence between RET-mediated autophagy inhibition and AML, and the fact that RET kinase is expressed in MOLM-13 and MOLM-14, which are AML cell lines (Rudat, et al., Leukemia 32, 2189-2202 (2018)), confirming the correlation between RET kinase activation mutations and AML induction. Summary of the Invention

[0005] Technical problems to be solved

[0006] The present invention is the result of the inventors’ efforts to find new compounds with inhibitory functions of FLT3 and RET kinase. It was found that indirubin derivative compounds with new heterobicyclic residues can effectively inhibit FLT3, especially mutant FLT3, compared with existing known indirubin derivative compounds, and also have excellent inhibitory function of RET kinase.

[0007] The purpose of this invention is to provide indirubin derivative compounds with novel heterocyclic residues that have inhibitory functions on FLT3 and RET kinases, as well as pharmaceutically acceptable salts thereof.

[0008] Another object of the present invention is to provide pharmaceutical compositions for the prevention or treatment of FLT3 and RET kinase-related diseases comprising indirubin derivative compounds having novel heterobicyclic residues, uses of said novel compounds for the prevention or treatment of FLT3 and RET kinase-related diseases, and methods for the prevention or treatment of FLT3 and RET kinase-related diseases comprising the step of administering said novel compounds to a subject in need of said novel compounds.

[0009] Technical solution

[0010] According to one embodiment of the present invention, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof is provided.

[0011] [Chemical Formula 1]

[0012]

[0013] In the chemical formula 1,

[0014] R1 is hydrogen or halogen.

[0015] R2 is or ,

[0016] R3 is a free choice , , as well as Any one of the groups formed,

[0017] Wherein, X1 is CR a R b X2 is CR c R d X3 is CR e R f ,

[0018] R4, R5, R a R b R c R d R e and R f Each can be either hydrogen or C1-C8 alkyl.

[0019] N, m, and l are each independent integers from 1 to 3.

[0020] The compounds disclosed in this application have high inhibitory activity against FLT3 and RET kinases.

[0021] The compounds and pharmaceutical compositions disclosed in this application are useful, for example, in the prevention or treatment of FLT3-related diseases or RET kinase-related diseases by administering the compounds or compositions to a subject.

[0022] Invention Effects

[0023] The indirubin derivative compounds of the present invention, which are novel compounds not previously known, can effectively inhibit the activity of FLT3 and RET kinases, and are useful in the prevention or treatment of FLT3-mutant and RET-mutant related diseases, especially acute myeloid leukemia. Attached Figure Description

[0024] Figures 1a-1d This is a graph showing changes in tumor size and body weight measured over a total of 28 days after mice were treated with the indirubin derivative compounds of the present invention and the existing known indirubin derivative compounds, respectively. Detailed Implementation

[0025] Definition of terminology

[0026] Unless otherwise defined, all technical terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, unless explicitly stated otherwise, numerical values ​​recorded in this application should be considered to include the meaning of "about".

[0027] The following provides definitions of residues and substituents used in this application. Unless otherwise specified, each residue has the following definition and is used as having the same meaning as commonly understood by one of ordinary skill in the art.

[0028] The substituents used in this application are intended to include all cases of further substitution or no substitution.

[0029] As used in this application, the terms "halogenated" and "halogen" refer to bromine, chlorine, fluorine, or iodine.

[0030] As used in this application, the term "alkyl" refers to a hydrocarbon having one, two, three, and / or four carbon atoms, including saturated aliphatic groups that can be linear, branched, cyclic, or combinations thereof. For example, an alkyl group can have 1 to 8 carbon atoms (i.e., C1-C8 alkyl), 1 to 6 carbon atoms (i.e., C1-C6 alkyl), or 1 to 3 carbon atoms (i.e., C1-C3 alkyl). Examples of suitable alkyl groups include methyl (Me, -CH3), ethyl (Et, -C2H5), 1-propyl (n-Pr, -CH2CH2CH3), 2-propyl (i-Pr, -CH(CH3)2), 1-butyl (n-Bu, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, -CH2CH(CH3)2), 2-butyl (s-Bu, -CH(CH3)CH2CH3), and 2-methyl-2-propyl 1-Pentyl (t-Bu, -C(CH3)3), 1-Pentyl (n-Pentyl, -CH2CH2CH2CH2CH3), 2-Pentyl (-CH(CH3)CH2CH2CH3), 3-Pentyl (-CH(CH2CH3)2), 2-Methyl-2-Butyl (-CH(CH3)2CH2CH3), 3-Methyl-2-Butyl (-CH(CH3)CH(CH3)2), 3-Methyl-1-Butyl (-CH2CH2CH(CH3)2), 2- Methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4 -Methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), and octyl (-(CH2)7CH3), but not limited thereto.

[0031] The phrase “pharmaceutically acceptable” is a common expression in the art, indicating that a substance or composition needs to be chemically and / or toxicologically compatible with other components constituting the formulation and / or with the mammals treated with the component.

[0032] The term "pharmaceutically acceptable salt" or simply "salt" is used to refer to the acid- or base-added salts suitable for treating patients or compatible with the present application. Exemplary inorganic acids for forming suitable salts include not only hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, but also metal salts such as sodium monohydrogen phosphate and potassium hydrogen sulfate. Exemplary organic acids for forming suitable salts include not only mono-, di-, and tricarboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, but also sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Monoacids or diacid salts can be formed, and these salts can exist in hydrated, solvated, or substantially anhydrous forms. Generally, the acid-added salts of the compounds of the present invention are more soluble in water and various hydrophilic organic solvents than their free base forms, and typically exhibit higher melting points. The selection of suitable salts is well known to those skilled in the art. Other non-pharmaceutical acceptable salts, such as oxalates, may be used, for example, in the isolation of compounds of the present invention for laboratory purposes or as a subsequent conversion to pharmaceutically acceptable acids. Exemplary inorganic bases for forming suitable salts include hydroxides of lithium, sodium, potassium, calcium, magnesium, or barium. Exemplary organic bases for forming suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and methylpyridine or ammonia. Thus, in some examples, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts may be included as salts contemplated in the present invention. In specific embodiments, salts considered as considerations for the present invention include, but are not limited to, L-arginine, phenethylbenzylamine, benzylamine, betaine, calcium hydroxide, choline, dianophenate, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In specific embodiments, salts considered as considerations for the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

[0033] The selection of suitable salts is well known to those skilled in the art.

[0034] Pharmaceutically acceptable acid-added salts can also exist as solvates of various solvates, such as water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. The source of this solvate can be from the crystallizing solvent, or inherent in the solvent used for preparation or crystallization, or incidentally occurring in such solvent.

[0035] The term "IC" used in this application50 "Refers to the concentration of an inhibitor or compound that produces 50% inhibition."

[0036] The term "GI" used in this application 50 "Refers to the maximum inhibition of cell proliferation, the concentration of an inhibitor or compound that produces 50% inhibition."

[0037] As used in this application, the terms “object,” “object body,” and “patient” refer to warm-blooded animals, such as pigs, cattle, chickens, horses, guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, monkeys, chimpanzees, and humans.

[0038] As used in this application, based on the terms “treatment,” “therapeutic,” and “treatment,” “improvement” and “improved” refer to any objective or subjective parameter, such as relief; improvement; reduction of symptoms or making symptoms, pain, pathology, or symptom more tolerable for the patient; or reduction of the frequency or duration of symptoms or symptom; or, in some cases, prevention of the onset of symptoms or symptom, any indication of success in the treatment or improvement of pain, pathology, symptom, or symptom (e.g., pain). Treatment or improvement of symptoms may, for example, be based on any objective or subjective parameter, including the results of a physical examination.

[0039] heterocyclic indigo derivative compounds

[0040] This invention provides novel heterobicyclic indirubin derivative compounds.

[0041] In one embodiment, the present invention relates to heterobicyclic indirubin compounds represented by the following chemical formula 1, or pharmaceutically acceptable salts thereof.

[0042] [Chemical Formula 1]

[0043]

[0044] In the chemical formula 1,

[0045] R1 is hydrogen or halogen.

[0046] R2 is or ,

[0047] R3 is a free choice , , as well as Any one of the groups formed,

[0048] Wherein, X1 is CR a R b X2 is CR c Rd X3 is CR e R f ,

[0049] R4, R5, R a R b R c R d R e and R f Each can be either hydrogen or C1-C8 alkyl.

[0050] n, m, and l are each independent integers from 1 to 3.

[0051] In one specific embodiment of the present invention, R4, R5, R a R b R c R d R e and R f Each is independently hydrogen or C1-C3 alkyl, and n, m and l are 1.

[0052] In one specific embodiment of the present invention, R3 is selected from... , , , , , as well as In any of the groups formed, n and m are each independent integers from 1 to 3.

[0053] In one specific embodiment of the present invention, R4, R5, R a R b R c and R d Each is independently hydrogen or C1-C3 alkyl, and n and m are 1.

[0054] In one specific embodiment of the present invention, R1 is hydrogen or a halogen, and R2 is selected from... , , , , , , , , , , , , as well as Any one of the groups formed.

