Derivatives of an aryl hydrocarbon receptor agonist

Abstract

Compounds having one of the following formulae (IV) or (V): or a stereoisomer, salt, or tautomer thereof, wherein R1, R2, R3, R4, R5, R6, R7, R 10 , R 11a , R 11b , R 12a , R 12b , X, Y and Z are as defined herein. Pharmaceutical compositions comprising said compounds and their use in methods of treating diseases are also described.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 254,052, filed October 8, 2021, which is incorporated herein by reference in its entirety. Technical Field

[0003] The subject matter described in this application relates to compounds, methods for preparing the compounds, pharmaceutical compositions comprising the compounds, and treatment methods, wherein the compounds are derivatives of aryl hydrocarbon receptor agonists, including indirubin. Background Technology

[0004] Aromatic hydrocarbon receptor (AhR) agonists are promising drug compounds; however, the potential use of many such compounds is limited by issues such as toxicology and / or pharmacokinetics. Oral formulations of AhR agonists sometimes lead to serious adverse events, while topical formulations for dermatological conditions are better tolerable. The exact cause of adverse events in oral formulations may be compound-specific and / or possibly related to their metabolic pathways, bioavailability, solubility, pharmacokinetics, or off-target activities. Generally, oral formulations result in greater systemic exposure than topical formulations because the skin provides a barrier to other parts of the body. It would be desirable to design AhR agonists that are released and retained in the gastrointestinal tract, entering the bloodstream with minimal absorption, thus resulting in minimal systemic exposure. AhR agonists that provide these desired properties offer a more effective and safer treatment option than known AhR agonists.

[0005] Indirubin is an example of an AhR agonist compound; AhR is a receptor that plays a role in immunity, barrier function, and metabolism. Indirubin has been used to treat human diseases, both as a minor component and as an enriched extract of a fermented plant product known as indigo naturalis. The purified form of indirubin has been observed to have very poor solubility and dissolution. This lack of solubility is thought to stem from its highly crystalline structure and intramolecular and intermolecular hydrogen bonds, leading to poor solubility (Meijer et al., Indirubin, the red shade of indig O (2006), Chapter 10. Indirubin: The relationship between chemical structure and physical properties, with particular reference to Colour. Christie RM 103-108). The lack of dissolution and solubility in indirubin results in poor bioavailability and limited selection of pharmacokinetic characteristics. There is a demand in the pharmaceutical industry for compounds that have therapeutic activity similar to indirubin but offer improved properties (e.g., improved dissolution and solubility at room and body temperatures) to enable the easy design and preparation of dosage forms for delivering indirubin and other AhR agonists and to improve their bioavailability. Summary of the Invention

[0006] In short, this disclosure provides compounds, including their stereoisomers, pharmaceutically acceptable salts, or tautomers, which can be used alone or in combination with other therapeutic agents as aryl hydrocarbon receptor (AhR) agonists.

[0007] In one embodiment, compounds having the structure of formula (IV) or (V) are provided:

[0008]

[0009] Or its stereoisomers, salts or tautomers, R1, R2, R3, R4, R5, R6, R7, R8, R 10 R 11a R 11b R 12a R 12b X, Y, and Z are as defined in this application.

[0010] Also provided are pharmaceutical compositions comprising one or more of the aforementioned (IV) or (V) compounds and a therapeutic agent.

[0011] In other embodiments, a treatment method is provided that treats a disease by administering a compound of formula (IV) or (V) or a pharmaceutical composition containing formula (IV) or (V) to a subject in need. In some embodiments, the disease is, for example, an inflammatory disease. In other embodiments, the disease is a gastrointestinal inflammatory disease, a dermatitis, pneumonia, a systemic inflammatory disease, acne, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, hidradenitis suppurativa, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, or xerosis. Syndrome, type 1 and type 2 diabetes, or vitiligo. In the implementation scheme, the disease is ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, cryptitis, mucositis, cancer, fibrotic disease, or graft-versus-host disease.

[0012] In other respects, compounds having the structure of formula (I) or formula (Ia) are described:

[0013]

[0014] Or its stereoisomers, salts or tautomers, wherein R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as defined in this application.

[0015] Compositions comprising compounds (I), (Ia), (IV) and (V) and methods of use are also described.

[0016] Methods for treating inflammatory and immune disorders are also described. In embodiments, the methods include administering compounds of the present application, including but not limited to compounds of formulas (I), (Ia), (IV) and / or (V), or administering a composition containing the compound, thereby facilitating its conversion into a therapeutically effective amount of indirubin.

[0017] Various aspects and implementations will be described more fully below. These aspects and implementations may take many different forms, and the exemplary forms disclosed in this application should not be construed as limiting; rather, these implementations are provided to make this disclosure comprehensive and complete and to fully communicate its scope to those skilled in the art.

[0018] Brief description of the attached figures

[0019] Figure 1A-1B To illustrate the effects of compound 8 (triangle), indirubin (square), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD; circle) (positive control), and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio; dashed line) (positive control) at nM concentrations in human Huh7 cells (… Figure 1A ) and rat H4IIE cells ( Figure 1B A diagram of AhR activation in ).

[0020] Figure 2 The disease activity index (see table in Example 6), a measure of ulcerative colitis in a mouse model, is plotted as a function of time in days after daily treatment with compound 8 (triangles) or placebo (distilled water; circles).

[0021] Figures 3A-3B It displays the mouse's initial body weight percentage ( Figure 3A ) and Disease Activity Index (DAI) score ( Figure 3B The graph is a function of time (in days) for mice suffering from induced acute colitis and treated twice daily with placebo (solid circle), synthetic indirubin (solid square), compound 9 (solid triangle), compound 10 (inverted triangle), compound 26 (hollow circle), compound 40 (hollow square), or compound 41 (hollow triangle) for 9 days, where the doses shown are in milligrams per kilogram (mpk).

[0022] Figure 3C It is a bar graph showing the average colon length (in centimeters, using SEM) of mice with ulcerative colitis treated for 9 days with placebo, synthetic indirubin, compound 9, compound 10, compound 26, compound 40, or compound 41, wherein the doses shown are in milligrams per kilogram (mpk). Detailed Implementation

[0023] I. definition

[0024] For convenience, certain terms used in the specification, embodiments, and claims are collected herein. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0025] If a numerical range is provided, it means that every intermediate value between the upper and lower limits of the range, as well as any other value or intermediate value within the range, is included in this disclosure. For example, if a range of 1 μm to 8 μm is described, it means that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm, as well as numerical ranges greater than or equal to 1 μm and numerical ranges less than or equal to 8 μm, are also explicitly disclosed.

[0026] The singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. Thus, for example, references to “polymer” include a single polymer as well as two or more identical or different polymers, and references to “excipient” include a single excipient as well as two or more identical or different excipients, and so on.

[0027] The term “about,” especially in relation to a given quantity, is intended to cover a deviation of plus or minus five percent.

[0028] The compositions disclosed herein may comprise, consist substantially of, or consist of the disclosed components.

[0029] Unless otherwise stated, all percentages, parts and ratios are based on the total weight of the composition, and all measurements are performed at about 25°C.

[0030] As used in the specification and appended claims, unless otherwise specified, the following terms shall have the meanings indicated:

[0031] "Amino" refers to the -NH2, -NHR, or -NR2 group.

[0032] "Cyano" refers to the -CN group.

[0033] "Hydroxy group" refers to the -OH group.

[0034] "Imine" refers to =NH or =NR substituents.

[0035] "Nitro" refers to the -NO2 group.

[0036] "Oxyto" refers to the =O substituent.

[0037] "Thio" refers to the =S substituent.

[0038] "Trifluoromethyl" refers to the -CF3 group.

[0039] Hydrazine or hydrazine group refers to NN substituent.

[0040] Each R is a compatible substituent as described in this disclosure. If the R group is chiral, isomers are considered and included in this application.

[0041] "alkyl" refers to a straight-chain, saturated, acyclic, monovalent hydrocarbon group or a branched, saturated, acyclic, monovalent hydrocarbon group having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms or 1 to 6 carbon atoms and being connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Optionally substituted alkyl groups are alkyl groups that are optionally substituted with one, two, three, four, or five substituents, provided that the valence allows, and said substituents are independently selected from halogens, cyano, nitro, aryl, cycloalkyl, heterocyclic, heteroaryl, oxo, trimethylsilyl, -OR', -OC(O)-R', -N(R')2, -C(O)R”, -C(O)OR', -C(O)N(R')2, -N(R')C(O)OR”', -N(R')C(O)R”', -N(R')S(O) t R”' (where t is 1 or 2), -S(O) t OR”' (where t is 1 or 2), -S(O) p R”' (where p is 0, 1, or 2) and -S(O) t N(R')2 (where t is 1 or 2), wherein each R' is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic or heteroaryl; each R” is independently hydrogen, cycloalkyl, aryl, heterocyclic or heteroaryl; and each R”' is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl.

[0042] "Alkoxy" refers to the formula -OR a The group, wherein R a It is an alkyl group containing one to twelve carbon atoms as defined above. The alkyl portion of the alkoxy group may optionally be substituted as defined above for alkyl groups.

[0043] "Alkoxyalkyl" refers to the formula -R a -OR b The group, wherein R a It is an alkylene group, R b Alkyl groups are as defined above. The alkyl and alkylene portions of the optionally substituted alkoxyalkyl groups are optionally substituted as defined above for the alkyl groups and alkylene chains, respectively.

[0044] "Aryl group" refers to the formula -R a -R b The group, wherein R a It is an alkylene group, R bThe aryl group is as described in this application. The alkylene and aryl portions of the optionally substituted aryl group are optionally substituted as defined herein.

[0045] "Aryl" refers to an aromatic monocyclic or polycyclic aromatic ring system containing 6 to 18 carbon atoms, wherein the polycyclic aromatic ring system is a bicyclic, tricyclic, or tetracyclic system. Aryl groups include, but are not limited to, fluorenyl, phenyl, and naphthyl groups. Optionally substituted aryl groups are those optionally substituted by one, two, three, four, or five substituents, said substituents being independently selected from alkyl, alkenyl, halogen, haloalkyl, haloalkenyl, cyano, nitro, aryl, heteroaryl, heteroarylalkyl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) p R″′ (where p is 0, 1, or 2) and -R″-S(O) t N(R′)2 (where t is 1 or 2), wherein each R′ is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl; each R″ is independently a direct bond or a linear or branched alkylene or alkenyl chain; and each R″′ is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclic or heteroaryl.

[0046] "Arylalkoxy" refers to a group of the formula –OR, where R is an aralkyl group. Optionally substituted arylalkoxy groups are arylalkoxy groups that are optionally substituted as described for aralkyl groups in this application. In some embodiments, the arylalkoxy group is a benzyloxy group.

[0047] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group having three to fifteen carbon atoms, preferably three to ten carbon atoms, and is either saturated or unsaturated, connected to the rest of the molecule by a single bond. Polycyclic hydrocarbon groups are bicyclic, tricyclic, or tetracyclic ring systems. Unsaturated cycloalkyl groups contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decahydronaphthyl, etc. The optionally substituted cycloalkyl group is a cycloalkyl group optionally substituted with one, two, three, four, or five substituents, said substituents being independently selected from alkyl, alkenyl, halogen, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclic, heteroaryl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) p R″′ (where p is 0, 1, or 2) and -R″-S(O) t N(R′)2 (where t is 1 or 2), wherein each R′ is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl; each R″ is independently a direct bond or a linear or branched alkylene or alkenyl chain; and each R″′ is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic or heteroaryl.

[0048] A "deuterated compound" is a compound in which one or more hydrogen atoms are replaced by deuterium atoms. Deuterated drugs can be derivatives of active compounds. Deuterated drugs can be prodrugs. Deuteration may alter the physical properties, metabolic properties, activity, or safety of a drug.

[0049] "Derivatives" are related chemical substances that can be derived from similar compounds through chemical reactions. They may include minor chemical modifications, substitution of atoms with deuterium atoms, substitution of atoms with stable or radioactive isotopes, or other modifications that give the compound the desired properties.

[0050] "Fused" refers to any ring system described in this application that is fused with an existing ring structure in the compounds of this invention. When the fused ring system is a heterocyclic or heteroaryl group, any carbon atom on the existing ring structure that is part of the fused ring system may be replaced by a nitrogen atom.

[0051] "Halogenated" refers to halogen substituents: bromine, chlorine, fluorine, and iodine.

[0052] "Haloalkyl" refers to an alkyl group as defined above that is further substituted with one or more halogen substituents. The number of halogen substituents contained in a haloalkyl group is from one to the total number of hydrogen atoms that can be replaced by halogen substituents (e.g., perfluoroalkyl). Non-limiting examples of haloalkyl groups include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, etc. For optionally substituted haloalkyl groups, the hydrogen atoms bonded to the carbon atom of the alkyl portion of the haloalkyl group may optionally be replaced by substituents as defined above for optionally substituted alkyl groups.

[0053] "Haloalkenyl" refers to an alkenyl group as defined above that is further substituted with one or more halogen substituents. The number of halogen substituents in a haloalkenyl group is from one to the total number of hydrogen atoms that can be substituted by the halogen substituents (e.g., perfluoroalkenyl). Non-limiting examples of haloalkenyl groups include 2,2-difluorovinyl, 3-chloropropen-1-alkenyl, etc. For optionally substituted haloalkenyl groups, the hydrogen atoms bonded to the carbon atom of the alkenyl portion of the haloalkenyl group may optionally be substituted with substituents as defined above for the optionally substituted alkenyl group.

[0054] "Haloynyl" refers to an ynyl group as defined above, which is further substituted with one or more halogen substituents. The number of halogen substituents in a haloynyl group is from 1 to the total number of hydrogen atoms that can be substituted by the halogen substituents (e.g., perfluoroynyl). Non-limiting examples of haloynyl groups include 3-chloroprop-1-ynyl, etc. The ynyl portion of a haloynyl group may also optionally be substituted as defined above for the ynyl group.

[0055] "Heteroarylalkyl" refers to the formula -R a -R b The group, wherein R a It is an alkylene group, R b The heteroaryl group is as described in this application. Optionally substituted alkylene and heteroaryl moieties of the heteroaryl alkyl group are optionally substituted as described in this application for the alkylene and heteroaryl groups respectively.

[0056] A "heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group having 2 to 12 carbon atoms and containing a total of 1 to 6 heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. Heterocyclic groups can be monocyclic, bicyclic, tricyclic, or tetracyclic. Bicyclic, tricyclic, or tetracyclic heterocyclic groups are fused, helical, and / or bridging ring systems. Heterocyclic groups can be saturated or unsaturated. Unsaturated heterocyclic groups contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. The optionally substituted heterocyclic group is a heterocyclic group optionally substituted with one, two, three, four, or five substituents, said substituents being independently selected from alkyl, alkenyl, halogen, haloalkyl, haloalkenyl, cyano, oxo, thio, nitro, aryl, aralkyl, cycloalkyl, heterocyclic, heteroaryl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) p R″′ (where p is 0, 1, or 2) and -R″-S(O) t N(R′)2 (where t is 1 or 2), wherein each R′ is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl; each R″ is independently a direct bond or a linear or branched alkylene or alkenylene chain; each R″′ is independently alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl. The nitrogen, carbon, or sulfur atom in the heterocyclic group may optionally be oxidized (when the substituent is oxo-substituted and present on the heteroatom); the nitrogen atom may optionally be quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclic, heteroaryl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) p R″′ (where p is 0, 1, or 2) and -R″-S(O) tWhen N(R′)2 (where t is 1 or 2), where R″ is a linear or branched alkylene or alkenylene chain, and R′ and R″′ are as defined above. Examples of optionally substituted heterocyclic groups include, but are not limited to, azacyclic butyl, dioxopentyl, thienyl[1,3]dithiaalkyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperidyl, oxazolyl, piperidinyl, piperazine, 4-piperidinoneyl, pyrrolyl, pyrazolyl, thiazoalkyl, tetrahydrofuranyl, trithiaalkyl, tetrahydropyranyl, thiomorpholinyl, thio-morpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl.

[0057] "Subheterocyclic group" refers to a heterocyclic group in which one hydrogen atom is substituted by a valence. Optionally substituted subheterocyclic groups may be substituted as described in this application for heterocyclic groups.

[0058] "Heteroaryl" refers to a 5- to 18-membered ring system containing at least one aromatic ring, having 1 to 17 carbon atoms, and a total of 1 to 10 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Heteroaryl groups are monocyclic, bicyclic, tricyclic, or tetracyclic ring systems. Bicyclic, tricyclic, or tetracyclic heteroaryl groups are fused and / or bridged ring systems. The optionally substituted heteroaryl group is a heteroaryl group optionally substituted with one, two, three, four, or five substituents, said substituents being independently selected from alkyl, alkenyl, alkoxy, halogen, haloalkyl, haloalkenyl, cyano, oxo, thio, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclic, heteroaryl or heteroarylalkyl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) t R″′ (where p is 0, 1, or 2) and -R″-S(O) tN(R′)2 (where t is 1 or 2), wherein each R′ is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl; each R″ is independently a direct bond or a linear or branched alkylene or alkenyl chain; and each R″′ is alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclic or heteroaryl. The nitrogen, carbon, or sulfur atom in the heterocyclic group may optionally be oxidized (when the substituent is oxo-substituted and present on the heteroatom), provided that at least one ring in the heteroaryl group retains aromaticity; the nitrogen atom may optionally be quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclic, heteroaryl, -R″-OR′, -R″-OC(O)-R′, -R″-N(R′)2, -R″-C(O)R′, -R″-C(O)OR′, -R″-C(O)N(R′)2, -R″-N(R′)C(O)OR″′, -R″-N(R′)C(O)R″′, -R″-N(R′)S(O) t R″′ (where t is 1 or 2), -R″-S(O) t OR″′ (where t is 1 or 2), -R″-S(O) p R″′ (where p is 0, 1, or 2) and -R″-S(O) t When N(R′)2 (where t is 1 or 2), and R″ is a linear or branched alkylene or alkenylene chain, R′ and R″′ are defined as above), the condition is that at least one ring in the heteroaryl group retains aromaticity. Examples of optionally substituted heteroaryl groups include, but are not limited to, aza-aryl groups. acridine, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxacyclopentadienyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxazolyl 1,4-benzodioxane-hexyl, benzonaphthofuranyl, benzooxazolyl, benzodioxane-pentadienyl, benzodioxane-hexenyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzothiopheneyl (benzothiophene), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazoleyl, cenolinyl, dibenzofuranyl, dibenzothiopheneyl, furanyl, furanoneyl, isothiazolyl, imidazoyl, indazoleyl, indoleyl, indazoleyl, isoindoleyl, indolinyl, isoindolinyl, isoquinolinyl, indoleazinyl, isoxazolyl, naphthyl, naphthidyl, oxadiazolyl, 2-oxoazonyl The following groups are listed: 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purineyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quininecycloyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thiophenyl).

[0059] As used in this application, the phrase "pharmaceutically acceptable" means compounds, salts, compositions, dosage forms, etc., that are suitable for contact with human and / or other mammalian tissues to the extent of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio. In some respects, "pharmaceutically acceptable" means approved by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals (e.g., animals), and more specifically, for use in humans.

[0060] A "prodrug" is a compound that is metabolized or otherwise chemically converted into its active form after administration. A prodrug can be a derivative of the active compound. A prodrug may or may not be active before being converted into its active form in the body.

[0061] The term "treatment" in this application is used, for example, to refer to methods of treating inflammatory diseases or gastrointestinal disorders, and generally includes administering a compound or composition that, relative to a subject not receiving the compound or composition, reduces the frequency of a medical condition (e.g., autoimmune disease, inflammatory condition, gastrointestinal disorder), delays its onset, or alleviates its symptoms in the subject. This may include reversing, reducing, or preventing symptoms, clinical signs, and underlying pathologies of the condition (e.g., symptom relief of autoimmune diseases or inflammatory diseases, such as improvement in MAYO scores in the treatment of ulcerative colitis) in a manner that improves or stabilizes the subject's condition.

[0062] The embodiments disclosed in this application cover all pharmaceutically acceptable compounds of (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb) that are isotopically labeled by substituting one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that may be introduced into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, respectively. 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. These radiolabeled compounds, by characterizing, for example, their site of action or mode of action, or their binding affinity to pharmacologically important sites of action, may be used to help determine or measure the efficacy of the compound. Certain isotope-labeled (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb) compounds, for example, those incorporating a radioisotope, can be used for drug and / or substrate tissue distribution studies. Radioisotope tritium (i.e., 3 H) and carbon-14 (i.e., ... 14 C) They are particularly suitable for this purpose because they are easy to introduce and convenient to detect.

[0063] Using heavier isotopes such as deuterium (i.e., 2 H) Alternatives can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and may therefore be preferred in some cases.

[0064] Using positron-emitting isotopes (e.g.) 11 C 18 F, 15 O and 13 (N) Substitutes may be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb) can typically be prepared using conventional techniques known to those skilled in the art or by methods similar to those described in the preparation examples and embodiments below, replacing the previously used unlabeled reagents.

[0065] The embodiments disclosed in this application cover in vivo metabolites of the disclosed compounds. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc., of the applied compound, primarily due to enzymatic processes. Therefore, this disclosure includes compounds produced by methods comprising administering the disclosed compound to a mammal for a sufficient period of time to produce its metabolites. Typically, such products are identified by administering a detectable dose of the radiolabeled compound of this disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism, and separating its metabolites from urine, blood, or other biological samples.

[0066] "Pharmaceutical acceptable salts" include acid addition salts and base addition salts.

[0067] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the bioavailability and properties of the free base, are not biologically or otherwise undesirable, and are salts formed with inorganic and organic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such organic acids as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethyl... Ethyl ethanesulfonic acid, formic acid, fumaric acid, galactosic acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, mesylic acid, mucoic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid (TFA), undecenoic acid, etc.

[0068] "Pharmaceutically acceptable base addition salts" refer to salts that retain the bioavailability and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of the following bases: primary amines, secondary amines, and tertiary amines. Substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylaminoethanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenylethylamine, N,N'-dibenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, aminobutanetriol, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0069] "Pharmaceutical composition" refers to a formulation of the compounds disclosed herein with a medium generally accepted in the art for the delivery of biologically active compounds to mammals (e.g., humans). Such media include all pharmaceutically acceptable carriers, diluents, and excipients.

[0070] "Effective amount" or "therapeutic effective amount" means the amount of the compound of this disclosure that is sufficient to achieve a therapeutic effect in a mammal, preferably a human, when administered to such a mammal. The amount of the compound constituting the "therapeutic effective amount" will vary depending on the compound, the disease and its severity, the method of administration, and the age of the mammal being treated, but can be conventionally determined by those skilled in the art in light of their own knowledge and this disclosure.

[0071] "Stereoisomers" are compounds composed of identical atoms bonded by the same bonds but with different three-dimensional structures, and they are not interchangeable. This disclosure considers various stereoisomers and mixtures thereof, including "enantiomers," which are two stereoisomers whose molecules are mirror images of each other that cannot be superimposed. This disclosure also considers "diastereomers," which are non-mirror images of non-identical stereoisomers. Diastereomers occur when two or more stereoisomers of a compound have different configurations at one or more equivalent stereocenters and are not mirror images of each other.

[0072] "Tautomerism" refers to a proton shift from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomerisms of any of the said compounds.

[0073] By reserving the right to exclude or remove any individual member of any such group (including any subranges or combinations of subranges within that group), protection may be claimed in accordance with the scope or in any similar manner, and protection may be claimed for any reason less than the full scope of this disclosure. Furthermore, by reserving the right to exclude or remove any individual substituent, analogue, compound, ligand, structure or any member of any group thereof or any group claimed, protection may be claimed for any reason less than the full scope of this disclosure.

