Aryl Hydrocarbon Receptor Agonist Derivatives

Abstract

The following structure of formula (IV) or (V): [Formula 1] JPEG2024537130000192.jpg59137 A compound having one of or a stereoisomer, salt or tautomer thereof [wherein 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 herein. Pharmaceutical compositions containing the compounds and their use in methods of treating disease are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 254,052, filed on October 8, 2021, the entire contents of which are incorporated by reference herein.

[0002] Technical Field FIELD OF THE DISCLOSURE

[0002] The subject matter described herein relates to compounds that are derivatives of aryl hydrocarbon receptor agonists, including indirubins, methods for preparing the compounds, pharmaceutical compositions containing the compounds, and methods of treatment. [Background technology]

[0003]

[0003] Aryl hydrocarbon receptor (AhR) agonists have promising activity as pharmaceutical compounds, but many such compounds have limited potential use due to, for example, toxicological and / or pharmacokinetic challenges. Oral dosage forms of AhR agonists sometimes result in severe adverse events, while topical dosage forms for skin diseases have been better tolerated. The exact reasons for the appearance of adverse events with oral dosage forms may be compound specific and / or may be related to their metabolic pathways, bioavailability, solubility, pharmacokinetics or off-target activity. Generally speaking, oral dosage forms result in more systemic exposure than topical dosage forms, since the skin provides a barrier to the rest of the body. AhR agonists designed to be released in the gastrointestinal tract and remain there, minimizing uptake into the bloodstream and resulting in minimal systemic exposure, would be desirable. AhR agonists that provide these desirable properties would provide an approach for treatment that is more effective and safer than known AhR agonists.

[0004]

[0004] Indirubin is an example of a compound that is an AhR agonist, a receptor that plays a role in immunity, barrier function and metabolism. Indirubin has been used to treat human diseases either as a minor component or concentrated extract from a fermented plant product known as Indigo Naturalis. In its purified form, indirubin is observed to have very poor solubility and poor solubility. The lack of solubility is believed to result from its highly crystalline structure and intra- and intermolecular hydrogen bonds, resulting in poor solubility (Indirubin, the red shade of indigo (2006), Meijer et al., Chapter 10. Indirubin: The relationship between chemical structure and physical properties, with particular reference to colour. Christie RM 103-108). This lack of solubility and dissolution of indirubin limits options for poor bioavailability and pharmacokinetic profile. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need in the pharmaceutical field for compounds that have therapeutic activity like indirubin, but provide improved properties such as improved solubility and dissolution at room and body temperatures, facilitating the design and preparation of dosage forms for the delivery of indirubin and other AhR agonists, as well as improving their bioavailability. [Means for solving the problem]

[0006]

[0005] In brief, the present disclosure provides compounds, including their stereoisomers, pharma- ceutically acceptable salts or tautomers, that can be used alone or in combination with other therapeutic agents as aryl hydrocarbon receptor (AhR) agonists.

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

[0008] [ka] A compound having the formula or its stereoisomer, salt or tautomer [R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , R 11a , R 11b , R 12a , R 12b , X, Y and Z are as defined herein.

[0009] Also provided are pharmaceutical compositions comprising one or more of the aforementioned compounds of formula (IV) or (V) and a therapeutic agent. In another embodiment, a method of treatment is provided by administering to a subject in need thereof a compound of formula (IV) or (V) or a pharmaceutical composition comprising a compound of formula (IV) or (V) as described above to treat a disease. In an embodiment, the disease is, for example, an inflammatory disease. In an embodiment, the disease is a gastrointestinal inflammatory disease, a skin inflammatory disease, a pulmonary inflammatory disease, a systemic inflammatory disease, acne, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, hidradenitis suppurativa, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 and type 2 diabetes, or vitiligo. In an embodiment, the disease is ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, pouchitis, mucositis, cancer, fibrotic disease, or graft-versus-host disease.

[0010] In another embodiment, the structure of formula (I) or formula (Ia):

[0011] [ka] A compound having the formula or a stereoisomer, salt or tautomer thereof, 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 is as defined herein.

[0012] Compositions comprising the compounds of formulas (I), (Ia), (IV) and (V), and methods of use are also disclosed.

[0013] Also disclosed is a method for treating inflammatory and immune disorders.In an embodiment, the method comprises administering a compound as described herein, including but not limited to those of formula (I), (Ia), (IV) and / or (V), or a composition comprising such a compound, whereby administering provides the conditions for its conversion into a therapeutically effective amount of indirubin.

[0014]

[0011] Various aspects and embodiments will be described more fully below. Such aspects and embodiments may take many different forms, and the illustrative examples disclosed herein are not to be construed as limiting, but rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art. [Brief description of the drawings]

[0015] [Figure 1A]

[0012] A plot showing activation of AhR in human Huh7 cells in the presence of compound 8 (triangles), indirubin (squares), 2,3,7,8-tetrachlorodibenzodioxin (TCDD; circles) (positive control) and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio; dashed line) (positive control) at nM concentrations. [Figure 1B] 1 is a plot showing activation of AhR in rat H4IIE cells in the presence of compound 8 (triangles), indirubin (squares), 2,3,7,8-tetrachlorodibenzodioxin (TCDD; circles) (positive control), and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio; dashed line) (positive control) at nM concentrations. [Diagram 2]

[0013] Plot of the disease activity index (see table in Example 6) as 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). [Figure 3A]

[0014] A graph showing the percentage of initial body weight as a function of time in days for mice in which acute colitis was induced and treated twice daily for 9 days with placebo (filled circle), synthetic indirubin (filled square), compound 9 (filled triangle), compound 10 (inverted triangle), compound 26 (open circle), compound 40 (open square) or compound 41 (open triangle), the indicated doses being in milligrams per kilogram (mpk). [Figure 3B] Graph showing Disease Activity Index (DAI) score as a function of time in days for mice in which acute colitis was induced and treated twice daily for 9 days with placebo (filled circle), synthetic indirubin (filled square), compound 9 (filled triangle), compound 10 (inverted triangle), compound 26 (open circle), compound 40 (open square) or compound 41 (open triangle), the indicated doses being in milligrams per kilogram (mpk). [Figure 3C]

[0015] A bar graph showing the average colon length (in cm with 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, the indicated doses being in milligrams per kilogram (mpk). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] I. Definition

[0016] For convenience, certain terms used in the specification, examples and claims are collected here. 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 belongs.

[0017]

[0017] When a range of values ​​is provided, each intervening value between the upper and lower limits of the range and any other stated or intervening value within the stated range is intended to be encompassed within the disclosure. For example, if a range of 1 μm to 8 μm is stated, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm, as well as ranges of values ​​above 1 μm and ranges of values ​​below 8 μm are also intended to be expressly disclosed.

[0018]

[0018] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "an excipient" includes a single excipient as well as two or more of the same or different excipients, etc.

[0019]

[0019] The term "about" is meant to encompass a deviation of plus or minus 5 percent, particularly with respect to a given quantity.

[0020]

[0020] The compositions of the present disclosure can comprise, consist essentially of, or consist of the disclosed components.

[0021]

[0021] All percentages, parts and ratios are based on the total weight of the composition and all measurements are made at about 25° C. unless otherwise specified.

[0022]

[0022] As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated: "Amino" is -NH 2 , -NHR, or -NR 2 Refers to radicals. "Cyano" refers to the -CN radical. "Hydroxyl" refers to the -OH radical. "Imino" refers to a =NH or =NR substituent. "Nitro" is -NO 2 Refers to radicals. "Oxo" refers to the =O substituent. "Thio" refers to the ═S substituent. "Trifluoromethyl" is -CF 3 Refers to radicals. Hydrazido or hydrazino refers to the NN substituent. where each R is a compatible substituent as described in this disclosure. When an R group is chiral, the isomers are contemplated and included herein.

[0023] "Alkyl" refers to a straight-chain, saturated, acyclic, monovalent hydrocarbon radical or a branched-chain, saturated, acyclic, monovalent hydrocarbon radical having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms or 1 to 6 carbon atoms, attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Optionally substituted alkyl radicals, where valences permit, include halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilanyl, -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''' (wherein p is 0, 1 or 2) and -S(O) t N(R') 2 (where t is 1 or 2), where each R' is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl; each R'' is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl; and each R''' is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl.

[0024] "Alkoxy" refers to a group of the formula -OR a where R ais an alkyl radical as defined above containing one to twelve carbon atoms. The alkyl portion of the optionally substituted alkoxy radical is optionally substituted as defined above for an alkyl radical.

[0025] "Alkoxyalkyl" refers to a group of the formula -R a -OR b where R a is alkylene, and R b is alkyl as defined above. The alkyl and alkylene portions of the optionally substituted alkoxyalkyl radical are optionally substituted as defined above for the alkyl radical and alkylene chain, respectively.

[0026] "Aralkyl" refers to a group of the formula -R a -R b where R a is alkylene, and R b is aryl as defined above. The alkylene and aryl portions of the optionally substituted aralkyl are optionally substituted as described herein for alkylene and aryl, respectively.

[0027] "Aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system radical containing 6 to 18 carbon atoms, where the polycyclic aryl ring system is a bicyclic, tricyclic or tetracyclic ring system. Aryl radicals include, but are not limited to, groups such as fluorenyl, phenyl and naphthyl. Optionally substituted aryl includes alkyl, alkenyl, halo, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2 (where t is 1 or 2), where each R' is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R'' is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R''' is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclyl, or heteroaryl.

[0028]

[0028] "Arylalkoxy" refers to a group of formula -OR, where R is aralkyl. Optionally substituted arylalkoxy is optionally substituted arylalkoxy as described herein for aralkyl. In some embodiments, the arylalkoxy is benzyloxy.

[0029] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond. Polycyclic hydrocarbon radicals are bicyclic, tricyclic, or tetracyclic ring systems. Unsaturated cycloalkyls contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, and the like. Optionally substituted cycloalkyl includes alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2 (where t is 1 or 2), where each R' is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R'' is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R''' is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0030]

[0030] A "deuterated compound" is a compound in which one or more hydrogen atoms have been replaced with deuterium atoms. A deuterated drug can be a derivative of an active compound. A deuterated drug can be a prodrug. Deuteration can alter the physical properties, metabolic properties, activity, or safety of a drug.

[0031]

[0031] "Derivatives" are related chemical species that can be derived from similar compounds through chemical reactions. They may include slight chemical modifications, replacement of atoms with deuterated atoms, replacement of atoms with stable or radioactive isotopes, or other modifications that impart desirable properties to the compound.

[0032]

[0032] "Fused" refers to any ring system described herein that is fused to an existing ring structure in the compounds of the present invention. When the fused ring system is a heterocyclyl or heteroaryl, any carbon atom on the existing ring structure that becomes part of the fused ring system may be replaced with a nitrogen atom.

[0033] "Halo" refers to the following halogen substituents: bromo, chloro, fluoro and iodo.

[0034]

[0034] "Haloalkyl" refers to an alkyl radical as defined above, further substituted by one or more halogen substituents. The number of halo substituents contained in a haloalkyl ranges from 1 to up to the total number of hydrogen atoms available for replacement by halo substituents (e.g., perfluoroalkyl). Non-limiting examples of haloalkyl include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the like. For an optionally substituted haloalkyl, a hydrogen atom bonded to a carbon atom of the alkyl portion of the haloalkyl radical may be optionally replaced with a substituent as defined above for an optionally substituted alkyl.

[0035]

[0035] "Haloalkenyl" refers to an alkenyl radical as defined above, further substituted by one or more halo substituents. The number of halo substituents contained in a haloalkenyl is from 1 up to the total number of hydrogen atoms available for replacement by halo substituents (e.g., perfluoroalkenyl). Non-limiting examples of haloalkenyl include 2,2-difluoroethenyl, 3-chloroprop-1-enyl, and the like. For an optionally substituted haloalkenyl, a hydrogen atom bonded to a carbon atom of the alkenyl portion of the haloalkenyl radical may be optionally replaced with a substituent as defined above for an optionally substituted alkenyl group.

[0036]

[0036] "Haloalkynyl" refers to an alkynyl radical as defined above which is further substituted by one or more halo substituents. The number of halo substituents contained in a haloalkynyl is from 1 up to the total number of hydrogen atoms available for replacement by the halo substituents (e.g., perfluoroalkynyl). Non-limiting examples of haloalkynyl include 3-chloroprop-1-ynyl, and the like. The alkynyl portion of the haloalkynyl radical may be further optionally substituted as defined above for an alkynyl group.

[0037] "Heteroarylalkyl" refers to a group of the formula -R a -R b where R a is alkylene, and R b is heteroaryl as described herein. The alkylene and heteroaryl portions of the optionally substituted heteroarylalkyl are optionally substituted as described herein for alkylene and heteroaryl, respectively.

[0038] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring system radical having 2-12 carbon atoms and containing a total of 1-6 heteroatoms independently selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. The bicyclic, tricyclic, or tetracyclic heterocyclyl is a fused, spiro, and / or bridged ring system. The heterocyclyl radical can be saturated or unsaturated. The unsaturated heterocyclyl contains one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Optionally substituted heterocyclyl includes alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, oxo, thioxo, nitro, aryl, aralkyl, cycloalkyl, heterocyclyl, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2(where t is 1 or 2), where each R' is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R" is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R"' is independently alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl. The nitrogen, carbon, or sulfur atom in the heterocyclyl radical may be optionally oxidized (when the substituent is oxo and present on the heteroatom); the nitrogen atom may be optionally quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclyl, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2(where t is 1 or 2), where R″ is a straight or branched alkylene or alkenylene chain, and R′ and R′″ are as defined above. Examples of optionally substituted heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0039]

[0039] "Heterocyclylene" refers to a heterocyclyl in which one hydrogen atom is replaced by a valence. Optionally substituted heterocyclylene may be substituted as described herein for heterocyclyl.

[0040] "Heteroaryl" refers to a 5-18 membered ring system radical containing at least one aromatic ring, having a carbon number of 1-17 carbon atoms, and containing a total of 1-10 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heteroaryl radicals are monocyclic, bicyclic, tricyclic, or tetracyclic ring systems. Bicyclic, tricyclic, or tetracyclic heteroaryl radicals are fused and / or bridged ring systems. Optionally substituted heteroaryl includes alkyl, alkenyl, alkoxy, halo, haloalkyl, haloalkenyl, cyano, oxo, thioxo, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2 (where t is 1 or 2), where each R' is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R'' is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R''' is alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl. The nitrogen, carbon, or sulfur atoms in a heterocyclyl radical may be optionally oxidized (when the substituent is oxo and present on the heteroatom), provided that at least one ring in a heteroaryl remains aromatic; the nitrogen atom may be optionally quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclyl, 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''' (wherein p is 0, 1 or 2) and -R''-S(O) t N(R') 2(where t is 1 or 2), where R″ is a straight or branched alkylene or alkenylene chain, and R′ and R′″ are as defined above), with the proviso that at least one ring in the heteroaryl remains aromatic. Examples of optionally substituted heteroaryl radicals are azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl. , indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thiophenyl (i.e., thienyl).

[0041]

[0041] The phrase "pharmacologically acceptable" is used herein to refer to compounds, salts, compositions, dosage forms, and the like that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and / or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some aspects, "pharmacologically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in mammals (e.g., animals), and more particularly in humans.

[0042]

[0042] A "prodrug" is a compound that is metabolized or otherwise chemically converted to an active moiety after administration. A prodrug can be a derivative of an active compound. A prodrug may or may not be active prior to conversion to the active form in vivo.

[0043] The term "treating" is used herein, for example, in relation to methods of treating inflammatory disease or gastrointestinal disease, and generally includes administration of a compound or composition that reduces the frequency or delays the onset of symptoms of a medical condition (e.g., autoimmune disease, inflammatory disorder, gastrointestinal disorder) in a subject compared to a subject not receiving the compound or composition. This can include reversing, reducing or arresting the symptoms, clinical signs and underlying pathology of the condition in a manner that improves or stabilizes the subject's condition (e.g., regression of symptoms of an autoimmune or inflammatory disease, such as improvement in MAYO score in the treatment of ulcerative colitis).

[0044]

[0044] The embodiments disclosed herein encompass all pharma ceutical acceptable compounds of compounds (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb) Which are isotopically labeled by replacing one or more atoms With an atom having a different atomic mass or mass number. Examples of isotopes that may be incorporated into the disclosed compounds are: 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, etc., each contain isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine. These radiolabeled compounds can be useful to help determine or measure the effectiveness of a compound, for example, by characterizing the site or mode of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C are particularly useful for this purpose given their ease of incorporation and ready means of detection.

[0045] Deuterium, i.e. 2Substitution with heavier isotopes, such as H, can give rise to certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0046]

[0046] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes such as N can be useful in Positron Emission Topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb) can generally be prepared by conventional techniques well known to those skilled in the art or by processes similar to those described in the preparations and examples set forth below, using the appropriate isotopically labeled reagent in place of the previously used non-labeled reagent.

[0047]

[0047] The embodiments disclosed herein encompass the in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily by enzymatic processes. Thus, the present disclosure includes compounds produced by a process comprising administering a compound of the present disclosure to a mammal for a period of time sufficient to produce its metabolic products. Such products are typically identified by administering a radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, monkey, etc., in a detectable dose, or to a human, allowing sufficient time for metabolism to occur, and isolating the conversion products from urine, blood, or other biological samples.

[0048]

[0048] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0049]

[0049] "Pharmaceutically acceptable acid addition salts" refer to those acids which retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and which are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, and the like. It refers to salts formed with organic acids such as, but not limited to, cronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic 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, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid (TFA), and undecylenic acid.

[0050]

[0050] "Pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0051]

[0051] "Pharmaceutical composition" refers to a formulation of a compound of the present disclosure and a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal, such as a human. Such a vehicle includes all pharma- ceutically acceptable carriers, diluents, and excipients therefor.

[0052]

[0052] "Effective amount" or "therapeutically effective amount" refers to the amount of a compound of the present disclosure that, when administered to a mammal, preferably a human, is sufficient to achieve treatment in the mammal, preferably a human. The amount of a compound that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art having regard to his or her knowledge and this disclosure.

[0053]

[0053] "Stereoisomers" refer to compounds that are composed of the same atoms bonded by the same bonds but have different three-dimensional structures and are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof, including "enantiomers" which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other. The present disclosure also contemplates "diastereomers" which refer to non-mirror images of non-identical stereoisomers. Diastereomers occur when two or more stereoisomers of a compound have different configurations at one or more of the equivalent stereocenters and are not mirror images of each other.

[0054]

[0054] "Tautomer" refers to a proton migration from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any and all such compounds.

[0055]

[0055] Less than the full scale of this disclosure may be claimed for any reason by reserving the right to proviso out or exclude any individual member of any such group, including any subrange or combination of subranges within a group, which may be claimed according to ranges or in any similar manner. Furthermore, less than the full scale of this disclosure may be claimed for any reason by reserving the right to proviso out or exclude any individual substituent, analog, compound, ligand, structure, or group thereof, or any member of a claimed group.

[0056]

[0056] Throughout this disclosure, various patents, patent applications and publications are referred to.The disclosures of these patents, patent applications and publications are incorporated by reference in their entirety into this disclosure in order to more fully describe the state of the art as known to those skilled in the art as of the date of this disclosure.In the event that there is any inconsistency between the cited patents, patent applications and publications and this disclosure, this disclosure shall prevail.

[0057] II. Compounds The compounds described herein are derivatives of AhR agonists, in which indirubin is used as a model AhR agonist, and are grouped into several classes for ease of disclosure.The grouping is for convenience of disclosure and is not intended to be limiting in any way.Indirubin derivative compounds may have modifications to individual atoms in the indirubin molecule that do not change or add to either the backbone or connectivity of indirubin (e.g., replacing one isotope with another isotope of the same atom, or replacing carbon with nitrogen or oxygen to create an isostere).Indirubin derivatives can be, for example, of formula (I):

[0058] [ka] The indirubin structure may have modifications that change the backbone of indirubin by adding additional carbon branches to the indirubin structure as exemplified by:

[0058] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently selected from the group consisting of hydrogen, deuterium, alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonylamido, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;

[0059] R 9 is selected from the group consisting of hydroxyl, alkoxyl, amino, amido, carbonate, carbamate, phosphate, aminal, and hemiaminal, together with the nitrogen atom to which it is attached;

[0060] R 10 is selected from the group consisting of carbonyl, imine, acetal, hemiacetal, aminal, hemiaminal, oxime, hydrazone, cyanoimine, and thiocarbonyl, together with the carbon atom to which it is attached.