[0055] In one specific embodiment of the present invention, R1 is hydrogen or a halogen, and R2 is selected from... , , , as well as Any one of the groups formed.

[0056] In one specific embodiment of the present invention, R1 is hydrogen or fluorine, and R2 is selected from... , as well as Any one of the groups formed.

[0057] Representative compounds of Formula 1 include, but are not limited to, compounds selected from the group consisting of compounds 1 to 20 below.

[0058]

[0059]

[0060]

[0061]

[0062] In one specific embodiment of the invention, the invention relates to pharmaceutical compositions comprising the indirubin derivative compound having the heterobicyclic residues of the invention or its pharmaceutically acceptable salts, for the prevention or treatment of FLT3 and RET-related diseases.

[0063] In one specific embodiment of the present invention, the pharmaceutical composition is used for the prevention or treatment of leukemia or lymphoma.

[0064] In one specific embodiment of the present invention, the pharmaceutical composition is used for the prevention or treatment of multiple myeloma, malignant plasma cell tumors, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Kallmann's disease and myeloma, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute... Chronic myeloid leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-cell lymphoblastic lymphoma, myeloid leukemia, Waldenström macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary ALL Mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone B-cell lymphoma, marginal zone splenic lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, T-cell / histiocytic large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammatory phase This includes diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma caused by HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and Burkitt lymphoma, or unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0065] In one specific embodiment of the present invention, the pharmaceutical composition is used for the prevention or treatment of acute myeloid leukemia (AML).

[0066] In one specific embodiment of the present invention, the acute myeloid leukemia (AML) is expressed by a mutant FLT3 or a mutant RET kinase.

[0067] Therapeutic uses

[0068] The indirubin derivative compounds having novel heterobicyclic residues or their pharmaceutically acceptable salts according to the present invention inhibit the activity of FLT3 or RET kinases, especially mutant TFL3 and mutant RET kinases, and show preventive or therapeutic effects in diseases related to their activity.

[0069] The mutated FLT3 can be a mutation in the tyrosine kinase domain (TKD) of the FLT3 amino acid sequence (FLT3-TKD). The mutated FLT3 may further include an internal tandem duplication (ITD). Examples of the mutated FLT3 include, but are not limited to, one or more of FLT3-ITD, FLT3-D835Y, FLT3-F691L, FLT3-F691L / D835Y, FLT3-ITD / D835Y, or FLT3-ITD / F691L.

[0070] Impaired regulation of RET kinase expression, activity, or level is accompanied by point mutations / insertions / deletions of one or more RET kinase proteins. Non-limiting examples of point mutations / insertions / deletions of RET kinase proteins include, but are not limited to, one or more of M918T, M918V, C634W, V804L, and V804M.

[0071] In one embodiment, the compounds of the present invention can be used in the treatment of FLT3- and RET kinase-related diseases. In some embodiments, FLT3- and RET kinase-related diseases include malignant cells that express their kinases, such as cancer. In some embodiments, the cancer includes hematopoietic cancer, pancreatic cancer, esophageal cancer, lung cancer, and thyroid cancer. In a specific embodiment, the cancer is a hematopoietic cancer, such as lymphoma or leukemia. In a more specific embodiment, the cancer is multiple myeloma, malignant plasma cell tumor, Hodgkin lymphoma, nodular lymphocytic-predominant Hodgkin lymphoma, Kallmann's disease and myeloma, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (AL). L), Chronic myeloid leukemia (CML), Follicular lymphoma, Burkitt lymphoma, Marginal zone lymphoma, Mantle cell lymphoma, Large cell lymphoma, Precursor B-cell lymphoblastic lymphoma, Myeloid leukemia, Waldenström macroglobulinemia, Diffuse large B-cell lymphoma, Follicular lymphoma, Marginal zone lymphoma, Mucosa-associated lymphoid tissue lymphoma, Small cell lymphoma, Mantle cell lymphoma, Burkitt lymphoma, Primary mediastinal (thymic) large B-cell lymphoma Large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone B-cell lymphoma of the lymph node, marginal zone lymphoma of the spleen, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, T-cell / histiocytic large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma. Lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma caused by HHV8-associated multicentric Kastmann disease, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, or other hematopoietic cell-related cancers, but not limited thereto. Preferably, the cancer is acute myeloid leukemia (AML), more preferably, AML expressed by mutant FLT3 or RET kinase.

[0072] The term "acute myeloid leukemia (AML)" as used in this application refers to a bone marrow cancer characterized by the rapid growth of abnormal blood cells originating from the bone marrow, which inhibits the growth of normal blood cells. The main symptoms of AML include fatigue, shortness of breath, cyanosis or bleeding, and frequent infections. Various presumed causes of AML have been identified, but the exact cause remains unclear. The compounds of this invention, in particular, exhibit inhibitory activity against mutant FLT3 and mutant RET kinases, and have been shown to possess strong antiproliferative activity in FLT3-ITD expressing MV4-11 cell lines, FLT3-ITD and RET expressing MOLM-13 cell lines, FLT3 / D835Y and RET expressing MOLM14-FLT3 / D835Y cell lines, thus confirming their potential use as therapeutic agents for AML.

[0073] Therefore, the present invention provides a pharmaceutical composition comprising, as an active ingredient, a compound according to the invention or a pharmaceutically acceptable salt thereof, for inhibiting FLT3 and RET kinases. In one specific example, the pharmaceutical composition is used for the prevention or treatment of FLT3- and RET kinase-related diseases, more specifically, the prevention or treatment of AML.

[0074] In addition, the present invention provides a method for the prevention or treatment of FLT3- and RET kinase-related diseases, the method comprising the step of administering to a subject requiring a compound according to the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof.

[0075] In addition, the present invention provides a method for inhibiting FLT3 and RET kinases, the method comprising administering a pharmaceutically effective amount of a compound according to the invention, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof to an individual suffering from AML or an individual at risk of developing AML.

[0076] Furthermore, the present invention provides a method for inhibiting FLT3 in which the pharmaceutical composition described above exhibits strong antiproliferative activity in FLT3-ITD-expressing MV4-11 cell lines, FLT3-ITD and RET-expressing MOLM-13 cell lines, FLT3 / D835Y and RET-expressing MOLM14-FLT3 / D835Y cell lines.

[0077] Furthermore, this invention provides the use of a compound or a pharmaceutically acceptable salt thereof according to the invention for inhibiting FLT3 or for preventing or treating FLT3-related diseases. This invention also provides the use of a compound or a pharmaceutically acceptable salt thereof according to the invention for preparing medicines for the prevention or treatment of AML.

[0078] In one specific embodiment, the indirubin derivative compound of the present invention can be an IC50 inhibitor of FLT3 and RET kinase. 50 Values ​​below 100 nM. More specifically, compounds represented by Formula 1 can be IC50 inhibitors of FLT3 and RET kinases. 50 The value is below 50nM, further specifically, it can be below 40nM, even further specifically, it can be below 35nM, even further specifically, it can be below 30nM, even further specifically, it can be below 25nM, even further specifically, it can be below 20nM, even further specifically, it can be below 15nM, and even further specifically, it can be below 10nM.

[0079] The present invention will now be described in more detail through embodiments. These embodiments are merely for illustrating the invention more specifically, and those skilled in the art should understand that the scope of the invention is not limited to these embodiments.

[0080] General synthesis methods

[0081] The compounds according to the invention can be prepared using readily available starting materials, with variations of the specific synthetic protocols described below that are well known to those skilled in the art.

[0082] Following the steps shown in reactions 1 and 2, indirubin analogues 6a-t (including the ethyl linker), 8a-t (including the ethyl ketone linker), and substituted derivative 10a-e were synthesized.

[0083]

Reaction Formula 1

[0084] General synthetic route for preparing indirubin derivatives with bicyclic residues

[0085]

[0086]

[0087] R2=

[0088] (i) 2-Ethyl-2,6-diazaspiro[3.3]heptane

[0089] (ii) (1R,5S,6S)-N-ethyl-3-azabicyclo[3.1.0]hexane-6-amine

[0090] (iii) (1R,5S,6S)-3-ethyl-3-azabicyclo[3.1.0]hexane-6-amine

[0091] (iv) (1R,4R)-2-ethyl-2,5-diazabicyclo[2.2.1]heptane

[0092] (v) (1S,4S)-2-ethyl-2,5-diazabicyclo[2.2.1]heptane

[0093] (vi) 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethane-1-one

[0094] (vii) N-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)-acetamide

[0095] (viii) 1-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)ethane-1-one

[0096] (ix) 1-((1R,4R)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethane-1-one

[0097] (x) 1-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethane-1-one

[0098] Reagents and conditions:

[0099] (a) Various indigo, sodium methoxide, methanol, room temperature, 2 hours, 63-65%;

[0100] (b) Hydroxylamine hydrochloride, KOH, sonication, 2 hours, 89-91%;

[0101] (c) 1,2-Dibromoethane, TEA, DMF, RT, overnight, 76-80%;

[0102] (d) Various boc-amines, DMF, 70℃, overnight, 85-87%;

[0103] (e) 10a-e, TEA, DMF, 70℃, 12 hours, 80-84%;

[0104] (f) 20% TFA in DCM, 0℃, 2 hours, 15-30%.