[0074] Throughout this disclosure, various patents, patent applications, and publications are cited. The disclosures of these patents, patent applications, and publications are incorporated herein by reference in their entirety to provide a more comprehensive description of the state of the art as of the date of this disclosure. In the event of any inconsistency between the cited patents, patent applications, and publications and this disclosure, this disclosure shall prevail.

[0075] II. compound

[0076] The compounds described in this application are derivatives of AhR agonists (where indirubin is used as a model AhR agonist) and are classified into several classes for ease of disclosure. This grouping is for convenience of disclosure and is not intended to be limiting in any way. Indirubin derivative compounds can modify individual atoms in the indirubin molecule without altering or increasing the indirubin skeleton or connectivity (e.g., replacing one isotope with another of the same atoms or replacing carbon with nitrogen or oxygen to produce isosteres). As shown in formula (I), indirubin derivatives can also have changes that do indeed alter the indirubin skeleton, for example, by adding more carbon branches to the indirubin structure:

[0077]

[0078] R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, alkyl, halogen, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonamide, acylsulfonamide, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0079] R9, along with the nitrogen atom it is attached to, is selected from hydroxyl, alkoxy, amino, amide, carbonate, carbamate, phosphate, acetal, and hemiacetal; and

[0080] R 10Together with the carbon atom it is bonded to, it is selected from carbonyl, imine, acetal, hemiacetal, acetalamine, hemiacetalamine, oxime, hydrazone, cyanoimine and thiocarbonyl.

[0081] In another embodiment, the compound has formula (Ia):

[0082]

[0083] R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, alkyl, halogen, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonamide, acylsulfonamide, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0084] In another embodiment, the compound has formula (Ib):

[0085]

[0086] R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, alkyl, halogen, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonamide, acylsulfonamide, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0087] In another embodiment, the compound has formula (II):

[0088]

[0089] R9, along with the nitrogen atom it is attached to, is selected from the group consisting of hydroxyl, alkoxy, amino, amide, carbonate, carbamate, phosphate, acetal, and hemiacetal.

[0090] In another embodiment, the compound has formula (III):

[0091]

[0092] Where R 10 Together with the carbon atom it bonds to, it is selected from the group consisting of carbonyl, imine, acetal, hemiacetal, acetal, hemiacetal, oxime, hydrazone, cyanoimine, and thiocarbonyl.

[0093] In another embodiment, the compound of formula (III) has formula (IIIa):

[0094]

[0095] Where R 10a Selected from alkyl, halogen, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonamide, acylsulfonamide, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, thioester, nitrile, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0096] A. Category A

[0097] Class A derivatives are characterized by the fact that the substitution of atoms in the molecule does not change the overall structure of the molecule. Examples of such substitution include molecules in which one atom is exchanged for another atom at an equivalent valence (e.g., an oxygen atom is exchanged for a sulfur atom), or in molecules in which one atom is exchanged for another atom at a different valence (e.g., a carbon atom is exchanged for a boron atom).

[0098] Another form of substitution involves replacing one isotope of an atom with another (i.e., radioactive labeling). As a non-limiting example, naturally occurring isotopic carbon or hydrogen atoms (e.g., 1 H: 2 D、 12 C: 13 C 12 C: 14 (e.g., C) can be replaced by non-natural isotopic distributions. In some embodiments, the preparation of the molecule can produce a mixture of isotopes. For example, the isotopic mixture may contain more than 50% (e.g., more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%) of substitutes or naturally occurring non-major isotopes (e.g., 2 D、 13 C 15 N、 18 (e.g., O). This radioactive labeling can be applied specifically to a single location or multiple locations within a molecule, such as a class (e.g., 2 D) or multiple categories (e.g., 2 D and 13 C) 1, 2, 3, 4, 5, 6, 7, 8 or more alternative isotopes.

[0099] Compounds 1-2 shown below are exemplary and non-limiting examples of class A molecules:

[0100]

[0101] Besides replacing hydrogen with deuterium, this can also be achieved by using isotopes with lower abundances that are not naturally abundant, such as those with higher natural proportions.13 C: 12 C. Unnaturally high ratio 14 C: 12 C. Unnaturally high ratio 15 N: 14 N, Unnaturally high ratio 18 O: 16 Indirubin derivatives are prepared using O and other methods to alter the natural distribution of carbon, nitrogen, and oxygen. By changing the isotope ratios, indirubin derivatives can utilize isotope effects, such as primary kinetic isotope effects and secondary kinetic isotope effects.

[0102] B. Category B

[0103] Type B derivatives are derivatives that add to or modify the core structure of indirubin, for example, by adding or removing atoms, or a combination of both (e.g., substitution), to produce a new structure. For example, a hydrogen atom bonded to a carbon or nitrogen atom can be removed, and one or more atoms (e.g., carbon, nitrogen, oxygen, halogen, etc.) can be added, resulting in a new structural framework. In some embodiments, the substitution in the Type B derivative has one or more aryl hydrogen atoms. In other embodiments, the substitution has one or both carbonyl oxygen atoms. In other embodiments, the substitution in the Type B derivative is located at the N1 and N1' positions, as shown in the following formula:

[0104]

[0105] In a preferred embodiment, the substitution in the class B derivative is located at the N1 position. Non-limiting examples include compounds 3-52:

[0106]

[0107]

[0108]

[0109]

[0110] C. Category C

[0111] Class C derivatives are a subset of Class B derivatives in which one or both oxygen atoms are substituted. In some embodiments, the oxygen at position 2 is substituted. In a preferred embodiment, the oxygen at position 3' is substituted. Non-limiting examples of Class C derivatives include compounds 3-6:

[0112]

[0113] In one implementation, the compound is In another embodiment, the compound is In another embodiment, the compound is

[0114] D. Category D

[0115] Class D derivatives are a subset of Class B derivatives in which one or both of the hydrogen atoms at the N1 and N1' positions are substituted. In some embodiments, the hydrogen atom at position N1' is substituted. In a preferred embodiment, the hydrogen atom at the N1 position is substituted. Non-limiting examples of Class D derivatives include compounds 7-53:

[0116]

[0117]

[0118]

[0119]

[0120] Exemplary Class D compounds include urea compounds. The following structures are non-limiting examples of urea-based indirubin derivatives of this disclosure:

[0121]

[0122]

[0123] Exemplary Class D compounds include carbamate compounds. The following structures are non-limiting examples of carbamate indirubin derivatives of this disclosure:

[0124]

[0125] Exemplary Class D compounds include amide compounds. The following structures are non-limiting examples of amide-based indirubin derivatives of this disclosure:

[0126]

[0127] Exemplary Class D compounds include acetals and hemiacetals. The following structures are non-limiting examples of indirubin derivatives of the acetals and hemiacetals disclosed herein:

[0128]

[0129]

[0130] Exemplary Class D compounds include compounds having other substitutions at the nitrogen atom. The following structures are non-limiting examples of other indirubin derivatives of the present invention:

[0131]

[0132] In one embodiment, the compound has one of the structures of formula (IV) or (V):

[0133]

[0134] Or its stereoisomers, salts or tautomers, wherein:

[0135] R1, R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, deuterium, alkyl, halogen, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonamide, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0136] R 11a It can be a halogen, a 3-8 membered heterocyclic alkyl group, a 3-8 membered heteroaryl group, -OH, -OX-, -SX-, -S(O)2X-, -NX2-, -OC(=O)X- or -OC(=O)NX2-;

[0137] R 11b It is a 3-8 membered heterocyclic alkyl group, a 3-8 membered heteroaryl group, -OX- or -NX2-;

[0138] X is independently hydrogen, C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C6 haloalkyl, C1-C 10 Heteroalkyl, -S(O)2OH, aryl or 3-8 membered heterocyclic alkyl,

[0139] Where C1-C 10 Alkyl and C1-C 10 The heteroalkyl group may be optionally substituted with a 3-8 membered heterocyclic alkyl group, a C1-C6 alkyl carboxylic acid, -NHS(=O)2Y-, -NHC(=O)OY- or -NHC(=O)Y-;

[0140] The 3-8 membered heterocyclic alkyl groups are optionally replaced by C1-C6 alkyl groups, C1-C4 amines, halogens, -C(=O)Y- or -C(=O)OY-;

[0141] Y can be H, C1-C6 perfluoroalkyl, or C1-C6 alkyl, and each of them can be independently H.

[0142] R 12a and R 12b Each is independently hydrogen or C1-C6 alkyl;

[0143] R 10 For O, NZ, or NNZ2; and

[0144] Z is independently hydrogen, hydroxyl, aryl or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted with aryl.

[0145] In one embodiment, R1, R2, R3, R4, R5R6, R7, and R8 are hydrogen. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are deuterium. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are alkyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are halogens. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are perfluoroalkyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are alkynyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are alkenyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are alkoxy groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are cycloalkoxy groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are thioalkyl groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are thiocycloalkoxy groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are perfluoroalkoxy groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are perfluorothioalkyl groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are hydroxyl groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are esters. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are amides. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are carboxylic acids. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are carbamates. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are sulfonamides. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are acylsulfonamides. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are sulfones. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are sulfoxides. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are ketones. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are carbonates. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are ureas. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are sulfonylureas. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are amino groups. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are thioesters.In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are nitriles. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are nitro groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are azide groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are cycloalkyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are heterocycloalkyl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are aryl groups. In some embodiments, R1, R2, R3, R4, R5R6, R7, and R8 are heteroaryl groups. In some embodiments, one of R1, R2, R3, R4, R5R6, R7, and R8 is hydrogen. In some implementations, each of R1, R2, R3, R4, R5, R6, R7, and R8 is hydrogen.

[0146] In one implementation, R 12a Or R 12b One of them is hydrogen. In some implementations, R 12a and R 12b Each one in it is hydrogen.

[0147] In one implementation, R 12a Or R 12b One of them is a C1-C6 alkyl group. In other embodiments, R 12a Or R 12b Each of the terms is a C1-C6 alkyl group. In some embodiments, the C1-C6 alkyl group is a straight-chain C1-C6 alkyl group. In some embodiments, the straight-chain C1-C6 alkyl group is, for example, methyl (-CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), n-butyl (-CH2CH2CH2CH3), n-pentyl (-CH2CH2CH2CH2CH3), or n-hexyl (-CH2CH2CH2CH2CH2CH3).

[0148] In one embodiment, the C1-C6 alkyl group is a branched C3-C6 alkyl group. In some embodiments, the branched C3-C6 alkyl group is, for example, 2-propyl, sec-butyl, isobutyl, tert-butyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl, or 2,2-dimethylbutyl.

[0149] In one implementation, R 12a Or R 12b One is hydrogen and the other is R 12a Or R 12b It is a C1-C6 alkyl group.

[0150] In one embodiment, the compound has one of the structures of formula (IVa) or (Va):

[0151]

[0152] Or its stereoisomers, salts, or tautomers.

[0153] In one implementation, R 10 It is O. In some implementations, R 10 It is NZ. In some implementations, R 10 It is NNZ2.

[0154] Z is independently hydrogen, hydroxyl, aryl, or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted with an aryl group. In some embodiments, when R 10 When it is NZ, Z is a hydroxyl group. In some embodiments, when R... 10 When it is NNZ2, Z is hydrogen and a C1-C4 alkyl group, wherein the C1-C4 alkyl group is substituted with an aryl group, such as a phenyl group. In some embodiments, for example, the C1-C4 alkyl group is methyl (C1 alkyl). In some embodiments, when R... 10 When it is NNZ2, Z is hydrogen and aryl. In some embodiments, when R... 10 When it is NNZ2, Z represents hydrogen and phenyl.

[0155] In one implementation, R 10 It has one of the following structures:

[0156]

[0157] In one embodiment, the compound has one of the structures of formula (IVb) or (Vb):

[0158]

[0159] Or its stereoisomers, salts, or tautomers.

[0160] In one implementation, R 11a It is a 3-8 membered heterocyclic alkyl group. In some embodiments, R 11b It is a 3-8 nucleotide heteroaryl group. In some implementation schemes, R 11b It is -OX-. In some implementations, R 11b It is OH. In some implementations, R 11a It is a halogen. In some implementations, R 11b It is -SX-. In some implementations, R 11b It is -S(O)2X-. In some implementations, R11b It is -NX2-. In some implementations, R 11b It is -OC(=O)X-. In some implementations, R 11b It is -OC(=O)NX2-.

[0161] In one embodiment, the compound has the formula (IVc):

[0162]

[0163] Or its stereoisomers, salts or tautomers, wherein This indicates a 3-8 membered heterocyclic alkyl group or a 3-8 membered heteroaryl group. In some embodiments, formula (IVc) It is a 3-8 membered heterocyclic alkyl group. In some embodiments, formula (IVc) It consists of 3-8 heteroaryl groups.

[0164] In one implementation, formula (IVc) It includes aziridine, diaziridine, pyrrolidine, imidazoline, pyrazolidine, piperidine, diaziridine, triaziridine, aziridine-heptane, diaziridine-heptane, aziridine-hexane, oxacyclobutane, dioxacyclobutane, tetrahydrofuran, dioxane, dioxane, trioxane, oxacycloheptane, oxacyclooctane, phospharidine-butane, phospharidine-pentane, phospharidine-hexane, thiocyclobutane, dithiocyclobutane, tetrahydrothiophene, dithiocyclopentane, thiocyclohexane, dithiocyclohexane, trithiocyclohexane, thiocycloheptane, thiocyclooctane, oxacyclohexane, isoxacyclohexane, oxazolidine, isoxazolidine, thiazoline, isothiazolidine, morpholine, thiomorpholine, or oxacyclohexane.

[0165] In one implementation, R 11a It is azacyclobutane, diazacyclobutane, pyrrolidine, imidazoline, pyrazolidine, piperidine, diazacyclohexane, triazacyclohexane, azacycloheptane, diazacycloheptane, azacyclooctane, oxacyclobutane, dioxacyclobutane, tetrahydrofuran, dioxopentane, oxane, dioxane, trioxane, oxacycloheptane, oxacyclooctane, phosphotycyclobutane, phosphotycyclopentane, phosphotycyclohexane, thiobutane, dithiobutane, tetrahydrothiophene, dithiobutane, thiobutane, dithiobutane, trithiobutane, thiobutane, thiohexane, thiohexane, thiohexane, thiohexane, thiohexane, oxacycloheptane, oxacyclooctane, oxacyclopentane, isoxacyclopentane, oxazolidine, isoxazolidine, thiazoline, isothiazolidine, morpholine, thiomorpholine, or oxacyclohexane. In some embodiments, R 11a It is a nitrogen-containing heterocyclic butane. In some embodiments, R 11a It is a diazacyclobutane. In some embodiments, R 11a It is pyrrolidine. In some embodiments, R11a It is an imidazolidine. In some embodiments, R 11a It is pyrazolidine. In some embodiments, R 11a It is piperidine. In some implementations, R 11a It is diazacyclohexane. In some embodiments, R 11a It is triazacyclohexane. In some embodiments, R 11a It is azaheptanane. In some embodiments, R 11a It is diazacycloheptane. In some embodiments, R 11a It is a nitrogen-containing heterocyclic octane. In some embodiments, R 11a It is an oxetane. In some embodiments, R 11a It is dioxane. In some embodiments, R 11a It is tetrahydrofuran. In some implementations, R 11a It is dioxolane. In some implementations, R 11a It is an oxane. In some implementations, R 11a It is dioxane. In some implementations, R 11a It is trioxane. In some implementations, R 11a It is an oxacycloheptane. In some embodiments, R 11a It is an oxacyclooctane. In some embodiments, R 11a It is a phosphorus heterocyclic butane. In some embodiments, R 11a It is phosphacene. In some embodiments, R 11a It is phosphacyclohexane. In some embodiments, R 11a It is a thiohexacyclic butane. In some embodiments, R 11a It is a dithioheterocyclic butane. In some embodiments, R 11a It is thiacyclohexane. In some embodiments, R 11a It is dithiocyclohexane. In some embodiments, R 11a It is trithionecyclohexane. In some implementations, R 11a It is a thioheptanine. In some embodiments, R 11a It is a thiohexacyclic octane. In some embodiments, R 11a It is oxothione hexane. In some embodiments, R 11a It is isothiacyclohexane. In some embodiments, R 11a It is an oxazolidine. In some implementations, R 11a It is isoxazolidine. In some implementations, R 11a It is a thiazolidinedione. In some embodiments, R 11a It is isothiazolidine. In some embodiments, R 11aIt is morpholine. In some implementations, R 11a It is thiomorpholine. In some implementations, R 11a It is oxosulfur heterocyclohexane.

[0166] In some implementations, R 11a It has one of the following structures:

[0167] In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for In some implementation schemes, R 11a for

[0168] In some specific implementations, R 11a for In some implementation schemes, R 11a for In some other implementations, R 11a for

[0169] In one implementation, formula (IVc) It has one of the following structures:

[0170]

[0171] In one implementation, R 11b X is -NX2-. In one embodiment, one of X is hydrogen. In other embodiments, one of X is C1-C. 10 Alkyl group. In other embodiments, one of X is C1-C. 10 Alkenyl. In other embodiments, one of X is a C1-C6 haloalkyl. In other embodiments, one of X is C1-C6 haloalkyl. 10Heteroalkyl. In other embodiments, one of X is -S(O)2OH. In other embodiments, one of X is aryl. In other embodiments, one of X is a 3-8 membered heterocyclic alkyl group.

[0172] In one embodiment, each of X is hydrogen. In other embodiments, each of X is C1-C. 10 Alkyl group. In other embodiments, each of X is C1-C. 10 Alkenyl. In other embodiments, each of X is a C1-C6 haloalkyl. In other embodiments, each of X is C1-C6 haloalkyl. 10 Heteroalkyl. In other embodiments, each of X is -S(O)2OH. In other embodiments, each of X is aryl. In other embodiments, each of X is a 3-8 membered heterocyclic alkyl group.

[0173] In one implementation, one of X is hydrogen, and the other X is C1-C2. 10 Alkyl group. In other embodiments, one of X is hydrogen and the other X is C1-C. 10 Alkenyl. In other embodiments, one of X is hydrogen, and the other X is a C1-C6 haloalkyl. In other embodiments, one of X is hydrogen, and the other X is C1-C6 haloalkyl. 10 Heteroalkyl. In other embodiments, one of X is hydrogen and the other X is -S(O)2OH. In other embodiments, one of X is hydrogen and the other X is aryl. In other embodiments, one of X is hydrogen and the other X is a 3-8 membered heterocyclic alkyl group.

[0174] In one implementation, X is C1-C 10 Alkyl group. In some embodiments, X is a C1 alkyl group. In some embodiments, X is a C2 alkyl group. In some embodiments, X is a C3 alkyl group. In some embodiments, X is a C4 alkyl group. In some embodiments, X is a C1-C4 alkyl group.

[0175] In one implementation, X is C2-C 10 Alkenyl. In some embodiments, X is C3 alkenyl. For example, in some embodiments, X is propenyl. In some embodiments, X is C2-C3 alkenyl.

[0176] In one embodiment, X is a C1-C6 haloalkyl. In some embodiments, X is a C1 haloalkyl. For example, in some embodiments, X is chloromethyl.

[0177] In one implementation, X is C1-C 10Heteroalkyl. In some embodiments, X is a C2-C6 heteroalkyl. For example, in some embodiments, X is a C2 heteroalkyl. In another example, in some embodiments, X is a C3 heteroalkyl. In another example, in some embodiments, X is a C4 heteroalkyl. In another example, in some embodiments, X is a C5 heteroalkyl. In another example, in some embodiments, X is a C6 heteroalkyl.

[0178] In one embodiment, X is hydrogen, a C1-C6 alkyl, a C2-C6 alkenyl, a C1-C6 haloalkyl, a C1-C6 heteroalkyl, -S(O)2OH, a phenyl, or a C5 heterocyclic alkyl. In some embodiments, X is hydrogen. In some embodiments, X is a C1-C6 alkyl. In some embodiments, X is a C2-C6 alkenyl. In some embodiments, X is a C1-C6 haloalkyl. In some embodiments, X is a C1-C6 heteroalkyl. In some embodiments, X is -S(O)2OH. In some embodiments, X is phenyl. In some embodiments, X is a C5 heterocyclic alkyl.

[0179] In one implementation, R 11b It has one of the following structures:

[0180] In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for In some implementation schemes, R 11b for

[0181] In one embodiment, the halogen is -F, -Cl, -Br, or -I. In some embodiments, the halogen is -F. In some embodiments, the halogen is -Cl. In some embodiments, the halogen is -Br. In some embodiments, the halogen is -I.

[0182] In one implementation, C1-C 10 Alkyl, C2-C 10 alkenyl, C1-C6 haloalkyl or C1-C 10 The heteroalkyl group is straight-chain. In some embodiments, C1-C 10 Alkyl, C2-C 10 alkenyl, C1-C6 haloalkyl or C1-C 10 Heteroalkyl groups are branched.

[0183] In one embodiment, the C1-C6 perfluoroalkyl group is tetrafluoroethyl or trifluoromethyl. In some embodiments, the C1-C6 perfluoroalkyl group is tetrafluoroethyl. In some embodiments, the C1-C6 perfluoroalkyl group is trifluoromethyl.

[0184] In one embodiment, the 3-8 membered heteroaryl group is azahexacyclopropene, oxahexacyclopropene, thiohexacyclopropene, azahexacyclobutadiene, oxahexacyclobutadiene, thiohexacyclobutadiene, pyrrole, furan, thiophene, pyridine, pyran, thiaran, azaheptenene, oxaheptenene, thioheptenene, azaoctatetraene, oxaoctatetraene, thioheptentraene, diazahexacyclobutene, dioxahexacyclobutene, dithiohexacyclobutene, imidazole, pyrazole, oxothiohexacyclopentene, isoxothiohexacyclopentene, oxazole, isoxazole, thiazole, isothiazine, diazine, thiazine, oxothiohexene, dioxahexene, dithiohexene, triazine, tetraazine, pentaazine, diazaheptenene, thioazheptenene, azaoctatetraene, oxaoctatetraene, or thiohexacyclopentatetraene.

[0185] In one implementation, formula (IVc) It includes azahexacyclopropylene, oxahexacyclopropylene, thiohexacyclopropylene, azahexacyclobutadiene, oxahexacyclobutadiene, thiohexacyclobutadiene, pyrrole, furan, thiophene, pyridine, pyran, thiaran, azaheptenene, oxaheptenene, thioheptenene, azaoctatetraene, oxaheptentraene, thioheptentraene, diazaheptenene, dioxaheptenene, dithioheptenene, imidazole, pyrazole, oxothioheptenene, isoxothioheptenene, oxazole, isoxazole, thiazole, diazine, oxazine, thiazine, oxothiohexene, dioxahexene, dithiohexene, triazine, tetraazine, pentaazine, diazaheptenene, thioazheptenene, azaoctatetraene, oxaheptentraene, or thioheptentraene.

[0186] In one implementation, R 10 For O. In some implementations, R 10 For NZ. In some implementations, R 10 For NNZ. Z is independently hydrogen, hydroxyl, aryl, or C1-C4 alkyl. In one embodiment, when R... 10 When R is NZ, Z represents a hydroxyl group. In some embodiments, when R... 10 When R is NZ, Z represents hydrogen. In some implementations, when R... 10 When Z is NNZ2, Z is

[0187] C1-C4 alkyl groups are optionally replaced by aryl groups.

[0188] In one implementation, R 10 It has one of the following structures:

[0189] In some implementation schemes, R 10 for In some implementation schemes, R 10 for In some embodiments, R 10 for In some implementation schemes, R 10 for

[0190] In one embodiment, the compound has one of the following structures:

[0191]

[0192]

[0193]

[0194] In one embodiment, the AhR agonist, upon administration, preferably orally, releases indirubin in the lower gastrointestinal tract or primarily in the colon. In another embodiment, upon administration, preferably orally, the AhR agonist releases indirubin primarily in the colon and is minimally or negligibly taken up into the bloodstream and / or surrounding peripheral tissues.