[0059]

[0061] In another embodiment, the compound has formula (Ia):

[0060] [ka] The

[0062] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently selected from the group consisting of hydrogen, deuterium, alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonylamido, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0061]

[0063] In another embodiment, the compound has formula (Ib):

[0062] [ka] The

[0064] In the formula, R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of hydrogen, deuterium, alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonylamido, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0063]

[0065] In another embodiment, the compound has formula (II):

[0064] [ka] The

[0066] In the formula, R 9is selected from the group consisting of hydroxyl, alkoxyl, amino, amido, carbonate, carbamate, phosphate, aminal, and hemiaminal, together with the nitrogen atom to which it is attached.

[0065]

[0067] In another embodiment, the compound has formula (III):

[0066] [ka] The

[0068] In the formula, R 10 is selected from the group consisting of carbonyl, imine, acetal, hemiacetal, aminal, hemiaminal, oxime, hydrazone, cyanoimine, and thiocarbonyl, together with the carbon atom to which it is attached.

[0067]

[0069] In another embodiment, the compound of formula (III) has the formula (IIIa):

[0068] [ka] The

[0070] In the formula, R 10a is selected from the group consisting of alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonylamido, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, thioester, nitrile, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0069] A. Class A

[0071] Class A derivatives feature molecules in which the substitution of atoms does not change the overall structure of the molecule. Examples of such substitutions include molecules in which one atom is replaced with another atom having the same valence (e.g., replacing an oxygen atom with a sulfur atom) or a different valence (e.g., replacing a carbon atom with a boron atom).

[0070]

[0072] Another form of substitution includes replacing one isotope of an atom with another isotope (i.e., radiolabeling). Non-limiting examples include replacing carbon or hydrogen atoms with the natural isotope distribution (e.g., 1 H: 2 D. 12 C: 13 C. 12 C: 14 C, etc.) may be replaced with a non-natural isotope distribution. In some embodiments, preparation of a molecule may result in a mixture of isotopes. For example, a mixture of isotopes 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 an alternative or naturally non-predominant isotope (e.g., 2 D. 13 C. 15 N, 18 The radiolabel may be at a single position in the molecule or at multiple positions, e.g., one type (e.g., 2 D) or multiple types (e.g. 2 D and 13 C) may be limited to 1, 2, 3, 4, 5, 6, 7, 8 or more alternative isotopes.

[0071]

[0073] Compounds 1-2 shown below are illustrative and non-limiting examples of Class A molecules:

[0072] [ka]

[0073]

[0074] In addition to replacing hydrogen with deuterium, the natural distribution of carbon, nitrogen and oxygen has been altered to include unnaturally high proportions of less abundant isotopes, e.g. 13 C: 12C. An unnaturally high ratio 14 C: 12 C. An unnaturally high ratio 15 N: 14 N, an unnaturally high ratio 18 O: 16 The isotope ratio can be changed by preparing a derivative of indirubin having O, etc. By changing the isotope ratio, the indirubin derivative can utilize isotope effects, such as first order kinetic isotope effect, second order kinetic isotope effect, etc.

[0074] B. Class B

[0075] Class B derivatives are those that have additions or changes to the indirubin core structure, for example, by adding or removing atoms or a combination of the two (e.g., substitutions) to create new structures. For example, hydrogen atoms connected to carbon or nitrogen atoms can be removed, and one or more atoms (e.g., carbon, nitrogen, oxygen, halogen, etc.) can be added to create new structural frameworks. In some embodiments, the substitutions in Class B derivatives are at one or more aryl hydrogen atoms. In other embodiments, the substitutions are at one or both carbonyl oxygen atoms. In other embodiments, the substitutions in Class B derivatives are at the formula:

[0075] [ka] As shown in FIG.

[0076] In a preferred embodiment, the substitution in the class B derivatives is at the N1 position. Non-limiting examples include compounds 3-52:

[0077] [ka]

[0078] [ka]

[0079] [ka]

[0080] [ka]

[0081] [ka] Includes.

[0082] C. Class C Class C derivatives are a subset of Class B derivatives in which one or both of the oxygen atoms are substituted. In some embodiments, the oxygen at the 2 position is substituted. In preferred embodiments, the oxygen at the 3' position is substituted. Non-limiting examples of Class C derivatives include compounds 3-6:

[0083] [ka] Includes.

[0084] In one embodiment, the compound is

[0085] [ka] In another embodiment, the compound is

[0086] [ka] In yet another embodiment, the compound is

[0087] [ka] It is.

[0088] D. Class D 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 the N1' position is substituted. In preferred embodiments, the hydrogen atom at the N1 position is substituted. Non-limiting examples of Class D derivatives include compounds 7-53:

[0089] [ka]

[0090] [ka]

[0091] [ka]

[0092] [ka] Includes.

[0093]

[0080] Exemplary Class D compounds include urea compounds. The following structures are non-limiting examples of urea indirubin derivatives of the present disclosure.

[0094] [ka]

[0095] [ka]

[0096]

[0081] Exemplary Class D compounds include carbamate compounds. The following structures are non-limiting examples of carbamate indirubin derivatives of the present disclosure.

[0097] [ka]

[0098]

[0082] Exemplary class D compounds include amide compounds. The following structures are non-limiting examples of amide indirubin derivatives of the present disclosure:

[0099] [ka]

[0100]

[0083] Exemplary Class D compounds include aminal and hemiaminal compounds. The following structures are non-limiting examples of aminal and hemiaminal indirubin derivatives of the present disclosure.

[0101] [ka]

[0102] [ka]

[0103]

[0084] Exemplary Class D compounds include compounds with other substitutions at the nitrogen atom. The following structures are non-limiting examples of other indirubin derivatives of the present disclosure.

[0104] [ka]

[0105] In one embodiment, the following structure of formula (IV) or (V):

[0106] [ka] A compound having one of or a stereoisomer, salt or tautomer thereof, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, deuterium, alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, ester, amide, carboxylic acid, carbamate, sulfonylamide, acylsulfonylamido, sulfone, sulfoxide, ketone, carbonate, urea, sulfonylurea, amino, thioester, nitrile, nitro, azide, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 11a is halo, 3- to 8-membered heterocycloalkyl, 3- to 8-membered heteroaryl, -OH, -OX-, -SX-, -S(O) 2 X-, -NX 2 -, -OC(=O)X- or -OC(=O)NX 2 - and R 11b is 3 to 8 membered heterocycloalkyl, 3 to 8 membered heteroaryl, -OX- or -NX 2 - and X is independently hydrogen, C 1 ~C 10 Alkyl, C 1 ~C 10 Alkenyl, C 1 ~C 6 Haloalkyl, C 1 ~C 10 Heteroalkyl, -S(O) 2 OH, aryl or 3-8 membered heterocycloalkyl; Here, C 1 ~C 10 Alkyl and C 1 ~C 10 Heteroalkyl is 3-8 membered heterocycloalkyl, C 1 ~C 6 Alkyl carboxylic acid, -NHS(=O) 2 optionally substituted with -Y-, -NHC(=O)OY- or -NHC(=O)Y-; Here, 3-8 membered heterocycloalkyl is C 1 ~C 6 Alkyl, C 1 ~C 4 optionally substituted with amine, halo, -C(=O)Y- or -C(=O)OY-; Y is independently H, C 1 ~C 6 Perfluoroalkyl or C 1 ~C 6 is alkyl, R 12a and R 12b are each independently hydrogen or C 1 ~C 6 is alkyl, R 10 is O, NZ or NNZ 2 and Z is independently hydrogen, hydroxyl, aryl or C 1 ~C 4 alkyl, where C 1 ~C 4 The alkyl may be substituted with an aryl.

[0107] In one embodiment, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R6 , R 7 and R 8 is deuterium. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is alkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is halo. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is perfluoroalkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is alkynyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is alkenyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is alkoxy. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R6 , R 7 and R 8 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is thioalkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is hydroxyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is an ester. In some embodiments, R 1 , R 2 , R 3 , R 4 , R5 , R 6 , R 7 and R 8 is an amide. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a carboxylic acid. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a carbamate. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a sulfonylamido. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is an acylsulfonyl amide. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a sulfone. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is sulfoxide. In some embodiments, R 1 , R 2 , R 3 , R 4 , R5 , R 6 , R 7 and R 8 is a ketone. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a carbonate. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is urea. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a sulfonylurea. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is amino. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a thioester. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a nitrile. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5, R 6 , R 7 and R 8 is nitro. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is azide. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is cycloalkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is heterocycloalkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is aryl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is heteroaryl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 In some embodiments, one of R 1 , R 2 , R 3 , R 4 , R 5, R 6 , R 7 and R 8 Each of is hydrogen.

[0108] In one embodiment, R 12a or R 12b is hydrogen. 12a and R 12b Each of is hydrogen.

[0109] In one embodiment, R 12a or R 12b One of them is C 1 ~C 6 In another embodiment, R 12a or R 12b Each of the 1 ~C 6 In certain embodiments, C 1 ~C 6 Alkyl is a linear C 1 ~C 6 In some embodiments, the linear C 1 ~C 6 Alkyl is, for example, methyl (-CH 3 ), ethyl (-CH 2 CH 3 ), n-propyl (-CH 2 CH 2 CH 3 ), n-Butyl (-CH 2 CH 2 CH 2 CH 3 ), n-pentyl (-CH 2 CH 2 CH 2 CH 2 CH 3 ), or n-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ).

[0110] In one embodiment, C 1~C 6 Alkyl is a branched C 3 ~C 6 In some embodiments, the branched C 3 ~C 6 Alkyl 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.

[0111] In one embodiment, R 12a or R 12b One of the R 12a or R 12b is C 1 ~C 6 It is an alkyl.

[0112] In one embodiment, the compound has the following structure of formula (IVa) or (Va):

[0113] [ka] On the other hand, or a stereoisomer, salt or tautomer thereof.

[0114] In one embodiment, R 10 is O. In some embodiments, R 10 is NZ. In some embodiments, R 10 is NNZ 2 It is.

[0115]

[0093] Each Z is independently hydrogen, hydroxyl, aryl or C 1 ~C 4 alkyl, where C 1 ~C 4 The alkyl may be substituted with an aryl. In some embodiments, R 10When R is NZ, Z is hydroxyl. 10 NNZ 2 When Z is hydrogen and C 1 ~C 4 alkyl, where C 1 ~C 4 The alkyl is substituted with an aryl, such as phenyl. In some embodiments, for example, C 1 ~C 4 Alkyl is methyl (C 1 In some embodiments, R 10 NNZ 2 When R is, Z is hydrogen and aryl. 10 NNZ 2 When Z is hydrogen and phenyl.

[0116] In one embodiment, R 10 has the following structure:

[0117] [ka] has one of the following:

[0118] In one embodiment, the compound has the following structure of formula (IVb) or (Vb):

[0119] [ka] On the other hand, or a stereoisomer, salt or tautomer thereof.

[0120] In one embodiment, R 11a is 3-8 membered heterocycloalkyl. In some embodiments, R 11b is 3-8 membered heteroaryl. In some embodiments, R 11b is -OX-. In some embodiments, R 11bis OH. In some embodiments, R 11a is halo. In some embodiments, R 11b In some embodiments, R 11b -S(O) 2 In some embodiments, R 11b Ha-NX 2 In some embodiments, R 11b is -OC(=O)X-. In some embodiments, R 11b -OC(=O)NX 2 -It is.

[0121] In one embodiment, the compound has formula (IVc):

[0122] [ka] or a stereoisomer, salt or tautomer thereof, wherein

[0123] [ka] represents a 3- to 8-membered heterocycloalkyl or a 3- to 8-membered heteroaryl. In some embodiments, the formula (IVc)

[0124] [ka] is 3-8 membered heterocycloalkyl. In some embodiments, the formula (IVc)

[0125] [ka] is a 3- to 8-membered heteroaryl.

[0126] In one embodiment, the compound of formula (IVc)

[0127] [ka] is azetidine, diazetidine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, diazinane, triazinane, azepane, diazepane, azocane, oxetane, dioxetane, tetrahydrofuran, dioxolane, oxane, dioxane, trioxane, oxepane, oxocane, phosphetane, phosphorane, phosphinane, thietane, dithietane, tetrahydrothiophene, dithiolane, thiane, dithiane, trithiane, thiepane, thiocane, oxathiolidine, isoxathiolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, or oxathiane.

[0128] In one embodiment, R 11a is azetidine, diazetidine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, diazinane, triazinane, azepane, diazepane, azocane, oxetane, dioxetane, tetrahydrofuran, dioxolane, oxane, dioxane, trioxane, oxepane, oxocane, phosphetane, phosphorane, phosphinane, thietane, dithietane, tetrahydrothiophene, dithiolane, thiane, dithiane, trithiane, thiepane, thiocane, oxathiolidine, isoxathiolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, or oxathiane. 11a is azetidine. In some embodiments, R 11a is diazetidine. In some embodiments, R 11a is pyrrolidine. In some embodiments, R 11a is imidazolidine. In some embodiments, R 11a is pyrazolidine. In some embodiments, R 11a is piperidine. In some embodiments, R 11a is diazinan. In some embodiments, R 11a is a triazinane. In some embodiments, R 11ais azepane. In some embodiments, R 11a is diazepane. In some embodiments, R 11a is azocane. In some embodiments, R 11a is oxetane. In some embodiments, R 11a is a dioxetane. In some embodiments, R 11a is tetrahydrofuran. In some embodiments, R 11a is dioxolane. In some embodiments, R 11a is oxane. In some embodiments, R 11a is dioxane. In some embodiments, R 11a is trioxane. In some embodiments, R 11a is oxepane. In some embodiments, R 11a is oxocane. In some embodiments, R 11a is a phosphetane. In some embodiments, R 11a is a phosphorane. In some embodiments, R 11a is a phosphinane. In some embodiments, R 11a is thietane. In some embodiments, R 11a is dithietane. In some embodiments, R 11a In some embodiments, R 11a is dithiane. In some embodiments, R 11a is trithiane. In some embodiments, R 11a is thiepane. In some embodiments, R 11a is thiocane. In some embodiments, R 11a is oxathiolysine. In some embodiments, R 11a is an isoxthiolidine. In some embodiments, R 11a is oxazolidine. In some embodiments, R 11a is an isoxazolidine. In some embodiments, R 11a is thiazolidine. In some embodiments, R 11a is an isothiazolidine. In some embodiments, R 11ais morpholine. In some embodiments, R 11a is thiomorpholine. In some embodiments, R 11a is an oxathiane.

[0129] In some certain embodiments, R 11a has the following structure:

[0130] [ka] In some embodiments, R 11a teeth,

[0131] [ka] In some embodiments, R 11a teeth,

[0132] [ka] In some embodiments, R 11a teeth,

[0133] [ka] In some embodiments, R 11a teeth,

[0134] [ka] In some embodiments, R 11a teeth,

[0135] [ka] In some embodiments, R 11ateeth,

[0136] [ka] In some embodiments, R 11a teeth,

[0137] [ka] In some embodiments, R 11a teeth,

[0138] [ka] In some embodiments, R 11a teeth,

[0139] [ka] In some embodiments, R 11a teeth,

[0140] [ka] It is.

[0141] In some particular embodiments, R 11a teeth,

[0142] [ka] In some embodiments, R 11a teeth,

[0143] [ka] In some other embodiments, R11a teeth,

[0144] [ka] It is.

[0145] In one embodiment, the compound of formula (IVc)

[0146] [ka] has the following structure:

[0147] [ka] has one of the following:

[0148] In one embodiment, R 11b Ha-NX 2 In one embodiment, one of X is hydrogen. In another embodiment, one of X is C 1 ~C 10 In another embodiment, one of X is C 1 ~C 10 In another embodiment, one of X is C 1 ~C 6 In another embodiment, one of X is C 1 ~C 10 In another embodiment, one of X is -S(O) 2 In other embodiments, one of X is aryl. In other embodiments, one of X is 3-8 membered heterocycloalkyl.

[0149] In one embodiment, each X is hydrogen. In another embodiment, each X is C 1 ~C 10 In another embodiment, each X is C 1 ~C 10In another embodiment, each X is C 1 ~C 6 In another embodiment, each X is C 1 ~C 10 In other embodiments, each X is -S(O) 2 In other embodiments, each X is aryl. In other embodiments, each X is 3-8 membered heterocycloalkyl.

[0150] In one embodiment, one of X is hydrogen and the other X is C 1 ~C 10 In another embodiment, one X is hydrogen and the other X is C 1 ~C 10 In another embodiment, one X is hydrogen and the other X is C 1 ~C 6 In another embodiment, one X is hydrogen and the other X is C 1 ~C 10 In another embodiment, one X is hydrogen and the other X is -S(O) 2 In another embodiment, one X is hydrogen and the other X is aryl. In another embodiment, one X is hydrogen and the other X is 3-8 membered heterocycloalkyl.

[0151] In one embodiment, X is C 1 ~C 10 In some embodiments, X is C 1 In some embodiments, X is C 2 In some embodiments, X is C 3 In some embodiments, X is C 4 In some embodiments, X is C 1 ~C 4 It is an alkyl.

[0152] In one embodiment, X is C 2 ~C10 In some embodiments, X is C 3 For example, in some embodiments, X is propenyl. In some embodiments, X is C 2 ~C 3 It is alkenyl.

[0153] In one embodiment, X is C 1 ~C 6 In some embodiments, X is C 1 For example, in some embodiments, X is chloromethyl.

[0154] In one embodiment, X is C 1 ~C 10 In some embodiments, X is C 2 ~C 6 For example, in some embodiments, X is C 2 In another example, in some embodiments, X is C 3 In another example, in some embodiments, X is C 4 In another example, in some embodiments, X is C 5 In another example, in some embodiments, X is C 6 It is heteroalkyl.

[0155] In one embodiment, X is hydrogen, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Heteroalkyl, -S(O) 2 OH, phenyl, or C 5 In some embodiments, X is heterocycloalkyl. In some embodiments, X is hydrogen. In some embodiments, X is C 1 ~C 6 In some embodiments, X is C2 ~C 6 In some embodiments, X is C 1 ~C 6 In some embodiments, X is C 1 ~C 6 In some embodiments, X is -S(O) 2 In some embodiments, X is OH. In some embodiments, X is phenyl. In some embodiments, X is C 5 It is a heterocycloalkyl.

[0156] In one embodiment, R 11b has the following structure:

[0157] [ka] In some embodiments, R 11b teeth,

[0158] [ka] In some embodiments, R 11b teeth,

[0159] [ka] In some embodiments, R 11b teeth,

[0160] [ka] In some embodiments, R 11b teeth,

[0161] [ka] In some embodiments, R 11bteeth,

[0162] [ka] In some embodiments, R 11b teeth,

[0163] [ka] In some embodiments, R 11b teeth,

[0164] [ka] In some embodiments, R 11b teeth,

[0165] [ka] In some embodiments, R 11b teeth,

[0166] [ka] In some embodiments, R 11b teeth,

[0167] [ka] In some embodiments, R 11b teeth,

[0168] [ka] In some embodiments, R 11b teeth,

[0169] [ka] In some embodiments, R 11b teeth,

[0170] [ka] In some embodiments, R 11b teeth,

[0171] [ka] In some embodiments, R 11b teeth,

[0172] [ka] It is.

[0173]

[0112] In one embodiment, halo is -F, -Cl, -Br or -I. In some embodiments, halo is -F. In some embodiments, halo is -Cl. In some embodiments, halo is -Br. In some embodiments, halo is -I.

[0174] In one embodiment, C 1 ~C 10 Alkyl, C 2 ~C 10 Alkenyl, C 1 ~C 6 Haloalkyl, or C 1 ~C 10 The heteroalkyl is linear. In some embodiments, C 1 ~C 10 Alkyl, C 2 ~C 10 Alkenyl, C 1 ~C 6 Haloalkyl, or C 1 ~C 10The heteroalkyl is branched.

[0175] In one embodiment, C 1 ~C 6 Perfluoroalkyl is tetrafluoroethyl or trifluoromethyl. 1 ~C 6 The perfluoroalkyl is tetrafluoroethyl. 1 ~C 6 Perfluoroalkyl is trifluoromethyl.

[0176] In one embodiment, the 3- to 8-membered heteroaryl is azirine, oxirene, thiylene, azeto, oxeto, thieto, pyrrole, furan, thiophene, pyridine, pyran, thiopyran, azepine, oxepine, thiepine, azocine, oxocine, thiocine, diazeto, dioxeto, dithieto, imidazole, pyrazole, oxathiol, isoxathiol, oxazole, isoxazole, thiazole, isothiazole, diazine, oxazine, thiazine, oxathiin, dioxin, dithiin, triazine, tetrazine, pentazine, diazepine, thiazepine, azocine, oxocine, or thiocine.