[0105] In reaction 1, under basic conditions, indoleacetic acid ester (compound 1) is coupled with various isatitins to prepare 5'-substituted indirubin derivatives 2a-d. 2a-d is then reacted with hydroxylamine to convert to an N-oxime group in a ketone, synthesizing 5'-substituted -3-indirubin oxime derivatives 3a-d. Compounds 4a-d are harvested from nucleophilic substitution reactions between 1,2-dibromoethane and each of the N-oxime compounds 3a-d. The terminal bromide of 4a-d is substituted under basic conditions with various boc-protected amines containing bicyclic residues to prepare compound 5a-t. Then, using 20% ​​trifluoroacetic acid in dichloromethane, each boc group of 5a-t is deprotected to prepare the final compound 6a-t with an ethyl linker. Using compounds 3a-d and 10a-e, compounds 7a-t containing an ethyl ketone linker between various indirubin N-oximes and boc-protected amines containing bicyclic groups were prepared under similar reaction conditions to those used in step d of the substitution of boc-protected heterocycles. The final compound 8a-t with the ethyl ketone linker was synthesized from the corresponding boc-protected compound 7a-t under the same conditions as described in step f.

[0106] Compounds 10a-e, which are various chloroacetamide reagents, are prepared under alkaline conditions by reacting 2-chloroacetyl chloride and five nitrogen-containing bicyclic residues, as shown in reaction formula 2.

[0107]

Reaction 2

[0108] General synthetic route for preparing 10a-e

[0109]

[0110] 10a: R = tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid

[0111] 10b: R=(1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid

[0112] 10c: R = tert-butyl((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate

[0113] 10d: R=(1R,4R)-tert-butyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid

[0114] 10e: R=(1S,4S)-tert-butyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid

[0115] Reagents and conditions: (a) various boc-amines, TEA, DMF, 0℃, 2 hours, 88-90%.

[0116] Synthesis method

[0117] All chemicals and solvents were purchased from chemical suppliers in their unpurified state. For purification, column chromatography was performed using pre-coated silica gel plates (MERCK silica gel 60; F254, 0.040–0.063 mm). NMR spectra were obtained at 1H frequencies of 400 MHz using a 400 M NMR spectrometer (JEOL ECS 400 NMR spectrometer; Tokyo Materials, Japan). For internal standards, proton chemical migration was determined in parts per million (ppm). Chemical migration, multiplicity, and coupling constant (J) were recorded and calculated using ACD NMR Processor Academic Edition editing software. Analysis was performed using an ultra-high performance liquid chromatograph (Waters ACQUITY UPLC) connected to a triple quadrupole mass spectrometer (Micromass Quattro Micro, Waters). During separation under isosolvent conditions, chromatographic separation was performed on a BEH C18 1.7 μm, 2.1 mm × 50 mm column (Waters) maintained at 40 °C (mobile phase A: mobile phase B = 20:80). Mobile phase A consisted of water (LC-MS grade) containing 0.1% formic acid (v / v), and mobile phase B consisted of acetonitrile (LC-MS grade) containing 0.1% formic acid (v / v). The flow rate was 0.2 mL / min.

[0118] Synthesis of (Z)-[2,3'-binindololinylidene]-2,3'-dione (2a)

[0119] Indigo (1.3 equivalents) was added to a 0.05 M solution of indoleacetic acid and compound 1 (1 equivalent) in methanol. The reaction mixture was stirred for 5 minutes at room temperature. Then, sodium methoxide powder (2 equivalents) was added to the reaction mixture, and stirring was continued for 2 hours. TLC (chloroform:methanol = 20:1, compound 1 Rf = 0.9, product Rf = 0.7) showed complete consumption of indoleacetic acid and the detection of a new spot. The reaction mixture was then diluted with cold water to give a purple precipitate. The solid was filtered and washed with a 2 / 1 water / methanol solution and hexane. The solid was dried under reduced pressure at 40°C for 5 hours to obtain compound 2a of excellent purity.

[0120] Synthesis of (Z)-5'-fluoro-[2,3'-biindolineylidene]-2,3'-dione (2b)

[0121] Compound 2b was synthesized from compound 1 and 5-fluoroindigo in the order described for compound 2a.

[0122] Synthesis of (Z)-5'-chloro-[2,3'-biindololinylidene]-2,3'-dione (2c)

[0123] Compound 2c was synthesized from compound 1 and 5-chloroindigo in the order described for compound 2a.

[0124] Synthesis of (Z)-5'-bromo-[2,3'-binindololinylidene]-2,3'-dione (2d)

[0125] Compound 2d was synthesized from compound 1 and 5-bromoindigo in the order described for compound 2a.

[0126] Synthesis of (2Z,3E)-3-(hydroxyimino)-[2,3'-binindololinylidene]-2'-one (3a)

[0127] 12 equivalents of KOH solid were added to the reactants, and the reaction vessel was sonicated at 30°C for 30 minutes. After the reaction was completed, monitored by TLC, TLC (chloroform:methanol = 20:1, compound 2a Rf = 0.7, product Rf = 0.6) showed that compound 2a was completely consumed and a new spot was detected. The reaction mixture was acidified with 1N HCl solution until the pH was between 1 and 3, and a red product precipitated. The solid was filtered and washed with warm water and hexane. The solid was then subjected to reduced pressure at 40°C for 4 hours and dried to obtain compound 3a.

[0128] Synthesis of (2Z,3E)-5'-fluoro-3-(hydroxyimino)-[2,3'-binindololinylidene]-2'-one (3b)

[0129] Compound 3b was synthesized from compound 2b in the order described for compound 3a.

[0130] Synthesis of (2Z,3E)-5'-chloro-3-(hydroxyimino)-[2,3'-binindololinylidene]-2'-one (3c)

[0131] Compound 3c was synthesized from compound 2c in the order described for compound 3a.

[0132] Synthesis of (2Z,3E)-5'-bromo-3-(hydroxyimino)-[2,3'-binindololinylidene]-2'-one (3d)

[0133] Compound 3d was synthesized from compound 2d in the order described for compound 3a.

[0134] Synthesis of (2Z,3E)-3-((2-bromoethoxy)imino)-[2,3'-binindololinylidene]-2'-one (4a)

[0135] Under an argon atmosphere, TEA (7 equivalents) and 1,2-dibromoethane (7 equivalents) were added to a solution of compound 4 in DMF. The reaction mixture was stirred overnight at room temperature. After the reaction was completed, as monitored by TLC, TLC (chloroform:methanol = 20:1, compound 3a Rf = 0.6, product Rf = 0.8) showed that compound 3a was completely consumed and a new spot was detected. The reaction mixture was diluted with cold water, accompanied by precipitation of the product. The precipitate was filtered and washed with water and hexane. The solid was subjected to reduced pressure at 40°C for 4 hours and dried to obtain compound 4a.

[0136] Synthesis of (2Z,3E)-3-((2-bromoethoxy)imino)-5'-fluoro-[2,3'-binindololinylidene]-2'-one (4b)

[0137] Compound 4b was synthesized from compound 3b in the order described for compound 4a.

[0138] Synthesis of (2Z,3E)-3-((2-bromoethoxy)imino)-5'-chloro-[2,3'-binindololinylidene]-2'-one (4c)

[0139] Compound 4c was synthesized from compound 3c in the order described for compound 4a.

[0140] Synthesis of (2Z,3E)-5'-bromo-3-((2-bromoethoxy)imino)-[2,3'-binindololinylidene]-2'-one (4d)

[0141] Compound 4d was synthesized from compound 3d in the order described for compound 4a.

[0142] Synthesis of tert-butyl-6-(2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (5a)

[0143] Under an argon atmosphere, 4 equivalents of tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid and 10 equivalents of TEA were added to a solution of compound 4a in DMF. The reaction mixture was stirred at 70°C for 12 hours. After the reaction was completed, monitored by TLC, TLC (chloroform:methanol = 20:1, compound 4a Rf = 0.8, product Rf = 0.75) showed that compound 4a was completely consumed and a new spot was detected. Cold distilled water was poured into the reaction mixture, accompanied by precipitation of the product. The solid was filtered, washed with water, and dried in a vacuum oven at 60°C for 5 hours to harvest compound 5a.

[0144] Synthesis of (1R,5S,6s)-tert-butyl-6-((2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (5b)

[0145] Compound 5b was synthesized from compound 4a and (1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid in the order described for compound 5a.

[0146] Synthesis of tert-butyl((1R,5S,6S)-3-(2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (5c)

[0147] Compound 5c was synthesized from compound 4a and tert-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate in the order described for compound 5a.