[0195] Table A. (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva). (IVb), (IVc),

[0196] List of compounds for (V), (Va), and (Vb)

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] It is understood that the indirubin derivatives described in this application can fine-tune the amplitude, site, and duration of AhR activation, thereby facilitating the development of novel therapies for diseases without requiring external chemicals or specialized reagents or manufacturing techniques. The indirubin derivative compounds enable the production of dosage forms that can be used to treat diseases, as described below. The methods described in this application using indirubin can be used with any known AhR agonist, including but not limited to indirubin, indigo, methylisodigo, linolamine (roquimet), 6-formylindodo[3,2-b]carbazole (FICZ), and laquimod, tapinarof, 4-(4-methyl-2-(4-(trifluoromethyl)phenyl)thiazolyl)pyrimidin-2-amine, or compounds listed in US2021 / 0147390.

[0206] Pharmaceutical Composition

[0207] Other embodiments involve pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises any (or more) of the foregoing compounds and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In still more embodiments, the pharmaceutical composition comprises a compound disclosed in this application and an additional therapeutic agent (e.g., an anticancer agent). Non-limiting examples of such additional therapeutic agents are described below.

[0208] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, nasal, and topical administration. Additionally, to name just a few examples, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intracardiac, intraperitoneal, intralymphatic, and intranasal injection.

[0209] In some embodiments, the compounds described herein are administered locally rather than systemically, such as by direct injection into an organ, typically in the form of a reservoir formulation or a sustained-release formulation. In specific embodiments, long-acting formulations are administered via implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the compounds are delivered in a targeted drug delivery system, such as in liposomes coated with organ-specific antibodies. In such embodiments, the liposomes target the organ and are selectively absorbed by the organ. In other embodiments, the compounds described herein are provided in the form of a rapid-release formulation, a prolonged-release formulation, or an intermediate-release formulation. In still other embodiments, the compounds described herein are administered locally.

[0210] In a treatment method according to an embodiment of this disclosure, an effective amount of at least one compound of formula (I)-(Vb) is given to a subject who has or has been diagnosed with such a disease, condition, or medical condition. The effective amount or dose can be determined by methods such as modeling, dose escalation studies, or clinical trials, including the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and duration of the disease, condition, or illness, the subject's prior or ongoing treatment, the subject's health status and response to the drug, and the judgment of the treating physician.

[0211] The compounds according to this disclosure are effective over a wide dosage range. For example, in treating adults, dosages of about 0.001 to 0.1 mg, 0.01 to 0.1 mg, 0.5 to 5 mg, 0.5 to 10 mg, 0.01 to 10 mg, 10 to 5000 mg, 100 to 5000 mg, 1000 mg to 4000 mg, or 1000 to 3000 mg daily are examples of dosages used in some embodiments. The exact dosage will depend on the route of administration, the form of the compound administered, the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician.

[0212] In some embodiments, the compounds of this disclosure are administered in a single dose. In one embodiment, a single dose is administered orally. In another embodiment, a single dose is administered topically. However, other routes are used when necessary. In some embodiments, the compounds of this disclosure are administered in multiple doses. In some embodiments, administration is performed about once, twice, three times, four times, five times, six times, or more than six times daily. In other embodiments, administration is performed about once a month, once every two weeks, once a week, or every other day. In another embodiment, the compounds of this disclosure are administered together with another agent (e.g., an anticancer agent) about once a day to about six times a day. In another embodiment, administration of the compounds and agents of this disclosure lasts for less than about seven days. In yet another embodiment, administration lasts for more than about six, ten, fourteen, twenty-eight days, two months, six months, or one year. In some cases, continuous administration is achieved and maintained as long as necessary.

[0213] Administration of the disclosed compound can be continuous whenever necessary. In some embodiments, the administration period of the disclosed compound exceeds 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, the administration period of the disclosed compound is less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the disclosed compound is administered in a continuous manner for a long period, for example, to treat chronic effects.

[0214] In some embodiments, the compounds of this disclosure are administered in individualized dosage forms. It is known in the art that individualized dosing regimens are necessary for optimal treatment due to inter-individual variability in the pharmacokinetics of compounds.

[0215] In some embodiments, the compounds described in this application are formulated into pharmaceutical compositions. In specific embodiments, the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers, said carriers comprising excipients and adjuvants that facilitate the processing of the disclosed compounds into pharmaceutically acceptable formulations. A suitable formulation depends on the chosen route of administration. Any pharmaceutically acceptable technology, carrier, and excipient can be suitably used to formulate the pharmaceutical compositions described in this application: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0216] This application provides pharmaceutical compositions comprising one or more compounds of formula (I)-(Vb) and a pharmaceutically acceptable carrier. This application also provides pharmaceutical compositions comprising one or more compounds selected from formula (I)-(Vb) and a pharmaceutically acceptable diluent, excipient, and carrier. In some embodiments, the compounds are administered as pharmaceutical compositions wherein one or more compounds selected from formula (I)-(Vb) are mixed with other active ingredients, as in combination therapies. This application covers all combinations of active ingredients listed below and throughout the combination therapies section of this disclosure. In specific embodiments, the pharmaceutical composition comprises one or more compounds of formula (I)-(Vb).

[0217] The pharmaceutical composition used in this application refers to a mixture of one or more compounds selected from formulas (I)-(Vb) with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. In some embodiments, the pharmaceutical composition facilitates the application of the compound to a living organism. In some embodiments, a therapeutically effective amount of one or more compounds selected from formulas (I)-(Vb) provided in this application is administered as a pharmaceutical composition to a mammal suffering from a disease, condition, or medical condition to be treated. In a specific embodiment, the mammal is a human. In some embodiments, the therapeutically effective amount varies depending on the severity of the disease, the age and relative health status of the subject, the potency of the compound used, and other factors. The compounds described in this application may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0218] In one embodiment, one or more compounds selected from formula (I)-(Vb) are formulated in an aqueous solution. In a specific embodiment, the aqueous solution is selected from, for example, physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer. In other embodiments, one or more compounds selected from formula (I)-(Vb) are formulated for transmucosal administration. In a specific embodiment, the transmucosal formulation comprises a permeabilizing agent suitable for the barrier to be penetrated. In other embodiments in which the compounds described herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In a specific embodiment, such solutions contain physiologically compatible buffers and / or excipients.

[0219] In another embodiment, the compounds described in this application are formulated for oral administration. The compounds described in this application are formulated by combining an active compound with, for example, a pharmaceutically acceptable carrier or excipient. In various embodiments, the compounds described in this application are formulated into oral dosage forms, including (by way of example only) tablets, powders, pills, sugar-coated pills, capsules, liquids, gels, syrups, elixirs, liquids, suspensions, etc.

[0220] In some embodiments, the oral pharmaceutical formulation is obtained by mixing one or more solid excipients with one or more compounds described in this application, optionally grinding the resulting mixture, and processing the granular mixture, if necessary, with the addition of suitable excipients to obtain tablets or sugar-coated pellet cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose formulations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; or others, such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In specific embodiments, a disintegrant is optionally added. By way of example only, disintegrants include cross-linked croscarmellose sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, or alginate or salts thereof, such as sodium alginate.

[0221] In one embodiment, an oral dosage form, such as pills, capsules, or tablets, comprises one or more suitable layers or coatings. In a specific embodiment, a concentrated sugar solution is used for the coating dosage form. The sugar solution optionally contains additional components, such as, by way of example only, gum arabic, talc, polyvinylpyrrolidone, carboplatin gel, polyethylene glycol and / or titanium dioxide, varnish solution, and suitable organic solvents or solvent mixtures. For identification purposes, dyes and / or pigments are also optionally added to the coating. Additionally, dyes and / or pigments are optionally used to characterize different combinations of active compound dosages.

[0222] In some embodiments, a therapeutically effective amount of at least one compound described in this application is formulated into other oral dosage forms. Oral dosage forms include push-in capsules made of gelatin and soft-sealable capsules made of gelatin and a plasticizer (e.g., glycerin or sorbitol). In a specific embodiment, the push-in capsule contains an active ingredient mixed with one or more fillers. Fillers include (by example only) lactose, binders (e.g., starch) and / or lubricants (e.g., talc or magnesium stearate), and optionally, stabilizers. In other embodiments, the soft capsule contains one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include (by example only) one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer is optionally added.

[0223] In other embodiments, the compounds described in this application are formulated for parenteral injection, including formulations suitable for bolus or continuous infusion. In specific embodiments, the formulations for injection are present in unit dose form (e.g., in ampoules) or in multi-dose containers. Optionally, a preservative is added to the injectable formulation. In other embodiments, the pharmaceutical composition is formulated for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous carrier. The parenteral injection formulation optionally contains formulation substances such as suspending agents, stabilizers, and / or dispersants. In specific embodiments, the pharmaceutical formulation for parenteral administration comprises an aqueous solution of the active compound in a water-soluble form. In further embodiments, a suspension of one or more compounds selected from formulas (I)-(Vb) is prepared into a suitable oily injectable suspension. Lipophilic solvents or carriers suitable for the pharmaceutical compositions described in this application include (by example only) fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides) or liposomes. In certain specific embodiments, the aqueous injection suspension contains a substance that increases the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension contains a suitable stabilizer or a reagent that increases the solubility of the compound to allow for the preparation of high-concentration solutions. Alternatively, in other embodiments, the active ingredient is in powder form for reconstitution with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0224] The pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, diluent, or excipient, and one or more compounds selected from formulas (I)-(Vb) as the active ingredient. The active ingredient is in the form of a free acid or free base, or in the form of a pharmaceutically acceptable salt. Furthermore, the methods and pharmaceutical compositions described in this application include N-oxides, crystalline forms (also called polymorphs), and active metabolites having the same type of activity using these compounds. All tautomers of the compounds described in this application are included within the scope of the compounds described in this application. Furthermore, the compounds described in this application include unsolvable forms and solvated forms with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.). The solvated forms of the compounds described in this application are also considered to be disclosed in this application. In addition, the pharmaceutical composition optionally includes other drugs or agents, carriers, adjuvants such as preservatives, stabilizers, wetting agents or emulsifiers, solubilizers, osmotic pressure regulating salts, buffers, and / or other substances of therapeutic value.

[0225] Methods of preparing compositions comprising the compounds described in this application include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semi-solid, or liquid compositions. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, pouches, and suppositories. Liquid compositions include solutions containing the compounds, emulsions containing the compounds, or solutions containing liposomes, micelles, or nanoparticles comprising the compounds disclosed in this application. Semi-solid compositions include, but are not limited to, gels, ointments, suspensions, and creams. The pharmaceutical compositions described in this application are in the form of liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid prior to use, or as emulsions. These compositions may also optionally contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, etc.

[0226] In some embodiments, pharmaceutical compositions comprising one or more compounds selected from formulas (I)-(Vb) are, for example, in liquid form, wherein the agent is present in a solution, suspension, or both. Typically, when the composition is administered in suspension form, a first portion of the agent is present in the solution, and a second portion of the agent is present in particulate form in a suspension within a liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

[0227] In some embodiments, the aqueous suspension contains one or more polymers as suspending agents. Polymers include water-soluble polymers, such as cellulose polymers, such as hydroxypropyl methylcellulose, and water-insoluble polymers, such as cross-linked carboxyl-containing polymers. Some pharmaceutical compositions described in this application contain mucosal adhesion polymers selected from, for example, carboxymethyl cellulose, carbomer (acrylic polymer), poly(methyl methacrylate), polyacrylamide, polycarbofil, acrylate / butyl acrylate copolymer, sodium alginate, and dextran.

[0228] The pharmaceutical composition may also optionally contain a solubilizer to aid in the dissolution of one or more compounds selected from those of formula (I)-(Vb). The term "solventizer" generally includes an agent that results in the formation of a micelle solution or a true solution of the pharmaceutical agent. Certain acceptable nonionic surfactants, such as polysorbate 80, may be used as solubilizers, as may ophthalmologically acceptable glycols, polyethylene glycols such as polyethylene glycol 400, and glycol ethers.

[0229] In addition, the pharmaceutical composition optionally includes one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate / glucose, sodium bicarbonate, and ammonium chloride. The amounts of these acids, bases, and buffers should be such that the pH of the composition is maintained within an acceptable range.

[0230] The composition may also optionally contain one or more salts in an amount sufficient to achieve an acceptable isotonic molar concentration of the composition. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0231] Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thimerosal; stable chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, hexadecyltrimethylammonium bromide, and hexadecylpyridine chloride.

[0232] The composition may contain one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octylphenol polyether 10 and octylphenol polyether 40.

[0233] The composition may contain one or more antioxidants to enhance chemical stability when needed. Suitable antioxidants include (by way of example only) ascorbic acid and sodium metabisulfite.

[0234] In some embodiments, the aqueous suspension composition is packaged in a single-dose, non-resealable container. Alternatively, a multi-dose, resealable container is used, in which case the composition typically contains a preservative.

[0235] In alternative embodiments, delivery systems for other hydrophobic pharmaceutical compounds are employed. Liposomes and emulsions are examples of delivery carriers or vessels useful in this application. In some embodiments, organic solvents, such as N-methylpyrrolidone, are also used. In other embodiments, the compounds described in this application are delivered using sustained-release systems, such as a semi-permeable matrix of a solid hydrophobic polymer containing the therapeutic agent. Various sustained-release materials are available in this application. In some embodiments, sustained-release capsules release the compound for weeks to over 100 days. Depending on the chemical properties and biological stability of the therapeutic agent, additional protein-stabilizing strategies are employed.

[0236] In some embodiments, the formulations described in this application comprise one or more antioxidants, metal chelators, thiol compounds, and / or other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0237] In some embodiments, the concentration of one or more compounds selected from compounds of formula (I)-(Vb) provided in the pharmaceutical composition is greater than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%. 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 1.25%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0. 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v. In another embodiment, the amount of the compound selected from compounds of formula (I)-(Vb) in the pharmaceutical composition is between about any two of the values ​​listed in the preceding sentence, for example, between about 2-70 w / w%, 3.5-80 w / w%, 1-30 w / w%, etc.

[0238] In some embodiments, the concentration of one or more compounds selected from formula (I)-(Vb) provided in the pharmaceutical compositions of this disclosure is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, and from about 0.07%. The range of approximately 1% to 24%, approximately 0.08% to 23%, approximately 0.09% to 22%, approximately 0.1% to 21%, approximately 0.2% to 20%, approximately 0.3% to 19%, approximately 0.4% to 18%, approximately 0.5% to 17%, approximately 0.6% to 16%, approximately 0.7% to 15%, approximately 0.8% to 14%, approximately 0.9% to 12%, approximately 1% to 10% w / w, w / v, or v / v.

[0239] In some embodiments, the amount of one or more compounds selected from formula (I)-(Vb) provided in the pharmaceutical compositions of this disclosure is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, or 0.35g. , 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006 g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

[0240] In some embodiments, the amount of one or more compounds selected from formula (I)-(Vb) provided in the pharmaceutical composition of this disclosure is in the range of 0.0001-10g, 0.0005-9g, 0.001-8g, 0.005-7g, 0.01-6g, 0.05-5g, 0.1-4g, 0.5-4g or 1-3g.

[0241] Packaging materials used for packaging the pharmaceutical compositions described in this application include, for example, those found in U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and the intended mode of administration and treatment. For example, a container may contain one or more compounds described in this application, optionally in a composition or in combination with another substance disclosed in this application. The container may optionally have a sterile access port (e.g., the container is an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such cassettes may optionally contain a compound and an identification description or label or instruction associated with its use in the methods described in this application.

[0242] For example, a pharmaceutical kit typically includes one or more additional containers, each containing one or more various materials (e.g., reagents, optionally in concentrated form, and / or devices) necessary from a commercial and user perspective for using the compounds described in this application. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials, and / or tube labels listing the contents and / or instructions for use, as well as packaging inserts with instructions for use. A set of instructions is also typically included. The label is optionally located on or associated with the container. For example, when the letters, numbers, or other characters forming the label are attached, molded, or etched into the container itself, the label is located on the container; when the label is present within a container or carrier that also contains the container, the label is associated with the container, for example, as a package insert. Furthermore, the label is used to indicate that the contents will be used for a specific therapeutic application. Additionally, the label indicates instructions for use of the contents, such as in the methods described in this application. In some embodiments, the pharmaceutical composition is present in a packaging or dispenser device containing one or more unit dosage forms of the compounds provided in this application. The packaging may contain, for example, metal or plastic foil, such as blister packs. Alternatively, the packaging or dispenser device may include instructions for administration. Alternatively, the packaging or dispenser may include a notification related to the container, in a form prescribed by the government agency regulating the manufacture, use, or sale of the drug, reflecting that agency's approval of the form of the drug for human or veterinary use. For example, such a notification is a prescription drug label approved by the U.S. Food and Drug Administration, or an approved product label. In some embodiments, a composition containing the compounds provided in this application is prepared, formulated in a compatible drug carrier, placed in a suitable container, and labeled for the treatment of a specified condition.

[0243] As described above, the compounds and compositions disclosed herein can be used for a wide range of diseases and conditions mediated by protein kinases, including those mediated by kinases. For example, such diseases may include, but are not limited to, cancers such as lung cancer, NSCLC (non-small cell lung cancer), oat cell carcinoma, bone cancer, pancreatic cancer, skin cancer, dermatofibrosarcoma protuberans, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, colorectal cancer, anal region cancer, gastric cancer, colon cancer, breast cancer, gynecological tumors (e.g., uterine sarcoma, fallopian tube cancer, endometrial cancer), cervical cancer, vaginal cancer, or vulvar cancer), Hodgkin's disease, hepatocellular carcinoma, esophageal cancer, small bowel cancer, endocrine system cancers (e.g., thyroid cancer, pancreatic cancer, parathyroid cancer, or adrenal cancer), soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer (especially hormone-resistant), chronic or acute. Leukemia, childhood solid tumors, eosinophilia, lymphocytic lymphoma, bladder cancer, cancerous kidney or ureteral cancer (such as renal cell carcinoma, renal pelvis carcinoma), pediatric malignancies, central nervous system tumors (such as primary central nervous system lymphoma, spinal axis tumors, medulloblastoma, brainstem glioma, or pituitary adenoma), Barrett's tumor, esophageal cancer (precancerous syndrome), neoplastic skin diseases, psoriasis, mycosis fungoides, benign prostatic hyperplasia, diabetes-related diseases such as diabetic retinopathy, retinal ischemia, retinal neovascularization, cirrhosis, angiogenesis, cardiovascular diseases such as atherosclerosis, immune diseases such as autoimmune diseases, and kidney diseases.

[0244] In some embodiments, the pharmaceutical composition has the above-described compounds and a pharmaceutically acceptable carrier, including, for example, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0245] In some embodiments, a method of treating a disease or condition includes administering an effective amount of the compound or pharmaceutical composition described in this application to a subject in need. In some embodiments, the disease or condition is a kinase-expressing cancer. In some specific embodiments, the cancer is bladder cancer. In some other specific embodiments, the cancer is prostate cancer. In some other specific embodiments, the cancer is a hematologic malignancy, such as acute myeloid leukemia. In some other specific embodiments, the disease or condition is an autoimmune disease or an inflammatory disease.

[0246] In one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the compound of formula (I)-(Vb) is converted in vitro to indirubin in simulated intestinal fluid or human intestinal fluid at 37°C for about 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 2 hours, or 1 hour.

[0247] In one embodiment, the pharmaceutical composition comprises a compound of formula (I)-(Vb) and a second therapeutic agent.

[0248] In one embodiment, the compound or pharmaceutical composition of formula (I)-(Vb) is used to treat immune diseases, autoimmune diseases, or inflammatory diseases. In other embodiments, the compound or pharmaceutical composition of formula (I)-(Vb) is used to treat inflammatory or immune diseases.

[0249] In one embodiment, a method of treating ulcerative colitis or inflammatory bowel disease includes administering a compound of formula (I)-(Vb) or a pharmaceutical composition to a subject in need. In another embodiment, a method of treating hidradenitis suppurativa or a type of psoriasis is considered, wherein the treatment includes administering a compound of formula (I)-(Vb) or a pharmaceutical composition containing a compound of formula (I)-(Vb) to a subject in need. In one embodiment, the type of psoriasis is selected from plaque psoriasis, psoriasis recurvatum, erythrodermic psoriasis, guttate psoriasis, and pustular psoriasis. In other embodiments, a method of treating an inflammatory disease includes administering a compound of formula (I)-(Vb) or a pharmaceutical composition to a subject in need.

[0250] In one implementation, the subject is an animal. In other implementations, the subject is a human. In some implementations, the subject has an inflammatory or immune disease. For example, in some implementations, the disease is an inflammatory gastroenteritis, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome, type 1 and type 2 diabetes, or vitiligo. In other implementations, the disease is ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, cryptitis, mucositis, cancer, fibrotic disease, or graft-versus-host disease.

[0251] In one embodiment, a method of administering indirubin to a subject includes administering a compound of formula (I)-(Vb), wherein the compound is converted to indirubin upon administration. In some embodiments, the compound of formula (I)-(Vb) is hydrolyzed to indirubin.

[0252] In some embodiments, the AhR agonist prodrug exhibits a solubility in simulated intestinal fluid that is at least 1.5, 1.75, 2, or 3 times higher than the solubility of synthetic indirubin in the same fluid at the same temperature (e.g., 37°C) and at the same time point. In other embodiments, the AhR agonist prodrug exhibits a solubility in simulated intestinal fluid that is at least 10, 50, 100, 200, or 300 times higher than the solubility of synthetic indirubin in the same fluid at the same temperature (e.g., 37°C) and at the same time point. In other embodiments, the compounds of formulas (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), or (Vb) exhibit a solubility in simulated intestinal fluid that increases by at least 1.75, 2, or 3 times over 24 hours. In some embodiments, the AhR agonist prodrug is more therapeutically effective than an equimolar amount of synthetic indirubin. In some embodiments, the amount of indirubin produced in lung tissue after oral administration of an AhR agonist prodrug (e.g., any compound of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb)) is less than an equimolar amount of synthetic indirubin. In some embodiments, the amount of indirubin produced in plasma after oral administration of an AhR agonist prodrug (e.g., any compound of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb)) is less than an equimolar amount of synthetic indirubin. In some embodiments, the AhR agonist prodrug (e.g., any compound of formulas (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb)) produces less indigo in plasma and / or lung tissue than an equimolar amount of synthetic indigo after oral administration, but more in the colon than an equimolar amount of synthetic indigo. In other embodiments, when the skin flux or permeability of the formulation, which is identical in all respects except for the prodrug or pharmaceutical compound (which are present in the formulation in an equimolar amount of indigo), is measured in vitro, the AhR agonist prodrug (e.g., any compound of formulas (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va), and (Vb)) provides greater skin permeability (permeability) compared to the skin permeability of synthetic indigo.

[0253] B. Preparation method

[0254] In the embodiments, the compounds disclosed in this application can be prepared using conventional methods known in the art. This application includes exemplary and non-limiting examples of methods for preparing the disclosed compounds.

[0255] In one embodiment, class A molecules can be prepared using known methods of indirubin synthesis with radiolabeled starting materials. Other radiolabeled compounds (e.g., radiolabeled forms of class B, C, or D compounds) can be prepared using the methods proposed in this application with radiolabeled precursors. The following examples are illustrative and are in no way intended to be limiting.