[0177] In one embodiment, the compound of formula (IVc)

[0178] [ka] is azirine, oxirene, thiylene, azeto, oxeto, thieto, pyrrole, furan, thiophene, pyridine, pyran, thiopyran, azepine, oxepine, thiepine, azocine, oxocine, thiocine, diazeto, dioxeto, dithieto, imidazole, pyrazole, oxathiol, isoxathiol, oxazole, isoxazole, thiazole, isothiazole, diazine, oxazine, thiazine, oxathiin, dioxin, dithiin, triazine, tetrazine, pentazine, diazepine, thiazepine, azocine, oxocine, or thiocine.

[0179] In one embodiment, R 10 is O. In some embodiments, R 10 is NZ. In some embodiments, R 10 is NNZ. Each Z is independently hydrogen, hydroxyl, aryl or C 1 ~C 4 In one embodiment, R 10 When R is NZ, Z is hydroxyl. 10 When R is NZ, Z is hydrogen. 10 If is NNZ2, then Z is

[0118] In the formula, C 1 ~C 4 The alkyl may be substituted with an aryl.

[0180] In one embodiment, R 10 has the following structure:

[0181] [ka] In some embodiments, R 10 teeth,

[0182] [ka] In some embodiments, R10 teeth,

[0183] [ka] In some embodiments, R 10 teeth,

[0184] [ka] In some embodiments, R 10 teeth,

[0185] [ka] It is.

[0186] In one embodiment, the compound has the following structure:

[0187] [ka]

[0188] [ka]

[0189] [ka] has one of the following:

[0190] In one embodiment, the AhR agonist after administration, preferably after oral administration, releases indirubin in the lower gastrointestinal tract, or releases indirubin mainly in the colon.In an embodiment, the AhR agonist after administration, preferably after oral administration, releases indirubin mainly in the colon, with minimal or no significant uptake into bloodstream and / or surrounding peripheral tissues.

[0191] [Table 1-1]

[0192] [Table 1-2]

[0193] [Table 1-3]

[0194] [Table 1-4]

[0195] [Table 1-5]

[0196] [Table 1-6]

[0197] [Table 1-7]

[0198] [Table 1-8]

[0199] [Table 1-9]

[0200] As can be seen, the indirubin derivatives described herein allow fine tuning of the magnitude, site and duration of AhR activation, facilitating the development of new therapies for disease without the need for either exotic chemistry or specialized reagents or manufacturing techniques. The indirubin derivative compounds allow the production of dosage forms that have utility in treating disease, as described below. The approach exemplified herein with indirubin can be used with any known AhR agonist, including, but not limited to, indirubin, indigo, meisoindigo, linomide (roquinimex), 6-formylindolo[3,2-b]carbazole (FICZ), and laquinimod, tapinarof, 4-(4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)pyrimidin-2-amine, or compounds listed in US2021 / 0147390.

[0201] Pharmaceutical Compositions

[0123] Other embodiments are directed to pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises any one (or more) of the aforementioned compounds and a pharma- ceutically 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 further embodiments, the pharmaceutical composition comprises a compound as disclosed herein and an additional therapeutic agent (e.g., an anti-cancer agent). Non-limiting examples of such additional therapeutic agents are described herein below.

[0202] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, mucosal, transdermal, intravaginal, optical, nasal and topical administration.In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic and intranasal injection.

[0203] In certain embodiments, the compounds as described herein are administered in a local rather than systemic manner, for example, via injection of the compound directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long-acting formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the compounds are delivered in targeted drug delivery systems, for example, liposomes coated with organ-specific antibodies. In such embodiments, the liposomes target and are selectively taken up by the organ. In still other embodiments, the compounds as described herein are provided in the form of rapid release formulations, sustained release formulations, or intermediate release formulations. In still other embodiments, the compounds as described herein are administered locally.

[0204] In the treatment methods according to embodiments of the present disclosure, an effective amount of at least one compound of Formula (I)-(Vb) is administered to a subject suffering from or diagnosed with such a disease, disorder, or medical condition. An effective amount or dose can be ascertained by methods such as modeling, dose escalation studies, or clinical trials, for example, by consideration of the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's prior or ongoing therapy, the subject's health status and response to the drug, and the judgment of the treating physician.

[0205]

[0127] The compounds according to the present disclosure are effective over a wide dosage range.For example, for the treatment of an adult, dosages of about 0.001-0.1 mg, 0.01-0.1 mg, 0.5-5 mg, 0.5-10 mg, 0.01-10 mg, 0.1-10 mg, 10-5000 mg, 100-5000 mg, 1000 mg-4000 mg, or 1000-3000 mg per day are examples of dosages used in some embodiments.The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician.

[0206] In some embodiments, the compound of the present disclosure is administered in a single dose. In some embodiments, the single dose is administered orally. In another embodiment, the single dose is administered topically. However, other routes are used as appropriate. In some embodiments, the compound of the present disclosure is administered in multiple doses. In some embodiments, the dosing is about 1, 2, 3, 4, 5, 6, or more than 6 times per day. In other embodiments, the dosing is about once a month, once every two weeks, once a week, or once every other day. In another embodiment, the compound of the present disclosure and another agent (e.g., an anticancer agent) are administered together about once a day to about 6 times a day. In another embodiment, the administration of the compound of the present disclosure and the agent continues for less than about 7 days. In yet another embodiment, the administration continues for more than about 6, 10, 14, 28 days, 2 months, 6 months, or 1 year. In some cases, continuous dosing is achieved and maintained for as long as necessary.

[0207]

[0129] The administration of the disclosed compound may continue as long as necessary. In some embodiments, the disclosed compound is administered for more than 1, 2, 3, 4, 5, 6, 7, 14 or 28 days. In some embodiments, the disclosed compound is administered for less than 28, 14, 7, 6, 5, 4, 3, 2 or 1 day. In some embodiments, the disclosed compound is administered continuously and chronically, for example, for the treatment of a chronic condition.

[0208]

[0130] In some embodiments, the compounds of the present disclosure are administered in individual dosage forms. It is known in the art that subject-to-subject variability in the pharmacokinetics of compounds requires individualization of dosing regimens for optimal therapy.

[0209] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions.In specific embodiments, the pharmaceutical compositions are formulated into medicament-usable preparations in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the disclosed compounds.The appropriate formulation depends on the route of administration selected. Any pharma- ceutically acceptable technique, carrier, and excipient may be suitably used to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA, and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999).

[0210]

[0132] Provided herein is a pharmaceutical composition comprising one or more compounds of formula (I)-(Vb) and a pharma- ceutically acceptable carrier. Also provided herein is a pharmaceutical composition comprising one or more compounds selected from the compounds of formula (I)-(Vb) and a pharma- ceutically acceptable diluent, excipient and carrier. In certain embodiments, the described compounds are administered as a pharmaceutical composition in which one or more compounds selected from the compounds of formula (I)-(Vb) are mixed with other active ingredients, such as combination therapy. All combinations of actives specified in the combination therapy section below and throughout this disclosure are encompassed herein. In a specific embodiment, the pharmaceutical composition comprises one or more compounds of formula (I)-(Vb).

[0211]

[0133] Pharmaceutical composition, as used herein, refers to a mixture of one or more compounds selected from the compounds of formulae (I)-(Vb) with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the compound to an organism. In some embodiments, a therapeutically effective amount of one or more compounds selected from the compounds of formulae (I)-(Vb) provided herein is administered in a pharmaceutical composition to a mammal having a disease, disorder or medical condition to be treated. In a specific embodiment, the mammal is a human. In certain embodiments, the therapeutically effective amount varies depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. The compounds described herein are used alone or in combination with one or more therapeutic agents as components of a mixture.

[0212] In one embodiment, one or more compounds selected from the compounds of formula (I)-(Vb) are formulated in an aqueous solution. In a specific embodiment, the aqueous solution is selected from a physiologically compatible buffer, such as, by way of example only, Hanks' solution, Ringer's solution, or physiological saline buffer. In another embodiment, one or more compounds selected from the compounds of formula (I)-(Vb) are formulated for transmucosal administration. In a specific embodiment, the transmucosal formulation comprises a penetrant suitable for penetrating the barrier. In yet another embodiment, where the compounds described herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In a specific embodiment, such solutions comprise physiologically compatible buffers and / or excipients.

[0213] In another embodiment, the compounds described herein are formulated for oral administration.The compounds described herein are formulated by combining active compounds with, for example, pharma-ceutically acceptable carriers or excipients.In various embodiments, the compounds described herein are formulated in oral dosage forms, including, by way of example only, tablets, powders, pills, sugar-coated tablets, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, etc.

[0214] In certain embodiments, pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more of the compounds described herein, optionally comminute the resulting mixture, and if desired, processing the mixture of granules after adding suitable auxiliaries to obtain tablets or sugar-coated tablet cores.Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others, such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.In specific embodiments, disintegrants are optionally added.Disintegrants include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate.

[0215] In one embodiment, oral dosage forms such as pills, capsules or tablets include one or more suitable layers or coatings. In a specific embodiment, a concentrated sugar solution is used to coat the dosage form. The sugar solution optionally contains additional components such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures, by way of example only. Dyes and / or pigments are also optionally added to the coating for identification purposes. In addition, dyes and / or pigments are optionally utilized to characterize different combinations of active compound doses.

[0216]

[0138] In certain embodiments, a therapeutically effective amount of at least one of the compounds described herein is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. In a specific embodiment, the push-fit capsules contain the active ingredient in admixture with one or more fillers. The fillers include, by way of example only, binders such as lactose, starch, and / or lubricants such as talc or magnesium stearate, and optionally, stabilizers. In other embodiments, the soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

[0217] In yet another embodiment, the compounds described herein are formulated for parental injection, including formulations suitable for bolus injection or continuous infusion. In a specific embodiment, the formulation for injection is provided in unit dosage form (e.g., in ampoules) or in multi-dose containers. Optionally, a preservative is added to the injection formulation. In yet another embodiment, the pharmaceutical composition is formulated in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle. The parenteral injection formulation optionally contains formulating agents such as suspending, stabilizing and / or dispersing agents. In a specific embodiment, the pharmaceutical formulation for parenteral administration comprises an aqueous solution of the active compound in water-soluble form. In an additional embodiment, a suspension of one or more compounds selected from the compounds of formula (I)-(Vb) is prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.In certain specific embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compound, allowing the preparation of highly concentrated solutions.Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0218]

[0140] The pharmaceutical composition comprises at least one pharma- ceutically acceptable carrier, diluent or excipient and one or more compounds selected from the compounds of formula (I)-(Vb) as active ingredients. The active ingredients are in free acid or free base form, or in pharma- ceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs) and active metabolites of the compounds having the same type of activity. All tautomers of the compounds described herein are included within the scope of the compounds presented herein. In addition, the compounds described herein encompass unsolvated and solvated forms with pharma- ceutically acceptable solvents such as water, ethanol, etc. The solvated forms of the compounds presented herein are also considered to be disclosed herein. In addition, the pharmaceutical composition optionally comprises other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solubility enhancers, salts for adjusting osmotic pressure, buffers, and / or other therapeutically valuable substances, for example.

[0219]

[0141] The method for preparing a composition comprising the compounds described herein includes formulating the compound with one or more inert pharma- ceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include liquids in which the compound is dissolved, emulsions containing the compound, or liquids containing liposomes, micelles, or nanoparticles containing the compound as disclosed herein. Semi-solid compositions include, but are not limited to, gels, ointments, suspensions, and creams. The forms of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid before use, or suitable as emulsions. These compositions also optionally contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0220]

[0142] In some embodiments, the pharmaceutical composition comprising one or more compounds selected from the compounds of formula (I)-(Vb) is illustratively in the form of a liquid formulation, in which the active agent is present in a liquid, in a suspension, or both. Typically, when the composition is administered as a suspension, a first portion of the active agent is present in a solution, and a second portion of the active agent is present in particulate form in suspension in a liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

[0221] In certain embodiments, aqueous suspension contains one or more polymers as suspending agents.Polymers include water-soluble polymers such as cellulose polymers, e.g. hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers.Certain pharmaceutical compositions described herein include mucoadhesive polymers, e.g., selected from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0222]

[0144] The pharmaceutical composition also optionally comprises a solubilizer to aid in the solubility of one or more compounds selected from the compounds of formula (I)-(Vb). The term "solubilizer" generally includes agents that result in the formation of a micellar or true solution of the agent. Certain acceptable non-ionic surfactants, such as polysorbate 80, are useful as solubilizers, as are ophthalmically acceptable glycols, polyglycols, such as polyethylene glycol 400, and glycol ethers.

[0223]

[0145] In addition, the pharmaceutical composition optionally includes one or more pH adjusting or buffering agents, 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-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amounts required to maintain the pH of the composition within an acceptable range.

[0224]

[0146] The composition also optionally contains one or more salts in the amount required to bring the osmolality of the composition into an acceptable range.Such salts include those with sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0225]

[0147] Other pharmaceutical compositions optionally contain one or more preservatives to inhibit microbial activity.Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0226]

[0148] The composition may contain one or more surfactants for enhancing physical stability or 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 octoxynol 10, octoxynol 40.

[0227]

[0149] The compositions may include, where required, one or more antioxidants to enhance chemical stability. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0228]

[0150] In certain embodiments, the aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.

[0229] In alternative embodiments, other delivery systems for hydrophobic pharmaceutical compounds are used. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also used. In additional embodiments, the compounds described herein are delivered using sustained release systems such as semipermeable matrices of solid hydrophobic polymers containing therapeutic agents. A variety of sustained release materials are useful herein. In some embodiments, sustained release capsules release the compound for several weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization are used.

[0230]

[0152] In certain embodiments, the formulations described herein include one or more antioxidants, metal chelators, thiol-containing compounds and / or other general stabilizing agents. 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) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0231] In some embodiments, the concentration of one or more compounds selected from the compounds of Formulae (I)-(Vb) provided in the pharmaceutical composition is 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%, 0.07%, 0.06%, 0.05%, 0.04%, 0.0 3%, 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 the compounds of Formulae (I)-(Vb) in the pharmaceutical composition is between about any two of the values ​​recited in the preceding sentence, such as between about 2-70 w / w%, 3.5-80 w / w%, 1-30 w / w%, etc.

[0232]

[0154] In some embodiments, the concentration of one or more compounds selected from the compounds of Formulae (I)-(Vb) provided in the pharmaceutical composition of the present 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%, from about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, about 1% to about 10% w / w, w / v or v / v.

[0233]

[0155] In some embodiments, the amount of one or more compounds selected from the compounds of Formulae (I)-(Vb) provided in the pharmaceutical composition of the present disclosure is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

[0234]

[0156] In some embodiments, the amount of one or more compounds selected from the compounds of Formulae (I)-(Vb) provided in the pharmaceutical composition of the present disclosure is within the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

[0235] Packaging materials for use in packaging the pharmaceutical compositions described herein 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 intended mode of administration and treatment. For example, a container contains one or more compounds described herein, optionally in a composition or in combination with another agent as disclosed herein. The container optionally has a sterile access port (e.g., the container is an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). Such a kit optionally contains the compound with an identifying description or label or instructions related to its use in the methods described herein.

[0236] For example, a kit typically includes one or more additional containers, each with one or more of various materials (optionally in concentrated form, reagents, and / or devices, etc.) that are desirable from a commercial and user standpoint for the use of the compounds described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carriers that contain the contents and / or instructions for use, packaging, containers, vial and / or tube labels, and package inserts with instructions for use. Typically, a set of instructions will also be included. The label is optionally on or associated with the container. For example, the label is on the container when the letters, numbers or other characters that form the label are attached, molded or etched into the container itself, and the label is associated with the container when they are present, for example, as a package insert, in a receptacle or carrier that also contains the container. In addition, the label is used to indicate that the contents are to be used for a specific therapeutic application. In addition, the label indicates instructions for the use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device that contains one or more unit dosage forms containing the compound provided herein. The pack contains, for example, metal or plastic foil, such as a blister pack. Alternatively, the pack or dispenser device is accompanied by an administration instruction. Alternatively, the pack or dispenser is accompanied by a notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects the approval by the agency of the form of the drug for human or veterinary administration. Such notice is, for example, the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. In some embodiments, a composition containing the compound provided herein that is formulated in a compatible pharmaceutical carrier is prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0237] As mentioned above, the compounds and compositions of the present disclosure will find utility in a wide range of protein kinase-mediated diseases and conditions, including kinase-mediated diseases and conditions. Such diseases include, by way of example and without limitation, lung cancer, NSCLC (non-small cell lung cancer), oat cell carcinoma, bone cancer, pancreatic cancer, skin cancer, dermatofibrosarcoma protuberans, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, anal 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 intestine cancer, endocrine system cancer (e.g., thyroid, pancreatic, parathyroid cancer, and thyroid cancer). cancer of the bladder, kidney, or adrenal gland, sarcoma of soft tissue, urethral cancer, penile cancer, prostate cancer (especially hormone resistant), chronic or acute leukemia, solid tumors of childhood, hypereosinophilia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer (e.g., renal cell carcinoma, renal pelvis carcinoma), childhood malignancies, neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumor, medulloblastoma, brain stem glioma or pituitary adenoma), Barrett's esophagus (premalignant syndrome), neoplastic skin diseases, cancers such as psoriasis, mycoses fungoides and benign prostatic hyperplasia, diabetes-related diseases such as diabetic retinopathy, retinal ischemia and retinal neovascularization, cirrhosis of the liver, angiogenesis, cardiovascular diseases such as atherosclerosis, immune diseases such as autoimmune diseases, and renal diseases.

[0238]

[0160] In some embodiments, the pharmaceutical composition comprises the compound described above and a pharma- ceutically acceptable carrier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine, including, for example, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent or emulsifier.

[0239] In some embodiments, a method of treating a disease or disorder, the method comprising administering an effective amount of a compound or pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the disease or disorder 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 hematological malignancy, such as acute myeloid leukemia. In some other specific embodiments, the disease or disorder is an autoimmune or inflammatory disease.

[0240]

[0162] In one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the compounds of formulae (I)-(Vb) are converted to indirubin in vitro in simulated or human intestinal fluid at 37°C within 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.

[0241]

[0163] In one embodiment, a pharmaceutical composition comprising a compound of Formulae (I)-(Vb) and a second therapeutic agent.

[0242] In one embodiment, the compound or pharmaceutical composition of Formula (I)-(Vb) is for use in treating an immune disease, an autoimmune disease, or an inflammatory disease. In another embodiment, the compound or pharmaceutical composition of Formula (I)-(Vb) is for use in treating an inflammatory or immune disease.

[0243] In one embodiment, a method for treating ulcerative colitis or inflammatory bowel disease is contemplated, comprising administering a compound of Formula (I)-(Vb) or a pharmaceutical composition to a subject in need thereof. In another embodiment, a method for treating hidradentitis suppurativa or a type of psoriasis is contemplated, wherein the treatment comprises administering a compound of Formula (I)-(Vb) or a pharmaceutical composition comprising a compound of Formula (I)-(Vb) to a subject in need thereof. In an embodiment, the type of psoriasis is selected from psoriasis vulgaris, inverse psoriasis, erythrodermic psoriasis, guttate psoriasis, and pustular psoriasis. In another embodiment, a method for treating an inflammatory disease is contemplated, comprising administering a compound of Formula (I)-(Vb) or a pharmaceutical composition to a subject in need thereof.

[0244] In one embodiment, the subject is an animal. In another embodiment, the subject is a human. In some embodiments, the subject has an inflammatory disease or an immune disease. For example, in some embodiments, the disease is gastrointestinal inflammatory disease, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 and type 2 diabetes, or vitiligo. In other embodiments, the disease is ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, pouchitis, mucositis, cancer, fibrotic disease, or graft-versus-host disease.

[0245] In one embodiment, a method for administering indirubin to a subject, comprising administering a compound of formula (I)-(Vb), wherein the compound is converted to indirubin after administering. In some embodiments, the compound of formula (I)-(Vb) is hydrolyzed to indirubin.