[0148] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5d)

[0149] Compound 5d was synthesized from compound 4a and tert-butyl(1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0150] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5e)

[0151] Compound 5e was synthesized from compound 4a and tert-butyl(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0152] Synthesis of tert-butyl 6-(2-((((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (5f)

[0153] Compound 5f was synthesized from compound 4b and tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid in the order described for compound 5a.

[0154] Synthesis of (1R,5S,6S)-tert-butyl-6-((2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (5g)

[0155] Compound 5g was synthesized from compound 4b and (1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid in the order described for compound 5a.

[0156] Synthesis of tert-butyl((1R,5S,6S)-3-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (5h)

[0157] Compound 5h was synthesized from compound 4b and tert-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate in the order described for compound 5a.

[0158] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5i)

[0159] Compound 5i was synthesized from compound 4b and tert-butyl(1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0160] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5j)

[0161] Compound 5j was synthesized from compound 4b and tert-butyl(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0162] Synthesis of tert-butyl-6-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)ethyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (5k)

[0163] Compound 5k was synthesized from compound 4c and tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid in the order described for compound 5a.

[0164] Synthesis of (1R,5S,6S)-tert-butyl-6-((2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)ethyl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (5l)

[0165] Compound 5l was synthesized from compound 4c and (1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid in the order described for compound 5a.

[0166] Synthesis of tert-butyl(1R,5S,6S)-3-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (5m)

[0167] Compound 5m was synthesized from compound 4c and tert-butyl-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate in the order described for compound 5a.

[0168] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5n)

[0169] Compound 5n was synthesized from compound 4c and tert-butyl(1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0170] Example 12: Synthesis of 1-((2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)indoline-2-ylidene)-6-chloro-1,3-dihydro-2H-inden-2-one (5o)

[0171]

[0172] Compound 5o was synthesized from compound 4c and tert-butyl(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0173] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.34 - 1.45 (m, 1 H), 1.59 - 1.70 (m,1 H), 2.40 - 2.47 (m, 1 H), 2.59 - 2.72 (m, 1 H), 2.90 - 3.01 (m, 2 H), 3.01- 3.17 (m, 2 H), 3.38 - 3.47 (m, 2 H), 4.63 (t, J=5.84 Hz, 2 H), 6.90 (d, J=8.24 Hz, 1 H), 7.00 - 7.12 (m, 1 H), 7.18 (dd, J=8.24, 2.29 Hz, 1 H), 7.36 -7.51 (m, 2 H), 8.15 (dt, J=7.56, 0.92 Hz, 1 H), 8.71 (d, J=2.06 Hz, 1 H), 10.46 - 11.27 (m, 1 H), 11.29 - 12.14 (m, 1 H). MS (ESI): [M + H]+ = 436.5.

[0174] Synthesis of tert-butyl-6-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (5p)

[0175] Compound 5p was synthesized from compound 4d and tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid in the order described for compound 5a.

[0176] Synthesis of (1R,5S,6s)-tert-butyl-6-((2-((((E)-((Z)-5'-bromo-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (5q)

[0177] Compound 5q was synthesized from compound 4d and (1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid in the order described for compound 5a.

[0178] Synthesis of tert-butyl((1R,5S,6S)-3-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)ethyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (5r)

[0179] Compound 5r was synthesized from compound 4d and tert-butyl-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate in the order described for compound 5a.

[0180] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (5S)

[0181] Compound 5s was synthesized from compound 4d and tert-butyl(1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0182] Example 14: Synthesis of 1-((2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)indoline-2-yl)-6-bromo-1,3-dihydro-2H-inden-2-one (5t)

[0183]

[0184] Compound 5t was synthesized from compound 4d and tert-butyl(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid, in the order described for compound 5a.

[0185] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.35 - 1.43 (m, 1 H), 1.59 - 1.71 (m,1 H), 2.40 - 2.48 (m, 1 H), 2.58 - 2.69 (m, 1 H), 2.88 - 3.01 (m, 2 H), 3.02- 3.16 (m, 2 H), 3.38 - 3.47 (m, 2 H), 4.63 (td, J=6.01, 0.80 Hz, 2 H), 6.86 (d, J=8.24 Hz, 1 H), 6.98 - 7.14 (m, 1 H), 7.27 - 7.35 (m, 1 H), 7.40 - 7.52 (m, 2 H), 8.15 (dt, J=7.79, 0.92 Hz, 1 H), 8.86 (d, J=2.06 Hz, 1 H), 10.54 -11.26 (m, 1 H), 11.36 - 12.11 (m, 1 H). MS (ESI): [M + H]+ = 480.4.

[0186] Example 1: Synthesis of (2Z,3E)-3-((2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-[2,3'-biindololinylidene]-2'-one (6a)

[0187]

[0188] 20% TFA in DCM was slowly added to a solution of compound 5a at 0°C for 2 hours. After the reaction was completed, monitored by TLC (ammonia-saturated chloroform:methanol = 10:1, compound 5a Rf = 0.9, product Rf = 0.4), TLC showed that compound 5a was completely consumed and a new spot was detected. After evaporation of the solution, the residue was resuspended using the minimum volume of methanol. For neutralization, the solution was slowly added to a saturated aqueous NaHCO3 solution. The solid was filtered, washed with water, and dried in a vacuum oven at 60°C for 5 hours to harvest compound 6a. The mixture was purified using a silica gel column (eluent:ammonia-saturated chloroform:methanol = 100:1).

[0189] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.87 (t, J=5.61 Hz, 2 H) 3.27 (s, 4H) 3.43 (s, 4 H) 4.54 (t, J=5.61 Hz, 2 H) 6.89 - 6.92 (m, 1 H) 6.98 (td, J=7.67, 1.15 Hz, 1 H) 7.02 - 7.07 (m, 1 H) 7.13 - 7.18 (m, 1 H) 7.40 - 7.47 (m,2 H) 8.14 (dt, J=7.67, 0.97 Hz, 1 H) 8.61 (dd, J=7.90, 0.57Hz, 1H) 10.79(br. s., 1 H). MS (ESI): [M + H]+ = 401.7.

[0190] Example 2: Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-ylamino)ethoxy)imino)-[2,3'-binindololinylidene]-2'-one (6b)

[0191]

[0192] Compound 6b was synthesized from compound 5b according to the steps described for compound 6a.

[0193] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.30 (s, 2 H) 2.03 (t, J=1.95 Hz, 1H) 2.58 (s, 1 H) 2.60 (s, 1 H) 2.79 (s, 1 H) 2.82 (s, 1 H) 3.08 (t, J=5.84Hz, 2 H) 4.62 (t, J=5.72 Hz, 2 H) 6.89 - 6.92 (m, 1 H) 6.97 - 7.06 (m, 2 H)7.12 - 7.18 (m, 1 H) 7.39 - 7.46 (m, 2 H) 8.16 (dt, J=7.67, 0.97 Hz, 1H)8.62 (d, J=8.01 Hz, 1 H) 10.74 - 10.80 (m, 1 H) 11.65 - 11.74 (m, 1 H). MS(ESI): [M + H]+ = 401.1.

[0194] Example 3: Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)ethoxy)imino)-[2,3'-binindololinylidene]-2'-one (6c)

[0195]

[0196] Compound 6c was synthesized from compound 5c according to the steps described for compound 6a.

[0197] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.15 - 1.28 (m, 2 H), 1.49 - 1.79 (m,2 H), 2.36 (t, J=1.95 Hz, 1 H), 2.40 (dt, J=8.47, 1.49 Hz, 2 H), 2.90 (t, J=5.80 Hz, 2 H), 3.04 (d, J=8.47 Hz, 2 H), 4.61 (t, J=5.72 Hz, 2 H), 6.86 -6.93 (m, 1 H), 6.98 (td, J=7.67, 1.15 Hz, 1 H), 7.01 - 7.08 (m, 1 H), 7.15(td, J=7.60, 1.10 Hz, 1 H), 7.37 - 7.49 (m, 2 H), 8.15 (dt, J=7.67, 0.97 Hz, 1 H), 8.60 (d, J=7.40 Hz, 1 H), 10.63 - 10.91 (m, 1 H), 11.31 - 12.03 (m, 1H). MS (ESI): [M + H]+ = 402.5.

[0198] Example 4: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-[2,3'-biindolineylidene]-2'-one (6d)

[0199]

[0200] Compound 6d was synthesized from compound 5d according to the steps described for compound 6a.

[0201] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.35 - 1.41 (m, 1 H) 1.58 - 1.63 (m,1 H) 2.40 (d, J=8.70 Hz, 1 H) 2.60 (dd, J=9.73, 1.03 Hz, 1 H) 2.91 - 2.98 (m,2 H) 2.99 - 3.12 (m, 2 H) 3.40 (br. s., 1 H) 4.58 - 4.65 (m, 2 H) 6.90 (d, J=7.79 Hz, 1 H) 6.96 - 7.06 (m, 2 H) 7.13 - 7.18 (m, 1 H) 7.39 - 7.46 (m, 2 H)8.16 (dd, J=7.67, 0.57 Hz, 1 H) 8.63 (d, J=7.79 Hz, 1 H) 10.74 - 10.80 (m, 1H) 11.66 - 11.73 (m, 1 H). MS (ESI): [M + H]+ = 401.5.