[0256] In some embodiments, class D compounds are prepared starting from indirubin. In these embodiments, indirubin reacts with a chloroformate followed by nucleophilic attack by a selected nucleophile. As non-limiting examples, the results include urethanes, ureas, and amide derivatives. This technique considers any base compatible with nucleophilic attack on chloroformates. Furthermore, this technique considers any chloroformate known in the art that is sensitive to subsequent nucleophilic attack. Preferred nucleophiles particularly include amines, alcohols, amides, carboxylic acids, and thiols. The preparation method can follow the following scheme:

[0257]

[0258] In other embodiments, chloroformates may be selected to obtain a final product that is not further attacked by nucleophiles, or it may be further derivatized. This technique considers any base compatible with nucleophilic attack on chloroformates. Preferred chloroformates include alkyl chloroformates, aryl chloroformates, cycloalkyl chloroformates, heterocyclic alkyl chloroformates, and heteroaryl chloroformates. In these embodiments, the resulting derivative is a carbamate derivative, as shown in the following schemes:

[0259]

[0260] In other embodiments, indirubin reacts with selected acyl chlorides. As a non-limiting example, the resulting derivatives include amide derivatives. Preferred acyl chlorides particularly include alkyl acyl chlorides, aryl acyl chlorides, cycloalkyl acyl chlorides, heterocyclic alkyl acyl chlorides, and heteroaryl acyl chlorides. The preparation method can follow the following scheme:

[0261]

[0262] In other embodiments, indirubin reacts with formaldehyde and a selected nucleophile. As non-limiting examples, the resulting derivatives include acetals and hemiacetals. Preferred nucleophiles particularly include alcohols and amines. The preparation method can follow the following scheme:

[0263]

[0264] In a further step of these embodiments, the alcohol derivative can be further derivatized. One embodiment is described below, but other chemical modifications known in the art are considered. Alternatively, in other embodiments, indirubin alcohol derivatives prepared by other means may also be used, as shown in the following embodiments:

[0265]

[0266] In these embodiments, the alcoholic indirubin derivative X is reacted with SOCl2 to prepare for nucleophilic attack on carbon. Selected nucleophiles are permitted to react with indirubin chloride derivatives to form other indirubin derivatives, including but not limited to hemiacetals and acetals. In specific embodiments, the hemiacetal can be an organophosphate ester, as shown in the following schemes:

[0267]

[0268] When necessary, or when testing the hydrolysis rate of indirubin derivatives in physiological models (e.g., intestinal fluid models, gastric fluid models) to demonstrate the function of the derivative as a prodrug, indirubin derivatives can also be reduced to indirubin. The following scheme illustrates such a hydrolysis method:

[0269]

[0270] III. How to use

[0271] Indirubin has been shown to have potential therapeutic activity in treating proliferative diseases, cancer, fibrotic diseases, inflammatory diseases, and autoimmune diseases, as a potent agonist of AhR. In some embodiments, the indirubin derivatives of this disclosure may have altered therapeutic or agonist efficacy compared to indirubin itself. The level of either efficacy may increase, decrease, or remain substantially unchanged. In a preferred embodiment, the efficacy is increased compared to indirubin.

[0272] The pharmacokinetic (PK) characteristics of a compound are one aspect to consider. These can be assessed as PK properties in the target tissue or plasma PK properties, which can represent total systemic exposure. As an effect parameter of PK properties, the metabolic rate of indirubin derivatives may differ from that of indirubin. The metabolic rate of the prodrug form can be faster, slower, or substantially the same. Changes in metabolism can lead to differences in PK properties, safety, efficacy, or dosage and dosing regimens to achieve the desired therapeutic effect. Alternatively, indirubin derivatives may differ from indirubin in terms of safety or efficacy, regardless of metabolic rate.

[0273] Another effect parameter of PK characteristics is the dissolution rate of the indirubin derivative. The dissolution rate of the indirubin derivative can be faster, slower, or substantially the same as that of indirubin. In a preferred embodiment, when using an indirubin prodrug, the concentration of indirubin in solution can be greater compared to a similar molar dose of indirubin. The prodrug may exhibit enhanced solubility due to the breaking of hydrogen bonds, resulting in a higher concentration of the prodrug in solution. If the prodrug is converted to indirubin in solution, the concentration of indirubin in solution can be increased compared to a similar molar dose of indirubin administered in a non-prodrug form.

[0274] Indirubin prodrugs can also be converted to indirubin in vivo at varying rates, depending on the route of administration, the drug product, and the dosage. The conversion can be based on chemical or enzymatic conversion. If the conversion of the indirubin prodrug to indirubin is chemical (e.g., without enzymatic activity), it may be caused by the presence of pH, temperature, light, water, oxygen, or other chemicals, or by conditions in the target organ or tissue. For example, conversion may be enhanced by or require a relatively high pH (such as that found in the intestine or colon). In some cases, conversion may not occur in a meaningful amount until the pH is below or above 7 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0275] In other cases, conversion may require contact with water or other bodily fluids, which can be mitigated by protecting the prodrug through encapsulation, tableting, or formulation with hydrophobic excipients. In such cases, protecting against contact with bodily fluids limits exposure to the prodrug / derivative / indirubin and minimizes systemic absorption. In one example, the prodrug can be formulated into a sustained-release capsule, minimizing exposure of indirubin to the oral mucosa, pharynx, stomach, and / or small intestine. Such capsules, tablets, or encapsulations can be made from pH-sensitive polymers, such as copolymers of acrylic acid and methacrylate esters or hydroxypropyl methylcellulose phthalate.

[0276] If the conversion of indirubin derivatives to indirubin is enzymatic, it can be caused by CYPp450s, esterases, carboxylesterases, azoreductases, carboxypeptidases, alkaline phosphatases, nitroreductases, acetylcholinesterases, butylcholinesterases, or any other naturally occurring enzyme. These enzymes may be expressed at higher levels in one tissue or site than in another, resulting in varying degrees of exposure to indirubin.

[0277] Indirubin derivatives can also be characterized by changes in other physical, chemical, or biological properties, such as solubility, dissolution rate, color, tissue penetration, transporter inflow or efflux, metabolism, and / or long-term stability. Better solubility or dissolution rate may result in greater local exposure of indirubin, thereby reducing the amount of drug required to activate AhR in target tissues. Greater local exposure may lead to lower costs, reduced systemic exposure, and / or minimized side effects.

[0278] The indirubin derivatives disclosed herein are also characterized by advantages, particularly in the formation processes of certain types of formulations, micronization, or solid amorphous dispersions.

[0279] Prodrugs may be easier to formulate than indirubin and may result in increased site-specific activity or systemic exposure. For example, the low solubility of indirubin may exclude aqueous topical formulations containing large doses. Prodrugs with better water solubility can enable dosage forms such as solutions, suspensions, ointments, creams, and gels to have higher drug loadings.

[0280] The compounds described in this application as derivatives of indirubin are prodrugs that are converted to indirubin. The indirubin prodrugs can be studied in vitro to see if they are converted to indirubin. For example, the indirubin prodrug can be added to a solution and evaluated later to determine the relative conversion to indirubin. This can be done, for example, in water, ethanol, simulated intestinal fluid (e.g., fasting simulated intestinal fluid (FaSSIF), simulated gastric fluid (e.g., fasting simulated gastric fluid (FaSSGF)), isolated intestinal fluid, isolated gastric fluid, plasma, blood, saliva, or other solutions. The solutions can contain various additives, including microorganisms, fecal samples, enzymes, or salts. These solutions can be buffered and / or their pH adjusted. This can be done at different temperatures (including body temperature). The amount of indirubin present in the solution can be determined by LC-MS, HPLC, or other laboratory techniques.

[0281] Indirubin derivatives can also be studied in vivo, for example, in laboratory animals. Animals can be healthy or used as disease models. Disease models can be models of inflammatory diseases, such as a sodium dextran sulfate model of colitis. In these models, indirubin derivatives can be administered prophylactically or therapeutically to assess their safety and / or efficacy. It can be administered via any route, such as orally, topically, or by injection. Administration of indirubin derivatives can reduce weight loss, improve disease activity index scores, reduce bloody stools, improve stool consistency, reduce intestinal motility, increase colon length, reduce inflammation, improve intestinal barrier function, increase intestinal regulatory T cells, reduce intestinal Th17 cells, alter the gut microbiota, reduce the expression of pro-inflammatory cytokines, increase the expression of anti-inflammatory cytokines, or reduce the expression of pro-inflammatory chemokines.

[0282] Indirubin derivatives can be studied to determine if they are AhR agonists. This can be done in vivo or in vitro. It can be accomplished by observing AhR translocation to the cell nucleus, searching for AhR protein complexes, and examining AhR transcriptional or translational activity. AhR activity can be assessed by searching for the expression of mRNAs or proteins known to be regulated by AhR. These may include metabolic enzymes, including CYP1A1 or CYP1A2. This can be done by searching for light produced by luciferase, which acts on luciferin and is controlled by genetic elements that respond to the dimerization of AhR / ARNT.

[0283] In some implementations, the treatment of a disease may benefit from local pharmacokinetic characteristics that differ from systemic pharmacokinetic characteristics. For example, to treat blood cancers (such as chronic myeloid leukemia) and autoimmune diseases manifesting in certain systems or the central nervous system, low concentrations of the active agent in the intestines and lungs but high plasma concentrations are required. To treat skin diseases (such as hidradenitis suppurativa, acne, psoriasis, or atopic dermatitis), high skin levels but low plasma levels are required. To treat idiopathic pulmonary fibrosis, low drug exposure in the intestines and plasma and high drug exposure in the lungs are required. To treat intestinal diseases such as ulcerative colitis, a significant portion of the administered drug needs to be in the lower gastrointestinal tract, with lower amounts in plasma and lung tissue. The compounds disclosed in this invention produce indirubin in the colon and / or lower gastrointestinal tract after oral administration, and the concentration of indirubin in the colon and / or lower gastrointestinal tract is greater than the concentration of indirubin in the lungs and / or blood (plasma).

[0284] Therefore, a method for treating a disease or condition associated with aryl hydrocarbon receptor activity is provided, the method comprising administering to a subject in need an amount of the compound disclosed herein or a pharmaceutical composition thereof, said amount being sufficient to provide an amount of indirubin modulating the aryl hydrocarbon receptor. It is understood that after oral administration, the compound produces indirubin at a dose or concentration of indirubin for therapeutic purposes. As mentioned, the compound described herein preferentially releases indirubin in the lower gastrointestinal tract, preferably in the colon, and the indirubin uptake or absorption into the bloodstream is minimal or negligible. Such methods include both therapeutic and preventative methods. In some embodiments, the disease or condition is associated with reduced aryl hydrocarbon receptor activity. In some embodiments, the disease or condition is associated with reduced aryl hydrocarbon receptor activity compared to the activity of aryl hydrocarbon receptors in normal (i.e., non-disease) cells.

[0285] Studies were conducted to support this disclosure, as illustrated in Examples 1-9 below. Example 1 details the preparation of the indirubin derivative compound (indirubin prodrug compound) described in this application. Example 2 shows the results of a study on the stability of the indicated indirubin prodrug compound in simulated intestinal and gastric fluids under fasting conditions. In this study, the test compound was placed in a simulated fluid at 37°C for 24 hours. The amount of the test compound (or starting material “SM”) and the amount of indirubin in the fluid were then analyzed by LCMS. Tables 2-1 in Example 2 report the percentage of the starting material and the percentage of indirubin in each simulated fluid, providing a measurement of the stability or ease of conversion of the prodrug compound to indirubin. This study demonstrates an in vitro method for determining whether and / or to what extent the prodrug produces indirubin.

[0286] Examples 3-4 describe studies of the time process or rate of conversion of the indirubin prodrug compound to indirubin. In these studies, the test compound was placed in a fasting-state simulated intestinal fluid (Example 3) or a fasting-state simulated gastric fluid (Example 4) at a controlled temperature, and the fractions of the starting material (test compound), indirubin methylene alcohol, and indirubin were measured over 24 hours. The results are shown in Tables 3-1 and 4-1 of Examples 3 and 4, respectively. It is understood that the prodrug compound can be tailored to produce indirubin at different rates. In one embodiment, an AhR agonist prodrug compound is provided, wherein the prodrug compound produces a therapeutically active AhR agonist compound by converting the prodrug compound to the AhR agonist compound, wherein at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the prodrug compound is converted to the AhR agonist compound within 24 hours in a fasting-state simulated intestinal fluid or a fasting-state simulated gastric fluid at 37°C. In another embodiment, an indirubin prodrug compound is provided, wherein the indirubin prodrug compound produces indirubin by converting the indirubin prodrug compound into indirubin, wherein at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the indirubin prodrug compound is converted into indirubin within 24 hours in simulated intestinal fluid under fasting conditions at 37°C or in simulated gastric fluid under fasting conditions.

[0287] Example 5 describes a study of the AhR activity of an exemplary indirubin derivative compound 8. In this study, solutions containing compound 8, indirubin, 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), or (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio) (the latter two serving as positive controls) were added to human Huh7 cells or rat H4IIE cell lines containing native AhR and the AhR / arnt-luciferase reporter vector. After culturing, luciferase assay reagents were added, and activity measurements were obtained in relative luminescent units. The results showed... Figure 1A-1B middle.

[0288] Figure 1A-1B The figures show the concentrations of compound 8 (triangle), indirubin (square), TCDD (circle; positive control), and MeBio (dashed line; positive control) in human Huh7 cells at nM concentrations. Figure 1A ) and rat H4IIE cells ( Figure 1B A diagram showing the activation of AhR in compound 8. EC50 in human Huh7 cells. 50 EC that is redder than indigo 50 Approximately 20% (71.47 nM vs. 58.82 nM). EC50 of indirubin in rat H4IIE cells. 50 The estimated value is 654 nM (estimated due to the lack of an upper plateau). Compound 8 in rat H4IIE cells EC50 50 The value was 25.62 nM. This data indicates that, compared to an equimolar amount of synthetic indirubin, compound 8 activates AhR to the same or greater extent at the same or lower concentrations. Given the similarity between the curves of compound 8 and indirubin, it is likely that compound 8 is substantially completely converted to indirubin (e.g., at least about 75%, 80%, 90%, or 100% conversion) within about 1, 2, or 24 hours, and therefore has a similar ability to activate AhR in this assay.

[0289] In Example 6, a study is described in which mice with induced acute colitis were treated with indirubin prodrug (exemplified by compound 8). Mice were orally administered compound 8 orally twice daily with distilled water. Body weight and disease activity index (DAI) scores were recorded daily to assess colitis. Results showed… Figure 2 middle.

[0290] Figure 2The graph shows the disease activity index (see Table 6-1 in Example 6), a measure of ulcerative colitis in a mouse model, as a function of time in days, after daily treatment with either compound 8 (triangles) or placebo (distilled water; circles). During the 9-day test period, mice treated with the indirubin prodrug had a lower disease activity index than mice treated with placebo (carrier only). This demonstrates that compound 8, as an example of an indirubin derivative described in this application, is converted to indirubin after oral administration to provide the therapeutic effect.

[0291] Example 7 details another study using a mouse model of ulcerative colitis. In this study, mice with induced acute colitis were treated by oral gavage with distilled water or with synthetic indirubin, compounds 9, 10, 26, 40, and 41. The results showed... Figures 3A-3C middle.

[0292] Figures 3A-3B This shows the percentage of initial body weight in mice treated twice daily for 9 days with placebo (solid circle), synthetic indirubin (solid square), compound 9 (solid triangle), compound 10 (inverted triangle), compound 26 (hollow circle), compound 40 (hollow square), or compound 41 (hollow triangle). Figure 3A ) and Disease Activity Index (DAI) score ( Figure 3B The figure is a graph representing time as a function of days. Dosage is expressed in milligrams per kilogram (mpk) and was designed to be equimolar with indirubin. All tested compounds were effective, with compound 41 being more effective than an equimolar amount of indirubin. Therefore, in one embodiment, the therapeutic effect provided by the indirubin derivative after oral administration to a subject is at least about 10%, 20%, 25%, 40%, 50%, or 75% greater than the therapeutic effect provided by an equimolar amount of orally administered synthetic indirubin.

[0293] Figure 3C This is a bar chart showing the average colon length (in centimeters, standard error of the mean (SEM)) of mice with ulcerative colitis and treated as described in Example 7. Colon length is correlated with inflammation; colons with more severe inflammation are shorter than those with less or no inflammation. Figure 3C Data showed that mice treated with the medium alone had shorter colons than mice treated with synthetic indirubin (positive control). Mice treated with the indirubin prodrug exhibited significantly longer colons than those treated with the medium alone, indicating that the prodrug-treated mice had milder colonic inflammation.

[0294] In Example 8, a study of animals treated in Example 7 is described to analyze the distribution of indirubin in plasma, colon, and lungs. Table 8-1 reports the mean indirubin levels in plasma, colon, and lungs of animals treated with the medium, synthetic indirubin, compound 9, compound 10, compound 26, compound 40, or compound 41. Treatment with compounds 9, 40, and 41 resulted in significant levels of indirubin in the colon compared to the levels in the lungs and / or plasma. For example, the ratio of indirubin in the colon to plasma was approximately 1003 for compound 40 (Table 8-2). The ratio of indirubin in the colon to plasma was approximately 824 for compound 9 (Table 8-2). The ratio of indirubin in the colon to plasma was approximately 170 for compound 41 (Table 8-2). Therefore, in one embodiment, 4, 6, 7, 8, 9, 10, 11, or 12 days after administration, at 4, 6, 8, or 12 hours after oral administration, the amount of indirubin provided in the colon by the indirubin derivative is at least 125, 150, 170, 250, or 300 times higher than the amount of indirubin in plasma. In another embodiment, approximately 6 hours after administration, at least 6 hours after oral administration, the amount of indirubin provided in plasma by the indirubin derivative is less than an equimolar amount of the orally administered synthetic indirubin.

[0295] The ratio of indirubin in the colon to indirubin in the lungs of compound 40 was approximately 4390 (Table 8-3). The ratio of indirubin in the colon to indirubin in the lungs of compound 9 was approximately 217 (Table 8-3). The ratio of indirubin in the colon to indirubin in the lungs of compound 41 was approximately 1.6 (Table 8-3). Therefore, in one embodiment, after treatment for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, oral administration of the indirubin derivative to the subject provided an amount of indirubin in the colon that was at least 1.3, 1.4, 1.5, 10, or more times higher than the amount of indirubin in the lungs.

[0296] In Example 9, the solubility of exemplary indirubin prodrug compounds 8, 9, and 10 in simulated fluids (e.g., simulated fasting gastric juice (FaSSGF) and simulated fasting intestinal juice (FaSSIF)) was measured. For comparison, the solubility of synthetic indirubin and indigo was also measured. Both compound 9 and compound 10 were soluble in the simulated fluids (Table 9-1).

[0297] In one embodiment, the compounds disclosed in this application are considered for treating immune diseases, autoimmune diseases, or inflammatory diseases. In one embodiment, the compounds are considered for treating ulcerative colitis or inflammatory bowel disease. In other embodiments, the compounds are considered for treating inflammatory gastrointestinal diseases, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjögren's syndrome, type 1 and type 2 diabetes, or vitiligo. In other embodiments, the compounds are considered for treating ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, cryptitis, or mucositis.

[0298] Derivatives of AhR agonists may differ from their parent compounds in many ways, resulting in improved and desired properties that make them more suitable for treating or preventing diseases. For the treatment of gastrointestinal diseases such as ulcerative colitis, an ideal oral AhR agonist produces AhR activity in the intestine without causing significant systemic exposure and / or systemic AhR activation. An ideal oral AhR agonist also clears rapidly from the intestine, resulting in non-sustained activation of extraintestinal AhR. For the treatment of skin diseases such as hidradenitis suppurativa or psoriasis, a topical AhR agonist produces AhR activity in the skin without causing significant systemic exposure and / or systemic AhR activation. Topical AhR agonists also clear rapidly from the skin, resulting in non-sustained activation of extradermal AhR. The strategies described in this application regarding indirubin derivatives can be used with any known AhR agonist, including but not limited to indigo, iso-indigo, linolamine (roquimet), 6-formylindolo[3,2-b]carbazole (FICZ), and laquimod, benvitimod, 4-(4-methyl-2-(4-(trifluoromethyl)phenyl)thiazolyl)pyrimidin-2-amine, compounds disclosed in US 2021 / 0147390A1, etc.

[0299] IV. Example

[0300] The following examples are merely illustrative and are not intended to be limiting.

[0301] Example 1

[0302] Preparation of indigo derivative compounds

[0303]

[0304] (Z)-1'-(morpholino-4-carbonyl)-[2,3'-biindolyl]-2',3-dione

[0305] Under a nitrogen atmosphere, at 0 °C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1M, THF solution) (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise to a solution of (3Z)-3-(3-oxoindolano-2-ylidene)indolano-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17.0 mL). The reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8.4 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h; TLC analysis showed that SM was completely consumed and an activating agent was produced. Morpholine (4.00 eq, 0.27 mL, 3.05 mmol) was added dropwise and the reaction mixture was stirred for 67 h. The reaction mixture was diluted with CH₂Cl₂ / iPrOH (9:1, 50.0 mL) and washed with water (2 × 50.0 mL) and brine (50.0 mL). The aqueous layer was extracted with ethyl acetate (2 × 50.0 mL), the organic matter was combined, and the mixture was dried over Na₂SO₄. 4) The crude substance was then concentrated under vacuum. The crude substance was purified by automated column chromatography on a silica (40 g column) by elution with an EtOAc gradient (5% to 60% v / v) in hexane to give the desired product (3Z)-1-(morpholino-4-carbonyl)-3-(3-oxoindolan-2-ylidene)indolan-2-one (122 mg, 0.322 mmol, 42.19% yield), as a deep purple solid.

[0306] UPLC-MS analysis (sample number: 142242-2): (4 min, alkaline): rt = 1.86 min, m / Z = 376.3 [M+H]+, purity 99%. ¹H NMR analysis (sample number: 142242-2, 400 MHz, CDCl3): δ 10.37 (s, ¹H), 8.88 (d, J = 7.9 Hz, ¹H), 7.69 (d, J = 7.6 Hz, ¹H), 7.47 (dd, J = 7.7, 7.7 Hz, ¹H), 7.28 (dd, J = 7.7, 7.7 Hz, ¹H), 7.21–7.11 (m, 2H), 7.03–6.91 (m, 2H), 3.98–3.24 (m, 8H).

[0307]

[0308] (Z)-2',3-Dioxo-N-(tetrahydro-2H-pyran-4-yl)-[2,3'-biindolyl]-1'-carboxamide

[0309] Under a nitrogen atmosphere, at 0 °C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1M, THF solution) (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise to a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17.0 mL). The reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8.4 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h; 4-aminotetrahydropyran (4.00 eq, 0.32 mL, 3.05 mmol) was added and the reaction mixture was stirred for 1 h, then concentrated under vacuum and stored in a refrigerator. Methanol (10.0 mL) was added to the reaction mixture, and then the reaction mixture was concentrated under vacuum. The crude substance was purified by automated column chromatography on a silica (40 g column) using a gradient elution with EtOAC (5% to 80% v / v) in isohexane to obtain the desired product. LCMS analysis showed that the product was contaminated with an unknown impurity, so the product was repurified. The product was dissolved in CH2Cl2 (150 mL) and washed with NaHCO3 (2 x 50.0 mL) and brine (50.0 mL), dried (Na2SO4), and then purified by automated column chromatography on a silica (40 g) using a gradient elution with methanol (0-15%) in CH2Cl2 to obtain the desired product (3Z)-2-oxo-3-(3-oxoindolan-2-ylidene)-N-tetrahydropyran-4-yl-indolan-1-carboxamide (277 mg, 0.676 mmol, yield 88.62%).