[0246] In some embodiments, the AhR agonist prodrug has a solubility in simulated intestinal fluid that is at least about 1.5, 1.75, 2 or 3 times higher than the solubility of synthetic indirubin in the same fluid at the same time and temperature (e.g., 37°C). In embodiments, the AhR agonist prodrug has a solubility in simulated intestinal fluid that is at least about 10, 50, 100, 200 or 300 times higher than the solubility of synthetic indirubin in the same fluid at the same time and temperature (e.g., 37°C). In other embodiments, the compound of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) or (Vb) has a solubility in simulated intestinal fluid that increases by at least about 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 AhR agonist prodrug (such as any of the compounds of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb)) produces a lower amount of indirubin in lung tissue after oral administration than an equimolar amount of synthetic indirubin.In some embodiments, the AhR agonist prodrug (such as any of the compounds of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb)) produces a lower amount of indirubin in plasma after oral administration than an equimolar amount of synthetic indirubin. In some embodiments, the AhR agonist prodrug (such as any of the compounds of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb)) produces lower amounts of indirubin in plasma and / or lung tissue after oral administration than an equimolar amount of synthetic indirubin, and higher amounts of indirubin in the colon.In other embodiments, the AhR agonist prodrug (such as any of the compounds of formula (I), (Ia), (Ib), (II), (III), (IIIa), (IV), (Iva), (IVb), (IVc), (V), (Va) and (Vb)) provides increased skin penetration (permeation) compared to the skin penetration of synthetic indurbin when permeation of skin flux or permeation is measured in vitro from a formulation that is identical in all respects except for the prodrug or drug compound present in the formulation at an equimolar amount of indirubin.

[0247] B. Preparation method

[0169] In embodiments, the compounds disclosed herein can be prepared using conventional means known in the art. Illustrative and non-limiting examples of the preparation methods of the disclosed compounds are contained herein.

[0248] In some embodiments, class A molecules can be prepared using known synthesis of indirubin using radiolabeled starting materials.Other radiolabeled compounds (e.g., radiolabeled versions of class B, C or D compounds) can be prepared using the methods presented herein using radiolabeled precursors.The following examples are illustrative in nature and are not intended to be limiting in any way.

[0249]

[0171] In some embodiments, class D compounds are prepared starting from indirubin. In these embodiments, indirubin is reacted with chloroformates, followed by nucleophilic attack by a selected nucleophile. As non-limiting examples, the results include carbamates, ureas, and amide derivatives. Any base compatible with nucleophilic attack on chloroformates is contemplated in the technology of the present invention. Furthermore, any chloroformates known in the art to be susceptible to subsequent nucleophilic attack are contemplated in the technology of the present invention. Preferred nucleophiles include amines, alcohols, amides, carboxylic acids, and thiols, among others. This preparation method may conform to the following scheme:

[0250] [ka]

[0251]

[0172] In other embodiments, the chloroformates may be selected to arrive at final products that are not further attacked by nucleophiles, or may be further derivatized. Any base compatible with nucleophilic attack on chloroformates is contemplated in the technology of the present invention. Preferred chloroformates include alkyl chloroformates, aryl chloroformates, cycloalkyl chloroformates, alkyl heterocyclic chloroformates, and heteroaryl chloroformates. In these embodiments, the resulting derivatives are carbamate derivatives as shown in the following scheme:

[0252] [ka]

[0253] In other embodiments, indirubin reacts with selected acyl chloride. As a non-limiting example, the resulting derivative includes amide derivative. Preferred acyl chlorides include, among others, alkyl acyl chloride, aryl acyl chloride, cycloalkyl acyl chloride, heterocycloalkyl acyl chloride, and heteroaryl acyl chloride. This preparation method may be based on the following scheme:

[0254] [ka]

[0255]

[0174] In another embodiment, indirubin reacts with formaldehyde and a selected nucleophile. As a non-limiting example, the resulting derivatives include aminal and hemiaminal derivatives. Preferred nucleophiles include, inter alia, alcohols and amines. This preparation method may conform to the following scheme:

[0256] [ka]

[0257] In a further step of these embodiments, the alcohol derivative may be further derivatized.Other embodiments are disclosed in the following scheme, but other known chemical modifications in the art are contemplated.Alternatively, in other embodiments, the alcohol derivative of indirubin prepared by other means may be used as in the following scheme:

[0258] [ka]

[0259] In these embodiments, the alcohol indirubin derivative X is reacted with SOCl in the preparation for nucleophilic attack at the carbon. 2 Selected nucleophiles are reacted with the indirubin chloride derivatives to form other indirubin derivatives, including but not limited to hemiaminals and aminals. In a specific embodiment, the hemiaminals can be organophosphates, as in the following scheme:

[0260] [ka]

[0261]

[0177] If desired, or to test the hydrolysis rate of indirubin derivatives in physiological models (e.g., intestinal fluid model, gastric fluid model) to prove the function of the derivatives as prodrugs, indirubin derivatives may be converted back to indirubin.The following scheme demonstrates one such hydrolysis method:

[0262] [ka]

[0263] III.How to use Indirubin has been shown to act as a strong agonist for AhR, and has potential therapeutic activity in treating proliferative diseases, cancer, fibrotic diseases, inflammatory diseases and autoimmune diseases.In some embodiments, the indirubin derivatives of the present disclosure may have altered therapeutic or agonist efficacy when compared with indirubin itself.The level of either efficacy may increase, decrease, or remain substantially the same.In preferred embodiments, efficacy is increased over indirubin.

[0264] The pharmacokinetic (PK) profile of the compound is an aspect to consider. These can be evaluated as the PK profile in the tissue of interest or as the plasma PK profile, which can be representative of the total systemic exposure. As an effector of the PK profile, the indirubin derivative can be metabolized at a different rate than indirubin. The metabolic rate in the prodrug form can be faster, slower, or substantially the same. The alteration of metabolism can lead to differences in PK profile, safety, efficacy, or dose and dosing regimen to achieve the intended therapeutic effect. Alternatively, the indirubin derivative can have differences in safety or efficacy compared to indirubin, independent of metabolic rate.

[0265] Another effector of PK profile is the dissolution rate of indirubin derivative. The dissolution rate of indirubin derivative may be faster, slower, or substantially the same as indirubin. In a preferred embodiment, the concentration of indirubin in solution may be greater when a prodrug of indirubin is used, compared to a similar molar dose of indirubin. Due to the disruption of hydrogen bonds, the prodrug may have enhanced dissolution, resulting in a greater concentration of the prodrug in solution. If the prodrug is converted to indirubin when in solution, the concentration of indirubin in solution may be increased, compared to a similar molar dose of indirubin administered in non-prodrug form.

[0266] Indirubin prodrugs may be converted to indirubin in vivo at different rates depending on the route of administration, drug product and administered dose. The conversion method may be based on chemical or enzymatic conversion. If the conversion of indirubin prodrugs to indirubin is chemical (e.g., there is no enzymatic activity), it may be due to pH, temperature, light, the presence of water, oxygen, or other chemicals or conditions found in the organ or tissue of interest. 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 less than or equal to 7, including values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0267] In other cases, conversion may require exposure to water or other body fluids, which may be inhibited by protecting the prodrug by encapsulation, tableting, or formulation with hydrophobic excipients. In such cases, protection from body fluids may limit the sites of exposure to the prodrug / derivative / indirubin and minimize systemic absorption. In one example, the prodrug may be formulated in a delayed release capsule that will minimize exposure of the indirubin in the oral mucosa, throat, stomach, and / or small intestine. This capsule or tablet or encapsulation may be done with a pH-sensitive polymer, such as a copolymer of esters of acrylic and methacrylic acid or hydroxypropyl methylcellulose phthalate.

[0268]

[0183] When the conversion of indirubin derivative to indirubin is enzymatic, it can be by CYPp450s, esterase, carboxylesterase, azoreductase, carboxypeptidase, alkaline phosphatase, nitroreductase, acetyl or butylcholinesterase or any other naturally occurring enzyme.These enzymes may be highly expressed in one tissue or site, besides causing different degrees of exposure to indirubin.

[0269] Indirubin derivatives may also feature changes in other physical, chemical or biological properties, such as solubility, dissolving ability, color, tissue penetration, transporter influx or efflux, metabolism and / or long-term stability.Better solubility or dissolving ability can result in greater local exposure to indirubin, reducing the amount of drug required to activate AhR in target tissue.Greater local exposure can result in reduced cost, reduced systemic exposure and / or minimized side effects.

[0270]

[0185] The indirubin derivatives of the present disclosure may feature, inter alia, advantages during certain types of formulation, micronization, or formation of solid amorphous dispersions.

[0271] Prodrugs can be formulated more easily than indirubin, and can lead to site-specific activity or increased systemic exposure.For example, the low solubility of indirubin can preclude substantial doses of aqueous topical formulations.Prodrugs with better aqueous solubility allow for higher drug loading in dosage forms such as solutions, suspensions, ointments, creams and gels.

[0272] The compounds described herein as derivatives of indirubin are prodrugs that are converted to indirubin. An indirubin prodrug can be studied in vitro to see if it is converted to indirubin. For example, an indirubin prodrug can be added to a solution and evaluated after a period of time to determine the relative conversion to indirubin. This can be done, for example, in water, ethanol, simulated intestinal fluid (e.g., fasted-state simulated intestinal fluid (FaSSIF)), simulated gastric fluid (e.g., fasted-state simulated gastric fluid (FaSSGF)), isolated intestinal fluid, isolated gastric fluid, plasma, blood, saliva, or other solutions. The solutions can have various additives, including microorganisms, fecal samples, enzymes, or salts. These solutions can be buffered and / or their pH can be adjusted. This can be done at various temperatures, including body temperature. The amount of indirubin present in the solution can be determined by LC-MS, HPLC, or other laboratory techniques.

[0273] Indirubin derivatives may be studied in vivo, such as in laboratory animals. The animals may be healthy or severe as models of disease. The disease model may be a model of inflammatory disease, such as the dextran sulfate sodium model of colitis. In this model, the indirubin derivative may be administered prophylactically or therapeutically to evaluate its safety and / or efficacy. It may be administered by any route, such as orally, topically or via injection. Administration of the indirubin derivative may reduce weight loss, improve disease activity index score, reduce bloody stool, improve stool consistency, reduce number of stools, increase colon length, reduce inflammation, improve intestinal barrier function, increase intestinal regulatory T cells, reduce intestinal Th17 cells, alter gut flora, reduce proinflammatory cytokine expression, increase anti-inflammatory cytokine expression or reduce proinflammatory chemokine expression.

[0274] Indirubin derivatives can be studied to see if they are AhR agonists. This can be done in vivo or in vitro. This can be done by looking at AhR translocation to the nucleus, by looking for AhR protein complexes, by looking for AhR transcription or translation activity. AhR activity can be assessed by looking for the expression of mRNA or proteins known to be regulated by AhR. These may include metabolic enzymes, including CYP1A1 or CYP1A2. This can be done by looking for the light produced by luciferase acting on luciferin, where luciferase is under the control of a genetic response element that responds to dimerized AhR / ARNT.

[0275]

[0190] Disease treatment may benefit in some embodiments from a local pharmacokinetic profile that differs from the systemic pharmacokinetic profile. For example, to treat blood cancers such as chronic myeloid leukemia and to treat certain autoimmune diseases that manifest systemically or in the central nervous system, low concentrations of active agent in the gastrointestinal tract and lungs but high plasma concentrations are desired. To treat skin diseases such as hidradenitis suppurativa, acne, psoriasis or atopic dermatitis, high skin levels but low plasma levels are desired. To treat idiopathic pulmonary fibrosis, low gastrointestinal and plasma exposure to the drug and higher lung exposure to the drug are desired. To treat intestinal diseases such as ulcerative colitis, a substantial proportion of the administered drug in the lower gastrointestinal tract is desired, along with a smaller amount in plasma and lung tissue. The compounds disclosed herein generate indirubin in the colon and / or lower gastrointestinal tract following oral ingestion of the compound, 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).

[0276]

[0191] Thus, provided is a method for treating a disease or condition associated with the activity of aryl hydrocarbon receptor, comprising administering a compound as disclosed herein or a pharmaceutical composition thereof to a subject in need thereof in an amount sufficient to provide an amount of indirubin to modulate aryl hydrocarbon receptor. As can be seen, the compound after oral administration produces indirubin at a therapeutic dose or concentration of indirubin. As mentioned, the compounds described herein release indirubin preferentially in the lower gastrointestinal tract and preferably in the colon, with minimal or no significant uptake or absorption of indirubin into the bloodstream. Such methods include therapeutic and protective (prophylactic) methods. In some embodiments, the disease or condition is associated with a reduction in the activity of aryl hydrocarbon receptor. In some embodiments, the disease or condition is associated with a reduction in the activity of aryl hydrocarbon receptor compared to the activity of aryl hydrocarbon receptor in normal (i.e., non-diseased) cells.

[0277] Studies were conducted as set forth in Examples 1-9 below in support of this disclosure. Example 1 details the preparation of the indirubin derivative compounds (indirubin prodrug compounds) described herein. Example 2 shows results from a study of the stability of the indicated indirubin prodrug compounds in fasted-state simulated intestinal fluid and fasted-state simulated gastric fluid. In this study, test compounds were placed in the simulated fluid at 37° C. for 24 hours. The fluid was then analyzed via LCMS for the amount of test compound (or starting material, “SM”) and the amount of indirubin. Table 2-1 in Example 2 reports the percentage of starting material and the percentage of indirubin in each of the simulated fluids, providing a measure of the stability or ease of conversion of the prodrug compounds to indirubin. The study demonstrates an in vitro approach to determine whether and / or to what extent a prodrug produces indirubin.

[0278]

[0193] Examples 3-4 describe studies on the time course or rate of conversion of indirubin prodrug compounds to indirubin. In these studies, test compounds were placed in fasted-state simulated intestinal fluid (Example 3) or fasted-state simulated gastric fluid (Example 4) at controlled temperatures, and the ratio of starting material (test compound), indirubin methylene alcohol, and indirubin was measured as a function of time over a 24-hour period. The results are shown in Tables 3-1 and 4-1 in Examples 3 and 4, respectively. As can be seen, prodrug compounds can be tailored to produce indirubin at different rates. In one embodiment, an AhR agonist prodrug compound is provided, wherein the prodrug compound generates a therapeutically active AhR agonist compound by conversion of 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 fasted state simulated intestinal fluid or fasted state simulated gastric fluid at 37° C. In another embodiment, an indirubin prodrug compound is provided, wherein the indirubin prodrug compound generates indirubin by conversion of the indirubin prodrug compound to indirubin, wherein at least about 60%, 70%, 75%, 80%, 85%, 90% or 95% of the indirubin prodrug compound is converted to indirubin within 24 hours in fasted state simulated intestinal fluid or fasted state simulated gastric fluid at 37° C.

[0279] Example 5 describes a study of the AhR activity of an exemplary indirubin derivative, Compound 8. In this study, solutions of Compound 8, indirubin, 2,3,7,8-tetrachlorodibenzodioxin (TCDD) or (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio) (the latter two as positive controls) were added to human Huh7 cells or rat H4IIE cell lines spiked with native AhR and AhR / arnt-luciferase reporter vector. After incubation, luciferase detection reagent was added and activity measurements were taken in relative luminescence units. The results are shown in Figures 1A-1B.

[0280]

[0195] Figures 1A-1B are plots showing the activation of AhR in human Huh7 cells (Figure 1A) and rat H4IIE cells (Figure 1B) in the presence of compound 8 (triangles), indirubin (squares), TCDD (circles; positive control) and MeBio (dashed line; positive control) at nM concentrations. EC 50 EC for indirubin 50 The EC for indirubin in rat H4IIE cells was approximately 20% greater than that in 50 The EC for compound 8 in rat H4IIE cells was estimated to be 654 nM (estimated by the absence of an upper plateau). 50 was 25.62 nM. The data demonstrate that compound 8 activates AhR to an equal or greater extent than equimolar synthetic indirubin, and at the same or lower concentration. Given the similarity between the curves of compound 8 and indirubin, compound 8 will essentially be completely converted to indirubin (e.g., at least about 75%, 80%, or 90% or 100% conversion) within about 1, 2 or 24 hours, and therefore will have a similar ability to activate AhR in this assay.

[0281]

[0196] In Example 6, a study is disclosed in which mice with acute colitis are induced and treated with an indirubin prodrug, exemplified by compound 8. Mice were treated with oral administration of compound 8 or with distilled water twice a day. Body weight and Disease Activity Index (DAI) soring were recorded daily to assess colitis. The results are shown in Figure 2.

[0282]

[0197] Figure 2 is a plot of the disease activity index (see Table 6-1 in Example 6) as 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). Mice treated with indirubin prodrugs had lower disease activity indexes than mice treated with placebo (vehicle only) over the 9-day test period. As an example of the indirubin derivatives described herein, compound 8 was demonstrated to be converted to indirubin after oral administration and provide a therapeutic effect.

[0283]

[0198] Example 7 details another study using a mouse model of ulcerative colitis. In this study, mice with acute colitis were treated by oral gavage with distilled water or synthetic indirubin, compound 9, compound 10, compound 26, compound 40, compound 41. The results are shown in Figures 3A-3C.

[0284] 3A-3B are graphs showing percent of initial body weight (FIG. 3A) and Disease Activity Index (DAI) scores (FIG. 3B) as a function of time in days for mice treated twice daily for 9 days with placebo (filled circle), synthetic indirubin (filled square), compound 9 (filled triangle), compound 10 (inverted triangle), compound 26 (open circle), compound 40 (open square) or compound 41 (open triangle). Doses are in milligrams per kilogram (mpk) and are designed to be equimolar to indirubin. All of the test compounds provided efficacy, with compound 41 being more effective than an equimolar amount of indirubin. Thus, in certain embodiments, the indirubin derivatives after oral administration to subjects provide a therapeutic effect that 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.

[0285]

[0200] Figure 3C is a bar graph showing the average colon length (in cm with standard error of the mean (SEM)) of mice with ulcerative colitis and treated as described in Example 7. Colon length correlates with inflammation, where colons with more inflammation are shorter than those with less or no inflammation. The data in Figure 3C show that mice treated with vehicle only have shorter colons than mice treated with synthetic indirubin (positive control). Mice treated with indirubin prodrugs exhibited significantly longer colons than mice treated with vehicle only, indicating less colonic inflammation in prodrug-treated mice.

[0286]

[0201] In Example 8, the study of animals treated in Example 7 is disclosed to analyze the distribution of indirubin in plasma, colon and lung. Table 8-1 reports the average indirubin levels in plasma, colon and lung for animals treated with vehicle, 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 colon compared to the levels of indirubin in lung and / or plasma. For example, the ratio of indirubin in colon to plasma for compound 40 was about 1003 (Table 8-2). The ratio of indirubin in colon to plasma for compound 9 was about 824 (Table 8-2). The ratio of indirubin in colon to plasma for compound 41 was about 170 (Table 8-2). Thus, in one embodiment, the indirubin derivative after oral administration to a subject provides an amount of indirubin in the colon that is at least 125, 150, 170, 250 or 300 times higher than the amount of indirubin in plasma at 4, 6, 8 or 12 hours after administration for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days.In another embodiment, the indirubin derivative after oral administration to a subject provides an amount of indirubin in plasma that is less than an equimolar amount of orally administered synthetic indirubin at about 6 hours after administration.

[0287] The ratio of indirubin in the colon to lung for compound 40 is about 4390 (Table 8-3). The ratio of indirubin in the colon to lung for compound 9 is about 217 (Table 8-3). The ratio of indirubin in the colon to lung for compound 41 is about 1.6 (Table 8-3). Thus, in some embodiments, the indirubin derivative after oral administration to subjects provides the amount of indirubin in the colon that is at least 1.3, 1.4, 1.5, 10 times higher than the amount of indirubin in the lung after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of treatment.

[0288] In Example 9, the solubility of exemplary indirubin prodrugs, Compound 8, Compound 9 and Compound 10, was measured in simulated fluid, such as simulated fasting gastric fluid (FaSSGF) and simulated fasting intestinal fluid (FaSSIF).For comparison, the solubility of synthetic indirubin and blue-green algae was also measured.Compound 9 and Compound 10 were both soluble in simulated fluid (Table 9-1).

[0289] In one embodiment, the compounds disclosed herein are intended for use in treating immune disease, autoimmune disease or inflammatory disease.In one embodiment, the compounds are intended for use in treating ulcerative colitis or inflammatory bowel disease.In other embodiments, the compounds are intended for use in treating gastrointestinal inflammatory disease, ankylosing spondylitis, atopic dermatitis, Alzheimer's disease, celiac disease, Graves' disease, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 and type 2 diabetes or vitiligo.In other embodiments, the compounds are intended for use in treating ulcerative colitis, Crohn's disease, celiac disease, inflammatory bowel disease, gastrointestinal graft-versus-host disease, pouchitis or mucositis.