[0202] Example 5: Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-[2,3'-biindolineylidene]-2'-one (6e)

[0203]

[0204] Compound 6e was synthesized from compound 5e according to the steps described for compound 6a.

[0205] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.37 - 1.47 (m, 1 H), 1.63 - 1.71 (m,1 H), 2.42 - 2.48 (m, 1 H), 2.67 (dd, J=10.08, 2.29 Hz, 1 H), 2.94 (dd, J=9.27, 2.40 Hz, 1 H), 2.97 - 3.15 (m, 3 H), 3.44 - 3.48 (m, 2 H), 4.53 - 4.68 (m, 2 H), 6.86 - 6.94 (m, 1 H), 6.94 - 7.08 (m, 2 H), 7.15 (td, J=7.61, 1.26Hz, 1H), 7.33 - 7.50 (m, 2 H), 8.10 - 8.20 (m, 1 H), 8.63 (d, J=7.30 Hz, 1H), 10.66 - 10.90 (m, 1 H), 11.69 (br. s., 1 H). MS (ESI): [M + H]+ = 402.5.

[0206] Example 6: Synthesis of (2Z,3E)-3-((2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (6f)

[0207]

[0208] Compound 6f was synthesized from compound 5f according to the steps described for compound 6a.

[0209] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.89 (t, J=5.50 Hz, 2 H) 3.27 (s, 2H) 3.29 (s, 4 H) 3.43 (s, 2 H) 4.54 (t, J=5.61 Hz, 2 H) 6.84 - 6.90 (m, 1 H)6.95 - 7.02 (m, 1 H) 7.07 (ddd, J=7.73, 5.21, 3.32 Hz, 1 H) 7.42 - 7.50 (m, 2H) 8.15 (dt, J=7.56, 0.92 Hz, 1 H) 8.45 (dd, J=11.33, 2.63Hz, 1H) 10.74 -10.86 (m, 1 H). MS (ESI): [M + H]+ = 420.2.

[0210] Example 7: Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-ylamino)ethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (6g)

[0211]

[0212] Compound 6g was synthesized from compound 5g according to the steps described for compound 6a.

[0213] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.28 - 1.31 (m, 2 H) 2.02 (t, J=1.95Hz, 1 H) 2.56 - 2.61 (m, 2 H) 2.80 (d, J=10.99 Hz, 2 H) 3.10 (t, J=5.84 Hz, 2H) 4.63 (t, J=5.84 Hz, 2 H) 6.87 (dd, J=8.47, 5.04 Hz, 1 H) 6.98 (td, J=8.82,2.75 Hz, 1 H) 7.06 (ddd, J=7.90, 5.15, 3.21 Hz, 1 H) 7.42 - 7.48 (m, 2 H)8.16 (d, J=7.56 Hz, 1 H) 8.45 (dd, J=11.33, 2.63 Hz, 1 H) 10.78 (br. s., 1H). MS (ESI): [M + H]+ = 420.3.

[0214] Example 8: Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)ethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (6h)

[0215]

[0216] Compound 6h was synthesized from compound 5h according to the steps described for compound 6a.

[0217] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.12 - 1.24 (m, 2 H), 1.42 - 2.02 (m,2 H), 2.35 (t, J=1.95 Hz, 1 H), 2.41 (dt, J=8.42, 1.52 Hz, 2 H), 2.92 (t, J=6.00 Hz, 2 H), 3.04 (d, J=8.50 Hz, 2 H), 4.61 (t, J=6.00 Hz, 2 H), 6.82 -6.91 (m, 1 H), 6.93 - 7.01 (m, 1 H), 7.02 - 7.12 (m, 1 H), 7.38 - 7.52 (m, 2H), 8.15 (dt, J=7.79, 0.92 Hz, 1 H), 8.43 (dd, J=11.22, 2.52 Hz, 1 H), 10.61- 10.90 (m, 1 H), 11.27 - 12.23 (m, 1 H). MS (ESI): [M + H]+ = 420.4.

[0218] Example 9: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (6i)

[0219]

[0220] Compound 6i was synthesized from compound 5i according to the steps described for compound 6a.

[0221] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (d, J=8.01 Hz, 1 H) 1.60 (d, J=8.70 Hz, 1 H) 2.40 (d, J=9.85 Hz, 1 H) 2.60 (dd, J=9.73, 2.18 Hz, 1 H) 2.90 -2.96 (m, 2 H) 2.99 - 3.15 (m, 3 H) 3.39 (s, 1 H) 4.62 (t, J=5.84 Hz, 2 H)6.84 - 6.89 (m, 1 H) 6.95 - 7.01 (m, 1 H) 7.03 - 7.09 (m, 1 H) 7.42 - 7.48(m, 2 H) 8.16 (dt, J=7.79, 0.92 Hz, 1 H) 8.47 (dd, J=11.33, 2.63 Hz, 1 H)10.74 - 10.83 (m, 1 H) 11.75 (br. s., 1 H). MS (ESI): [M + H]+ = 420.2.

[0222] Example 10: Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (6j)

[0223]

[0224] Compound 6j was synthesized from compound 5j according to the steps described for compound 6a.

[0225] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.37 - 1.45 (m, 1 H), 1.60 - 1.68 (m,1 H), 2.39 - 2.47 (m, 1 H), 2.64 (dd, J=9.85, 2.06 Hz, 1 H), 2.93 (dd, , 6.92 - 7.02 (m, 1H), 7.02 - 7.11 (m, 1 H), 7.39 - 7.51 (m, 2 H), 8.11 - 8.23 ​​(m, 1 H), 8.46 (dd, J=11.20, 2.80 Hz, 1 H), 10.69 - 10.98 (m, 1 H), 11.65 - 11.94 (m, 1 H).MS (ESI): [M + H]+ = 420.5.

[0226] Synthesis of (2Z,3E)-3-((2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-5'-chloro-[2,3'-biindolineylidene]-2'-one (6k)

[0227] Compound 6k was synthesized from compound 5k according to the steps described for compound 6a.

[0228] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-ylamino)ethoxy)imino)-5'-chloro-[2,3'-biindolineylidene]-2'-one (6l)

[0229] Compound 6l was synthesized from compound 5l according to the steps described for compound 6a.

[0230] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)ethoxy)imino)-5'-chloro-[2,3'-biindolineylidene]-2'-one (6m)

[0231] Compound 6m was synthesized from compound 5m according to the steps described for compound 6a.

[0232] Example 11: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-chloro-[2,3'-biindolineylidene]-2'-one (6n)

[0233]

[0234] Compound 6n was synthesized from compound 5n according to the steps described for compound 6a.

[0235] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.31 - 1.46 (m, 1 H), 1.57 - 1.71 (m,1 H), 2.43 (d, J=9.80 Hz, 1 H), 2.63 (dd, J=9.85, 2.29 Hz, 1 H), 2.86 - 3.00(m, 2 H), 3.01 - 3.17 (m, 2 H), 3.36 - 3.45 (m, 2 H), 4.63 (t, J=6.20 Hz, 2H), 6.90 (d, J=8.24 Hz, 1 H), 6.99 - 7.12 (m, 1 H), 7.18 (dd, J=8.24, 2.29Hz, 1H), MS (ESI): [M +H]+ = 436.4.

[0236] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-chloro-[2,3'-biindololinylidene]-2'-one (6o)

[0237] Compound 6o was synthesized from compound 5o according to the steps described for compound 6a.

[0238] Synthesis of (2Z,3E)-3-((2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-5'-bromo-[2,3'-biindololinylidene]-2'-one (6p)

[0239] Compound 6p was synthesized from compound 5p according to the steps described for compound 6a.

[0240] Synthesis of (2Z,3E)-3-((2-((1R,5S,6s)-3-azabicyclo[3.1.0]hexane-6-ylamino)ethoxy)imino)-5'-bromo-[2,3'-biindolineylidene]-2'-one (6q)

[0241] Compound 6q was synthesized from compound 5q according to the steps described for compound 6a.

[0242] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)ethoxy)imino)-5'-bromo-[2,3'-biindolineylidene]-2'-one (6r)

[0243] Compound 6r was synthesized from compound 5r according to the steps described for compound 6a.

[0244] Example 13: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-bromo-[2,3'-biindolineylidene]-2'-one (6S)

[0245]

[0246] Compound 6s was synthesized from compound 5s according to the steps described for compound 6a.

[0247] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 - 1.43 (m, 1 H), 1.55 - 1.66 (m,1 H), 2.41 (dd, J=9.04, 1.03 Hz, 1 H), 2.60 (dd, J=9.85, 2.29 Hz, 1 H), 2.90- 3.00 (m, 2 H), 3.02 - 3.17 (m, 2 H), 3.36 - 3.43 (m, 2 H), 4.63 (td, J=6.01, 0.80 Hz, 2 H), 6.86 (d, J=8.24 Hz, 1 H), 7.02 - 7.11 (m, 1 H), 7.30(dd, J=8.20, 2.10 Hz, 1 H), 7.41 - 7.50 (m, 2 H), 8.15 (dt, J=7.61, 1.00 Hz,1 H), 8.86 (d, J=2.06 Hz, 1 H), 10.60 - 11.27 (m, 1 H), 11.34 - 12.09 (m, 1H). MS (ESI): [M + H]+= 482.3.