[0310] UPLC-MS analysis (sample number: 142295-1) (4 min, alkaline): rt = 2.14 min, m / Z = 390.3 [M+H]+, purity 95%. ¹H NMR analysis (sample number: 142295-1) (400 MHz, CDCl₃): δ 10.41 (s, ¹H), 8.92 (d, J = 7.9 Hz, ¹H), 8.72 (d, J = 7.4 Hz, ¹H), 8.27 (d, J = 8.2 Hz, ¹H), 7.70 (d, J = 7.6 Hz, ¹H), 7.53–7.44 (m, ¹H), 7.35–7.26 (m, 1H), 7.23–7.15 (m, 1H), 7.05–6.93 (m, 2H), 4.10–3.99 (m, 1H), 3.95 (dt, J=11.9, 3.9Hz , 2H), 3.51 (td, J=11.8, 11.3, 2.4Hz, 2H), 2.04–1.95 (m, 2H), 1.60 (qd, J=10.7, 4.2Hz, 2H).

[0311]

[0312] (Z)-1'-Undecanoyl-[2,3'-Biindole-2',3-dione

[0313] Under a nitrogen atmosphere, at 0 °C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1 M, THF solution) (1.05 eq, 0.80 mL, 0.801 mmol) was added dropwise to a solution of (3Z)-3-(3-oxoindolano-2-ylidene)indolano-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17 mL). The reaction mixture was stirred for 10 min, then undecanoyl chloride (1.20 eq, 0.20 mL, 0.915 mmol) was added, and the reaction mixture was heated to room temperature and stirred for 2 h, followed by concentration under vacuum. The crude substance was purified by automated column chromatography on a silica (40 g column) by elution with a gradient of MeOH (0% to 10% v / v) in CH2Cl2 to give the desired product (3Z)-3-(3-oxoindolman-2-ylidene)-1-undecanoyl-indolman-2-one (298 mg, 0.664 mmol, 87.13% yield) as a purple solid.

[0314] UPLC-MS analysis (sample number: 142535-2) (4 min, alkaline): rt = 3.08 min, m / Z = 431.4 [M+H]+, purity 96%. ¹H NMR analysis (sample number: 142535-2) (400 MHz, CDCl₃): δ 10.50 (s, ¹H), 8.95 (d, J = 7.6 Hz, ¹H), 8.24 (d, J = 8.2 Hz, ¹H), 7.68 (d, J = 7.6 Hz, ¹H), 7.46 (t, J = 7.7 Hz, ¹H), 7.29 (t, J = 7.6 Hz, ¹H). Hz, 1H), 7.24–7.16 (m, 1H), 6.99 (t, J) = 7.5Hz, 1H), 6.93 (d, J = 8.0Hz, 1H), 3.09 (t , J=7.5Hz, 2H), 1.72 (p, J=7.5Hz, 2H), 1.43–1.14 (m, 14H)), 0.81 (t, J=6.6Hz, 3H).

[0315]

[0316] (Z)-N-(2-morpholinoethyl)-2',3-dioxo-[2,3'-biindolyl]-1'-formamide

[0317] Under a nitrogen atmosphere at 0°C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1M in THF) (1.50 eq, 1.1 mL, 1.14 mmol) was added to a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17.0 mL). The reaction mixture was stirred for 10 min, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (6.00 mL) was added. The reaction mixture was stirred for 1 h and then allowed to warm to room temperature. A solution of N-(2-aminoethyl)morpholine (2.50 eq, 0.32 mL, 1.91 mmol) in THF (2.40 mL) was added, and the reaction mixture was stirred for 1 h. The mixture was then concentrated under vacuum and stored in a refrigerator. The crude substance was purified by automated column chromatography using silica (40 g column) and eluted with a gradient of methanol in CH2Cl2 (0% to 5%; v / v) to give the desired product (3Z)-N-(2-morpholinoethyl)-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carboxamide (210 mg, 0.487 mmol, yield 63.83%) as a dark purple solid.

[0318] UPLC-MS analysis (sample number: 143019-2) (4 min, alkaline): rt = 2.02 min, m / Z = 419.4 [M+H]+, purity 97%. ¹H NMR analysis (sample number: 143019-2) (400MHz, CDCl₃) δ 10.37 (s, ¹H), 8.94–8.78 (m, 2H), 8.25 (d, ¹H), 7.67 (d, J = 7.7 Hz, 1H), 7.46 (dd, J = 7.7, 7.7 Hz, 1H), 7.28 (dd, J = 7.9, 7.9 Hz, 1H), 7.21–7.10 (m, 1H), 7.04–6.91 (m, 2H), 3.76–3.68 (m, 4H), 3.54–3.45 (m, 2H), 2.62–2.40 (m, 6H).

[0319]

[0320] (Z)-N-(4-(dimethylamino)butyl)-2',3-dioxo-[2,3'-biindolyl]-1'-formamide

[0321] Under a nitrogen atmosphere at 0 °C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1 M, THF solution) (1.50 eq, 1.1 mL, 1.14 mmol) was added to a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17.0 mL); the reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8.40 mL) was added. The reaction mixture was allowed to warm to room temperature and stirred for 1 h; then N',N'-dimethylbutane-1,4-diamine (2.50 eq, 0.26 mL, 1.91 mmol) was added and the reaction mixture was stirred for 1 h. The crude reaction mixture was concentrated under vacuum, and then purified by automated column chromatography on a silica (40 g column) using a gradient elution of MeOH (0% to 20% v / v) in CH2Cl2 to give the desired product (3Z)-N-[4-(dimethylamino)butyl]-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide (270 mg, 0.648 mmol, 84.91% yield), as a deep purple crystalline solid.

[0322] UPLC-MS analysis (sample number: 143194-1) (4 min, alkaline): rt = 2.05 min, m / Z = 405.4 [M+H]+, purity 97%. ¹H NMR analysis (sample number: 143194-1) (400 MHz, CDCl₃): δ 10.53 (s, ¹H), 8.98 (d, J = 7.9 Hz, ¹H), 8.87–8.79 (m, ¹H), 8.32 (d, J = 8.2 Hz, ¹H), 7.76 (d, J = 7.7 Hz, ¹H), 7.55 (dd, J = 7.8 Hz, ¹H). 7, 7.7Hz, 1H), 7.36 (dd, J=7.9, 7.9Hz, 1H), 7.29–7.21 (m, 1H), 7.12–7.04 (m, 2H) , 3.49 (dt, J=6.6, 6.6Hz, 2H), 2.79-2.67 (m, 2H), 2.54 (s, 6H), 1.89-1.68 (m, 4H).

[0323]

[0324] (Z)-(2-(2',3-dioxo-[2,3'-biindolan-1'-yl-formylamino)ethyl)(methyl)carbamate tert-butyl ester

[0325] Under a nitrogen atmosphere at 0 °C, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1M, THF solution) (1.50 eq, 1.10 mL, 1.14 mmol) was added to a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (17.0 mL); the reaction mixture was stirred for 10 min, then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8.40 mL) was added, and the reaction mixture was allowed to warm to room temperature and stirred for 30 min. N-(2-aminoethyl)-N-methyl-tert-butyl carbamate (2.50 eq, 0.34 mL, 1.91 mmol) was added, and the reaction mixture was stirred for 1 h. The crude material was purified by automated column chromatography on a silica (40 g column) by gradient elution with MeOH (0% to 15% v / v) in CH2Cl2 to give the desired product, N-methyl-N-[2-[[(3Z)-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carbonyl]amino]ethyl]tert-butyl carbamate (551 mg, 0.774 mmol, 101.54% yield), as a deep purple viscous oil. This product was used without further purification.

[0326]

[0327] (Z)-2-(2',3-dioxo-[2,3'-biindolan-1'-yl]-1'-formylamino)-

[0328] N-Methylethane-1-ammonium chloride

[0329] At room temperature, hydrochloric acid solution (4.00 eq, 1.20 mL, 4.80 mmol) was added to a solution of N-methyl-N-[2-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]ethyl]carbamate (1.00 eq, 551 mg, 0.774 mmol) in DCM (12.0 mL); the reaction mixture was stirred for 22 hours. The reaction mixture was filtered, and the filter cake was washed successively with CH2Cl2 and diethyl ether to give the desired product methyl-[2-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]ethyl]ammonium chloride (264 mg, 0.662 mmol, yield 85.47%) as a deep purple solid.

[0330] UPLC-MS analysis (sample number: 143976-1) (4 min, alkaline): rt = 1.79 min, m / Z = 363.2 [M+H]+, purity 100% (mass corresponds to product without chloride anions). ¹H NMR analysis (sample number: 143976-1): δ 11.26 (s, 1H), 9.01 (bs, 2H), 8.92 (d, J = 7.9 Hz, 1H), 8.86 (t, J = 5.9 Hz, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.62 (dd, J = 7.6, 7.6 Hz, 1H). , 7.44 (d, J=8.0Hz, 1H), 7.37 (dd, J=7.9, 7.9Hz, 1H), 7.24 (dd, J=7.7, 7.7Hz, 1H), 7.09 ( dd, J=7.4, 7.4Hz, 1H), 3.69 (dt, J=6.0Hz, 2H), 3.18-3.09 (m, 2H), 2.58 (t, J=5.4Hz, 3H).

[0331]

[0332] (Z)-1'-((4-(dimethylamino)piperidin-1-yl)methyl)-[2,3'-biindolyl]-2',3-dione

[0333] A solution of (3Z)-3-(3-oxoindolman-2-ylidene)indolman-2-one (1.00 eq, 200 mg, 0.763 mmol), 4-(dimethylamino)piperidine (1.95 eq, 0.17 mL, 1.49 mmol), and formaldehyde (37% aqueous solution) (1.50 eq, 0.085 mL, 1.14 mmol) in THF (7.6 mL) was heated at 55 °C for 20 h. The reaction mixture was concentrated under vacuum and the solid was washed with a minimal amount of hexane to give the desired product (3Z)-1-[[4-(dimethylamino)-1-piperidinyl]methyl]-3-(3-oxoindolman-2-ylidene)indolman-2-one (271 mg, 0.673 mmol, 88.29% yield) as a deep red solid.

[0334] UPLC-MS analysis (sample number: 146220-1) (4 min, alkaline): rt = 1.96 min, m / Z = 403.4 [M+H]+, purity 28%. The product degraded under LCMS conditions. ¹H NMR analysis (sample number: 146220-1): δ 10.57 (s, ¹H), 8.91 (d, J = 7.9 Hz, ¹H), 7.74 (d, J = 7.6 Hz, ¹H), 7.51 (dd, J = 7.6 Hz, ¹H), 7.31 (dd, J = 7.8 Hz, ¹H), 7.14 (dd, J = 7.7 Hz, ¹H), 7.10–6.93 (m, 3H), 4.59 (s, 2H), 3.13–3.06 (m, 2H), 2.34–2.20 (m, 8H), 2.13–2.02 (m, 1H), 1.84–1.77 (m, 2H), 1.65–1.44 (m, 2H).

[0335]

[0336] (Z)-1'-(azacyclobutane-1-ylmethyl)-[2,3'-biindolyl]-2',3-dione

[0337] A solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol), aziridine (1.95 eq, 0.10 mL, 1.49 mmol), and formaldehyde (37% aqueous solution) (1.50 eq, 0.085 mL, 1.14 mmol) in THF (7.6 mL) was stirred at 45 °C for 71 h. The reaction mixture was concentrated under vacuum and the solid was washed with a minimal amount of hexane to give the desired product (3Z)-1-(aziridine-1-ylmethyl)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (240 mg, 0.688 mmol, 90.22% yield) as a deep red solid (the desired product contains 5% methylene alcohol byproduct).

[0338] UPLC-MS analysis (sample number: 146352-1) (4 min, alkaline): rt = 2.01 min, m / Z = 332.2 [M+H]+, purity 64%. The product degraded under LCMS conditions. 1H NMR analysis (sample number: 146352-1) δ 10.56 (s, 1H), 8.91 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.51 (dd, J = 7.7, 7.7 Hz, 1H), 7.32 (dd, J = 7.6, 7.6 Hz, 1H), 7.15 (dd, J = 7.6, 7.6 Hz, 1H), 7.07-6.94 (m, 3H), 4.51 (s, 2H), 3.40 (t, J = 7.0 Hz, 4H), 2.04 (p, J = 7.0 Hz, 2H).

[0339]

[0340] (Z)-1'-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-[2,3'-biindolyl]-2',3-dione

[0341] A solution of (3Z)-3-(3-oxoindolman-2-ylidene)indolman-2-one (1.00 eq, 200 mg, 0.763 mmol), formaldehyde (37% aqueous solution) (1.50 eq, 0.085 mL, 1.14 mmol), and N,N,N'-trimethyl-1,2-ethylenediamine (1.95 eq, 0.19 mL, 1.49 mmol) in THF (7.6 mL) was stirred at 45 °C for 71 h. The reaction mixture was concentrated under vacuum, and the solid was washed with a minimal amount of hexane to give the desired product (3Z)-1-[[2-(dimethylamino)ethyl-methyl-amino]methyl]-3-(3-oxoindolman-2-ylidene)indolman-2-one (258 mg, 0.624 mmol, yield 81.78%) as a deep red solid (the desired product contains about 9% methylene alcohol byproduct).

[0342] UPLC-MS analysis (sample number: 146353-1) showed that the product was completely degraded under LCMS conditions. ¹H NMR analysis (sample number: 146353-1) (400MHz, DMSO) δ 11.11 (s, 1H), 8.82 (d, J = 7.8Hz, 1H), 7.66 (d, J = 7.6Hz, 1H), 7.58 (dd, J = 7.7, 7.7Hz, 1H), 7.42 (d, J = 8.0Hz, 1H), 7.32 (dd, J = 7.8, 7.8Hz, 1H), 7.23 (d, J = 7.9Hz, 1H), 7.15–6.99 (m, 2H), 4.57 (s, 2H), 2.62 (t, J = 6.9Hz, 2H), 2.36 (t, J = 6.8Hz, 3H), 2.27 (s, 3H), 2.12 (s, 6H).

[0343]

[0344] (Z)-1'-(morpholinomethyl)-[2,3'-biindolyl]-2',3-dione

[0345] A solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol), formaldehyde (37% aqueous solution) (1.50 eq, 0.085 mL, 1.14 mmol), and morpholine (1.95 eq, 0.13 mL, 1.49 mmol) in THF (7.6 mL) was stirred at 85 °C for 108 hours. The crude product was purified by automated column chromatography (40 g column) using a gradient elution of MeOH (0% to 15% v / v) in CH2Cl2 to give the desired product (3Z)-1-(morpholinomethyl)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (270 mg, 0.747 mmol, yield 97.97%) as a deep purple solid.

[0346] UPLC-MS analysis (sample number: 146704-3) (4 min, alkaline): rt = 1.99 min, m / Z = 362.3 [M+H]+, purity 84%. The product degraded under LCMS conditions. 1H-NMR analysis (sample number: 146704-3) (400MHz, DMSO) δ 11.13 (s, 1H), 8.83 (d, J = 7.8Hz, 1H), 7.67 (d, J = 7.6Hz, 1H), 7.59 (dd, J = 7.7, 7.7Hz, 1H), 7.42 (d, J = 8.1Hz, 1H), 7.34 (dd, J = 7.6, 7.6Hz, 1H), 7.24 (d, J = 7.9Hz, 1H), 7.11 (dd, J = 7.6, 7.6Hz, 1H), 7.04 (dd, J = 7.4, 7.4Hz, 1H), 4.57 (s, 2H), 3.55 (t, J = 4.5Hz, 4H), 2.57 (t, J = 4.5Hz, 4H).

[0347]

[0348] (3Z)-N-[2-(dimethylamino)ethyl]-2-oxo-3-(3-oxoindolome-2-ethylene)indolome-

[0349] 1-Formamide

[0350] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthyl / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. N,N-dimethylethylenediamine (4.00 eq, 0.33 mL, 3.05 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. The reaction mixture was concentrated to dryness, and the residue was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purified by rapid column chromatography, the product (3Z)-N-[2-(dimethylamino)ethyl]-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carboxamide (123 mg, 0.327 mmol, yield 42.85%) was obtained by gradient elution with 0 to 20% (v / v) DCM in methanol, as a dark purple solid.

[0351] UPLC-MS analysis (sample number: 142297-2): (4 min, alkaline): rt = 2.07 min, m / Z = 377.3 [M+H]+, purity 98%. ¹H-NMR analysis (sample number: 142297-2): (400MHz, CDCl₃) δ 10.51 (s, ¹H), 9.02–8.92 (m, 2H), 8.35 (d, J = 8.2Hz, ¹H), 7.77 (d, J = 7.6Hz, 1H), 7.56 (t, J = 7.7Hz, 1H), 7.38 (t, J = 7.8Hz, 1H), 7.26 (t, J = 7.7Hz, 1H), 7.13–7.01 (m, 2H), 3.63 (q, J = 5.9Hz, 2H), 2.70 (t, J = 6.2Hz, 2H), 2.44 (s, 6H). Methanol 0.28% w / w.

[0352]

[0353] (3Z)-N-[2-(4-methylpiperazin-1-yl)ethyl]-2-oxo-3-(3-oxoindolome-2-yl)indolome-1-

[0354] formamide

[0355] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthalene / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature (rt) and stirred for 1 h. 2-(4-methylpiperazin-1-yl)ethylamine (4.00 eq, 0.46 mL, 3.05 mmol) was added dropwise and the reaction mixture was stirred for 1 h. The reaction mixture was concentrated to dryness to obtain a residue, which was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purified by rapid column chromatography, eluted with a 0-20% (v / v) DCM gradient in MeOH, the desired product (3Z)-N-[2-(4-methylpiperazin-1-yl)ethyl]-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carboxamide (195 mg, 0.452 mmol, 59.26% yield) was obtained as a deep purple solid.

[0356] UPLC-MS analysis (sample number: 142297-2): (4 min, alkaline): rt = 1.94 min, m / Z = 432.4 [M+H]+, 100% purity. 1H-NMR analysis (sample number: 142298-2) (400 MHz, CDCl3): δ 10.50 (s, 1H), 9.01 (d, J = 7.9 Hz, 1H), 8.90 (t, J = 5.2 Hz, 1H), 8.36 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H). =7.7Hz, 1H), 7.40 (t, J = 7.8Hz, 1H), 7.29 (d, J = 8.0Hz, 1H), 7.11 (t, J = 7.4Hz, 1H) , 7.07 (d, J=8.0Hz, 1H), 3.62 (q, J=5.9Hz, 2H), 2.90–2.71 (m, 10H), 2.54 (s, 3H). Methanol 0.52% w / w.

[0357]

[0358] (3Z)-N-(2-acetamidoethyl)-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide

[0359] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthalene / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. N-acetylated ethylenediamine (4.00 eq, 0.29 mL, 3.05 mmol) was added dropwise, and the reaction mixture was stirred for 30 min. The reaction was concentrated to dryness to obtain a residue, which was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purified by rapid column chromatography, the initial product was eluted with a 0-20% (v / v) DCM gradient in MeOH to obtain a deep purple solid. The initial product was dissolved in EtOAC (50 mL) and washed successively with water (50 mL × 2) and saturated salt solution (50 mL). The organic matter was then separated and dried (Na₂SO₄), and then concentrated to dryness to recover the washing product as a deep purple solid. This substance was dried in a vacuum oven to constant mass to give the final product (3Z)-N-(2-acetamidoethyl)-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide (147 mg, 0.377 mmol, yield 49.38%) as a deep purple solid.

[0360] UPLC-MS analysis (sample number: 142383-5): (4 min, alkaline): rt = 1.74 min, m / Z = 391.3 [M+H]+, purity 98%. 1H-NMR analysis (sample number: 142383-5): (400MHz, CDCl3) δ 10.46 (s, 1H), 9.02–8.91 (m, 2H), 8.34–8.28 (m, 1H), 7.77 (d, J = 7.7Hz, 1H), 7.55 (td, J = 7.7, 1.3Hz, 1H), 7.37 (td, J = 7.9, 1.4Hz, 1H), 7.26 (s, 1H), 7.09 (td, J = 7.5, 0.8Hz, 1H), 7.04 (d, J = 8.0Hz, 1H), 6.13 (s, 1H), 3.57 (dtd, J = 27.5, 7.2, 6.6, 3.6Hz, 4H), 2.02 (s, 3H). 0.43% DCM impurities w / w. CDCl3 masking 1× aromatic CH.

[0361]

[0362] (3Z)-N-(1-Methyl-2-oxo-4-piperidinyl)-2-oxo-3-(3-oxoindolan-2-ylide)indolan-1-carboxamide

[0363] To a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in 12.0 mL of THF, sodium bis(trimethylsilyl)amino / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 minutes, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in 6 mL of THF was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. During this process, N,N-diisopropylethylamine (DIPEA) (5.00 eq, 0.66 mL, 3.81 mmol) was added dropwise to a solution of (1-methyl-2-oxo-4-piperidinyl)ammonium chloride (2.50 eq, 314 mg, 1.91 mmol) in a mixture of THF (5 mL) and DMF (2 mL) stirred in a separate flask. The resulting free base was added to the main reaction mixture and stirred at room temperature for 30 min, followed by vacuum concentration. The crude purple residue was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purification was performed by rapid column chromatography, eluting with a gradient of 0 to 20% (v / v) DCM in MeOH to give 413 mg of a deep purple solid. The crude product was dissolved in EtOAC (100 mL) and washed sequentially with water (100 mL x 3). The organic matter was then separated and dried (Na2SO4), and then concentrated to dryness to give (3Z)-N-(1-methyl-2-oxo-4-piperidinyl)-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carboxamide (206 mg, 0.495 mmol, yield 64.87%), as a dark purple solid.

[0364] UPLC-MS analysis (sample number: 142675-3): (4 min, alkaline): rt = 1.87 min, m / Z = 417.3 [M+H]+, purity 99%. 1H-NMR analysis (sample number: 142675-3): (400 MHz, CDCl3) δ 10.43 (s, 1H), 8.97 (d, J = 7.9 Hz, 1H), 8.87 (d, J = 6.9 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H). 7.08 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 4.42–4.29 (m, 1H), 3.43 (t, J = 6.2 Hz, 2H), 3.02 (s, 3H), 2.87 (dd, J = 17.1, 5.5 Hz, 1H), 2.48 (dd, J = 17.2, 8.2 Hz, 1H), 2.31–2.22 (m, 1H), 2.07–1.92 (m, 1H). DCM 1.1% w / w impurities. Slight oily peak at 1.26 ppm.

[0365]

[0366] (3Z)-N-[3-(dimethylamino)propyl]-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide

[0367] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthyl / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. 3-(dimethylamino)-1-propylamine (2.50 eq, 0.24 mL, 1.91 mmol) was added dropwise, and the reaction mixture was stirred for 30 min, then concentrated under vacuum. (3Z)-N-[3-(dimethylamino)propyl]-2-oxo-3-(3-oxoindolman-2-yl)indolman-1-carboxamide (69 mg, 0.171 mmol, yield 22.48%) was purified by preparative HPLC under alkaline conditions (water + 0.1% ammonia, acetonitrile + 0.1% ammonia) as a dark purple glassy solid.

[0368] UPLC-MS analysis (sample number: 143027-3): (4 min, alkaline): rt = 2.05 min, m / Z = 391.3 [M+H]+, purity 97%. 1H-NMR analysis (sample number: 143027-3): (400 MHz, DMSO) δ 11.24 (s, 1H), 8.91 (d, J = 7.9 Hz, 1H), 8.74 (t, J = 5.7 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H). ), 7.45 (d, J = 8.1 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.22 (t, J = 7.7 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 3.39–3.37 (m, 2H), 2.32 (t, J = 7.0 Hz, 2H), 2.17 (s, 6H), 1.71 (p, J = 7.0 Hz, 2H). Contains 3.25% indigo red (by weight). A clear MeOD of CH2 is observed, which is obscured by the H2O peak. ¹H-NMR analysis (sample number: 143027-3): (400MHz, MeOD) δ 8.90 (d, J = 7.9Hz, 1H), 8.15 (d, J = 8.2Hz, 1H), 7.64 (d, J = 7.6Hz, 1H), 7.54 (t, J = 7.8Hz, 1H), 7.26 (t, J = 7.8Hz, 1H), 7.14 (t, J = 8.1Hz, 2H), 7.04 (t, J = 7.6Hz, 1H), 3.44 (t, J = 7.0Hz, 2H), 2.50 (t, J = 7.8Hz, 2H), 2.32 (s, 6H), 1.87 (p, J = 7.2Hz, 2H). DMSO ¹H-NMR of exchangeable compounds was observed.