[0290] Derivatives of AhR agonists may differ in many ways from their parent compounds, resulting in improved and desirable prosperities that make them more suitable for use in treating or preventing disease. For the treatment of gastrointestinal diseases such as ulcerative colitis, an ideal oral AhR agonist would provide AhR activity in the gastrointestinal tract without significant systemic exposure and / or systemic AhR activation. An ideal oral AhR agonist would also be rapidly cleared in the gastrointestinal tract, resulting in non-persistent activation of AhR outside the gastrointestinal tract. For the treatment of dermatological diseases such as hidradenitis suppurativa or psoriasis, a topically applied AhR agonist would provide AhR activity in the skin without significant systemic exposure and / or systemic AhR activation. A topical AhR agonist would also be rapidly cleared in the skin, resulting in non-persistent activation of AhR outside the skin. These strategies described herein for derivatives of indirubin can be used with any known AhR agonist, including, but not limited to, indigo, meisoindigo, linomide (roquinimex), 6-formylindolo[3,2-b]carbazole (FICZ) and laquinimod, tapinarof, 4-(4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)pyrimidin-2-amine, compounds disclosed in US2021 / 0147390A1, and the like. EXAMPLES

[0291] IV. Working Examples

[0206] The following examples are illustrative in nature and are not intended to be limiting in any way.

[0292] Example 1 Preparation of indirubin derivative compounds

[0293] [ka] (Z)-1'-(morpholine-4-carbonyl)-[2,3'-bindolinylidene]-2',3-dione To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17.0 mL) at 0° C. under nitrogen atmosphere, sodium bis(trimethylsilyl)amide / NaHMDS (1 M in THF) (1.50 equiv., 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8.4 mL) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h; TLC analysis indicted complete consumption of SM and generation of active species. Morpholine (4.00 equiv, 0.27 mL, 3.05 mmol) was added dropwise and the reaction mixture was stirred for 67 h. The reaction mixture was diluted with CH 2 Cl 2 The mixture was diluted with 100% ethyl acetate (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) and the organics were combined and dried (Na 2 SO 4 ) and concentrated in vacuo. The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of EtOAc in hexanes (5% to 60%; v / v) to give the desired product (3Z)-1-(morpholine-4-carbonyl)-3-(3-oxoindolin-2-ylidene)indolin-2-one (122 mg, 0.322 mmol, 42.19% yield) as a dark purple solid.

[0208] UPLC-MS analysis (sample reference number: 142242-2): (4 minutes, basic): rt=1.86 minutes, m / z=376.3[M+H]+, purity 99%. 1H NMR analysis (sample reference number: 142242-2,400MHz, CDCl 3)δ10.37(s,1H),8.88(d,J=7.9Hz,1H),7.69(d,J=7.6Hz,1H),7.47(dd,J=7.7,7.7Hz,1H ),7.28(dd,J=7.7,7.7Hz,1H),7.21-7.11(m,2H),7.03-6.91(m,2H),3.98-3.24(m,8H).

[0294] [ka]

[0295] (Z)-2',3-Dioxo-N-(tetrahydro-2H-pyran-4-yl)-[2,3'-bindolinylidene]-1'-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17.0 mL) at 0° C. under nitrogen atmosphere, sodium bis(trimethylsilyl)amide / NaHMDS (1M in THF) (1.50 equiv., 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8.4 mL) at 0° C. was added quickly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h; 4-aminotetrahydropyran (4.00 equiv., 0.32 mL, 3.05 mmol) was added, and the reaction mixture was stirred for 1 h before being concentrated in vacuo and stored in a freezer. Methanol (10.0 mL) was added to the reaction mixture, and then the reaction mixture was concentrated in vacuo. The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of EtOAc in iso-hexane (5% to 80%; v / v) to give the desired product. LCMS analysis indicated that the product was contaminated with an unidentified impurity, so the product was repurified; the product was then purified by HCl. 2 Cl 2 (150 mL) and dissolved in NaHCO 3 (2×50.0 mL) and brine (50.0 mL), dried (Na 2SO 4 ), then CH 2 Cl 2 Purification by automated column chromatography on silica (40 g cartridge) eluting with a gradient of methanol (0-15%) in water afforded the desired product (3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)-N-tetrahydropyran-4-yl-indoline-1-carboxamide (277 mg, 0.676 mmol, 88.62% yield).

[0210] UPLC-MS analysis (sample reference number: 142295-1) (4 minutes, basic): rt=2.14 minutes, m / z=390.3[M+H]+, purity 95%. 1H NMR analysis (sample reference number: 142295-1) (400MHz, CDCl 3 )δ10.41(s,1H),8.92(d,J=7.9Hz,1H),8.72(d,J=7.4Hz,1H),8.27(d,J=8.2Hz ,1H),7.70(d,J=7.6Hz,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).

[0296] [ka]

[0297] (Z)-1'-Undecanoyl-[2,3'-bindolinylidene]-2',3-dione To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17 mL) at 0° C. under nitrogen atmosphere was added sodium bis(trimethylsilyl)amide / NaHMDS (1 M in THF) (1.05 equiv., 0.80 mL, 0.801 mmol) dropwise. The reaction mixture was stirred for 10 min, then undecanoyl chloride (1.20 equiv., 0.20 mL, 0.915 mmol) was added and the reaction mixture was allowed to warm to room temperature and stirred for 2 h before being concentrated in vacuo. CH 2 Cl 2 The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH (0% to 10%; v / v) to give the desired product (3Z)-3-(3-oxoindolin-2-ylidene)-1-undecanoyl-indolin-2-one (298 mg, 0.664 mmol, 87.13% yield) as a purple solid.

[0212] UPLC-MS analysis (sample reference number: 142535-2) (4 minutes, basic): rt=3.08 minutes, m / z=431.4[M+H]+, purity 96%. 1H NMR analysis (sample reference number: 142535-2) (400MHz, CDCl 3 )δ10.50(s,1H),8.95(d,J=7.6Hz,1H),8.24(d,J=8.2Hz,1H),7.68(d,J=7.6Hz,1H),7.46(t,J=7.7Hz,1H),7.29(t,J=7.6Hz,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).

[0298] [ka]

[0299] (Z)-N-(2-morpholinoethyl)-2',3-dioxo-[2,3'-bindolinylidene]-1'-carboxamide

[0213] To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17.0 mL) at 0°C under a nitrogen atmosphere was added sodium bis(trimethylsilyl)amide / NaHMDS (1 M in THF) (1.50 equiv., 1.1 mL, 1.14 mmol); the reaction mixture was stirred for 10 minutes, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (6.00 mL) was added and the reaction mixture was stirred for 1 hour and allowed to warm to room temperature. A solution of N-(2-aminoethyl)morpholine (2.50 equiv, 0.32 mL, 1.91 mmol) in THF (2.40 mL) was added and the reaction was stirred for 1 h before being concentrated in vacuo and stored in the freezer. 2 Cl 2 The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH in water (0% to 5%; v / v) to give the desired product (3Z)-N-(2-morpholinoethyl)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (210 mg, 0.487 mmol, 63.83% yield) as a dark purple solid.

[0214] UPLC-MS analysis (sample reference number: 143019-2) (4 minutes, basic): rt=2.02 minutes, m / z=419.4[M+H]+, purity 97%. 1H NMR analysis (sample reference number: 143019-2) (400MHz, CDCl 3 )δ10.37(s,1H),8.94-8.78(m,2H),8.25(d,1H),7.67(d,J=7.7Hz,1H),7.46(dd,J=7.7,7.7Hz,1H),7.28(dd,J= 7.9,7.9Hz,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).

[0300] [ka]

[0301] (Z)-N-(4-(dimethylamino)butyl)-2',3-dioxo-[2,3'-bindolinylidene]-1'-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17.0 mL) at 0° C. under nitrogen atmosphere, sodium bis(trimethylsilyl)amide / NaHMDS (1M in THF) (1.50 equiv., 1.1 mL, 1.14 mmol) was added; the reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 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; N′,N′-dimethylbutane-1,4-diamine (2.50 equiv., 0.26 mL, 1.91 mmol) was added and the reaction mixture was stirred for 1 h. The crude reaction mixture was concentrated in vacuo and then diluted with CH 2 Cl 2 The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH in water (0% to 20%; v / v) to give the desired product (3Z)-N-[4-(dimethylamino)butyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (270 mg, 0.648 mmol, 84.91% yield) as a dark purple crystalline solid.

[0216] UPLC-MS analysis (sample reference number: 143194-1) (4 minutes, basic): rt=2.05 minutes, m / z=405.4[M+H]+, purity 97%. 1H NMR analysis (sample reference number: 143194-1) (400MHz, CDCl 3)δ10.53(s,1H),8.98(d,J=7.9Hz,1H),8.87-8.79(m,1H),8.32(d,J=8.2Hz,1H),7.76(d,J=7.7Hz,1H),7.55(dd,J=7.7,7.7Hz,1H),7.36(d d,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).

[0302] [ka]

[0303] tert-Butyl (Z)-(2-(2',3-dioxo-[2,3'-biindoline-1'-yl-carboxamido)ethyl)(methyl)carbamate To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17.0 mL) at 0° C. under nitrogen atmosphere, sodium bis(trimethylsilyl)amide / NaHMDS (1M in THF) (1.50 equiv., 1.10 mL, 1.14 mmol) was added; the reaction mixture was stirred for 10 minutes, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8.40 mL) was added, the reaction was allowed to warm to room temperature and stirred for 30 minutes. tert-Butyl N-(2-aminoethyl)-N-methyl-carbamate (2.50 equiv., 0.34 mL, 1.91 mmol) was added, and the reaction mixture was stirred for 1 hour. CH 2 Cl 2The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH in water (0% to 15%; v / v) to give the desired product tert-butyl N-methyl-N-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]carbamate (551 mg, 0.774 mmol, 101.54% yield) as a dark purple gummy oil. The product of the reaction was used without further purification.

[0304] [ka]

[0305] (Z)-2-(2',3-dioxo-[2,3'-biindoline-1'-yl]-1'-carboxamido)-N-methylethane-1-aluminum chloride To a solution of tert-butyl N-methyl-N-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]carbamate (1.00 equiv., 551 mg, 0.774 mmol) in DCM (12.0 mL) at room temperature was added hydrogen chloride solution (4 M in 1,4-dioxane) (4.00 equiv., 1.20 mL, 4.80 mmol); the reaction mixture was stirred for 22 h. The reaction mixture was filtered and the filter cake was separated by HCl. 2 Cl 2 and ether to give the desired product methyl-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]ammonium chloride (264 mg, 0.662 mmol, 85.47% yield) as a dark purple solid. UPLC-MS analysis (sample reference number: 143976-1) (4 min, basic): rt = 1.79 min, m / z = 363.2 [M+H] +, 100% purity (mass corresponds to product without chloride anion). 1H NMR analysis (sample reference 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).

[0306] [ka]

[0307] (Z)-1'-((4-(dimethylamino)piperidin-1-yl)methyl)-[2,3'-bindolinylidene]-2',3-dione A solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol), 4-(dimethylamino)piperidine (1.95 equiv., 0.17 mL, 1.49 mmol) and formaldehyde (37% in water) (1.50 equiv., 0.085 mL, 1.14 mmol) in THF (7.6 mL) was heated at 55° C. for 20 h. The reaction mixture was concentrated in vacuo and the solid was washed with a minimum of hexane to yield the desired product (3Z)-1-[[4-(dimethylamino)-1-piperidyl]methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one (271 mg, 0.673 mmol, 88.29% yield) as a dark red solid. UPLC-MS analysis (sample reference number: 146220-1) (4 min, basic): rt = 1.96 min, m / z = 403.4 [M+H] + , purity 28%. The product decomposes under LCMS conditions. 1H NMR analysis (sample reference number: 146220-1) δ10.57(s,1H),8.91(d,J=7.9Hz,1H),7.74(d,J=7.6Hz,1H),7.51(dd,J=7.6Hz,1H),7.31(dd,J=7.8Hz,1H),7.14(dd ,J=7.7Hz,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).

[0308] [ka]

[0309] (Z)-1'-(azetidin-1-ylmethyl)-[2,3'-bindolinylidene]-2',3-dione A solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv, 200 mg, 0.763 mmol), azetidine (1.95 equiv, 0.10 mL, 1.49 mmol) and formaldehyde (37% in water) (1.50 equiv, 0.085 mL, 1.14 mmol) in THF (7.6 mL) at 45° C. was stirred for 71 h. The reaction mixture was concentrated in vacuo and the solid was washed with a minimal amount of hexane to give the desired product (3Z)-1-(azetidin-1-ylmethyl)-3-(3-oxoindolin-2-ylidene)indolin-2-one (240 mg, 0.688 mmol, 90.22% yield) as a dark red solid (desired product contains 5% methylene alcohol by-product). UPLC-MS analysis (sample reference number: 146352-1) (4 min, basic): rt=2.01 min, m / z=332.2 [M+H]+, 64% purity. The product decomposes under LCMS conditions. 1H NMR analysis (sample reference number: 146352-1) δ10.56(s,1H),8.91(d,J=7.8Hz,1H),7.75(d,J=7.6Hz,1H),7.51(dd,J=7.7,7.7Hz,1H),7.32(dd, J=7.6,7.6Hz,1H),7.15(dd,J=7.6,7.6Hz,1H),7.07-6.94(m,3H),4.51(s,2H),3.40(t,J=7.0Hz,4H),2.04(p,J=7.0Hz,2H).

[0310] [ka]

[0311] (Z)-1'-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-[2,3'-biindolinylidene]-2',3-dione A solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol), formaldehyde (37% in water) (1.50 equiv., 0.085 mL, 1.14 mmol) and N,N,N'-trimethyl-1,2-ethanediamine (1.95 equiv., 0.19 mL, 1.49 mmol) in THF (7.6 mL) at 45°C was stirred for 71 h. The reaction mixture was concentrated in vacuo 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-oxoindolin-2-ylidene)indolin-2-one (258 mg, 0.624 mmol, 81.78% yield) as a dark red solid (the desired product contains approximately 9% methylene alcohol by-product). UPLC-MS Analysis (Sample Reference Number: 146353-1) The product decomposes completely under LCMS conditions. 1H NMR analysis (sample reference number: 146353-1) (400MHz, DMSO) δ11.11(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).

[0312] [ka]

[0313] (Z)-1'-(morpholinomethyl)-[2,3'-bindolinylidene]-2',3-dione A solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol), formaldehyde (37% in water) (1.50 equiv., 0.085 mL, 1.14 mmol) and morpholine (1.95 equiv., 0.13 mL, 1.49 mmol) in THF (7.6 mL) at 85° C. was stirred for 108 h. 2 Cl 2 The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH in water (0% to 15%; v / v) to give the desired product (3Z)-1-(morpholinomethyl)-3-(3-oxoindolin-2-ylidene)indolin-2-one (270 mg, 0.747 mmol, 97.97% yield) as a dark purple solid. UPLC-MS analysis (sample reference number: 146704-3) (4 min, basic): rt=1.99 min, m / z=362.3 [M+H]+, 84% purity. The product decomposes under LCMS conditions. 1H-NMR analysis (sample reference number: 146704-3) (400MHz, DMSO) δ11.13(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).

[0314] [ka]

[0315] (3Z)-N-[2-(dimethylamino)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. N,N-dimethylethylenediamine (4.00 equiv., 0.33 mL, 3.05 mmol) was added dropwise and the reaction mixture was stirred for 1 h. The reaction was concentrated to dryness to give a residue which was dissolved in a minimum of DCM and added 40 g of SiO 2The crude product was poured onto the column and purified by flash column chromatography eluting with a gradient of 0–20% (v / v) DCM in MeOH to give the desired product (3Z)-N-[2-(dimethylamino)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (123 mg, 0.327 mmol, 42.85% yield) as a dark purple solid.

[0229] UPLC-MS analysis (sample reference number: 142297-2): (4 minutes, basic): rt=2.07 minutes, m / z=377.3[M+H]+, purity 98%. 1H-NMR analysis (sample reference number: 142297-2): (400MHz, CDCl3) δ10.51(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).MeOH 0.28%w / w.

[0316] [ka]

[0317] (3Z)-N-[2-(4-methylpiperazin-1-yl)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature (rt) and stirred for 1 h. 2-(4-methylpiperazin-1-yl)ethanamine (4.00 equiv., 0.46 mL, 3.05 mmol) was added dropwise and the reaction mixture was stirred for 1 h. The reaction was concentrated to dryness to give a residue which was dissolved in a minimum of DCM and added 40 g of SiO 2 The crude product was poured onto the column and purified by flash column chromatography eluting with a gradient of 0–20% (v / v) DCM in MeOH to give the desired product (3Z)-N-[2-(4-methylpiperazin-1-yl)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (195 mg, 0.452 mmol, 59.26% yield) as a dark purple solid.

[0231] UPLC-MS analysis (sample reference number: 142297-2): (4 minutes, basic): rt=1.94 minutes, m / z=432.4[M+H]+, purity 100%. 1H-NMR analysis (sample reference number: 142298-2) (400MHz, CDCl3) δ10.50(s,1H),9.01(d,J=7.9Hz,1 H),8.90(t,J=5.2Hz,1H),8.36(d,J=8.2Hz,1H),7.79(d,J=7.6Hz,1H),7.58(t,J= 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).MeOH 0.52% w / w.

[0318] [ka]

[0319] (3Z)-N-(2-acetamidoethyl)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. N-acetylethylenediamine (4.00 equiv., 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 give a residue which was dissolved in a minimum of DCM and added 40 g of SiO 2 The column was loaded. Purification by flash column chromatography eluting with a gradient of 0-20% (v / v) DCM in MeOH gave the initial product as a dark purple solid. The initial product was dissolved in EtOAc (50 mL) and washed successively with water (50 mL x 2) and saturated brine solution (50 mL). The organics were then separated and dried (Na 2 SO 4 ) and concentrated to dryness to recover the washed product as a dark purple solid. This material was dried to constant weight in a vacuum oven to give the final product (3Z)-N-(2-acetamidoethyl)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (147 mg, 0.377 mmol, 49.38% yield) as a dark purple solid.

[0233] UPLC-MS analysis (sample reference number: 142383-5): (4 minutes, basic): rt=1.74 minutes, m / z=391.3[M+H]+, purity 98%. 1H-NMR analysis (sample reference number: 142383-5): (400MHz, CDCl3) δ10.46(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 impurity w / w. CDCl3 masks one aromatic CH.

[0320] [ka]

[0321] (3Z)-N-(1-methyl-2-oxo-4-piperidyl)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (12.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (6 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. During this time, N,N-diisopropylethylamine (DIPEA) (5.00 equiv., 0.66 mL, 3.81 mmol) was added dropwise to a stirred solution of (1-methyl-2-oxo-4-piperidyl)ammonium chloride (2.50 equiv., 314 mg, 1.91 mmol) in a mixture of THF (5 mL) and DMF (2 mL) in a separate flask. The resulting free base was added to the main reaction mixture, stirred at room temperature for 30 min, then concentrated in vacuo. The crude purple residue was dissolved in a minimum of DCM and diluted with 40 g of SiO 2 The column was loaded. Purification by flash column chromatography eluting with a gradient of 0-20% (v / v) DCM in MeOH afforded 413 mg of a dark purple solid. The crude product was dissolved in EtOAc (100 mL) and washed successively with water (100 mL x 3). The organics were then separated and dried (Na 2 SO 4 ) and then concentrated to dryness to give (3Z)-N-(1-methyl-2-oxo-4-piperidyl)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (206 mg, 0.495 mmol, 64.87% yield) as a dark purple solid.

[0235] UPLC-MS analysis (sample reference number: 142675-3): (4 minutes, basic): rt=1.87 minutes, m / z=417.3[M+H]+, purity 99%. 1H-NMR analysis (sample reference number: 142675-3): (400MHz, CDCl3) δ10.43(s,1H),8.97(d,J=7.9Hz,1H),8.87(d,J=6.9Hz,1H), 8.29(d,J=8.2Hz,1H),7.75(d,J=7.6Hz,1H),7.55(t,J=7.7Hz,1H),7.36(t,J=7.8Hz,1H),7.25(t,J=7.7Hz,1 H), 7.08(t,J=7.5Hz,1H), 7.03(d,J=8.0Hz,1H), 4.42-4.29(m,1H), 3.43(t,J=6.2Hz,2H), 3.02(s,3H), 2.87(dd,J=17.1,5.5Hz,1H), 2.48(dd,J=17.2,8.2Hz,1H), 2.31-2.22(m,1H), 2.07-1.92(m,1H).DCM 1.1% w / w impurity. A slight grease peak at 1.26ppm.

[0322] [ka]

[0323] (3Z)-N-[3-(dimethylamino)propyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. 3-(Dimethylamino)-1-propylamine (2.50 equiv., 0.24 mL, 1.91 mmol) was added dropwise and the reaction mixture was stirred for 30 min, then concentrated in vacuo. Purification via preparative HPLC using basic conditions (water + 0.1% ammonia, acetonitrile + 0.1% ammonia) afforded (3Z)-N-[3-(dimethylamino)propyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (69 mg, 0.171 mmol, 22.48% yield) as a dark purple glass-like solid.