[0248] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)ethoxy)imino)-5'-bromo-[2,3'-biindololinylidene]-2'-one (6t)

[0249] Compound 6t was synthesized from compound 5t according to the steps described for compound 6a.

[0250] Synthesis of tert-butyl-6-(2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (7a)

[0251] Under an argon atmosphere, 4 equivalents of 10a and 10 equivalents of TEA were added to a solution of compound 3a in DMF. The reaction mixture was stirred at 70°C for 12 hours. After the reaction was completed, monitored by TLC, TLC (chloroform:methanol = 20:1, compound 3a Rf = 0.8, product Rf = 0.75) showed that compound 3a was completely consumed and a new spot was detected. Cold distilled water was poured into the reaction mixture, accompanied by precipitation of the product. The solid was filtered, washed with water, and dried in a vacuum oven at 60°C for 5 hours to harvest compound 7a.

[0252] Synthesis of (1R,5S,6s)-tert-butyl-6-(2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetamido)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (7b)

[0253] Compound 7b was synthesized from compounds 3a and 10b in the order described for compound 7a.

[0254] Synthesis of tert-butyl(1R,5S,6S)-3-(2-(((E)-((Z)-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (7c)

[0255] Compound 7c was synthesized from compounds 3a and 10c in the order described for compound 7a.

[0256] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7d)

[0257] Compound 7d was synthesized from compounds 3a and 10d in the order described for compound 7a.

[0258] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7e)

[0259] Compound 7e was synthesized from compounds 3a and 10e in the order described for compound 7a.

[0260] Synthesis of tert-butyl-6-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (7f)

[0261] Compound 7f was synthesized from compounds 3b and 10a in the order described for compound 7a.

[0262] Synthesis of (1R,5S,6s)-tert-butyl-6-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamido)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (7g)

[0263] Compound 7g was synthesized from compounds 3b and 10b in the order described for compound 7a.

[0264] Synthesis of tert-butyl((1R,5S,6S)-3-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (7h)

[0265] Compound 7h was synthesized from compounds 3b and 10c in the order described for compound 7a.

[0266] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7i)

[0267] Compound 7i was synthesized from compounds 3b and 10d in the order described for compound 7a.

[0268] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7j)

[0269] Compound 7j was synthesized from compounds 3b and 10e in the order described for compound 7a.

[0270] Synthesis of tert-butyl-6-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (7k)

[0271] Compound 7k was synthesized from compounds 3c and 10a in the order described for compound 7a.

[0272] Synthesis of (1R,5S,6S)-tert-butyl-6-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamido)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (7l)

[0273] Compound 7l was synthesized from compounds 3c and 10b in the order described for compound 7a.

[0274] Synthesis of tert-butyl(1R,5S,6S)-3-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (7m)

[0275] Compound 7m was synthesized from compounds 3c and 10c in the order described for compound 7a.

[0276] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7n)

[0277] Compound 7n was synthesized from compounds 3c and 10d in the order described for compound 7a.

[0278] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7o)

[0279] Compound 7o was synthesized from compounds 3c and 10e in the order described for compound 7a.

[0280] Synthesis of tert-butyl-6-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (7p)

[0281] Compound 7p was synthesized from compounds 3d and 10a in the order described for compound 7a.

[0282] Synthesis of (1R,5S,6s)-tert-butyl-6-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamido)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (7q)

[0283] Compound 7q was synthesized from compounds 3d and 10b in the order described for compound 7a.

[0284] Synthesis of tert-butyl((1R,5S,6S)-3-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-binindololinylidene]-3-ylidene)amino)oxy)acetyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (7r)

[0285] Compound 7r was synthesized from compounds 3d and 10c in the order described for compound 7a.

[0286] Synthesis of (1R,4R)-tert-butyl5-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7S)

[0287] Compound 7s was synthesized from compounds 3d and 10d in the order described for compound 7a.

[0288] Synthesis of (1S,4S)-tert-butyl5-(2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (7t)

[0289] Compound 7t was synthesized from compounds 3d and 10e in the order described for compound 7a.

[0290] Example 15: Synthesis of (2Z,3E)-3-((2-oxo-2-(2,6-diazaspiro[3.3]heptane-2-yl)oxy)imino)-[2,3'-binindololinylidene]-2'-one (8a)

[0291]

[0292] Compound 8a was synthesized from compound 7a in the order described for compound 6a.

[0293] 1H NMR (400 MHz, DMSO-d6) δ ppm 3.49 - 3.54 (m, 4 H) 4.01 (s, 2 H)4.33 (s, 2 H) 5.05 (s, 2 H) 6.87 - 6.93 (m, 2 H) 7.02 - 7.07 (m, 1 H) 7.15(td, J=7.67, 1.14 Hz, 1 H) 7.41 - 7.48 (m, 2 H) 8.18 - 8.21 (m, 1 H) 8.42 -8.46 (m, 1 H). MS (ESI): [M + H]+ = 415.7.

[0294] Example 16: Synthesis of N-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)-2-(((E)-((Z)-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamide (8b)

[0295]

[0296] Compound 8b was synthesized from compound 7b in the order described for compound 6a.

[0297] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.46 (s, 2 H) 2.59 (dt, J=4.24, 2.00Hz, 1 H) 2.61 (s, 1 H) 2.64 (s, 1 H) 2.88 (s, 1 H) 2.91 (s, 1 H) 4.91 (s, 2H) 6.87 - 6.90 (m, 1 H) 6.93 (td, J=7.67, 1.14 Hz, 1 H) 7.01 - 7.06 (m, 1 H)7.15 (td, J=7.67, 1.14 Hz, 1 H) 7.40 - 7.47 (m, 2 H) 8.19 (dt, J=7.61, 1.00Hz, 1 H) 8.29 (d, J=4.12 Hz, 1 H) 8.43 - 8.47 (m, 1 H) 10.77 (br. s., 1 H)11.66 (br. s., 1 H). MS (ESI): [M + H]+ = 415.9.

[0298] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)-2-oxoethoxy)imino)-[2,3'-binindololinylidene]-2'-one (8c)

[0299] Compound 8c was synthesized from compound 7c in the order described for compound 6a.

[0300] Example 17: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-[2,3'-binindololinylidene]-2'-one (8d)

[0301]

[0302] Compound 8d was synthesized from compound 7d in the order described for compound 6a.

[0303] 1 H NMR (400 MHz, DMSO-d6, room temperature) δ ppm 1.54 - 1.71 (m, 2 H) 2.77 (d, J=9.62 Hz, 1 H) 2.84 (dt, J=9.79, 2.20 Hz, 1 H) 3.16 (d, J=10.76 Hz, 0.5 H)3.40 (d, J=8.70 Hz, 0.5 H) 3.53 (dd, J=9.04, 2.18 Hz, 0.5 H) 3.58 (s, 0.5 H)3.68 (s, 0.5 H) 4.64 (d, J=13.97 Hz, 1 H) 5.05 - 5.15 (m, 1 H) 5.25 (s, 1 6.88 (d, J=7.10 Hz, 1 H) 6.93 - 6.98 (m, 1 H) 7.02 - 7.07 (m, 1 H) 7.11 -7.17 (m, 1 H) 7.40 - 7.47 (m, 2 H) 8.22 (t, J=6.98 Hz, 1 H) 8.47 (t, J=8.01Hz, 1 H) 10.76 (br. s., 1 H). 8-day Hz at room temperature. 1 The 1H NMR spectrum showed a 2-fold increase in a portion of the signal, indicating that two rotational conformational isomers exist in DMSO-d6 in a ~1:1 ratio. MS (ESI): [M + H] + = 415.9.

[0304] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-[2,3'-biindololinylidene]-2'-one (8e)

[0305] Compound 8e was synthesized from compound 7e in the order described for compound 6a.

[0306] Example 18: Synthesis of (2Z,3E)-5'-fluoro-3-((2-oxo-2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-[2,3'-binindolinyl]-2'-one (8f)

[0307]

[0308] Compound 8f was synthesized from compound 7f in the order described for compound 6a.

[0309] 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.53 (s, 3 H) 3.62 (br. s., 1 H) 3.98- 4.03 (m, 2 H) 4.33 (br. s., 2 H) 5.05 (s, 2 H) 6.83 - 6.88 (m, 1 H) 6.94 -7.00 (m, 1 H) 7.07 (td, J=7.27, 1.95 Hz, 1 H) 7.42 - 7.50 (m, 2 H) 8.18 (dd,J=7.79, 0.92 Hz, 1 H) 8.20 - 8.25 (m, 1 H) 10.80 (br. s., 1 H). MS (ESI): [M+ H]+ = 434.6.