[0369]

[0370] (3Z)-N-[2-(methanesulfonylamino)ethyl]-2-oxo-3-(3-oxoindoman-2-methylene)indoman-1-

[0371] formamide

[0372] To a magnetically stirred solution of (3Z)-3-(3-oxoindolan-2-yl)indolan-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (10.0 mL), sodium bis(trimethylsilyl)amino / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added. The reaction mixture was stirred for 10 minutes, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (6 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. During this process, N,N-diisopropylethylamine (DIPEA) (5.00 eq, 0.66 mL, 3.81 mmol) was added dropwise to a solution of 2-(methanesulfonylamino)ethylammonium chloride (2.50 eq, 333 mg, 1.91 mmol) in a mixture of THF (4 mL) and DMF (4 mL) in a separate flask under stirring. The resulting free base was added to the main reaction mixture and stirred at room temperature for 30 minutes, followed by vacuum concentration. The crude purple residue was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purified by rapid column chromatography, the product (3Z)-N-[2-(methanesulfonylamino)ethyl]-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carboxamide (65 mg, 0.152 mmol, 19.99% yield) was obtained as a dark purple solid by elution with a 0-20% (v / v) DCM gradient in MeOH.

[0373] UPLC-MS analysis (sample number: 143028-3): (4 min, alkaline): rt = 1.83 min, m / Z = 427.3 [M+H]+, purity 97%. UPLC-MS analysis (sample number: 143028-3): ELSD (4 min, alkaline): rt = 1.87 min, m / Z = 427.2 [M+H]+, purity 94%. Note: 6% unknown substance not observed by LC-MS: rt = 2.71 min, m / Z = 282.3 [M+H]+. 1H-NMR analysis (sample number: 143028-3): 1H-NMR (400MHz, DMSO) δ 11.31 (s, 1H), 8.94 (d, J = 7.9Hz, 1H), 8.86 (t, J = 5.8Hz, 1H), 8.21 (d, J = 8.1Hz, 1H), 7.70 (d, J = 7.5Hz, 1H), 7.62 (d The concentrations were: 7.46 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.23 (q, J = 7.3 Hz, 2H), 7.09 (t, J = 7.5 Hz, 1H), 3.49 (q, J = 6.0 Hz, 2H), 3.18 (q, J = 6.0 Hz, 2H), and 2.94 (s, 3H). The sample contained 14.7% indigo red and was unstable in DMSO.

[0374]

[0375] (3Z)-N-[2-(methanesulfonylamino)ethyl]-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide

[0376] To a magnetically stirred solution of (3Z)-3-(3-oxoindolan-2-yl)indolan-2-one (1.00 eq, 200 mg, 0.763 mmol) in 10.0 mL of THF, sodium bis(trimethylsilyl)amino / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 minutes, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in 6 mL of THF was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. During this process, N,N-diisopropylethylamine (DIPEA) (5.00 eq, 0.66 mL, 3.81 mmol) was added dropwise to a solution of 2-(methanesulfonylamino)ethylammonium chloride (2.50 eq, 333 mg, 1.91 mmol) in a mixture of THF (4 mL) and DMF (4 mL) under stirring in a separate flask. The resulting free base was added to the main reaction mixture and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure to give a crude, deep purple oil. Purification by preparative HPLC under alkaline conditions (water + 0.1% ammonia, acetonitrile + 0.1% ammonia) yielded (3Z)-N-[2-(methanesulfonylamino)ethyl]-2-oxo-3-(3-oxoindolman-2-yl)indolman-1-carboxamide (38 mg, 0.0864 mmol, yield 11.33%) as a fluffy purple solid.

[0377] UPLC-MS analysis (sample number: 143334-2): (4 min, alkaline): rt = 1.84 min, m / Z = 427.1 [M+H]+, purity 97%. 1H-NMR analysis (sample number: 143334-2): (400MHz, DMSO) δ 11.30 (s, 1H), 8.91 (s, 2H), 8.77 (d, J = 7.8Hz, 1H), 8.19 (d, J = 8.2Hz, 1H), 7.65 (d, J = 7.4Hz, 2H), 7.59 (d, J = 8.4Hz, 1H), 7.42 (d, J = 8.1Hz, 2H), 7.24 (d, J = 10.5Hz, 4H), 7.03 (q, J = 10.0, 7.6Hz, 3H), 3.48 (q, J = 6.1Hz, 2H), 3.18 (s, 2H), 2.94 (s, 4H). NB 68% desired product 32% indirubin, sample immediately run.

[0378]

[0379] (3Z)-2-oxo-3-(3-oxoindolan-2-ylide)indolan-1-carboxylic acid ethyl ester

[0380] Under a nitrogen atmosphere, sodium bis(trimethylsilyl)amino sodium / NaHMDS (1M THF solution) (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise to a THF (17 mL) solution of (3Z)-3-(3-oxoindolan-2-ylidene)indolan-2-one (1.00 eq, 200 mg, 0.763 mmol) at 0 °C. The reaction mixture was stirred for 10 min, and then ethyl chloroformate (1.20 eq, 0.087 mL, 0.915 mmol) was added. The reaction mixture was then heated to room temperature and stirred for 30 min. The reaction mixture was concentrated to dryness to give a crude purple residue, which was dissolved in EtOAC (50 mL). The resulting solution was washed successively with saturated sodium bicarbonate solution (2 × 50 mL) and water (50 mL). The organic matter was then separated and dried (Na2SO4), then filtered and concentrated to dryness to obtain the desired product (3Z)-2-oxo-3-(3-oxoindolman-2-methylene)indolman-1-carboxylic acid ethyl ester (203 mg, 0.607 mmol, yield 79.62%), as a dark purple solid.

[0381] UPLC-MS analysis (sample number: 143594-2): (4 min, alkaline): rt = 2.18 min, m / Z = 335.1 [M+H]+, purity 100%. ¹H-NMR analysis (sample number: 143594-2): (400MHz, CDCl₃) δ 10.66 (s, ¹H), 9.04 (d, J = 7.9Hz, ¹H), 7.99 (d, J = 8.2Hz, ¹H), 7.76 (d, J = 7.6Hz, ¹H), 7.53 (t, J = 7.7Hz, ¹H), 7.39 (t, J = 7.8Hz, ¹H), 7.29 (d, J = 7.7Hz, ¹H), 7.06 (t, J = 7.5Hz, ¹H), 6.98 (d, J = 7.9Hz, ¹H), 4.54 (q, J = 7.1Hz, 2H), 1.51 (t, J = 7.1Hz, 3H). EtOAC 1.99% w / w. The NBNMR error is marked as 143594_1 on the PDF.

[0382]

[0383] N-[2-(trifluoromethylsulfonylamino)ethyl]carbamate tert-butyl ester

[0384] Trifluoromethanesulfonyl chloride (1.00 eq, 0.67 mL, 6.24 mmol) was added dropwise to a magnetically stirred solution of N-(2-aminoethyl)carbamate tert-butyl ester (1.00 eq, 0.99 mL, 6.24 mmol) in DCM (63.6 mL) at 0 °C. The reaction mixture was slowly heated to room temperature and stirred overnight for 20 hours. The reaction mixture was concentrated to dryness to obtain a residue, which was dissolved in EtOAC (100 mL). The resulting solution was washed successively with water (2 × 100 mL) and saturated saline solution (100 mL). The organic matter was then separated and dried over Na2SO4, filtered, and concentrated to dryness to give the crude product. Purified by rapid column chromatography, the product N-[2-(trifluoromethylsulfonylamino)ethyl]carbamate tert-butyl ester (1.68 g, 5.75 mmol, 92.09% yield) was obtained by elution with a gradient of 0 to 50% (v / v) EtOAC in isohexane, as a white solid.

[0385] ¹H-NMR analysis (sample number: 145427-2): (400 MHz, CDCl₃) δ 6.55 (s, ¹H), 4.94 (s, ¹H), 3.43–3.38 (m, 2H), 3.34 (q, J = 5.6) Hz, 2H), 1.45 (s, 9H). Note: Due to the lack of chromophores and weak / disordered IPCs, the product structure was confirmed by comparison with literature NMR data. Reference: J Am Chem Soc. 2018 Mar 7; 140(9):3202-3205.

[0386]

[0387] 2-(trifluoromethylsulfonylamino)ethylammonium; chloride

[0388] A solution of 4N HCl in dioxane (4.00 eq, 5.7 mL, 23.0 mmol) was added dropwise to a magnetically stirred solution of N-[2-(trifluoromethanesulfonylamino)ethyl]carbamate tert-butyl (1.00 eq, 1.68 g, 5.75 mmol) in 36.5 mL of DCM at room temperature. The reaction was stirred overnight, and then another 4N HCl solution in dioxane (4.00 eq, 5.7 mL, 23.0 mmol) was added. The reaction was stirred for another 24 hours until complete, and then concentrated to dryness to give a crude residue. The crude residue was concentrated twice from diethyl ether to give the desired product, 2-(trifluoromethanesulfonylamino)ethylammonium chloride (1.14 g, 4.99 mmol, yield 86.75%), as a white solid.

[0389] 1H-NMR analysis (sample number: 145775-2): (400MHz, DMSO) δ 9.90 (s, 1H), 8.23 ​​(s, 3H), 3.43 (t, J = 6.8Hz, 2H), 2.94 (t, J = 6.8Hz, 2H).

[0390]

[0391] (3Z)-2-oxo-3-(3-oxoindolome-2-yl)-N-[2-(trifluoromethylsulfonylamino)ethyl]indolome

[0392] -1-Formamide

[0393] To a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in 16.0 mL of THF, sodium bis(trimethylsilyl)amino / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 minutes, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in 8 mL of THF was rapidly added. The reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. Simultaneously, N,N-diisopropylethylamine (DIPEA) (10.0 eq, 1.3 mL, 7.63 mmol) was added dropwise to a stirred solution of 2-(trifluoromethylsulfonylamino)ethylammonium chloride (2.50 eq, 436 mg, 1.91 mmol) in DMF (4 mL) and THF (4 mL). The free base product was added to previously activated indirubin and stirring continued for 30 minutes. The reaction was concentrated to dryness to obtain a crude purple residue, which was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purification was performed by rapid column chromatography, eluting with a gradient of 0 to 15% (v / v) DCM in MeOH to obtain 325 mg of the desired product, with a purity of less than 95% as determined by LC-MS. The crude product was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. A second purification was performed by rapid column chromatography, eluting with a gradient of 0 to 15% (v / v) DCM in MeOH to give the desired product (3Z)-2-oxo-3-(3-oxoindolman-2-ylidene)-N-[2-(trifluoromethylsulfonylamino)ethyl]indolman-1-carboxamide (203 mg, 0.376 mmol, yield 49.31%), as a dark purple solid.

[0394] UPLC-MS analysis (sample number: 146019-3): (4 min, alkaline): rt = 1.71 min, m / Z = 481.2 [M+H]+, purity 89%. 1H-NMR analysis (sample number: 146019-3): (400MHz, DMSO) δ 11.23 (s, 1H), 9.68–9.53 (m, 1H), 8.94–8.84 (m, 2H), 8.17 (d, J) = 8.1Hz, 1H), 7.66 (d, J = 7.6Hz, 1H), 7.60 (t, J = 7.7Hz, 1H), 7.43 (d, J = 8.0Hz, 1H), 7.34 (t, J = 7.8Hz, 1H), 7.21 (t, J = 7.7Hz, 1H), 7.07 (t, J = 7.4Hz, 1H), 3.50 (q, J = 6.1Hz, 2H), 3.40 (d, J = 6.0Hz, 2H). Due to the co-elution of phenolic esters, the purity of NB is <90%.

[0395]

[0396] N-[2-[[(3Z)-2-oxo-3-(3-oxoindolome-2-yl)indolome-1-carbonyl]amino]ethyl]tert-butyl carbamate

[0397] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthyl / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrophenyl chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. N-(2-aminoethyl)carbamate tert-butyl ester (2.50 eq, 305 mg, 1.91 mmol) was added dropwise, and the reaction mixture was stirred for 30 min. The reaction mixture was concentrated to dryness to give a crude purple residue, which was dissolved in a minimal amount of DCM and injected into a 40 g SiO2 column. Purified by rapid column chromatography, the product N-[2-[[(3Z)-2-oxo-3-(3-oxoindolman-2-ylidene)indolman-1-carbonyl]amino]ethyl]carbamate tert-butyl ester (608 mg, 1.13 mmol, yield 147.55%) was obtained as a dark purple solid by gradient elution with 0 to 10% (v / v) DCM in methanol.

[0398] UPLC-MS analysis (sample number: 145405-2): (2 min, alkaline): rt = 1.28 min, m / Z = 447.2 [MH]-, purity 83%.

[0399]

[0400] 2-[[(3Z)-2-oxo-3-(3-oxoindo-2-subunit)indo-1-

[0401] [Carbonyl]amino]ethylammonium; chloride

[0402] A solution of dioxane in 4N HCl (4.00 eq, 1.1 mL, 4.50 mmol) was added dropwise to a magnetically stirred solution of N-[2-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]ethyl]carbamate (1.00 eq, 608 mg, 1.13 mmol) in DCM (13.6 mL) at room temperature. The reaction was stirred overnight, resulting in a deep purple precipitate. The precipitate was collected by vacuum filtration and washed with DCM (10 mL) and diethyl ether (2 x 10 mL) to give 2-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]ethylammonium chloride (158 mg, 0.386 mmol, yield 34.30%) as a deep purple solid.

[0403] UPLC-MS analysis (sample number: 145541-1): (4 min, alkaline): rt = 1.77 min, m / Z = 349.0 [M+H]+[-HCl], purity 94%. ¹H NMR analysis (sample number: 145541-1): (400 MHz, DMSO) δ 11.27 (s, 1H), 8.94 (d, J = 7.9 Hz, 1H), 8.83 (t, J = 5.9 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.10 (s, 3H), 7.70 (d, J = 7.6 Hz, 1H). ), 7.63 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 3.63 (q, J = 6.1 Hz, 2H), 3.04 (q, J = 5.9 Hz, 2H). Unknown trace impurities were present at NB 3.09, 3.15, and 8.37 ppm.

[0404]

[0405] N-[3-[[(3Z)-2-oxo-3-(3-oxoindo-2-subunit)indo-1-

[0406] [Carbonyl]amino]propyl]tert-butyl carbamate

[0407] To a solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (16.0 mL), sodium bis(trimethylsilyl)aminonaphthalene / NaHMDS (1.50 eq, 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, and then a solution of 4-nitrobenzene chloroformate (1.80 eq, 277 mg, 1.37 mmol) in THF (8 mL) was rapidly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. N-(3-aminopropyl)carbamate tert-butyl ester (2.50 eq, 0.33 mL, 1.91 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. The reaction mixture was concentrated to dryness to give a crude purple residue, which was dissolved in a minimal amount of DCM and injected onto a 40 g SiO2 column. Purified by rapid column chromatography, eluted with a 0 to 10% (v / v) DCM gradient in MeOH, the desired product N-[3-[[(3Z)-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carbonyl]amino]propyl] tert-butyl carbamate (522 mg, 0.880 mmol, 115.44% yield) was obtained as a dark purple solid.

[0408] UPLC-MS analysis (sample number: 145891-2): (2 min, alkaline): rt = 1.32 min, m / Z = 461.2 [MH]-, 78% purity.

[0409]

[0410] 3-[[(3Z)-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carbonyl]amino]propylammonium; chloride

[0411] A solution of dioxane in 4NHCl (4.00 eq, 1.4 mL, 5.42 mmol) was added dropwise to a magnetically stirred solution of N-[3-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]propyl]carbamate (1.00 eq, 522 mg, 1.13 mmol) in DCM (13.6 mL) at room temperature. The reaction was stirred overnight to produce a deep purple precipitate. The precipitate was collected by vacuum filtration and washed with DCM (10 mL) and diethyl ether (2 × 10 mL) to give 3-[[(3Z)-2-oxo-3-(3-oxoindolome-2-ylidene)indolome-1-carbonyl]amino]propylammonium chloride (83 mg, 0.200 mmol, 27.00% yield).

[0412] UPLC-MS analysis (sample number: 145967-1): (4 min, alkaline): rt = 1.71 min, m / Z = 363.2 [M+H]+[-HCl], purity 96%. 1H-NMR analysis (sample number: 145967-1): 400 MHz, DMSO) δ 11.24 (s, 1H), 8.89 (d, J = 7.9 Hz, 1H), 8.73 (t, J = 5.7 Hz, 1H), 8.12 (t, J = 7.2 Hz, 4H), 7.66 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.7 Hz). z, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 3.44 (q, J = 6.5 Hz, 2H), 2.89 (h, J = 6.3 Hz, 2H), 1.90 (p, J = 7.1 Hz, 2H). NB Indigo Red 2.57% w / w. DCM 0.08% w / w.

[0413]

[0414] (3Z)-1-[(4,4-difluoro-1-piperidinyl)methyl]-3-(3-oxoindolan-2-ethylene)indolan-2-one

[0415] Formaldehyde, 37 wt.% H₂O solution (1.50 eq, 0.084 mL, 1.14 mmol) and 4,4-difluoropiperidine (1.90 eq, 0.16 mL, 1.45 mmol) were added to a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (7.6 mL). The reaction mixture was heated to 55 °C for 96 hours, then cooled and concentrated under vacuum. The resulting crude residue was suspended in hexane (20 mL) and collected by suction filtration. The initial product was washed with a further fraction of hexane (2 × 20 mL), collected, and dried on a rotary evaporator. The obtained viscous purple solid was sonicated into diethyl ether (20 mL) and dried again under reduced pressure to give (3Z)-1-[(4,4-difluoro-1-piperidinyl)methyl]-3-(3-oxoindolman-2-ylidene)indolman-2-one (234 mg, 0.592 mmol, 77.60% yield), a free-flowing dark purple solid.

[0416] UPLC-MS analysis (sample number: 146375-1): (4 min, alkaline): rt = 2.27 min, m / Z = 396.1 [M+H]+, purity 81% [Note 1]. ¹H-NMR analysis (sample number: 146375-1): 400 MHz, CDCl₃) δ 10.54 (s, ¹H), 8.92 (d, J = 7.8 Hz, ¹H), 7.75 (d, J = 7.6 Hz, ¹H), 7.51 (t, J = 7.7 Hz, ¹H), 7.32 (t, J = 7.8 Hz, ¹H), 7.16 (t, J = 7.7 Hz, ¹H), 7.03 (t, J = 6.6 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 4.61 (s, 2H), 2.79 (t, J = 5.8 Hz, 4H), 2.00 (tt, J = 13.0, 5.6 Hz, 4H). No 1x exchangeability was observed in NB.

[0417]

[0418] (3Z)-3-(3-oxoindolome-2-yl)-1-(1-piperidinylmethyl)indolome-2-one

[0419] Formaldehyde, 37 wt.% H₂O solution (1.50 eq, 0.084 mL, 1.14 mmol), and piperidine (1.90 eq, 0.14 mL, 1.45 mmol) were added to a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (7.6 mL). The reaction mixture was heated to 55 °C for 96 hours, then cooled and concentrated under vacuum. The resulting crude residue was suspended in hexane (20 mL) and collected by suction filtration. The initial product was washed with a further fraction of hexane (2 × 20 mL), collected, and dried on a rotary evaporator. The obtained viscous red solid was sonicated into diethyl ether (20 mL) and dried again under reduced pressure to obtain (3Z)-3-(3-oxoindolman-2-ylidene)-1-(1-piperidinylmethyl)indolman-2-one (259 mg, 0.721 mmol, yield 94.49%), a free-flowing dark red solid.

[0420] UPLC-MS analysis (sample number: 146391-1): (4 min, alkaline): rt = 1.76 min, m / Z = 261.1 [MH]-, purity 96%. ¹H-NMR analysis (sample number: 146391-1): (400 MHz, CDCl₃) δ 10.58 (s, ¹H), 8.91 (d, J = 7.8 Hz, ¹H), 7.74 (d, J = 7.6 Hz, ¹H), 7.50 (t, J = 7.6 Hz, ¹H), 7.31 (t, J = 7.7 Hz, ¹H), 7.14 (t, J = 7.7 Hz, ¹H). =7.7Hz, 1H), 7.07(d, J = 7.9Hz, 1H), 7.01(t, J = 7.5Hz, 1H), 6.96(d, J = 8.0Hz, 1H), 4.56(s, 2H), 2.62(t, J = 5.3Hz, 4H), 1.58(d, J = 5.8Hz, 4H), 1.41(q, J = 5.9Hz, 2H). No 1x commutativity was observed in NB.

[0421]

[0422] (3Z)-3-(3-oxoindolan-2-yl)-1-(pyrrolidone-1-ylmethyl)indolan-2-one

[0423] Formaldehyde, 37 wt.% H₂O solution (1.50 eq, 0.084 mL, 1.14 mmol), and pyrrolidine (1.90 eq, 0.12 mL, 1.45 mmol) were added to a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (7.6 mL). The reaction mixture was heated to 55 °C for 96 hours, then cooled and concentrated under vacuum. The resulting crude residue was suspended in hexane (20 mL) and collected by suction filtration. The initial product was washed with additional hexane (2 x 20 mL), collected, and dried on a rotary evaporator. The resulting viscous red solid was sonicated into ether (20 mL) and dried again under reduced pressure to obtain (3Z)-3-(3-oxoindolman-2-ylidene)-1-(pyrrolidine-1-ylmethyl)indolman-2-one (255 mg, 0.738 mmol, yield 96.81%), a free-flowing dark red solid.

[0424] UPLC-MS analysis (sample number: 146393-1): (4 min, alkaline): rt = 1.76 min, m / Z = 261.1 [MH]-, purity 96%. ¹H-NMR analysis (sample number: 146393-1): (400 MHz, CDCl₃) δ 10.58 (s, ¹H), 8.91 (d, J = 7.8 Hz, ¹H), 7.74 (d, J = 7.6 Hz, ¹H), 7.50 (t, J = 7.7 Hz, ¹H), 7.31 (t, J = 7.7 Hz, ¹H), 7.14 (t, J = 7.7 Hz, ¹H), 7.08 (d, J = 7.9 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 4.74 (s, 2H), 2.75 (d, J = 6.1 Hz, 4H), 1.84–1.72 (m, 4H). No 1x exchangeability was observed in NB.

[0425]

[0426] (3Z)-1-[(4-acetylpiperazin-1-yl)methyl]-3-(3-oxoindolman-2-ethylene)indolman-2-one

[0427] Formaldehyde, 37 wt.% H₂O solution (1.50 eq, 0.084 mL, 1.14 mmol) and 1-acetylpiperazine (1.90 eq, 0.19 mL, 1.45 mmol) were added to a magnetically stirred solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (7.6 mL). The reaction mixture was heated to 55 °C for 72 h, then cooled and concentrated under vacuum. The resulting crude residue was suspended in hexane (20 mL) and collected by filtration. The initial product was washed with additional hexane (2 x 20 mL), collected, and dried on a rotary evaporator. The resulting residue was sonicated to diethyl ether (20 mL) and re-dried under reduced pressure to give a crude solid. The crude material was dissolved in a minimal amount of DCM and injected into a 40 g SiO₂ column. Purified by rapid column chromatography, the product (3Z)-1-[(4-acetylpiperazin-1-yl)methyl]-3-(3-oxoindolman-2-ylidene)indolman-2-one (147 mg, 0.334 mmol, yield 43.78%) was obtained as a dark purple solid by gradient elution with 0 to 15% (v / v) DCM in methanol.