[0237] UPLC-MS analysis (sample reference number: 143027-3): (4 minutes, basic): rt=2.05 minutes, m / z=391.3[M+H]+, purity 97%. 1H-NMR analysis (sample reference number: 143027-3): (400MHz, DMSO) δ11.24(s,1H),8.91(d,J=7.9Hz,1H),8.74( t,J=5.7Hz,1H),8.16(d,J=8.1Hz,1H),7.68(d,J=7.7Hz,1H),7.61(t,J=7.6Hz,1H),7.45(d ,J=8.1Hz,1H), 7.35(t,J=7.8Hz,1H), 7.22(t,J=7.7Hz,1H), 7.08(t,J=7.4Hz,1H), 3.39-3.37(m,2H), 2.32(t,J=7.0Hz,2H), 2.17(s,6H), 1.71(p,J=7.0Hz,2H). Contains 3.25% w / w indirubin. Clear CH 2see MeOD for details, obscured here by H2O peak. 1H-NMR analysis (sample reference: 143027-3): (400MHz, MeOD) δ 8.90 (d, J = 7.9 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 8.1 Hz, 2H), 7.04 (t, J = 7.6 Hz, 1H), 3.44 (t, J = 7.0 Hz, 2H), 2.50 (t, J = 7.8 Hz, 2H), 2.32 (s, 6H), 1.87 (p, J = 7.2 Hz, 2H). See DMSO 1H-NMR for interchangeable.

[0324] [ka]

[0325] (3Z)-N-[2-(methanesulfonamido)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (10.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (6 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. During this time, N,N-diisopropylethylamine (DIPEA) (5.00 equiv., 0.66 mL, 3.81 mmol) was added dropwise to a stirred solution of 2-(methanesulfonamido)ethylammonium chloride (2.50 equiv., 333 mg, 1.91 mmol) in a mixture of THF (4 mL) and DMF (4 mL) in a separate flask. The resulting free base was added to the main reaction mixture, stirred at room temperature for 30 min, then concentrated in vacuo. The crude purple residue was dissolved in a minimum of DCM and diluted with 40 g of SiO 2 The crude product was poured onto the column and purified by flash column chromatography eluting with a gradient of 0–20% (v / v) DCM in MeOH to give the desired product (3Z)-N-[2-(methanesulfonamido)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (65 mg, 0.152 mmol, 19.99% yield) as a dark purple solid. UPLC-MS Analysis (Sample Reference No: 143028-3): (4 min, basic): rt=1.83 min, m / z=427.3[M+H]+, 97% purity. UPLC-MS Analysis (Sample Reference No: 143028-3): ELSD (4 min, basic): rt=1.87 min, m / z=427.2[M+H]+, 94% purity. Note: 6% of unknowns not seen by LC-MS: rt=2.71 min, m / z=282.3[M+H]+. 1H-NMR analysis (sample reference number: 143028-3): 1H-NMR (400MHz, DMSO) δ11.31 (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,J=7.6Hz,1H), 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), 2.94 (s, 3H). Contains 14.7% indirubin, sample is unstable in DMSO.

[0326] [ka]

[0327] (3Z)-N-[2-(methanesulfonamido)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (10.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (6 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. During this time, N,N-diisopropylethylamine (DIPEA) (5.00 equiv., 0.66 mL, 3.81 mmol) was added dropwise to a stirred solution of 2-(methanesulfonamido)ethylammonium chloride (2.50 equiv., 333 mg, 1.91 mmol) in a mixture of THF (4 mL) and DMF (4 mL) 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 dark purple oil. Purification via preparative HPLC using basic conditions (water + 0.1% ammonia, acetonitrile + 0.1% ammonia) afforded (3Z)-N-[2-(methanesulfonamido)ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide (38 mg, 0.0864 mmol, 11.33% yield) as a fluffy purple solid.

[0241] UPLC-MS analysis (sample reference number: 143334-2): (4 minutes, basic): rt=1.84 minutes, m / z=427.1[M+H]+, purity 97%. 1H-NMR analysis (sample reference number: 143334-2): (400MHz, DMSO) δ 11.30 (s, 1H), 8.91 (s, 2H), 8.77 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 10.5 Hz, 4H), 7.03 (q, J = 10.0, 7.6 Hz, 3H), 3.48 (q, J = 6.1 Hz, 2H), 3.18 (s, 2H), 2.94 (s, 4H). Note: 68% desired product, 32% indirubin, sample was converted immediately.

[0328] [ka]

[0329] Ethyl (3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxylate To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (17 mL) at 0° C. under nitrogen atmosphere, sodium bis(trimethylsilyl)amide / NaHMDS (1M in THF) (1.50 equiv., 1.1 mL, 1.14 mmol) was added dropwise. The reaction mixture was stirred for 10 min, then ethyl chloroformate (1.20 equiv., 0.087 mL, 0.915 mmol) was added and the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The reaction 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 organics were then separated and dried (Na 2 SO 4) followed by filtration and concentration to dryness to give the desired product ethyl (3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxylate (203 mg, 0.607 mmol, 79.62% yield) as a dark purple solid.

[0243] UPLC-MS analysis (sample reference number: 143594-2): (4 minutes, basic): rt=2.18 minutes, m / z=335.1[M+H]+, purity 100%. 1H-NMR analysis (sample reference: 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. Note: NMR incorrectly labeled as 143594_1 in pdf.

[0330] [ka]

[0331] tert-Butyl N-[2-(trifluoromethylsulfonylamino)ethyl]carbamate To a magnetically stirred solution of tert-butyl N-(2-aminoethyl)carbamate (1.00 equiv, 0.99 mL, 6.24 mmol) in DCM (63.6 mL) at 0° C. was added trifluoromethanesulfonyl chloride (1.00 equiv, 0.67 mL, 6.24 mmol) dropwise. The reaction mixture was allowed to warm slowly to room temperature and stirred overnight for 20 h. The reaction was concentrated to dryness to give a residue which was dissolved in EtOAc (100 mL). The resulting solution was washed successively with water (2×100 mL) and saturated brine solution (100 mL). The organics were then separated and Na 2 SO 4After drying at 40° C., filtration and concentration to dryness afforded the crude product. Purification by flash column chromatography eluting with a gradient of 0 to 50% (v / v) EtOAc in isohexane afforded the desired product tert-butyl N-[2-(trifluoromethylsulfonylamino)ethyl]carbamate (1.68 g, 5.75 mmol, 92.09% yield) as a white solid. 1H-NMR analysis (sample reference: 145427-2): (400MHz, CDCl3) δ 6.55 (s, 1H), 4.94 (s, 1H), 3.43-3.38 (m, 2H), 3.34 (q, J=5.6Hz, 2H), 1.45 (s, 9H). Note: Product structure confirmed by comparison with literature NMR data due to lack of chromophore and weak / disordered IPC. Reference: J Am Chem Soc. 2018 Mar 7;140(9):3202-3205.

[0332] [ka]

[0333] 2-(Trifluoromethylsulfonylamino)ethylammonium chloride To a magnetically stirred solution of tert-butyl N-[2-(trifluoromethylsulfonylamino)ethyl]carbamate (1.00 equiv, 1.68 g, 5.75 mmol) in DCM (36.5 mL) at room temperature, 4N HCl in dioxane (4.00 equiv, 5.7 mL, 23.0 mmol) was added dropwise. The reaction was allowed to stir overnight, and then 4N HCl in dioxane (4.00 equiv, 5.7 mL, 23.0 mmol) was added. The reaction was stirred for an additional 24 hours until complete, and then concentrated to dryness to give the crude residue. The crude material was concentrated from diethyl ether x2 to give the desired product 2-(trifluoromethylsulfonylamino)ethylammonium chloride (1.14 g, 4.99 mmol, 86.75% yield) as a white solid.

[0247] 1H-NMR analysis (sample reference number: 145775-2): (400MHz, DMSO) δ9.90(s,1H),8.23(s,3H),3.43(t,J=6.8Hz,2H),2.94(t,J=6.8Hz,2H).

[0334] [ka]

[0335] (3Z)-2-Oxo-3-(3-oxoindolin-2-ylidene)-N-[2-(trifluoromethylsulfonylamino)ethyl]indoline-1-carboxamide To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. Concurrently, N,N-diisopropylethylamine (DIPEA) (10.0 equiv., 1.3 mL, 7.63 mmol) was added dropwise to a stirred solution of 2-(trifluoromethylsulfonylamino)ethylammonium chloride (2.50 equiv., 436 mg, 1.91 mmol) in DMF (4 mL) and THF (4 mL) at room temperature. The free base product was added to the preactivated indirubin and stirring was continued for 30 min. The reaction was concentrated to dryness to give a crude purple residue which was dissolved in a minimum amount of DCM and diluted with 40 g of SiO. 2 Purification by flash column chromatography eluting with a gradient of 0-15% (v / v) DCM in MeOH afforded 325 mg of the desired product with a purity of less than 95% by LC-MS. The crude product was dissolved in a minimum amount of DCM and washed with 40 g of SiO 2A second purification by flash column chromatography eluting with a gradient of 0-15% (v / v) DCM in MeOH afforded the desired product (3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)-N-[2-(trifluoromethylsulfonylamino)ethyl]indoline-1-carboxamide (203 mg, 0.376 mmol, 49.31% yield) as a dark purple solid.

[0249] UPLC-MS analysis (sample reference number: 146019-3): (4 minutes, basic): rt=1.71 minutes, m / z=481.2[M+H]+, purity 89%. 1H-NMR analysis (sample reference number: 146019-3): (400MHz, DMSO) δ11.23(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,1 H),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).Note: Purity <90% due to co-elution of phenolate.

[0336] [ka]

[0337] tert-Butyl N-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]carbamate To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. tert-Butyl N-(2-aminoethyl)carbamate (2.50 equiv., 305 mg, 1.91 mmol) was added dropwise and the reaction mixture was stirred for 30 min. The reaction was concentrated to dryness to give a crude purple residue which was dissolved in a minimum of DCM and added 40 g of SiO 2 The crude product was poured onto the column. Purification by flash column chromatography eluting with a gradient of 0–10% (v / v) DCM in MeOH afforded the desired product tert-butyl N-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]carbamate (608 mg, 1.13 mmol, 147.55% yield) as a dark purple solid.

[0251] UPLC-MS analysis (sample reference number: 145405-2): (2 minutes, basic): rt=1.28 minutes, m / z=447.2[MH]-, purity 83%.

[0338] [ka]

[0339] 2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethylammonium chloride To a magnetically stirred solution of tert-butyl N-[2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethyl]carbamate (1.00 equiv, 608 mg, 1.13 mmol) in DCM (13.6 mL) at room temperature was added dropwise 4N HCl in dioxane (4.00 equiv, 1.1 mL, 4.50 mmol). The reaction was allowed to stir overnight producing a dark purple precipitate. The precipitate was collected by vacuum filtration and washed with DCM (10 mL) and diethyl ether (2×10 mL) to give 2-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]ethylammonium chloride (158 mg, 0.386 mmol, 34.30% yield) as a dark purple solid.

[0253] UPLC-MS analysis (sample reference number: 145541-1): (4 minutes, basic): rt=1.77 minutes, m / z=349.0 [M+H]+[-HCl], purity 94%. 1H NMR analysis (sample reference number: 145541-1): (400MHz, DMSO) δ11.27(s,1H),8.94(d,J=7.9Hz,1H),8.83(t, J=5.9Hz,1H),8.19(d,J=8.2Hz,1H),8.10(s,3H),7.70(d,J=7.6Hz,1H),7.63(t,J=7.7Hz, 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). Note: Small amounts of unknown impurities present at 3.09, 3.15, and 8.37 ppm.

[0340] [ka]

[0341] tert-Butyl N-[3-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]propyl]carbamate To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (16.0 ml) at 0° C. was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 1.1 mL, 1.14 mmol) dropwise. The reaction mixture was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 277 mg, 1.37 mmol) in THF (8 ml) at 0° C. was added rapidly. The reaction mixture was allowed to warm to room temperature and stirred for 30 min. tert-Butyl N-(3-aminopropyl)carbamate (2.50 equiv., 0.33 mL, 1.91 mmol) was added dropwise and the reaction mixture was stirred for 1 h. The reaction was concentrated to dryness to give a crude purple residue which was dissolved in a minimum of DCM and added 40 g of SiO 2 The crude product was poured onto the column. Purification by flash column chromatography eluting with a gradient of 0–10% (v / v) DCM in MeOH afforded the desired product tert-butyl N-[3-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]propyl]carbamate (522 mg, 0.880 mmol, 115.44% yield) as a dark purple solid.

[0255] UPLC-MS analysis (sample reference number: 145891-2): (2 minutes, basic): rt=1.32 minutes, m / z=461.2[MH]-, purity 78%.

[0342] [ka]

[0343] 3-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]propylammonium chloride To a magnetically stirred solution of tert-butyl N-[3-[[(3Z)-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]propyl]carbamate (1.00 equiv, 522 mg, 1.13 mmol) in DCM (13.6 mL) at room temperature was added dropwise 4N HCl in dioxane (4.00 equiv, 1.4 mL, 5.42 mmol). The reaction was allowed to stir overnight producing a dark 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-oxoindolin-2-ylidene)indoline-1-carbonyl]amino]propylammonium chloride (83 mg, 0.200 mmol, 27.00% yield).

[0257] UPLC-MS analysis (sample reference number: 145967-1): (4 minutes, basic): rt=1.71 minutes, m / z=363.2[M+H]+[-HCl], purity 96%. 1H-NMR analysis (sample reference number: 145967-1): 400MHz, DMSO) δ11.24 (s, 1H), 8.89 (d, J = 7.9Hz, 1H), 8.73 (t,J=5.7Hz,1H),8.12(t,J=7.2Hz,4H),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), 3.44(q,J=6.5Hz,2H),2.89(h,J=6.3Hz,2H),1.90(p,J=7.1Hz,2H).Note: Indirubin 2.57%w / w. DCM 0.08%w / w.

[0344] [ka]

[0345] (3Z)-1-[(4,4-difluoro-1-piperidyl)methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (7.6 mL) was added formaldehyde, H 2 37 wt.% in 0 (1.50 equiv., 0.084 mL, 1.14 mmol) and 4,4-difluoropiperidine (1.90 equiv., 0.16 mL, 1.45 mmol) were added. The reaction mixture was heated to 55° C. for 96 h, then cooled and concentrated in vacuo. The resulting crude residue was suspended in hexanes (20 mL) and collected via suction filtration. The initial product was washed with an additional portion of hexanes (2×20 mL) before being collected and dried on a rotary evaporator. The resulting sticky purple solid was sonicated in diethyl ether (20 mL) and redried under reduced pressure to give (3Z)-1-[(4,4-difluoro-1-piperidyl)methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one (234 mg, 0.592 mmol, 77.60% yield) as a free-flowing dark purple solid.

[0259] UPLC-MS analysis (sample reference number: 146375-1): (4 minutes, basic): rt=2.27 minutes, m / z=396.1[M+H]+, purity 81% [Note 1]. 1H-NMR analysis (sample reference 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). Note: No single interchangeables are recognized.

[0346] [ka]

[0347] (3Z)-3-(3-oxoindolin-2-ylidene)-1-(1-piperidylmethyl)indolin-2-one To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (7.6 mL) was added formaldehyde, H 2 37 wt.% in 0 (1.50 equiv., 0.084 mL, 1.14 mmol) and piperidine (1.90 equiv., 0.14 mL, 1.45 mmol) were added. The reaction mixture was heated to 55° C. for 96 h, then cooled and concentrated in vacuo. The resulting crude residue was suspended in hexanes (20 mL) and collected via suction filtration. The initial product was washed with an additional portion of hexanes (2×20 mL) before being collected and dried on a rotary evaporator. The resulting sticky red solid was sonicated in diethyl ether (20 mL) and redried under reduced pressure to give (3Z)-3-(3-oxoindolin-2-ylidene)-1-(1-piperidylmethyl)indolin-2-one (259 mg, 0.721 mmol, 94.49% yield) as a free-flowing dark red solid.

[0261] UPLC-MS analysis (sample reference number: 146391-1): (4 minutes, basic): rt=1.76 minutes, m / z=261.1[MH]-, purity 96%. 1H-NMR analysis (sample reference number: 146391-1): (400MHz, CDCl3) δ10.58(s,1H),8.91(d,J=7.8Hz,1H),7 .74(d,J=7.6Hz,1H),7.50(t,J=7.6Hz,1H),7.31(t,J=7.7Hz,1H),7.14(t,J=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).Note: No single interchangeables are permitted.

[0348] [ka]

[0349] (3Z)-3-(3-oxoindolin-2-ylidene)-1-(pyrrolidin-1-ylmethyl)indolin-2-one To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (7.6 mL) was added formaldehyde, H 2 37 wt.% in 0 (1.50 equiv., 0.084 mL, 1.14 mmol) and pyrrolidine (1.90 equiv., 0.12 mL, 1.45 mmol) were added. The reaction mixture was heated to 55° C. for 96 h, then cooled and concentrated in vacuo. The resulting crude residue was suspended in hexanes (20 mL) and collected via suction filtration. The initial product was washed with an additional portion of hexanes (2×20 mL) before being collected and dried on a rotary evaporator. The resulting sticky red solid was sonicated in diethyl ether (20 mL) and redried under reduced pressure to give (3Z)-3-(3-oxoindolin-2-ylidene)-1-(pyrrolidin-1-ylmethyl)indolin-2-one (255 mg, 0.738 mmol, 96.81% yield) as a free-flowing dark red solid.

[0263] UPLC-MS analysis (sample reference number: 146393-1): (4 minutes, basic): rt=1.76 minutes, m / z=261.1[MH]-, purity 96%. 1H-NMR analysis (sample reference 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). Note: No single interchangeables are allowed.

[0350] [ka]

[0351] (3Z)-1-[(4-acetylpiperazin-1-yl)methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (7.6 mL) was added formaldehyde, H 2 37 wt.% in 20 (1.50 equiv., 0.084 mL, 1.14 mmol) and 1-acetylpiperazine (1.90 equiv., 0.19 mL, 1.45 mmol) were added. The reaction mixture was heated to 55° C. for 72 h, then cooled and concentrated in vacuo. The crude residue obtained was suspended in hexanes (20 mL) and collected via suction filtration. The initial product was washed with additional portions of hexanes (2×20 mL), then collected and dried on a rotary evaporator. The obtained residue was sonicated in diethyl ether (20 mL) and redried under reduced pressure to obtain a crude solid. The crude material was dissolved in a minimum amount of DCM and diluted with 40 g of SiO 2 The crude product was poured onto the column. Purification by flash column chromatography eluting with a gradient of 0–15% (v / v) DCM in MeOH afforded the desired product (3Z)-1-[(4-acetylpiperazin-1-yl)methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one (147 mg, 0.334 mmol, 43.78% yield) as a dark purple solid.

[0265] UPLC-MS analysis (sample reference number: 146684-2): (4 minutes, basic): rt=1.81 minutes, m / z=403.3[M+H]+, purity 82%. 1H-NMR analysis (sample reference number: 146684-2): (400MHz, DMSO) δ11.12(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 = 7.9 Hz, 1H), 7.10 (t, J = 7.7 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 4.63 (s, 2H), 3.40 (q, J = 4.9 Hz, 4H), 2.64-2.51 (m, 4H), 1.94 (s, 3H). Note: Contains 8.6% w / w of associated methylene alcohol and 4.15% residual DCM.

[0352] [ka]

[0353] (3Z)-1-[[bis[2-(dimethylamino)ethyl]amino]methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one To a magnetically stirred solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 200 mg, 0.763 mmol) in THF (7.6 mL) was added formaldehyde, H 237 wt.% in O (1.50 equiv., 0.084 mL, 1.14 mmol) and N-[2-(dimethylamino)ethyl]-N',N'-dimethyl-ethane-1,2-diamine (1.90 equiv., 0.28 mL, 1.45 mmol) were added. The reaction mixture was heated to 55° C. for 72 h, then cooled and concentrated in vacuo. The resulting crude residue was suspended in hexane (20 mL) and collected via suction filtration. The initial product was washed with additional portions of hexane (2×20 mL) before being collected and dried on a rotary evaporator. The obtained residue was sonicated, taken up in diethyl ether (20 mL) and redried under reduced pressure to give (3Z)-1-[[bis[2-(dimethylamino)ethyl]amino]methyl]-3-(3-oxoindolin-2-ylidene)indolin-2-one (114 mg, 0.263 mmol, 34.48% yield) as a dark purple solid.