[0310] Example 19: Synthesis of N-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)-2-(((E)-((Z)-5'-fluoro-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamide (8 g)

[0311]

[0312] Compound 8g was synthesized from compound 7g in the order described for compound 6a.

[0313] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.45 (s, 2 H) 2.54 - 2.57 (m, 1 H)2.61 (br. s., 1 H) 2.87 (s, 1 H) 2.90 (s, 1 H) 4.94 (s, 2 H) 6.82 - 6.89 (m,1 H) 6.97 (td, J=8.82, 2.29 Hz, 1 H) 7.04 - 7.10 (m, 1 H) 7.42 - 7.50 (m, 2H) 8.18 - 8.27 (m, 3 H) 10.79 (br. s., 1 H) 11.75 (br. s., 1 H). MS (ESI): [M+ H]+ = 434.6.

[0314] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6-amino-3-azabicyclo[3.1.0]hexane-3-yl)-2-oxoethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (8h)

[0315] Compound 8h was synthesized from compound 7h in the order described for compound 6a.

[0316] Example 20: Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-fluoro-[2,3'-biindolineylidene]-2'-one (8i)

[0317]

[0318] Compound 8i was synthesized from compound 7i in the order described for compound 6a.

[0319] 1H NMR (400 MHz, DMSO-d6, room temperature) δ ppm 1.55 - 1.72 (m, 2 H) 2.77 - 2.86 (m, 2 H) 3.15 - 3.19 (m, 0.5 H) 3.53 (dd, J=8.93, 2.06 Hz, 0.5 H) 3.58 (s,0.5 H) 3.68 (s, 0.5 H) 4.60 - 4.64 (m, 1 H) 5.06 - 5.17 (m, 1 H) 5.23 (d, J=14.88 Hz, 1 H) 5.32 (d, J=14.88 Hz, 1 H) 6.82 - 6.87 (m, 1 H) 6.92 - 6.98 (m, 1 H) 7.07 (ddd, J=8.01, 6.07, 2.18 Hz, 1 H) 7.42 - 7.50 (m, 2 H) 8.21 (dd, J=7.79, 3.43 Hz, 1 H) 8.27 (ddd, J=11.22, 6.98, 2.63 Hz, 1 H) 10.77 (br. s., 1 H) 11.72 (br. s., 1 H). 8i at room temperature 1 The 1H NMR spectrum shows a 2-fold increase in a portion of the signal, suggesting the presence of two rotational conformational isomers in DMSO-d6 at a ~1:1 ratio. MS (ESI): [M + H] + = 434.6.

[0320] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-fluoro-[2,3'-biindololinylidene]-2'-one (8j)

[0321] Compound 8j was synthesized from compound 7j in the order described for compound 6a.

[0322] Synthesis of (2Z,3E)-5'-chloro-3-((2-oxo-2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-[2,3'-biindololinylidene]-2'-one (8k)

[0323] Compound 8k was synthesized from compound 7k in the order described for compound 6a.

[0324] Synthesis of N-((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)-2-(((E)-((Z)-5'-chloro-2'-oxo-[2,3'-biindololinylidene]-3-ylidene)amino)oxy)acetamide (8l)

[0325] Compound 8l was synthesized from compound 7l in the order described for compound 6a.

[0326] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6amino-3-azabicyclo[3.1.0]hexane-3-yl)-2-oxoethoxy)imino)-5'-chloro-[2,3'-biindolineylidene]-2'-one (8m)

[0327] Compound 8m was synthesized from compound 7m in the order described for compound 6a.

[0328] Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-chloro-[2,3'-biindololinylidene]-2'-one (8n)

[0329] Compound 8n was synthesized from compound 7n in the order described for compound 6a.

[0330] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-chloro-[2,3'-biindololinylidene]-2'-one (8o)

[0331] Compound 8o was synthesized from compound 7o in the order described for compound 6a.

[0332] Synthesis of (2Z,3E)-5'-bromo-3-((2-oxo-2-(2,6-diazaspiro[3.3]heptane-2-yl)ethoxy)imino)-[2,3'-biindolinyl]-2'-one (8p)

[0333] Compound 8p was synthesized from compound 7p in the order described for compound 6a.

[0334] Synthesis of N-((1R,5S,6s)-3-azabicyclo[3.1.0]hexane-6-yl)-2-(((E)-((Z)-5'-bromo-2'-oxo-[2,3'-biindolineylidene]-3-ylidene)amino)oxy)acetamide (8q)

[0335] Compound 8q was synthesized from compound 7q in the order described for compound 6a.

[0336] Synthesis of (2Z,3E)-3-((2-((1R,5S,6S)-6amino-3-azabicyclo[3.1.0]hexane-3-yl)-2-oxoethoxy)imino)-5'-bromo-[2,3'-biindolineylidene]-2'-one (8r)

[0337] Compound 8r was synthesized from compound 7r in the order described for compound 6a.

[0338] Synthesis of (2Z,3E)-3-((2-((1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-bromo-[2,3'-biindololinylidene]-2'-one (8S)

[0339] Compound 8s was synthesized from compound 7s in the order described for compound 6a.

[0340] Synthesis of (2Z,3E)-3-((2-((1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-yl)-2-oxoethoxy)imino)-5'-bromo-[2,3'-biindololinylidene]-2'-one (8t)

[0341] Compound 8t was synthesized from compound 7t in the order described for compound 6a.

[0342] Synthesis of tert-butyl-6-(2-chloroacetyl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid (10a)

[0343] Under an argon atmosphere, TEA (2 equivalents) was added to a solution of tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid in DCM. The reaction mixture was stirred at 0°C for 10 hours. Then, chloroacetyl chloride (1.5 equivalents) was added dropwise, and the mixture was stirred at 0°C for 2 hours. After the reaction was completed as monitored by TLC, TLC (chloroform:methanol = 20:1, tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid Rf = 0.1, product Rf = 0.6) showed that tert-butyl-2,6-diazaspiro[3.3]heptane-2-carboxylic acid was completely consumed, and a new spot was detected by ninhydrin staining. After evaporation of the solvent, the mixture was purified using a silica gel column (elution: chloroform:methanol = 150:1).

[0344] Synthesis of (1R,5S,6S)-tert-butyl-6-(2-chloroacetamide)-3-azabicyclo[3.1.0]hexane-3-carboxylic acid (10b)

[0345] Compound 10b was synthesized from chloroacetyl chloride and (1R,5S,6S)-tert-butyl-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylic acid in the order described for compound 10a.

[0346] Synthesis of tert-butyl((1R,5S,6S)-3-(2-chloroacetyl)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate (10c)

[0347] Compound 10c was synthesized from chloroacetyl chloride and tert-butyl((1R,5S,6S)-3-azabicyclo[3.1.0]hexane-6-yl)carbamate in the order described for compound 10a.

[0348] Synthesis of (1R,4R)-tert-butyl-5-(2-chloroacetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (10d)

[0349] Compound 10d was synthesized from chloroacetyl chloride and (1R,4R)-tert-butyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid in the order described for compound 10a.

[0350] Synthesis of (1S,4S)-tert-butyl-5-(2-chloroacetyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid (10e)

[0351] Compound 10e was synthesized from chloroacetyl chloride and (1S,4S)-tert-butyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid in the order described for compound 10a.

[0352] Experimental Example 1. Evaluation of the kinase-inhibiting activity of the compounds according to the present invention

[0353] In this experimental example, the anticancer effect of the compound of the present invention was confirmed by FLT enzyme inhibition assay and cell proliferation inhibition assay in cancer cell lines.

[0354] FLT3 activity inhibition was assessed using the ADP-Glo ​​kinase assay (Promega; CAT#V9101). The ADP-Glo ​​kinase assay is a luminescent ADP detection method that quantifies the amount of ADP generated during a kinase reaction, thereby determining kinase activity and providing a uniform, high-throughput screening method.

[0355] The analysis was conducted in two steps. The first step was after the kinase reaction, followed by the addition of the same volume of ADP-Glo. TMThe first step involves using reagents to terminate the kinase reaction and deplete the remaining ATP. The second step involves adding kinase detection reagents to convert ADP to ATP simultaneously. This allows for the measurement of newly synthesized ATP via a luciferase / luciferin reaction, with the resulting light measured using a photometer. The measured light emission can then be applied to an ATP→ADP standard conversion curve to confirm its correlation with ADP concentration.

[0356] FLT3 and recombinant proteins containing the FLT3 (D835Y) domain were purchased from Promega (cat# V4064, cat# V4514). Following the manufacturer's instructions, ADP-Glo ​​was used... TM The kinase assay method (Promega, USA) was used to establish optimal enzyme, ATP, and substrate concentrations. The kinase reaction buffer 5X (200mM Tris-HCl, pH 7.5, 100mM MgCl2, 0.5mg / ml BSA) was diluted for use and then mixed sequentially with a diluted compound of the present invention (5X, 1μl), FLT3 enzyme or FLT3 / D835Y enzyme (2.5X, 2μl), and a substrate / ATP mixture (2.5X, 2μl). The reaction was carried out at room temperature for 120 minutes. Then, the mixture was placed in ADP-Glo... TM The reagent (5 μl) was added and reacted for another 40 minutes at room temperature. Finally, the detection reagent (kinase assay reagent, 10 μl) was added, and the reaction was continued for another 30 minutes at room temperature. The luciferase signal (integration time 0.5–1 sec) was then measured using a SpectraMax ID5 reader (Molecular Devices LLC., San Jose, CA, USA) to determine the phosphorylation of the matrix peptide. The IC50 was calculated using nonlinear regression analysis with data processing software (Prism version 5.01; GraphPad). 50 .