[0428] UPLC-MS analysis (sample number: 146684-2): (4 min, alkaline): rt = 1.81 min, m / Z = 403.3 [M+H]+, purity 82%. 1H-NMR analysis (sample number: 146684-2): (400MHz, DMSO) δ 11.12 (s, 1H), 8.82 (d, J = 7.8Hz, 1H), 7.66 (d, J = 7.6Hz, 1H), 7.59 (t, J = 7.7Hz, 1H), 7.42 (d, J = 8.0Hz, 1H), 7.33 (t, J = 7.4Hz, 1H), 7.25 (d, J = 7.9Hz, 1H), 7.10 (t, J = 7.7Hz, 1H), 7.03 (t, J = 7.4Hz, 1H), 4.63 (s, 2H), 3.40 (q, J = 4.9Hz, 4H), 2.64–2.51 (m, 4H), 1.94 (s, 3H). NB contains 8.6% of the corresponding methylene alcohol w / w and 4.15% of residual DCM.

[0429]

[0430] (3Z)-1-[[bis[2-(dimethylamino)ethyl]amino]methyl]-3-(3-oxoindolan-2-ethylene]indolan-2-one

[0431] To a magnetically stirred solution of (3Z)-3-(3-oxoindolman-2-ylidene)indolman-2-one (1.00 eq, 200 mg, 0.763 mmol) in THF (7.6 mL), 37 wt.% aqueous formaldehyde solution (1.50 eq, 0.084 mL, 1.14 mmol) and N-[2-(dimethylamino)ethyl]-N',N'-dimethyl-ethane-1,2-diamine (1.90 eq, 0.28 mL, 1.45 mmol) were added. The reaction mixture was heated to 55 °C for 72 hours, then cooled and concentrated under vacuum. The resulting crude residue was suspended in hexane (20 mL) and collected by suction filtration. The initial product was washed with additional hexane (2 x 20 mL), collected, and dried on a rotary evaporator. The resulting residue was sonicated into 20 mL of ether and dried again under reduced pressure to give (3Z)-1-[[bis[2-(dimethylamino)ethyl]amino]methyl]-3-(3-oxoindolman-2-ylidene)indolman-2-one (114 mg, 0.263 mmol, yield 34.48%), a dark purple solid.

[0432] UPLC-MS analysis (sample number: 146685-1): (4 min, alkaline): rt = 2.27 min, m / Z = 434.4 [M+H]+, purity 3%. 1H-NMR analysis (sample number: 146685-1): (400 MHz, DMSO) δ 11.08 (s, 1H), 8.81 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H). 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 4.64 (s, 2H), 2.69 (t, J = 7.0 Hz, 4H), 2.33 (t, J = 7.0 Hz, 4H), 2.10 (d, J = 2.2 Hz, 12H). NB contains 4.5% methylene alcohol (w / w).

[0433]

[0434] N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]tert-butyl carbamate

[0435] A solution of 4-nitrobenzene-1-sulfonyl chloride (1.05 eq, 1.45 g, 6.55 mmol) in CH2Cl2 (58.1 mL) was added dropwise to a solution of N-(2-aminoethyl)carbamate tert-butyl ester (1.00 eq, 0.99 mL, 6.24 mmol) and triethylamine (2.00 eq, 1.7 mL, 12.5 mmol) in CH2Cl2 (150 mL). The reaction mixture was slowly heated to room temperature and stirred overnight for 20 hours. The reaction mixture was concentrated under vacuum to a volume of about 100 mL, washed with water (3 x 50.0 mL), dried (Na2SO4), and concentrated under vacuum to give a crude product as a colorless solid. The crude substance was purified by automated column chromatography using silica gel (40 g column) and eluted with EtOAC (0% to 100%; v / v) gradient in isohexane to give colorless solid N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]carbamate tert-butyl ester (1.79 g, 5.18 mmol, yield 83.03%).

[0436] UPLC-MS analysis (sample number: 143957-2) (2 min, alkaline): rt = 1.03 min, m / Z = 344.2 [MH]-, 100% purity. ¹H NMR analysis (sample number: 143957-2): δ 8.36 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 5.70 (bs, 1H), 4.79 (bs, 1H), 3.25 (dt, J = 5.7, 5.7 Hz, 2H), 3.14 (dt, J = 5.4, 5.4 Hz, 2H), 1.43 (s, 9H).

[0437]

[0438] 2-[(4-nitrophenyl)sulfonylamino]ethylammonium; chloride

[0439] At room temperature, hydrogen chloride solution (4.00 eq, 2.9 mL, 1.00 g, 2.90 mmol) was added to a DCM suspension of N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]carbamate (1.00 eq, 1.00 g, 2.90 mmol) (19.3 mL, 4 M 1,4-dioxane solution) (4.00 eq, 2.9 mL, 11.6 mmol). The reaction mixture was filtered, and the filter cake was washed successively with CH2Cl2 and diethyl ether to give the desired product 2-[(4-nitrophenyl)sulfonylamino]ethylammonium chloride (816 mg, 2.90 mmol, 100.04% yield) as a colorless powder.

[0440] UPLC-MS analysis (sample number: 145404-1) (2 min, alkaline): rt = 0.72 min, m / Z = 246.2 [M+H]+, purity 100% (mass corresponds to product without chloride anions). ¹H NMR analysis (sample number: 145404-1) (400 MHz, DMSO): δ 8.56 (t, J = 5.8 Hz, 1H), 8.44 (d, J = 8.4 Hz, 2H), 8.27–8.06 (m, 5H), 3.06 (q, J = 6.3 Hz, 2H), 2.86 (h, J = 6.1 Hz, 2H).

[0441]

[0442] (3Z)-N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]-2-oxo-3-(3-oxoindolan-2-ylidene)indolan-1-carboxamide

[0443] Under a nitrogen atmosphere, sodium bis(trimethylsilyl)aminosodium / NaHMDS (1.50 eq, 0.57 mL, 0.572 mmol) was added to a THF (7.40 mL) solution of (3Z)-3-(3-oxoindolomellon-2-yl)indolomellon-2-one (1.00 eq, 100 mg, 0.381 mmol) at 0 °C; the reaction was stirred for 10 minutes, and then a THF (5.00 mL) solution of 4-nitrophenyl chloroformate (1.80 eq, 138 mg, 0.686 mmol) was added at 0 °C, and the reaction mixture was stirred for 30 minutes. A suspension of N-(2-aminoethyl)-4-nitrobenzenesulfonamide; hydrochloride (2.33 eq, 250 mg, 0.887 mmol) and N,N-diisopropylethylamine (DIPEA) (5.00 eq, 0.33 mL, 1.91 mmol) in THF (6.00 mL) was stirred for 25 minutes, then added to a mixture containing indirubin; the reaction mixture was stirred for 1 hour. The reaction mixture was concentrated under vacuum, and the crude product was purified by automated column chromatography on a silica gel (25 g column) using a gradient elution of methanol (0% to 15%; v / v) in CH2Cl2 to give a deep purple solid. The purity of the product was below the threshold required for compound submission, therefore the crude material was purified by automated column chromatography on a silica gel (40 g column) using a gradient elution of MeOH (0% to 15%; v / v) in CH2Cl2 to give a deep purple solid.

[0444]

[0445] (2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-carboxylic acid methyl ester

[0446] Chloromethyl chloroformate (4.00 mL, 45.0 mmol) was added dropwise to a slurry of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (5.36 g, 20.4 mmol; lot number = 2009-BML-4B) in tetrahydrofuran (100 mL, 1230 mmol) and triethylamine (6.3 mL, 45 mmol). After 30 minutes, HPLC showed a conversion of 97%. The slurry was heated to room temperature and stirred for another hour. HPLC showed a conversion of 99%. The reaction was stirred overnight.

[0447] HPLC showed a conversion of the starting material >99%. The mixture was filtered through glass frit. The solid was washed with water (200 mL). The solid was dried on glass frit for 1 hour, then transferred to a flask and dried in a vacuum oven (50 °C) for 24 hours. HPLC analysis of the solid showed the presence of <1 area % indirubin. [B](2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-carboxylic acid methyl ester (4.81 g; yield = 66.3%; lot number = 2009-B ML-22A) was obtained as a purple solid.

[0448]

[0449] (2Z)-1-acetyl-2,3'-biindole-2',3(1H,1'H)-dione and (2Z)-1'-acetyl-2,3'-biindole

[0450] -2',3(1H,1'H)-dione

[0451] A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (1.21 g, 4.61 mmol; lot number = 1951-BML-31C) in acetic anhydride (2.18 mL, 23.1 mmol) and sulfuric acid (0.04 mL, 0.8 mmol) was heated for 10 min at 140 °C in an aluminum heating block. HPLC showed high conversion, so the mixture was cooled to room temperature and filtered through a glass frit. The solid was washed with water (4 x 50 mL). NMR showed only one product. The substance was dried overnight in a vacuum oven (~50 °C). [B](2Z)-1'-acetyl-2,3'-biindole-2',3(1H,1'H)-dione (1.25 g; yield = 89.0%; lot number = 2009-B ML-12A) was given as a purple solid.

[0452]

[0453] (2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione

[0454] A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (10.12 g, 38.59 mmol; lot number = 2009-BML-6B) in an aqueous formaldehyde solution (6.0 mL, 8.0E1 mmol), tetrahydrofuran (250 mL, 3.0E3 mmol), and triethylamine (6.3 mL, 45 mmol) was heated overnight in an oil bath at 55 °C. After 26 hours, the mixture was filtered to give [B](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (10.45 g; yield = 92.65%; lot number = 2009-B ML-82A) as a purple solid.

[0455]

[0456] [(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl acetate

[0457] Triethylamine (3.5 g, 34 mmol) and acetic anhydride (1.6 mL, 17 mmol) were added to a mixture of [A](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (3.34 g, 11.4 mmol; batch number = 2009-B ML-62A) in dichloromethane (100 mL, 1560 mmol). The mixture was stirred overnight at room temperature. After 69 hours, HPLC showed complete conversion to the product. The slurry was filtered to give a purple solid, dried under nitrogen pressure, and then dried in a vacuum oven at 30 °C. A purple solid, [B][(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl acetate (2.98 g; yield = 78.0%; batch number = 2009-B ML-84A), was obtained. The solid was dried overnight in a vacuum drying oven.

[0458]

[0459] (2Z)-1'-[(diethylamino)methyl]-2,3'-biindol-2',3(1H,1'H)-dione

[0460] A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (4.95 g, 0.0189 mol; batch number = 2009-B ML-6B), 37% formaldehyde (37:63, formaldehyde:water, 2.11 mL, 0.0283 mol), and N-ethylethylamine (3.81 mL, 0.0368 mol) in tetrahydrofuran (200 mL, 2.46 mol) was heated overnight in a pressure tube in an oil bath at 55 °C.

[0461] The reaction mixture was concentrated under vacuum. NMR analysis of the solid showed no remaining starting material; the solid was a product containing residual THF and excess diethylamine. The solid was carefully washed with ethanol. The solid was dried under high vacuum for 4 hours. [B](2Z)-1'-[(diethylamino)methyl]-2,3'-biindole-2',3(1H,1'H)-dione (6.45 g; yield = 98.4%; lot number = 2009-B ML-69A) was given as a purple solid.

[0462]

[0463] (2Z)-1'-[(diethylamino)methyl]-2,3'-biindol-2',3(1H,1'H)-dione-4-methylbenzenesulfonate

[0464] In a 100 mL flask, [A](2Z)-1'-[(diethylamino)methyl]-2,3'-biindole-2',3(1H,1'H)-dione (0.35 g, 1.0 mmol; lot number = 2009-B ML-35A) and p-toluenesulfonic acid monohydrate (0.21 g, 1.1 mmol) were dissolved in tetrahydrofuran (6 mL, 70 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under vacuum. The solid was slurried in acetone and the suspension was filtered. The solid was air-dried for about 60 hours to give [B](2Z)-1'-[(diethylamino)methyl]-2,3'-biindole-2',3(1H,1'H)-dione 4-methylbenzenesulfonate (0.42 g; yield = 8.0E1%; lot number = 2009-BML-70A), as a purple solid.

[0465]

[0466] (2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindol-2',3(1H,1'H)-dione

[0467] A mixture of [A](2Z)-2,3'-biindol-2',3(1H,1'H)-dione (8.05 g, 0.0307 mol; batch number = 2009-BML-4B), 37% formaldehyde (37:63, formaldehyde:water, 3.4 mL, 0.046 mol), and [B]piperazine, 1-methyl- (6.8 mL, 0.061 mol) in tetrahydrofuran (200 mL, 2.46 mol) was heated in an aluminum block at 55 °C for 3 days. NMR showed completion, and the mixture was then concentrated under vacuum. The solid was washed with EtOH and dried to give [C](2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindol-2',3(1H,1'H)-dione (10.21 g; yield = 88.8%; lot number = 2009-B ML-73A).

[0468]

[0469] (2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindol-2',3(1H,1'H)-dione dihydrochloride

[0470] Acetyl chloride (0.04 mL, 0.6 mmol) was added to a mixture of methanol (0.03 mL, 0.7 mmol) and ethyl acetate (4.00 mL, 41.0 mmol). After 30 minutes, [A](2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione (0.100 g, 0.267 mmol; lot number = 2009-B ML-40A) was added. A precipitate formed. Filtration yielded [B](2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione dihydrochloride (0.07 g; yield = 60%; lot number = 2009-B ML-58A) as a purple solid.

[0471]

[0472] 4-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}piperazine-

[0473] 1-Tertiaryl carboxylate

[0474] A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (0.561 g, 0.00214 mol; batch number = 2009-B ML-4B), 37% formaldehyde (37:63, formaldehyde:water, 0.31 mL, 0.0042 mol), and [B]1-piperazincarnate tert-butyl ester (0.79 g, 0.0042 mol) in tetrahydrofuran (15 mL, 0.18 mol) was heated in an aluminum block at 55 °C for approximately 60 hours. The mixture was then concentrated under vacuum. The solid was washed with ethanol and filtered to give [C]4-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}piperazine-1-carboxylic acid tert-butyl ester (0.80 g; yield = 81%; batch = 2009-B ML-41A), a dark purple solid.

[0475]

[0476] (2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione

[0477] In a 100 mL round-bottom flask, a mixture of [A](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (1.11 g, 3.80 mmol; batch number = 2009-B ML-46A) and thionyl chloride (6.7 mL, 92 mmol) was heated under reflux for 2 hours. The reaction mixture was concentrated under vacuum to give [B](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (1.18 g; yield = 102%; batch number = 2009-B ML-61cr) as a purple solid.

[0478]

[0479] 1-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}-

[0480] L-proline tert-butyl ester

[0481] Add [B]L-proline tert-butyl ester (0.15 mL, 0.87 mmol) and triethylamine (0.12 mL, 0.86 mmol) to a solution of [A](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (0.17 g, 0.55 mmol; batch number = 2009-BML-61cr) in dichloromethane (6.5 mL, 100 mmol). Stir the mixture overnight at room temperature. Concentrate the mixture under vacuum. Rapid chromatography (5–8% EtOAc in DCM solution) yielded [C]1-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}-L-proline tert-butyl ester (0.10 g; yield = 41%; lot number = 2009-B ML-63A) as a purple solid.

[0482]

[0483] (2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione and [(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-yl]ditert-butyl methylphosphate

[0484] In a 100 mL round-bottom flask, a mixture of [A](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (1.66 g, 5.68 mmol; lot number = 2009-B ML-62A) and thionyl chloride (8.0 mL, 110 mmol) was heated under reflux for 2 hours. The reaction mixture was concentrated under vacuum to give [B](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (lot number = 2009-B ML-86-INT) as a purple solid.

[0485] The intermediate (2009-B ML-86-INT) was dissolved in ethyl acetate (34.2 mL, 350 mmol), potassium di-tert-butyl phosphate (1.69 g, 6.82 mmol) was added, followed by dropwise addition of sodium iodide (0.170 g, 1.14 mmol). The mixture was stirred overnight. When left at room temperature, the product slowly reverted to the starting material (approximately 80% reversion after 72 hours).

[0486]

[0487] [(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl dihydrophosphate and (2Z)-1'-(hydroxymethyl)-2,3'-biindol-2',3(1H,1'H)-dione and (2Z)-1'-(ethoxymethyl)-2,3'-biindol-

[0488] 2',3(1H,1'H)-dione

[0489] A solution of 1,4-dioxane in 4.0 M hydrogen chloride in 1,4-dioxane (3.0 mL, 12 mmol) was added to a 1,4-dioxane solution (9.0 mL, 120 mmol) of [A][(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-yl]methyl ditert-butyl phosphate (0.55 g, 1.1 mmol). The reaction mixture was stirred at room temperature for 1 hour. HPLC showed that the starting material was completely consumed. The solvent was removed under vacuum, and the solid was dried overnight in a vacuum oven. The product quickly reverted to [C](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione. Exposure to ethanol yields a mixture of (2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione and [D](2Z)-1'-(ethoxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione.

[0490]

[0491] (Z)-2',3-dioxo-[2,3'-biindolyl]-1'-carboxylic acid allyl ester

[0492] LiHMDS (0.84 mL, 1.1 equivalence) was added to a solution of (Z)-[2,3'-biindolyl-2',3-dione (200 mg, 0.19 mmol, 1 eq) in THF (10 mL) at 0 °C. After stirring at 0 °C for 1 hour, allyl chloroformate was added. The mixture was stirred overnight at room temperature. The reaction mixture was poured into water (20 mL) and extracted with EtOAC (10 × 3 mL). The combined organic phases were dried over Na₂SO₄ and concentrated. The solidified product was purified by column chromatography (PE / EA = 5 / 1, v / v) to give the desired product (200 mg, 0.57 mmol, 76%) as a purple solid.

[0493] HNMR: (400MHz, DMSO-d6) δppm: 10.54 (s, 1H), 8.93-8.91 (m, 1H), 7.93-7.91 (m, 1H), 7.65 (d, J=7.2Hz, 1H), 7.48-7.40 (m, 1H), 7.32-7.28 (m, 1H), 7.26-7.18 (m, 1H), 7.17 -6.99 (m, 1H), 6.90 (d, J=8Hz, 1H), 6.14-6.04 (m, 1H), 5.59-5.55 (m, 1H), 5.40-5.37 (m, 1H), 4.93-4.92 (m, 2H).

[0494] HPLC (Agilent-1200-A2): Retention time: 15.26 min, purity 92%.

[0495]

[0496] (Z)-(2',3-dioxo-[2,3'-biindolyl]-1'-yl)methyl hydrosulfate

[0497] Triethylamine (940 mg, 9.2 mmol, 1.2 eq) was added to a solution of (Z)-[2,3'-biindole-2',3-dione (2.02 g, 7.6 mmol, 1 eq) and HCHO (1.3 g, 16 mmol, 2.1 eq) in THF (50 mL) at room temperature. After stirring at 55 °C for 16 hours, the reaction mixture was filtered to give the desired product (1.8 g, 6.16 mmol, 81%) as a purple solid.

[0498] HNMR: (400MHz, DMSO-d6) δppm: 11.09 (s, 1H), 8.83 (d, J = 7.2Hz, 1H), 7.67 (d, J = 7.6Hz, 1H), 7.59 (t, J) = 7.6Hz, 1H), 7.43 (d, J = 8.0Hz, 1H), 7.35 (t, J=7.6Hz, 1H), 7.19 (d, J=8.0Hz, 1H), 7.11 (t, J=8.0)Hz, 1H), 7.04 (t, J=7.2Hz, 1H), 6.32 (t, J=6.8Hz, 1H), 5.22 (d, J=6.8Hz, 2H).

[0499] Add N,N-dimethylformamide sulfur trioxide complex (306 mg, 2 mmol, 10 eq) to a solution of (Z)-1'-(hydroxymethyl)-[2,3'-biindolyl]-2',3-dione (58 mg, 0.2 mmol, 1 eq) in pyridine (1 mL) and DMF (1 mL). Stir the mixture overnight at room temperature. Filter the solid and wash with EtOAC (30 mL) to give the desired product (20 mg, 0.05 mmol, 25%) as a purple solid.

[0500] HNMR (400MHz, DMSO-d6): δppm: 11.12 (s, 1H), 8.88 (d, J = 5.2Hz, 2H), 8.83 (d, J = 7.6Hz, 1H), 8.51-8.47 (m, 1H), 8.00-7.97 (m, 2H), 7.67 (d, J = 7.6Hz, 1H) ), 7.61-7.57(m, 1H), 7.44(d, J=8.0Hz, 1H), 7.37-7.33(m, 1H), 7.28-7.26( d, J=7.6Hz, 1H), 7.13 (t, J=7.6Hz, 1H), 7.04 (t, J=7.6Hz, 1H), 5.51 (s, 2H).

[0501] HPLC (Agilent-1100-A2): 214nm: Retention time: 12.41 min, purity 46%; 254nm: 12.41 min, purity 96%.

[0502]

[0503] (Z)-1'-((phenylthio)methyl)-[2,3'-biindolyl]-2',3-dione

[0504] Sodium thiophene (1.28 g, 9.7 mmol, 5 eq) was added to a THF (30 mL) solution of (Z)-1'-(chloromethyl)-[2,3'-biindolyl-2',3-dione (600 mg, 1.9 mmol, 1 eq) at room temperature. After stirring overnight at room temperature for 16 hours, the reaction mixture was concentrated under vacuum and purified by preparative TLC (DCM / MeOH = 20 / 1, v / v) to give the desired product (35 mg, 0.09 mmol, 5%) as a purple solid.

[0505] HNMR (400MHz, DMSO-d6): δppm: 11.03 (s, 1H), 8.81 (d, J=7.6Hz, 1H), 7.65 (d, J=7.2Hz, 1H), 7.58 (t, J= 7.2Hz, 1H), 7.47-7.40(m, 3H), 7.32-7.23(m, 4H), 7.14-7.11(m, 2H), 7.05-7.01(m, 1H), 5.40(s, 2H).

[0506]

[0507] (Z)-1'-(phenoxymethyl)-[2,3'-biindolyl]-2',3-dione

[0508] NaH (149 mg, 3.7 mmol, 2 eq) was added to a solution of phenol (351 mg, 3.7 mmol, 2 eq) in anhydrous THF (20 mL) at 0 °C. The mixture was stirred at this temperature for 10 min. Then, (Z)-1'-(chloromethyl)-[2,3'-biindolyl]-2',3-dione (580 mg, 1.9 mmol, 1 eq) was added to the reaction mixture. After stirring at room temperature for 16 hours, the reaction mixture was extracted with DCM (30 mL × 3) and washed with water and brine. The organic layer was concentrated and purified by preparative TLC (petroleum ether / EtOAC = 5 / 1, v / v) to give the desired product (40 mg, 0.11 mmol, 6%) as a purple solid.

[0509] LCMS (Agilent-S12): Rt 4.39min; [M+1] + =369.15

[0510] HNMR (400MHZ, DMSO-d6): δppm: 11.15 (s, 1H), 8.83 (d, J=8.0Hz, 1H), 7.66 (d, J=7.6Hz, 1H), 7.60 (t, J=7.2Hz, 1H) , 7.44 (d, J=8.4Hz, 1H), 7.39-7.35 (m, 1H), 7.32-7.24 (m, 3H), 7.17-7.12 (m, 3H), 7.08-6.98 (m, 2H), 5.89 (s, 2H).

[0511] HPLC (Agilent-1200-A1): 214nm: Rt: 10.95 min, purity 75%; 254nm: 10.95 min, purity 92%.