[0267] UPLC-MS analysis (sample reference number: 146685-1): (4 minutes, basic): rt=2.27 minutes, m / z=434.4[M+H]+, purity 3%. 1H-NMR analysis (sample reference number: 146685-1): (400MHz, DMSO) δ11.08 (s, 1H), 8.81 (d, J = 7.7Hz, 1H), 7.6 5(d,J=7.6Hz,1H),7.58(t,J=7.8Hz,1H),7.42(d,J=7.8Hz,1H),7.32(t,J=7.8Hz,1H),7 .25 (d,J=7.8Hz,1H), 7.08 (t,J=7.8Hz,1H), 7.02 (t,J=7.5Hz,1H), 4.64 (s,2H), 2.69 (t,J=7.0Hz,4H), 2.33 (t,J=7.0Hz,4H), 2.10 (d,J=2.2Hz,12H).Note: Contains 4.5% w / w methylene alcohol.

[0354] [ka]

[0355] tert-Butyl N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]carbamate

[0268] CH at 5℃ 2 Cl 2To a solution of tert-butyl N-(2-aminoethyl)carbamate (1.00 equiv, 0.99 mL, 6.24 mmol) and triethylamine (2.00 equiv, 1.7 mL, 12.5 mmol) in 150 mL of CH 2 Cl 2 A solution of 4-nitrobenzene-1-sulfonyl chloride (1.05 equiv., 1.45 g, 6.55 mmol) in 10 ml of ethyl acetate (58.1 mL) was added dropwise; the reaction mixture was allowed to warm slowly to room temperature and stirred overnight for 20 h. The reaction mixture was concentrated in vacuo to a volume of approximately 100 mL, then washed with water (3×50.0 mL), dried (Na 2 SO 4 ) and concentrated in vacuo to give the crude product as a colorless solid. The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of EtOAc in iso-hexane (0% to 100%; v / v) to give tert-butyl N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]carbamate (1.79 g, 5.18 mmol, 83.03% yield) as a colorless solid.

[0269] UPLC-MS analysis (sample reference number: 143957-2) (2 minutes, basic): rt=1.03 minutes, m / z=344.2[MH]-, purity 100%. 1H NMR analysis (sample reference number: 143957-2) δ8.36(d,J=8.4Hz,2H),8.05(d,J=8.4Hz,2H),5.70(bs,1H) ,4.79(bs,1H),3.25(dt,J=5.7,5.7Hz,2H),3.14(dt,J=5.4,5.4Hz,2H),1.43(s,9H).

[0356] [ka]

[0357] 2-[(4-nitrophenyl)sulfonylamino]ethylammonium chloride To a suspension of tert-butyl N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]carbamate (1.00 equiv., 1.00 g, 2.90 mmol) in DCM (19.3 mL) at room temperature was added hydrogen chloride solution (4 M in 1,4-dioxane) (4.00 equiv., 2.9 mL, 11.6 mmol). The reaction mixture was filtered and the filter cake was separated by HCl. 2 Cl 2 and ether to give the desired product 2-[(4-nitrophenyl)sulfonylamino]ethylammonium chloride (816 mg, 2.90 mmol, 100.04% yield) as a colorless powder. UPLC-MS analysis (sample reference number: 145404-1) (2 min, basic): rt = 0.72 min, m / z = 246.2 [M+H] + , 100% purity (mass corresponds to product without chloride anion). 1H NMR analysis (sample reference 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).

[0358] [ka]

[0359] (3Z)-N-[2-[(4-nitrophenyl)sulfonylamino]ethyl]-2-oxo-3-(3-oxoindolin-2-ylidene)indoline-1-carboxamide

[0272] To a solution of (3Z)-3-(3-oxoindolin-2-ylidene)indolin-2-one (1.00 equiv., 100 mg, 0.381 mmol) in THF (7.40 mL) at 0°C under a nitrogen atmosphere was added sodium bis(trimethylsilyl)amide / NaHMDS (1.50 equiv., 0.57 mL, 0.572 mmol); the reaction was stirred for 10 min, then a solution of 4-nitrophenyl chloroformate (1.80 equiv., 138 mg, 0.686 mmol) in THF (5.00 mL) at 0°C was added and the reaction mixture was stirred for 30 min. A suspension of N-(2-aminoethyl)-4-nitro-benzenesulfonamide; hydrochloride salt (2.33 equiv, 250 mg, 0.887 mmol) and N,N-diisopropylethylamine (DIPEA) (5.00 equiv, 0.33 mL, 1.91 mmol) in THF (6.00 mL) was stirred for 25 min and then added to the indirubin-containing mixture; the reaction mixture was stirred for 1 h. The reaction mixture was concentrated in vacuo and then diluted with CH 2 Cl 2 The crude material was purified by automated column chromatography on silica (25 g cartridge) eluting with a gradient of MeOH in water (0% to 15%; v / v) to give a dark purple solid. The purity of the product was below the threshold required for compound submission, so CH 2 Cl 2 The crude material was purified by automated column chromatography on silica (40 g cartridge) eluting with a gradient of MeOH in water (0% to 15%; v / v) to give a dark purple solid.

[0360] [ka]

[0361] Chloromethyl (2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-carboxylate To a slurry of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (5.36 g, 20.4 mmol; batch=2009-BML-4B) in tetrahydrofuran (100 mL, 1230 mmol) and triethylamine (6.3 mL, 45 mmol) was added chloromethyl chloroformate (4.00 mL, 45.0 mmol) dropwise. After 30 min, HPLC showed 97% conversion. The slurry was allowed to warm to room temperature and stirred for an additional hour. HPLC showed 99% conversion. The reaction was allowed to stir overnight.

[0362] HPLC showed >99% conversion of starting material. The mixture was filtered through a glass frit. The solid was washed with water (200 mL). The solid was dried on the frit for 1 h, then transferred to a bottle and dried in a vacuum oven (50° C.) for 24 h. HPLC of the solid showed less than 1 area % of indirubin was present. [B]Chloromethyl (2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindole-1'(2'H)-carboxylate (4.81 g; yield=66.3%; batch=2009-BML-22A) was obtained as a purple solid.

[0363] [ka]

[0364] (2Z)-1-Acetyl-2,3'-biindole-2',3(1H,1'H)-dione and (2Z)-1'-Acetyl-2,3'-biindole-2',3(1H,1'H)-dione A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (1.21 g, 4.61 mmol; batch=1951-BML-31C) in acetic anhydride (2.18 mL, 23.1 mmol) and sulfuric acid (0.04 mL, 0.8 mmol) was heated in an aluminum heating block at 140° C. for 10 min. HPLC showed high conversion so the mixture was cooled to room temperature and filtered through a glass frit. The solid was washed with water (4×50 mL). NMR showed only one product. The material was dried in a vacuum oven overnight (approximately 50° C.). [B](2Z)-1'-acetyl-2,3'-biindole-2',3(1H,1'H)-dione (1.25 g; yield = 89.0%; batch = 2009-BML-12A) was obtained as a purple solid.

[0365] [ka]

[0366] (2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (10.12 g, 38.59 mmol; batch=2009-BML-6B) in aqueous formaldehyde (6.0 mL, 8.0E1 mmol) and tetrahydrofuran (250 mL, 3.0E3 mmol) and triethylamine (6.3 mL, 45 mmol) was heated overnight in an oil bath at 55° C. The mixture was filtered after 26 hours to give [B](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (10.45 g; yield=92.65%; batch=2009-BML-82A) as a purple solid.

[0367] [ka]

[0368] [(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl acetate To a mixture of [A](2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione (3.34 g, 11.4 mmol; batch=2009-BML-62A) in methylene chloride (100 mL, 1560 mmol) was added triethylamine (3.5 g, 34 mmol) and acetic anhydride (1.6 mL, 17 mmol). The mixture was stirred at room temperature overnight. HPLC showed complete conversion to product after 69 hours. The slurry was filtered to give a purple solid, which was then dried by nitrogen pressure in a vacuum oven at 30°C. [B][(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl acetate (2.98 g; yield = 78.0%; batch = 2009-BML-84A) was obtained as a purple solid. The solid was dried in a vacuum oven overnight.

[0369] [ka]

[0370] (2Z)-1'-[(diethylamino)methyl]-2,3'-biindole-2',3(1H,1'H)-dione A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (4.95 g, 0.0189 mol; batch=2009-BML-6B), 37% formaldehyde (37:63, formaldehyde:water, 2.11 mL, 0.0283 mol) and N-ethylethanamine (3.81 mL, 0.0368 mol) in tetrahydrofuran (200 mL, 2.46 mol) in a pressure tube was heated in a 55°C oil bath overnight.

[0371] The reaction mixture was concentrated in vacuo. NMR of the solid showed no starting material remaining and the solid was product with residual THF and excess diethylamine. The solid was carefully rinsed 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%; batch=2009-BML-69A) was obtained as a purple solid.

[0372] [ka]

[0373] (2Z)-1'-[(diethylamino)methyl]-2,3'-biindole-2',3(1H,1'H)-dione 4-methylbenzenesulfonate 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; batch=2009-BML-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 in vacuo. The solid was slurried in acetone and the suspension was filtered. The solid was air-dried for approximately 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%; batch = 2009-BML-70A) as a purple solid.

[0374] [ka]

[0375] (2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (8.05 g, 0.0307 mol; batch=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 so the mixture was concentrated in vacuo. The solid was washed with EtOH to give, after drying, [C](2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione (10.21 g; yield=88.8%; batch=2009-BML-73A).

[0376] [ka]

[0377] (2Z)-1'-[(4-Methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione dihydrochloride To a mixture of methanol (0.03 mL, 0.7 mmol) in ethyl acetate (4.00 mL, 41.0 mmol) was added acetyl chloride (0.04 mL, 0.6 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; batch=2009-BML-40A) was added. A precipitate formed. Filtration gave [B](2Z)-1'-[(4-methylpiperazin-1-yl)methyl]-2,3'-biindole-2',3(1H,1'H)-dione dihydrochloride (0.07 g; yield = 60%; batch = 2009-BML-58A) as a purple solid.

[0378] [ka]

[0379] tert-Butyl 4-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}piperazine-1-carboxylate A mixture of [A](2Z)-2,3'-biindole-2',3(1H,1'H)-dione (0.561 g, 0.00214 mol; batch=2009-BML-4B), 37% formaldehyde (37:63, formaldehyde:water, 0.31 mL, 0.0042 mol) and [B]tert-butyl 1-piperazinecarboxylate (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 concentrated in vacuo. The solid was washed with ethanol and filtered to give [C]tert-butyl 4-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}piperazine-1-carboxylate (0.80 g; yield = 81%; batch = 2009-BML-41A) as a dark purple solid.

[0380] [ka]

[0381] (2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione 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=2009-BML-46A) and thionyl chloride (6.7 mL, 92 mmol) was heated to reflux for 2 h. The reaction mixture was concentrated in vacuo to give [B](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (1.18 g; yield=102%; batch=2009-BML-61cr) as a purple solid.

[0382] [ka]

[0383] tert-Butyl 1-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}-L-prolinate To a solution of [A](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (0.17 g, 0.55 mmol; batch=2009-BML-61cr) in methylene chloride (6.5 mL, 100 mmol), [B]L-proline tert-butyl ester (0.15 mL, 0.87 mmol) and triethylamine (0.12 mL, 0.86 mmol) were added. The mixture was stirred at room temperature overnight. The mixture was concentrated in vacuo. Flash chromatography (5–8% EtOAc in DCM) afforded [C]tert-butyl 1-{[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl}-L-prolinate (0.10 g; yield = 41%; batch = 2009-BML-63A) as a purple solid.

[0384] [ka]

[0385] (2Z)-1'-(Chloromethyl)-2,3'-biindol-2',3(1H,1'H)-dione and di-tert-butyl[(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl phosphate 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; batch=2009-BML-62A) and thionyl chloride (8.0 mL, 110 mmol) was heated to reflux for 2 h. The reaction mixture was concentrated in vacuo to give [B](2Z)-1'-(chloromethyl)-2,3'-biindole-2',3(1H,1'H)-dione (batch=2009-BML-86-INT) as a purple solid.

[0386] The intermediate (2009-BML-86-INT) was dissolved in ethyl acetate (34.2 mL, 350 mmol) and potassium di-t-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. The product slowly reverted to starting material on standing at room temperature (approximately 80% reversion after 72 hours).

[0387] [ka]

[0388] [(2Z)-2',3-Dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl dihydrogen phosphate and (2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione and (2Z)-1'-(ethoxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione To a solution of [A] di-tert-butyl [(2Z)-2',3-dioxo-1,3-dihydro-2,3'-biindol-1'(2'H)-yl]methyl phosphate (0.55 g, 1.1 mmol) in 1,4-dioxane (9.0 mL, 120 mmol) was added 4.0 M hydrogen chloride in 1,4-dioxane (3.0 mL, 12 mmol). The reaction was stirred at room temperature for 1 h. HPLC showed complete consumption of starting material. The solvent was removed in vacuo and the solid was dried in a vacuum oven overnight. The product readily reverted to [C] (2Z)-1'-(hydroxymethyl)-2,3'-biindole-2',3(1H,1'H)-dione. Exposure to ethanol gave 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.

[0389] [ka]

[0390] Allyl (Z)-2',3-dioxo-[2,3'-bindolinylidene]-1'-carboxylate To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (200 mg, 0.19 mmol, 1 equiv.) in THF (10 mL) was added LiHMDS (0.84 mL, 1.1 equiv.) at 0°C. After stirring at 0°C for 1 h, allyl carbonochloridate was added. The mixture was stirred at room temperature overnight. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (10 x 3 mL). The combined organic phase was washed with Na 2 SO 4 The crude was purified by column (PE / EA=5 / 1, v / v) to give the desired product (200 mg, 0.57 mmol, 76%) as a purple solid.

[0290] HNMR:(400MHz,DMSO-d 6)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).

[0291] HPLC (Agilent-1200-A2): Rt: 15.26 min, purity 92%.

[0391] [ka]

[0392] (Z)-(2',3-dioxo-[2,3'-bindolinylidene]-1'-yl)methyl hydrogen sulfate To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (2.02 g, 7.6 mmol, 1 equiv.) and HCHO (1.3 g, 16 mmol, 2.1 equiv.) in THF (50 mL) was added triethylamine (940 mg, 9.2 mmol, 1.2 equiv.) at room temperature. After stirring at 55°C for 16 h, the reaction mixture was filtered to give the desired product (1.8 g, 6.16 mmol, 81%) as a purple solid.

[0293] HNMR:(400MHz,DMSO-d 6 )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=8.0Hz,1H),7.04(t,J=7.2Hz,1H),6.32(t,J=6.8Hz,1H),5.22(d,J=6.8Hz,2H).

[0393] To a solution of (Z)-1'-(hydroxymethyl)-[2,3'-bindolinylidene]-2',3-dione (58 mg, 0.2 mmol, 1 equiv.) in pyridine (1 mL) and DMF (1 mL), sulfur trioxide N,N-dimethylformamide complex (306 mg, 2 mmol, 10 equiv.) was added. The mixture was stirred at room temperature overnight. The solid was filtered and washed with EtOAc (30 mL) to give the desired product (20 mg, 0.05 mmol, 25%) as a purple solid. HNMR (400MHz, DMSO-d 6 ):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),7.61-7.5 7(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).

[0296] HPLC (Agilent-1100-A2): 214 nm: Rt: 12.41 min, purity 46%; 254 nm: 12.41 min, purity 96%.

[0394] [ka]

[0395] (Z)-1'-((phenylthio)methyl)-[2,3'-bindolinylidene]-2',3-dione To a solution of (Z)-1'-(chloromethyl)-[2,3'-bindolinylidene]-2',3-dione (600 mg, 1.9 mmol, 1 equiv.) in THF (30 mL) was added sodium thiophenoxide (1.28 g, 9.7 mmol, 5 equiv.) at room temperature. After stirring at room temperature overnight for 16 h, the reaction mixture was concentrated in vacuo 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. HNMR (400MHz, DMSO-d 6 ):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).

[0396] [ka]

[0397] (Z)-1'-(Phenoxymethyl)-[2,3'-bindolinylidene]-2',3-dione To a solution of phenol (351 mg, 3.7 mmol, 2 equiv.) in dry THF (20 mL) was added NaH (149 mg, 3.7 mmol, 2 equiv.) at 0° C. The mixture was stirred at this temperature for 10 min, and then (Z)-1′-(chloromethyl)-[2,3′-bindolinylidene]-2′,3-dione (580 mg, 1.9 mmol, 1 equiv.) was added to the reaction mixture. After stirring at room temperature for 16 h, 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.

[0300] LCMS (Agilent-S12): Rt4.39 min; [M+1] + =369.15

[0301] HNMR (400MHz, DMSO-d 6 ):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).

[0302] HPLC (Agilent-1200-A1): 214 nm: Rt: 10.95 min, purity 75%; 254 nm: 10.95 min, purity 92%.

[0398] [ka]

[0399] (2Z,3E)-3-(2-phenylhydrazono)-[2,3'-bindolinylidene]-2'-one To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (200 mg, 0.76 mmol, 1 equiv.) and phenylhydrazine (164 mg, 1.52 mmol, 2 equiv.) in dry THF (8 mL) was added tetraisopropyl orthotitanate (1.3 g, 4.58 mmol, 6 equiv.) at 0° C. The mixture was stirred at 80° C. overnight. 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.

[0304] LCMS (Agilent-S12): Rt3.89 min; [M+1]+=353.2

[0305] 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).

[0400] [ka]

[0401] (2Z,3E)-3-(2-benzylhydrazono)-[2,3'-bindolinylidene]-2'-one To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (250 mg, 0.95 mmol, 1 equiv.) in THF (10 mL), benzylhydrazine (349 mg, 2.86 mmol, 3 equiv.) and tetraisopropyl orthotitanate (813 mg, 2.86 mmol, 3.0 equiv.) were added. After stirring at 80°C overnight, 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.

[0307] LCMS (Agilent-S12): Rt1.89 min; [M+1]+=367.1

[0308] HNMR:(400MHz,DMSO-d 6 )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),7.41-7.32(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).

[0309] HPLC (Agilent-1200-A2): Rt: 12.87 min, purity 84%.

[0402] [ka]

[0403] (2Z,3E)-3-(2-Methylhydrazono)-[2,3'-bindolinylidene]-2'-one To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (200 mg, 0.76 mmol, 1 equiv.) in pyridine (3 mL), methylhydrazine (93 mg, 1.98 mmol, 2.6 equiv.) was added. After stirring at 120°C for 24 h, 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.

[0311] LCMS (Agilent-S12): Rt3.16 min; [M+1]+=291.1

[0312] 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),

[0313] HPLC (Agilent-1200-A2): Rt: 10.90 min, purity 92%.

[0404] [ka]

[0405] (Z)-1'-((phenylsulfonyl)methyl)-[2,3'-bindolinylidene]-2',3-dione To a solution of (Z)-1'-(chloromethyl)-[2,3'-bindolinylidene]-2',3-dione (600 mg, 1.93 mmol, 1 equiv.) in dry THF (30 mL) was added benzenesulfinic acid sodium salt (1.68 g, 10.2 mmol, 5 equiv.). After stirring at room temperature for 16 h, the reaction mixture was concentrated in vacuo 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.

[0315] 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),7.66 -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).

[0406] [ka]

[0407] (Z)-(2',3-dioxo-[2,3'-bindolinylidene]-1'-yl)methylphenylcarbamate To a solution of (Z)-1'-(hydroxymethyl)-[2,3'-bindolinylidene]-2',3-dione (180 mg, 0.6 mmol, 1 equiv.) and (amd) phenyl isocyanate (71.5 mg, 0.6 mmol, 1 equiv.) in THF (10 mL) was added triethylamine (182 mg, 1.8 mmol, 3 equiv.). After stirring at room temperature for 16 h, the reaction mixture was concentrated in vacuo and purified by preparative TLC (DCM / MeOH=20 / 1, v / v) to give the desired product (15 mg, 0.04 mmol, 6%) as a purple solid.

[0317] 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).

[0318] HPLC (Agilent-1200-N1): Rt: 9.11 min, purity 96%.

[0408] [ka]

[0409] (Z)-1'-(Methoxymethyl)-[2,3'-bindolinylidene]-2',3-dione (Z)-1'-(hydroxymethyl)-[2,3'-bindolinylidene]-2',3-dione (58 mg, 0.2 mmol, 1 equiv.) and SO in DMF (1 mL). 3To a solution of DMF (306 mg, 2 mmol, 10 equiv) was added pyridine (15.8 mg, 0.2 mmol, 1 equiv) at room temperature. After stirring at room temperature for 16 h, 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.

[0320] LCMS (Agilent-S12): Rt1.97 min; [M+1]+=307.0

[0321] 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=7.6Hz,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).