[0357] RET wt, RET (M918T), and recombinant proteins containing the RET (V804L) domain were purchased from Promega (cat# V8061, cat# V7274, cat# V4473). Following the manufacturer's instructions, ADP-Glo ​​was used... TMA kinase assay (Promega, USA) was performed to establish optimal enzyme, ATP, and substrate concentrations. The kinase reaction buffer was diluted 5X (200mM Tris-HCl, pH 7.5, 100mM MgCl2, 0.5mg / ml BSA) and used in conjunction with the diluted compound of this invention (5X, 1μl), FLT3 enzyme, or FLT3 / D835Y enzyme (2.5X, 2μl), and the substrate / ATP mixture was mixed sequentially (mix; 2.5X, 2μl), and reacted at room temperature for 120 minutes. Then, it was placed in ADP-Glo... TM The reagent (5 μl) was added and reacted for another 40 minutes at room temperature. Finally, the detection reagent (kinase detection reagent, 10 μl) was added, and the reaction was continued for another 30 minutes at room temperature. The phosphorylation of the matrix peptide was then measured using a multi-functional microplate reader (SpectraMax ID5 reader; Molecular Devices LLC., San Jose, CA, USA) at an integration time of 0.5–1 minute (second). IC50 was calculated using nonlinear regression analysis with data processing software (Prism version 5.01; GraphPad). 50 .

[0358] The measured enzyme activities (IC50) are shown in Table 1 below. 50 (nM).

[0359] Table 1

[0360] Evaluation of FLT3 WT / D835Y and RET WT / V804L / M918T kinase inhibition activity

[0361]

[0362]

[0363] As confirmed in Table 1, the compounds of the present invention exhibit excellent inhibitory activity against FLT3, FLT3 mutation, RET, and RET mutation.

[0364] Experimental Example 2. Evaluation of Cell Proliferation Inhibition in AML Cell Lines

[0365] The cell proliferation inhibition experiment of acute myeloid leukemia (AML) cell lines was conducted using MV4-11, Molm-13, and Molm-14 cells, which are FLT3-ITD expressing AML cell lines, to test the FLT3 inhibitory activity. All cell lines were derived from human cells, and the purchase method and culture conditions of each cell line are as follows.

[0366] MV4-11 cells were purchased from ATCC (American Type Culture Collection; Rockville, MD, USA, cat #CRL-9591) in the United States. Molm-13 (DSMZ, cat #ACC554) and Molm-14 (DSMZ, cat #ACC777) were also purchased. The cells were cultured in RPMI 1640 medium (Corning Co. 10-040-CV) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin and 4 ml of glutamate (Life Technology, Grand Island, NY).

[0367] Cell viability was evaluated using the Alamar Blue Assay kit (Invitrogen, USA) and by a resazurin-based redox assay. Specifically, 8000 cells of MV4-11, Molm-13, and Molm-14 cells were spread into 384-well plates in 45 mg of medium. As a negative control, the cells were treated with dimethyl sulfoxide (DMSO). Three days (72 hours) after adding the compound, the Alamar Blue kit reagent was added to each well of the 384-well plates, and the plates were incubated at 37°C for 2 hours in a humidified CO2 incubator. After incubation, fluorescence density (Flu) was measured at wavelengths of 544 nm to 590 nm using a SpectraMax iD3 Multi Mode Microplate Reader (Molecular Devices, ID3-STD). The GI50 was calculated using nonlinear regression analysis with data processing software (Prism version 5.01; GraphPad, LaJolla, CA, USA).

[0368] Three indirubin compounds known in existing literature were used as comparative examples.

[0369]

[0370] The measured cell proliferation inhibitory activity (GI) is shown in Table 2 below. 50 , nM) results.

[0371] Table 2

[0372] Cell proliferation inhibition activity (GI) in AML cell lines 50 (nM)

[0373]

[0374]

[0375] fM: femtomolar

[0376] pM: Picomolar

[0377] As confirmed in Table 2, the compounds of the present invention exhibit strong antiproliferative activity in acute leukemia cell lines, and in particular, strong antiproliferative activity can be confirmed in FLT3-ITD-expressing MV4-11 cell lines, FLT3-ITD and RET-expressing Molm-13 cell lines, and FLT3 / D835Y and RET-expressing Molm-14 cell lines.

[0378] Experimental Example 3. Evaluation of In vivo tumor suppression

[0379] In vivo tumor suppression evaluation experiments were conducted on experimental animals using compound 4 of the present invention and the compound of Comparative Example 3. BALB / c nude mice (7-9 weeks old, female, Shanghai Lingchang Biotech Co. LTD.) were used as experimental animals. To enhance tumor development, MV4-11 tumor cells (10 × 10⁻⁶ cells) were subcutaneously inoculated into the right flank of each mouse using 0.2 mL of an artificial basement membrane / PBS (1:1) mixture. 6 (ATCC-CRL-9591). Animals were randomly treated when the average tumor volume reached 100-150 mm. 3 At that time, the experiment began.

[0380] Of the randomly treated animals, a total of 64 animals (8 groups, 8 animals per group) were selected for tumor suppression evaluation experiments. Mixture 4 consisted of 4 experimental groups: a 5 mg / kg administration group, a 10 mg / kg administration group, a 20 mg / kg administration group, and a vehicle control group. Comparative Example 3 also consisted of 4 experimental groups: a 5 mg / kg administration group, a 10 mg / kg administration group, a 20 mg / kg administration group, and a vehicle control group. Drug administration was once daily orally, using 100% distilled water as the excipient.

[0381] Weight measurement and tumor suppression were observed in all individuals over a 24-day period. Tumor size was measured using calipers and recorded in mm. 3 Indicates volume.

[0382] Figures 1a-1d The results of the comparative experiment with Comparative Example 3 are shown below. For example, from... Figures 1a-1d As confirmed, the compounds of the present invention did not show significant changes in body weight after administration, but exhibited a superior tendency to reduce tumor volume compared to the compounds of Comparative Example 3. In particular, the tumor suppression effect in the experiment with administration of the compounds of the present invention at 5 mg / kg was confirmed to be superior and significant compared to the tumor suppression effect obtained in the experiment with administration of the compounds of Comparative Example 3 at 40 mg / kg.

[0383] It is thus confirmed that the compounds of the present invention have excellent inhibitory activity against FTL3 and RET kinases, especially FLT3 mutations and mutant RET kinases, and their associated preventive or therapeutic effects on leukemia or lymphoma.

Claims

1. A compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the chemical formula 1, R1 is hydrogen or halogen. R2 is , R3 is a free choice and Any one of the groups formed, in, X1 is CR a R b X2 is CR c R d , R4, R a R b R c and R d Each is independently hydrogen. n and m are both 1.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, R1 is hydrogen, and R2 is selected from... and Any one of the groups formed.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R1 is a halogen, and R2 is a selected... and Any one of the groups formed.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, The compounds of chemical formula 1 are selected from the group consisting of compounds 4, 5, 9, 10, 11, and 13: 。 5. A pharmaceutical composition for the prevention or treatment of FLT3 and RET-related diseases, said pharmaceutical composition comprising any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition for the prevention or treatment of leukemia or lymphoma, said pharmaceutical composition comprising any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

7. A method for the prevention or treatment of multiple myeloma, malignant plasma cell tumors, Hodgkin lymphoma, nodular lymphocytic-predominant Hodgkin lymphoma, Kaller's disease and myeloma, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma, acute myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-cell lymphoblastic lymphoma, myeloid leukemia, Waldenström macroglobulinemia, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphoma, primary mediastinal large B-cell lymphoma, lymphoplasmacytic lymphoma, and intravascular large B-cell lymphoma. Pharmaceutical compositions comprising the following: primary exudative lymphoma, lymphomatoid granulomatosis, T-cell / histiocytic large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma caused by HHV8-associated multicentric Castrmann disease, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and Burkitt lymphoma, or unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, wherein the pharmaceutical compositions comprise the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, wherein, The pharmaceutical composition is used for the prevention or treatment of marginal zone B-cell lymphoma of the lymph nodes and marginal zone lymphoma of the spleen.

9. The pharmaceutical composition according to claim 7, wherein, The pharmaceutical composition is used for the prevention or treatment of acute myeloid leukemia.

10. The pharmaceutical composition according to claim 9, wherein, The acute myeloid leukemia is caused by the expression of mutated FLT3 or mutated RET kinase.

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