[0512]

[0513] (2Z,3E)-3-(2-phenylhydrazinyl)-[2,3'-biindolyl]-2'-one

[0514] Tetraisopropoxide titanium (1.3 g, 4.58 mmol, 6 eq) was added to an anhydrous THF (8 mL) solution of (Z)-[2,3'-biindole-1,3-dione (200 mg, 0.76 mmol, 1 eq) and phenylhydrazine (164 mg, 1.52 mmol, 2 eq). The mixture was stirred overnight at 80 °C. The reaction mixture was concentrated under vacuum and purified by preparative TLC (petroleum ether / EtOAC = 3 / 1, v / v) to give the desired product (10 mg, 0.03 mmol, 4%) as a purple solid.

[0515] LCMS (Agilent-S12): Rt 3.89min; [M+1]+=353.2

[0516] HNMR (400MHz, DMSO-d6): δppm: 12.04 (s, 1H), 10.81 (s, 1H), 10.61 (s, 1H), 8.93 (d, J=7.6Hz, 1H), 8 .31(d, J=7.6Hz, 1H), 7.68-7.66(m, 2H), 7.45-7.37(m, 4H), 7.13-7.05(m, 3H), 6.90-6.82(m, 2H).

[0517]

[0518] (2Z,3E)-3-(2-benzylhydrazinyl)-[2,3'-biindolyl]-2'-one

[0519] To a solution of (Z)-[2,3'-biindole-1,3-dione (250 mg, 0.95 mmol, 1 eq) in THF (10 mL), phenylmethylhydrazine (349 mg, 2.86 mmol, 3 eq) and tetraisopropoxide titanium (813 mg, 2.86 mmol, 3.0 eq) were added. After stirring overnight at 80 °C, the reaction mixture was concentrated and purified by preparative TLC (PE / EtOAC = 1 / 1, v / v) to give the desired product (60 mg, 0.16 mmol, 17.2%) as a purple solid.

[0520] LCMS (Agilent-S12): Rt 1.89 minutes; [M+1]+ = 367.1

[0521] HNMR: (400MHz, DMSO-d6) δppm: 12.17 (s, 1H), 10.47 ( s, 1H), 9.47 (s, 1H), 8.40 (d, J=8Hz, 1H), 8.22 (d, J=8Hz, 1H), 7.53-7.51 (m, 2H), 7.41-7.3 2 (m, 4H), 7.29-7.27 (m, 1H), 7.08 (t, J=7.2Hz, 1H), 6.99-6.95 (m, 1H), 6.82-6.74 (m, 2H).

[0522] HPLC (Agilent-1200-A2): Retention time: 12.87 min, purity 84%.

[0523]

[0524] (2Z,3E)-3-(2-methylhydrazinyl)-[2,3'-biindolyl]-2'-one

[0525] A solution of (Z)-[2,3'-biindole-1,3-dione (200 mg, 0.76 mmol, 1 eq) in pyridine (3 mL) was added with methylhydrazine (93 mg, 1.98 mmol, Equation 2.6). After stirring at 120 °C for 24 hours, the reaction mixture was concentrated and purified by preparative TLC (DCM / MeOH = 20 / 1, v / v) to give the desired product (10 mg, 0.03 mmol, 4.5%) as a purple solid.

[0526] LCMS (Agilent-S12): Retention time 3.16 minutes; [M+1]+=291.1

[0527] HNMR: (400MHz, DMSO-d6) δppm: 12.22 (s, 1H), 10.48 (s, 1H), 8.91 (d, J=4.4Hz, 1H), 8.61 (d , J=7.6Hz, 1H), 8.11 (d, J=7.6Hz, 1H), 7.41-7.30 (m, 2H), 7.08-6.85 (m, 4H), 3.45 (s, 3H),

[0528] HPLC (Agilent-1200-A2): Retention time: 10.90 min, purity 92%.

[0529]

[0530] (Z)-1'-((benzenesulfonyl)methyl)-[2,3'-biindolyl]-2',3-dione

[0531] Sodium benzenesulfinate (1.68 g, 10.2 mmol, 5 eq) was added to a solution of (Z)-1'-(chloromethyl)-[2,3'-biindolyl-2',3-dione (600 mg, 1.93 mmol, 1 eq) in anhydrous THF (30 mL). After stirring at room temperature for 16 hours, the reaction mixture was concentrated under vacuum and purified by preparative TLC (petroleum ether / EtOAC = 3 / 1, v / v) to give the desired product (18 mg, 0.04 mmol, 2%) as a purple solid.

[0532] HNMR (400MHz, DMSO-d6): δppm: 10.98 (s, 1H), 8.80 (d, J=8.0Hz, 1H), 7.82 (d, J=7.2Hz, 2H), 7.76-7.72 (m, 1H)), 7.6 6-7.56 (m, 4H), 7.40 (d, J=8.0Hz, 1H), 7.24 (t, J=7.6Hz, 1H), 7.11 (t, J=7.6Hz, 1H), 7.13-7.02 (m, 2H), 5.56 (s, 2H).

[0533]

[0534] (Z)-(2',3-dioxo-[2,3'-biindolyl]-1'-yl)methylphenylcarbamate

[0535] Triethylamine (182 mg, 1.8 mmol, 3 eq) was added to a solution of (Z)-1'-(hydroxymethyl)-[2,3'-biindolyl]-2',3-dione (180 mg, 0.6 mmol, 1 eq) and phenyl isocyanate (71.5 mg, 0.6 mmol, 1 eq) in THF (10 mL). After stirring at room temperature for 16 hours, the reaction mixture was concentrated under vacuum and purified by preparative TLC (DCM / MeOH = 20 / 1, v / v) to give the target product (15 mg, 0.04 mmol, 6%) as a purple solid.

[0536] HNMR (400MHz, DMSO-d6): δppm: 11.17 (s, 1H), 9.84 (s, 1H), 8.86 (d, J = 8.0Hz, 1H), 7.68 (d, J = 7.2Hz, 1H), 7.60 (t , J=8.0Hz, 1H), 7.46-7.33 (m, 5H), 7.28 (t, J=8.0Hz, 2H), 7.17 (t, J=7.6Hz, 1H), 7.07-6.99 (m, 2H), 5.98 (s, 2H).

[0537] HPLC (Agilent-1200-N1): Rt: 9.11 min, purity 96%.

[0538]

[0539] (Z)-1'-(methoxymethyl)-[2,3'-biindolyl]-2',3-dione

[0540] Pyridine (15.8 mg, 0.2 mmol, 1 eq) was added to a solution of (Z)-1'-(hydroxymethyl)-[2,3'-biindolyl-2',3-dione (58 mg, 0.2 mmol, 1 eq) and SO3DMF (306 mg, 2 mmol, 10 eq) in DMF (1 mL) at room temperature. After stirring at room temperature for 16 hours, the reaction mixture was concentrated and washed with MeOH (10 mL) to give the desired product (30 mg, 0.1 mmol, 49%) as a purple solid.

[0541] LCMS (Agilent-S12): Retention time 1.97 minutes; [M+1]+=307.0

[0542] HNMR (400MHz, DMSO-d6): δppm: 11.12 (s, 1H), 8.83 (d, J = 7.6Hz, 1H), 7.66 (d, J = 7.6Hz, 1H), 7.59 (t, J = 7.6)Hz, 1H) , 7.41 (d, J=7.6Hz, 1H), 7.33 (t, J=7.6Hz, 1H), 7.16-7.11 (m, 2H), 7.04 (d, J=7.6Hz, 1H), 5.22 (s, 2H), 3.26 (s, 3H).

[0543] HPLC (Agilent-1100-A2): Retention time: 12.42 min, purity 94%.

[0544]

[0545] (2Z,3E)-3-(hydroxyimino)-[2,3'-biindolyl]-2'-one

[0546] Hydroxylamine hydrochloride (154 mg, 2.4 mmol, 10 eq) was added to a solution of (Z)-[2,3'-biindolyl-2',3-dione (63 mg, 0.24 mmol, 1 eq) in pyridine (2.5 mL). After stirring under reflux for 2.5 hours, the reaction mixture was poured into water (20 mL), filtered, and the solid was recrystallized from MeOH / water (10 mL, 2 / 1, v / v) to give the desired product (40 mg, 0.1 mmol, 60%) as a red solid.

[0547] LCMS (Agilent-S12): Retention time 3.00 minutes; [M+1]+=278.1

[0548] HNMR (400MHz, DMSO-d6): δppm: 13.47 (brs, 1H), 11.73 (s, 1H), 10.70 (s, 1H), 8.65 (d, J=8.0Hz, 1H), 8.23 ​​((d, 1H), 8.23 ​​((d, 1H)J=7.9Hz, 1H), 7.40-7.39 (m, 2H), 7.15-7.11 (m, 1H), 7.06-7.01 (m, 1H), 6.97-6.89 (m, 2H).

[0549] HPLC (Agilent-1260C-B2): Retention time: 13.63 min, 100% purity.

[0550]

[0551] (Z)-4-((2',3-dioxo-[2,3'-biindolyl]-1'-yl)methoxy)-4-oxobutyric acid

[0552] Succinyl chloride (52 mg, 0.34 mmol, 1 eq) was added to a pyridine (5 mL) solution of (Z)-1'-(hydroxymethyl)-[2,3'-biindolyl-2',3-dione (200 mg, 0.68 mmol, 2 eq). After stirring at 100 °C for 16 hours, the reaction mixture was concentrated and purified by silica gel column chromatography (DCM / MeOH = 10 / 1, v / v) to give the target product (8 mg, 0.02 mmol, 6%) as a purple solid.

[0553] LCMS (Agilent-S12): Rt 3.41 minutes; [M+23]+=415.2

[0554] HNMR (400MHz, DMSO-d6): δppm: 12.23 (brs, 1H), 11.15 (s, 1H), 8.84 (d, J=7.6Hz, 1H), 7.67 (d, J=7.6Hz, 1H), 7.60 (t, J=8.0Hz, 1H), 7.43 ( d, J=8.0Hz, 1H), 7.36 (t, J=7.6Hz, 1H), 7.22-7.14 (m, 2H), 7.05 (t, J=7.6Hz, 1H), 5.89 (s, 2H), 2.56-2.53 (m, 2.5H), 2.48-2.46 (m, 1.5).

[0555]

[0556] Ethyl (Z)-4-(2',3-dioxo-[2,3'-biindolyl]-1'-yl)-4-oxobutyrate

[0557] LiHMDS (0.38 mL, 1 M, 2 eq) was added to a THF (5 mL) solution of (Z)-[2,3'-biindole-1,3-dione]-2',3-dione (200 mg, 0.19 mmol, 1 eq). After stirring at 0 °C for 1 hour, ethyl 4-chloro-4-oxobutyrate (63 mg, 0.38 mmol, 2 eq) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was poured into water (20 mL) and extracted with EtOAC (10 x 3 mL). The combined organic phases were dried over Na₂SO₄ and concentrated. The crude product was purified by column chromatography (PE / EA = 5 / 1, v / v) to give the desired product (130 mg, 0.57 mmol, 44%) as a purple solid.

[0558] LCMS (Agilent-S12): Rt 2.76 minutes; [M+1]+ = 391.2

[0559] HNMR: (400MHz, DMSO-d6) δppm: 11.34 (s, 1H), 8.96 (d, J = 8Hz, 1H), 8.19 (d, J = 8Hz, 1H), 7.68- 7.60(m, 2H)), 7.45-7.25(m, 3H), 7.06(t, J=7.6Hz, 1H), 4.12-4.07(m, 2H), 3.43(t, J=6.4Hz 2H), 2.71 (t, J=6.4Hz, 2H), 1.20 (t, J=7.2Hz, 3H).

[0560] HPLC (Agilent-1200-A1): Retention time: 10.21 min, purity 97%.

[0561] Example 2

[0562] Stability of indigo prodrug

[0563] Dissolve 10.41 g of fasting-state simulated intestinal fluid (FaSSIF) buffer concentrate in 240.3 g of deionized water, and add 560 mg of FaSSIF powder. Stir the mixture until complete dissolution is observed. Add the test compound (approximately 1–3 mg) to 5.00 mL of FaSSIF solution. After incubating at 37°C (oven-controlled temperature) for 24 hours, collect a sample of the solution containing the test compound.

[0564] Dissolve 36.78 g of fasting-state simulated gastric juice (FaSSGF) buffer concentrate in 961.9 g of deionized water, and add 60 mg of FaSSGF powder. Stir the mixture until complete dissolution is observed. Add the test compound (approximately 1–3 mg) to 5.00 mL of FaSSGF solution. After incubating at 37°C (oven-controlled temperature) for 24 hours, collect a sample of the solution containing the test compound.

[0565] Samples acquired over 24 hours were analyzed by UPLC-MS on a Waters Acquity UPLC system, which consisted of an Acquity I-Class sample manager (FL), an Acquity I-Class binary solvent manager, and an Acquity UPLC column manager. UV detection was performed using an Acquity UPLC PDA detector (scan range 210–400 nm), mass detection was performed using an Acquity QDa detector (mass scan from 100–1250 Da; both positive and negative modes were used), and ELS detection was performed using an Acquity UPLC ELS detector. A Waters Acquity UPLC BEH C18 column was used. Analytes were separated at 50 mm and 1.7 mm. Samples were prepared by dissolving in 1 mL of 50% (v / v) MeCN aqueous solution (with or without sonication). The resulting solution was then filtered through a 0.2 mm syringe filter before analysis. All solvents (including formic acid and 36% ammonia solution) were purchased as HPLC grade.

[0566] The results are listed in Table 2-1.

[0567] Table 2-1: Fractions (percentages) of starting material (SM) and indirubin in the simulated fluids (FaSSIF or FaSSGF) after 24 hours at 37°C.

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575] Example 3

[0576] At 37℃ in F A Transformation time process of indigo in SSIF

[0577] The study was conducted as described in Example 2 to analyze the rate and extent of prodrug conversion to indirubin in fasting-state simulated intestinal fluid (FaSSIF). Each test compound was added to the FaSSIF in the amount reported in Example 2. Solution samples were taken at 15 min, 30 min, 1 h, 2 h, and 24 h and analyzed by UPLC-MS (as described in Example 2). The study was conducted at 37 °C (oven-controlled temperature). The results are shown in Table 3-1, where the numbers in each column report the fractions (percentage of total amount) of the test compound (starting material), indirubin methylene alcohol, and indirubin.

[0578] Table 3-1: Rate of conversion of prodrug compound to indirubin in FaSSIF at 37°C over 24 hours, reported as the amount of starting material (prodrug compound) / indirubin methylene alcohol / indirubin.

[0579]

[0580]

[0581]

[0582] Example 4

[0583] The conversion time of indigo in FASSGF at 37℃

[0584] The study was conducted as described in Example 2 to analyze the rate and extent of prodrug conversion to indirubin in fasting-simulated gastric juice (FaSSGF). Each test compound was added to FaSSGF in the quantities reported in Example 2. Solution samples were taken at 15 minutes, 30 minutes, 1 hour, 2 hours, and 24 hours and analyzed by UPLC-MS (as described in Example 2). The study was conducted at 37°C (oven-controlled temperature). The results are shown in Table 4-1, where the numbers in each column report the fractions (percentage of total amount) of the test compound (starting material), indirubin methylene alcohol, and indirubin.

[0585] Table 4-1: Rate of conversion of prodrug compound to indirubin in FaSSIF at 37°C over 24 hours, reported as the amount of starting material (prodrug compound) / indirubin methylene alcohol / indirubin.

[0586]

[0587]

[0588]

[0589]

[0590] Example 5

[0591] In vitro AHR activity of exemplary compounds

[0592] Compound 8, indirubin, 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio) were each mixed with dimethyl sulfoxide (DMSO) to produce a 1000X stock solution. TCDD and MeBio were included as positive controls in the study. They were added to cell culture medium containing fetal bovine serum desorbed by activated charcoal to produce a 2X stock solution, which was then added to reporter cells. Reporter cells were human Huh7 cells or rat H4IIE cell lines with native AhR and AhR / arnt-luciferase reporter vectors. The final concentration of DMSO was 0.1%. Cells were cultured at 37°C for 22 hours, then luciferase assay reagent was added, and the activity of relative luminescent units was measured. Samples were tested in duplicate, and the average readings are shown in the figure. Figures 1A to 1B middle.

[0593] Figure 1A-1BThe figures show the effects of compound 8 (triangle), indirubin (square), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD; circle) (positive control), and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio; dashed line) (positive control) concentrations (in nM) on human Huh7 cells. Figure 1A ) and rat H4IIE cells ( Figure 1B Activation of AhR in α-indorubicin. EC50 in human Huh7 cells. 50 The concentration was 58.82 nM. Compound 8 in human Huh7 cells EC50... 50 The concentration was 71.47 nM. Indirubin EC in rat H4IIE cells. 50 The estimated value is 654 nM (estimated due to lack of an upper plateau). Compound 8 in rat H4IIE cells EC50 50 It is 25.62 nM.

[0594] Example 6

[0595] Activity of Exemplary Compound 8 in a Mouse Model of Ulcerative Colitis

[0596] Eight-week-old female C57BL / 6J mice, weighing approximately 20 grams, purchased from Charles River Laboratories, were housed in specific pathogen-free IVC cages (4 mice per cage) in a temperature-controlled (20±2℃) room with a 12-hour light-dark cycle. Feed pellets and tap water were readily available. All experimental protocols were approved by the Institutional Animal Care and Use Committee. Mice were acclimatized to the animal facility for at least three days prior to the experiment.

[0597] To induce acute colitis induced by sodium dextran sulfate (DSS), mice were given 3% DSS (molecular weight 36,000–50,000; MP Biomedicals, Santa Ana, California, USA) in their drinking water for 7 consecutive days, followed by the use of regular water. The DSS solution was freshly prepared and changed daily. The mice were euthanized on day 8 (day 9 of the experiment).

[0598] Mice were divided into groups of eight. From day 0 to day 9, mice were administered compound 8 at a dose of 2 mg / kg / day in 200 μL of distilled water (pH = 7.0) twice daily via gavage. Mice in the mediator group were administered 200 μL of distilled water via gavage only.

[0599] Weight and Disease Activity Index (DAI) were recorded daily to assess colitis. The DAI score is the sum of the weight loss score, stool score, and bleeding sub-score. A blinded scoring system was used to assess colitis. The DAI scorer was unaware of the group information and animal ID and was responsible for assessing stool consistency and bleeding. The DAI scoring criteria are as follows.

[0600] Table 6-1: DAI Scoring System

[0601]

[0602] The results are as follows Figure 2 As shown in the image. Figure 2 This is a graph of the disease activity index (see Table 6-1 above), a measure of ulcerative colitis in a mouse model, as a function of time in days, after daily treatment with compound 8 (triangles) or placebo (distilled water; circles).

[0603] Example 7

[0604] Activity of exemplary compounds in a mouse model of ulcerative colitis

[0605] The study was conducted as described in Example 6. Briefly, experimental mice were randomly assigned to the study group (n = 10) and treated with indirubin, compound 9 (20 mg / kg (mpk)), compound 10 (25 mpk), compound 26 (24.5 mpk), compound 40 (20.5 mpk), or compound 41 (20 mpk). Each compound was administered orally twice daily via tube feeding to mice in 200 μL of distilled water (pH = 7.0) at a dose providing a molar equivalent of 15 mg / kg indirubin, from day 0 to day 9. Mice in the mediator group were administered 200 μL of distilled water via tube feeding only.

[0606] The results show Figures 3A-3C In this study, compound 41 was more effective than an equimolar amount of indirubin.

[0607] Example 8

[0608] Pharmacokinetics of the compound in a mouse model of ulcerative colitis

[0609] In Example 7, five animals in each group were euthanized six hours after the last oral dose. Plasma, lungs, and colons were then harvested. The colons were washed three times with cold phosphate-buffered saline (PBS) to remove colonic contents.

[0610] Indirubin was detected using LC-MS / MS. The limit of quantification for this method was 0.05 ng / mL in plasma, 0.05 ng / mL in lung, and 0.10 ng / g in colon.

[0611] To prepare the samples, 20 μL of the unknown sample, calibration standard, quality control sample, and aliquots of single-blank and double-blank samples were added to a 96-well plate. Plasma samples (except for double-blank samples) were quenched with 100 μL of internal standard (double-blank samples were quenched with 100 μL of acetonitrile (ACN), while liver, lung, and colon samples (except for double-blank samples) were quenched with 80 μL of internal standard (double-blank samples were quenched with 80 μL of ACN). The samples were then vortexed at 800 rpm for 5 min, centrifuged at 3220 g (4000 rpm) for 15 min at 4 °C, and 50 μL of the supernatant was transferred to another clean 96-well plate. This supernatant was then directly injected for LC-MS / MS analysis.

[0612] The bioanalytical methods are as follows:

[0613] The results are shown in Tables 8-1, 8-2 and 8-3.

[0614] Table 8-1

[0615]

[0616] Table 8-2

[0617]

[0618] Table 8-3

[0619]

[0620] The ratios of indirubin in the colon to plasma and in the lungs for the mediator and compound 26 were not shown because they were undetectable above the lower limit of quantitation. The prodrug released indirubin to a greater extent in the colon than an equimolar amount of synthetic indirubin. Some compounds had favorable drug levels in the colon and lower systemic exposure to indirubin. Compound 9 had less plasma exposure than an equimolar amount of indirubin. Compound 10 had more plasma exposure than an equimolar amount of indirubin.

[0621] Example 9

[0622] Solubility of Indirubin in biologically relevant media

[0623] Samples of synthetic indirubin, indigo, or indirubin derivatives (at a concentration of 5 μg / mL indirubin or molar equivalents of indirubin) were evaluated in two bioassociated media: simulated fasting gastric juice (FaSSGF) and simulated fasting intestinal juice (FaSSIF). The bioassociated media were prepared using FaSSIF / FeSSIF / FaSSGF powder according to the supplier's (Biorelevant) method. Briefly, the FaSSGF medium was prepared using 0.06 g / L FaSSIF / FeSSIF / FaSSGF powder in a 2 g / L NaCl solution, with the pH adjusted to 1.6 using 1 N HCl. The FaSSIF medium was prepared using 2.24 g / L FaSSIF / FeSSIF / FaSSGF powder in sodium phosphate buffer, with the pH adjusted to 6.5 using 1 N NaOH. (The sodium phosphate buffer was prepared as a 0.42 g / L NaOH, 3.95 g / L NaH₂PO₄·H₂O, 6.19 g / L NaCl solution).

[0624] Solubility assessment was performed as follows: A separate flask was prepared for each culture medium and each time point. The flask was placed in a shaking water bath maintained at 37°C. At specific time points of 1 hour, 4 hours, and 24 hours, the flask was removed from the water bath, allowed to cool, and then an aliquot was collected and filtered.

[0625] The concentration of indirubin was determined using a validated HPLC method. The method is as follows:

[0626]

[0627] Instrument parameters

[0628]

[0629]

[0630] Indigo red is preserved for approximately 20.0 minutes.

[0631] gradient condition

[0632]

[0633] Indigo red recovery rate calculation:

[0634]

[0635] Concentration of indirubin from the prodrug:

[0636]

[0637] The calculation assumes that 1 mole of prodrug is equivalent to 1 mole of indirubin. The percentage recovery of prodrug molecular weight converted to indirubin is based on the assumption that 100% of the prodrug can be converted to indirubin.

[0638] The results are shown in Table 9-1.

[0639] Table 9-1: Evaluation of prodrug solubility in FaSSIF and FaSSGF media at 1 hour, 4 hours, and 24 hours. Shown as the percentage of indirubin recovered in solution.

[0640]

[0641] ND = Not detected.

[0642] While several exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain modifications, arrangements, additions, and sub-combinations thereof. Therefore, it is intended that the appended claims and the claims introduced herein be construed as including all such modifications, arrangements, additions, and sub-combinations within their true spirit and scope.

Claims

1. Selected from the following compounds: , , and The use of its salts in the preparation of a medicine for the treatment of ulcerative colitis.

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