[0322] HPLC (Agilent-1100-A2): Rt: 12.42 min, purity 94%.

[0410] [ka]

[0411] (2Z,3E)-3-(hydroxyimino)-[2,3'-bindolinylidene]-2'-one To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (63 mg, 0.24 mmol, 1 equiv.) in pyridine (2.5 mL) was added hydroxylamine hydrochloride (154 mg, 2.4 mmol, 10 equiv.). After stirring to reflux for 2.5 h, 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.

[0324] LCMS (Agilent-S12): Rt3.00 min; [M+1]+=278.1

[0325] 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,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).

[0326] HPLC (Agilent-1260C-B2): Rt: 13.63 min, purity 100%.

[0412] [ka]

[0413] (Z)-4-((2',3-dioxo-[2,3'-bindolinylidene]-1'-yl)methoxy)-4-oxobutanoic acid To a solution of (Z)-1'-(hydroxymethyl)-[2,3'-bindolinylidene]-2',3-dione (200 mg, 0.68 mmol, 2 equiv.) in pyridine (5 mL), succinyl chloride (52 mg, 0.34 mmol, 1 equiv.) was added. After stirring at 100°C for 16 h, the reaction mixture was concentrated and purified by silica gel column (DCM / MeOH=10 / 1, v / v) to give the desired product (8 mg, 0.02 mmol, 6%) as a purple solid.

[0328] LCMS (Agilent-S12): Rt3.41 min; [M+23]+=415.2

[0329] 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).

[0414] [ka]

[0415] Ethyl (Z)-4-(2',3-dioxo-[2,3'-bindolinylidene]-1'-yl)-4-oxobutanoate To a solution of (Z)-[2,3'-bindolinylidene]-2',3-dione (200 mg, 0.19 mmol, 1 equiv.) in THF (5 mL) was added LiHMDS (0.38 mL, 1 M, 2 equiv.). After stirring at 0°C for 1 h, ethyl 4-chloro-4-oxobutanoate (63 mg, 0.38 mmol, 2 equiv.) was added. The mixture was stirred at room temperature for 3 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (10 x 3 mL). The combined organic phase was washed with Na 2 SO 4 The crude was purified by column (PE / EA=5 / 1, v / v) to give the desired product (130 mg, 0.57 mmol, 44%) as a purple solid.

[0331] LCMS (Agilent-S12): Rt2.76 min; [M+1]+=391.2

[0332] 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).

[0333] HPLC (Agilent-1200-A1): Rt: 10.21 min, purity 97%.

[0416] Example 2 Stability of indirubin prodrugs

[0334] 10.41 g of Fasted State Simulated Intestinal Fluid (FaSSIF) buffer concentrate was dissolved in 240.3 g of deionized water and 560 mg of FaSSIF powder was added. The mixture was stirred until complete dissolution was observed. Test compound (approximately 1-3 mg) was placed in 5.00 mL of FaSSIF solution. A sample of the solution with test compound was taken after 24 hours at 37°C (oven controlled temperature).

[0417]

[0335] 36.78 g of Fasted State Simulated Gastric Fluid (FaSSGF) buffer concentrate was dissolved in 961.9 g of deionized water and 60 mg of FaSSGF powder was added. The mixture was stirred until complete dissolution was observed. Test compound (approximately 1-3 mg) was placed in 5.00 mL of FaSSGF solution. A sample of the solution with test compound was taken after 24 hours at 37°C (oven controlled temperature).

[0418] UPLC-MS analysis of samples collected at 24 hours was performed on a Waters Acquity UPLC system consisting of Acquity I Class Sample Manager FL, Acquity I Class Binary Solvent Manager and Acquity UPLC Column Manager. UV detection was performed using Acquity UPLC PDA detector (scanning 210-400 nm), whereas mass detection was achieved using Acquity QDa detector (mass scan 100-1250 Da; positive and negative mode simultaneously) and ELS detection was achieved using Acquity UPLC ELS detector. Analytes were separated using a Waters Acquity UPLC BEH C18 column (2.1 Å ca. 50 mm, 1.7 mm). Samples were prepared by dissolution in 1 mL of 50% (v / v) MeCN in water (with or without sonication). The resulting solution was then filtered through a 0.2 mm syringe filter before being submitted for analysis. All solvents, including formic acid and 36% ammonia solution, were purchased as HPLC grade.

[0419]

[0337] The results are set forth in Table 2-1.

[0420] [Table 2-1]

[0421] [Table 2-2]

[0422] [Table 2-3]

[0423] [Table 2-4]

[0424] [Table 2-5]

[0425] [Table 2-6]

[0426] [Table 2-7]

[0427] Example 3 Time course of conversion to indirubin in FaSSIF at 37 °C A study to analyze the rate and extent of prodrug conversion to indirubin in fasted-state simulated intestinal fluid (FaSSIF) was performed as described in Example 2. Each test compound was placed in FaSSIF in the amount 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 performed at 37°C (oven controlled temperature). The results are shown in Table 3-1, where the numbers in each column report the percentage of test compound (starting material), indirubin methylene alcohol, and indirubin (total percent).

[0428] [Table 3-1]

[0429] [Table 3-2]

[0430] [Table 3-3]

[0431] Example 4 Time course of conversion to indirubin in FaSSGF at 37 °C

[0339] A study to analyze the rate and extent of prodrug conversion to indirubin in fasted state simulated gastric fluid (FaSSGF) was carried out as described in Example 2. Each test compound was placed in FaSSGF in the amount 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 carried out at 37°C (oven controlled temperature). The results are shown in Table 4-1, where the numbers in each column report the percentage of test compound (starting material), indirubin methylene alcohol, and indirubin (total percent).

[0432] [Table 4-1]

[0433] [Table 4-2]

[0434] [Table 4-3]

[0435] Example 5 In vitro AhR activity of example compounds Compound 8, indirubin, 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio) were each mixed with dimethyl sulfoxide (DMSO) to generate a 1000x stock solution. TCDD and MeBio were included in the study as positive controls. These were added to cell culture medium containing charcoal-stripped fetal bovine serum to generate 2x stocks, which were then added to reporter cells. Reporter cells were human Huh7 or rat H4IIE cell lines spiked with native AhR and AhR / arnt-luciferase reporter vectors. The final concentration of DMSO was 0.1%. Cells were incubated at 37°C for 22 hours, after which luciferase detection reagent was added and activity was measured in relative luminescence units. Samples were run in duplicate and the average of the readings is shown in Figures 1A-1B.

[0436] 1A-1B are plots showing activation of AhR in human Huh7 cells (FIG. 1A) and rat H4IIE cells (FIG. 1B) in the presence of compound 8 (triangles), indirubin (squares), 2,3,7,8-tetrachlorodibenzodioxin (TCDD; circles) (positive control), and (2'Z,3'E)-6-bromo-1-methylindirubin-3'-oxime (MeBio; dashed line) (positive control) at nM concentrations. EC for indirubin in human Huh7 cells 50 The EC for compound 8 in human Huh7 cells was 58.82 nM. 50 The EC value for indirubin in rat H4IIE cells was 71.47 nM. 50 The EC for compound 8 in rat H4IIE cells was estimated to be 654 nM (estimated by the absence of an upper plateau). 50 was 25.62 nM.

[0437] Example 6 Activity of Example Compound 8 in a Mouse Model of Ulcerative Colitis Eight-week-old female C57BL / 6J mice, weighing approximately 20 g, purchased from Charles River Laboratories, were housed in specific pathogen-free IVC cages (four mice per cage) in a temperature-controlled (20±2°C) room with a 12-hour light / dark cycle. Chow pellets and tap water were available ad libitum. All experimental protocols were approved by the Animal Care and Use Committee. Mice were acclimated to the animal facility for at least 3 days prior to the experiment.

[0438] To generate dextran sulfate sodium (DSS)-induced acute colitis, mice were induced with 3% DSS (molecular weight 36,000-50,000; MP Biomedicals, Santa Ana, CA, USA) in their drinking water for 7 consecutive days and then switched to plain water. The DSS solution was freshly prepared and replaced daily. The mice were euthanized on day 8 (day 9 of the experiment).

[0439] Mice were divided into groups with 8 mice per group. Mice were dosed twice daily by gavage with either Compound 8 at 2 mg / kg / day in 200 μL of distilled water (pH=7.0) from day 0 to day 9. Mice in the vehicle group were given only 200 μL of distilled water by gavage.

[0440] Body weight and Disease Activity Index (DAI) scoring were recorded daily to assess colitis. The DAI score was the sum of the body weight loss score, fecal score and bleeding subscore. A blinded scoring system was used to assess colitis. A DAI scorer blinded to group information and animal ID was responsible for assessing stool consistency and bleeding. The DAI scoring criteria will be followed as follows:

[0441] [Table 5]

[0442] The results are shown in Figure 2, which is a plot of the Disease Activity Index (see Table 6-1 above) as 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).

[0443] Example 7 Activity of Example Compounds in a Mouse Model of Ulcerative Colitis The study was carried out as specified in Example 6. Briefly, test mice were randomized into study groups (n=10) for treatment with indirubin, Compound 9 (20 milligrams per kilogram (mpk)), Compound 10 (25 mpk), Compound 26 (24.5 mpk), Compound 40 (20.5 mpk) or Compound 41 (20 mpk). Mice were dosed with each compound in an amount to provide an indirubin molar equivalent of 15 mg / kg in 200 μL of distilled water (pH=7.0) and administered orally by gavage twice daily from day 0 to day 9. Mice in the vehicle group were given only 200 μL of distilled water by gavage.

[0444]

[0348] The results are shown in Figures 3A-3C. Compound 41 is more effective than an equimolar amount of indirubin.

[0445] Example 8 Pharmacokinetics of the compounds in a mouse model of ulcerative colitis Five animals from each group in Example 7 were euthanized 6 hours after the last oral dose. At this time, plasma, lungs and colons were harvested. The colons were washed three times in cold phosphate buffered saline (PBS) to remove colonic contents.

[0446]

[0350] Indirubin was detected using LC-MS / MS. The method had a lower limit of quantification of 0.05 ng / mL in plasma, 0.05 ng / mL in lung, and 0.10 ng / g in colon.

[0447] To prepare samples, 20 μL aliquots of unknown samples, calibration standards, quality controls, single blanks and double blanks were added to a 96-well plate. Plasma samples (except double blanks) were quenched with 100 μL internal standard (double blanks were quenched with 100 μL acetonitrile (ACN)), liver, lung and colon samples (except double blanks) were quenched with 80 μL internal standard (double blanks were quenched with 80 μL ACN), and the mixtures were then vortex mixed at 800 rpm for 5 min and centrifuged at 3220 g (4000 rpm) for 15 min at 4° C. 50 μL of the supernatant was transferred to another clean 96-well plate and the supernatant was directly injected for LC-MS / MS analysis.

[0448]

[0352] The biological analysis methods were as follows:

[0449] [Table 6]

[0450]

[0353] The results are shown in Tables 8-1, 8-2 and 8-3.

[0451] [Table 7]

[0452] [Table 8]

[0453] [Table 9]

[0454] The ratios of indirubin in colon to plasma and in colon to lung for vehicle and compound 26 are not shown because they were undetectable above the lower limit of quantification.Prodrugs liberate indirubin to a greater extent in the colon than equimolar synthetic indirubin.Some compounds have favorable levels of drug in the colon and low systemic exposure to indirubin.Compound 9 has less plasma exposure than equimolar indirubin.Compound 10 has more plasma exposure than equimolar indirubin.

[0455] Example 9 Indirubin solubility in biorelevant media

[0355] Samples of synthetic indirubin, sappan or indirubin derivatives (concentrates of 5 μg / mL indirubin or molar equivalents of indirubin) were evaluated in two biorelevant media, simulated fasted gastric fluid (FaSSGF) and simulated fasted intestinal fluid (FaSSIF). Biorelevant media was prepared using FaSSIF / FeSSIF / FaSSGF powder as per the supplier's method (Biorelevant). Briefly, FaSSGF media was prepared using 0.06 g / L FaSSIF / FeSSIF / FaSSGF powder in 2 g / L NaCl solution and pH adjusted to 1.6 with 1N HCl. FaSSIF medium was prepared using 2.24 g / L FaSSIF / FeSSIF / FaSSGF powder in sodium phosphate buffer and pH adjusted to 6.5 with 1 N NaOH (sodium phosphate buffer is 0.42 g / L NaOH, 3.95 g / L NaH 2 PO 4 .H 2 O, prepared as a 6.19 g / L NaCl solution).

[0456]

[0356] Solubility assessments were performed as follows: Individual flasks were prepared for each vehicle and time point. The flasks were placed in a shaking water bath maintained at 37°C. Flasks were removed from the bath at designated time points of 1, 4 and 24 hours, allowed to cool, and aliquots were taken and filtered.

[0457]

[0357] Indirubin concentrations were determined using a validated HPLC method for the detection of indirubin. The method was as follows: Instrument: UPLC Waters or HPLC Alliance Waters Separation module: Aquity H-class Detector: TUV or PDA Analytical column: Kinetex® F5 2.6 μm, 100 Å, 4.6×150 mm Instrument parameters Flow rate: 0.6mL / min Detector wavelength: 290nm Injection volume: 10μL Column temperature: 25℃ Sample temperature: 20℃ Run Time: 40 minutes The retention time of indirubin was approximately 20.0 min.

[0458] [Table 10] Indirubin recovery % calculation:

[0459]

number

[0460]

number

[0461]

[0358] The calculation assumes that 1 mole of prodrug is equivalent to 1 mole of indirubin. The percent recovery for the molecular weight conversion of prodrug to indirubin is based on the assumption that 100% of the prodrug can be converted to indirubin.

[0462]

[0359] The results are shown in Table 9-1.

[0463] [Table 11]

[0464]

[0360] While a number of exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. Accordingly, it is intended that the following appended claims, and any claims hereafter introduced, be construed to include all such modifications, permutations, additions, and subcombinations as are within their true spirit and scope.

Claims

1. The following structure of formula (IV) or (V): 【Chemistry 1】 or a stereoisomer, salt or tautomer thereof, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are each independently hydrogen, deuterium, alkyl, halo, perfluoroalkyl, alkynyl, alkenyl, alkoxy, cycloalkoxy, thioalkyl, thiocycloalkoxy, perfluoroalkoxy, perfluorothioalkyl, hydroxyl, carboxyl, carbamoyl, acylsulfonylamido, amino, nitrile, nitro, azido, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R １１ａ is halo, 4- to 8-membered heterocycloalkyl, 5- to 8-membered heteroaryl, -OH, -OX, -SX, -S(O) ２ X, -NX ２ , -OC(=O)X or -OC(=O)NX2; R １１ｂ is a 4- to 8-membered heterocycloalkyl, a 5- to 8-membered heteroaryl, -NX2 or the following structure: 【Chemistry 2】 It is one of the Here, R １１ａ Or R １１ｂ wherein said 4-8 membered heterocycloalkyl is optionally substituted; X is independently hydrogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenyl, C １ ~C ６ Haloalkyl, -S(O) ２ OH, aryl or 3-8 membered heterocycloalkyl; Here, the C １ ~C １０ Alkyl is carbonyl, -NHS(=O) ２ -NHC(=O)Y, -NHC(=O)Y, wherein the 3-8 membered heterocycloalkyl of X is C １ ~C ６ Alkyl, C １ ~C ４ optionally substituted with amino, halo, -C(=O)Y or -C(=O)OY; Y is independently H, C １ ~C ６ Perfluoroalkyl or C １ ~C ６ is alkyl, R １２ａ and R １２ｂ are each independently hydrogen or C １ ~C ６ is alkyl, R １０ is O, NZ or NNZ ２ and Each Z is independently hydrogen, hydroxyl, aryl or C １ ~C ４ alkyl, where C １ ~C ４ The alkyl may be substituted with an aryl.

2. R １１ａ Or R １１ｂ 2. The compound of claim 1, wherein said 4-8 membered heterocycloalkyl is azetidinyl, diazetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, diazinyl, triazinyl, azepanyl, diazepanyl, azocanyl, oxetanyl, dioxetanyl, tetrahydrofuranyl, dioxolanyl, oxanyl, dioxanyl, trioxanyl, oxepanyl, oxocanyl, phosphetanyl, phosphoranyl, phosphinanyl, thietanyl, dithietanyl, tetrahydrothiophenyl, dithiolanyl, thianyl, dithianyl, trithianyl, thiepanyl, thiocanyl, oxathiolidinyl, isoxathiolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl, thiomorpholinyl, or oxathianyl.

3. R １１ａ Or R １１ｂ 2. The compound of claim 1, wherein said 5-8 membered heteroaryl is pyrrole, furan, thiophene, pyridine, azonine, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, diazine, triazine, tetrazine, pentazine or diazepine.

4. R １１ａ has the following structure: 【Chemistry 3】 The compound of claim 1 having one of the following:

5. R １１ｂ The compound of claim 1, wherein is -NX2.

6. R １１ｂ has the following structure: 【Chemistry 4】 The compound of claim 1 having one of the following:

7. R １２ａ and R １２ｂ The compound of claim 1 , wherein each of is hydrogen.

8. R １０ has the following structure: 【Chemistry 5】 The compound of claim 1 having one of the following:

9. R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ The compound of claim 1 , wherein each is independently hydrogen, deuterium, alkyl, halo, or perfluoroalkyl. 【Request 10】 【Chemical 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 A compound selected from the group consisting of:

11. The following structure of formula (IVa) or (Va): 【Chemistry 7】 or a stereoisomer, salt or tautomer thereof, R １１ａ is halo, 4- to 8-membered heterocycloalkyl, 5- to 8-membered heteroaryl, -OH, -OX, -SX, -S(O) ２ X, -NX2, -OC(=O)X or -OC(=O)NX2; R １１ｂ is 4- to 8-membered heterocycloalkyl, 5- to 8-membered heteroaryl, -NX ２ or the following structure: 【Chemistry 8】 It is one of the Here, R １１ａ Or R １１ｂ wherein said 4-8 membered heterocycloalkyl is optionally substituted; X is independently hydrogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenyl, C １ ~C ６ Haloalkyl, -S(O) ２ OH, aryl or 3-8 membered heterocycloalkyl; Here, the C １ ~C １０ Alkyl is carbonyl, -NHS(=O) ２ -NHC(=O)Y, -NHC(=O)Y, wherein the 3-8 membered heterocycloalkyl of X is C １ ~C ６ Alkyl, C １ ~C ４ optionally substituted with amino, halo, -C(=O)Y or -C(=O)OY; Y is independently H, C １ ~C ６ Perfluoroalkyl or C １ ~C ６ is alkyl, R １０ is O, NZ or NNZ ２ and Each Z is independently hydrogen, hydroxyl, aryl or C １ ~C ４ alkyl, where C １ ~C ４ The alkyl may be substituted with an aryl.

12. The following structure of formula (IVb) or (Vb): 【Chemistry 9】 12. The compound of claim 11, or a stereoisomer, salt or tautomer thereof, having one of:

13. R １１ａ The compound of claim 12, wherein is a 4-8 membered heterocycloalkyl or a 5-8 membered heteroaryl.

14. R １１ａ 14. The compound of claim 13, wherein said 4-8 membered heterocycloalkyl is azetidinyl, diazetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, diazinyl, triazinyl, azepanyl, diazepanyl, azocanyl, oxetanyl, dioxetanyl, tetrahydrofuranyl, dioxolanyl, oxanyl, dioxanyl, trioxanyl, oxepanyl, oxocanyl, phosphetanyl, phosphoranyl, phosphinanyl, thietanyl, dithietanyl, tetrahydrothiophenyl, dithiolanyl, thianyl, dithianyl, trithianyl, thiepanyl, thiocanyl, oxathiolidinyl, isoxathiolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl, thiomorpholinyl, or oxathianyl.

15. R １１ａ 14. The compound of claim 13, wherein said 5-8 membered heteroaryl is pyrrole, furan, thiophene, pyridine, azonine, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, diazine, triazine, tetrazine, pentazine or diazepine.

16. Formula (IVc): 【Chemistry 10】 14. The compound of claim 13, or a stereoisomer, salt or tautomer thereof, wherein 【Chemistry 11】 represents a 4- to 8-membered heterocycloalkyl or a 5- to 8-membered heteroaryl.

17. 【Chemical 12】 has the following structure: 【Chemistry 13】 17. The compound of claim 16, having one of the following:

18.

14. but, 【Chemistry 15】 17. The compound of claim 16,

19. R １１ｂ The compound of claim 12, wherein is -NX2.

20. R １１ｂ has the following structure: 【Chemistry 16】 13. The compound of claim 12, having one of the following:

21. The structure: 【Chemistry 17】 The compound of claim 11 having one of the following:

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