Patent Application
20240307415
The present invention claims priority to the following applications: Chinese Patent Application No. 202110706921.X filed with China National Intellectual Property Administration on Jun. 24, 2021 and entitled “STEROID COMPOUND AND USE THEREOF”, Chinese Patent Application No. 202210101639.3 filed with China National Intellectual Property Administration on Jan. 27, 2022 and entitled “STEROID COMPOUND AND USE THEREOF”, and Chinese Patent Application No. 202210340109.4 filed with China National Intellectual Property Administration on Apr. 1, 2022 and entitled “STEROID COMPOUND AND USE THEREOF”. The prior applications described above are incorporated herein by reference in their entirety.
The present invention relates to a novel glucocorticoid receptor agonist, a preparation method therefor, a pharmaceutical composition containing the same, and use thereof in treating tumors, inflammatory diseases, or autoimmune diseases.
The glucocorticoid receptor (GR) is a part of the nuclear receptor family. GR is expressed in almost every cell in the human body and regulates genes that control development, metabolism, and immune response. When the glucocorticoid receptor binds to a glucocorticoid, its main mechanism of action is to regulate gene transcription. The direct route: glucocorticoids enter the cytoplasm via the cell membrane and bind to GR within the cytoplasm, and the GR is activated, enters the nucleus, and binds to GR response elements (GRE) on DNA, which initiates or inhibits mRNA transcription of the corresponding gene. For example, mRNA transcription of TNFα, IL-1β, IL-2, IL-6 and the like is inhibited, and mRNA transcription of IκB (capable of inhibiting the activation of NF-κB) is started. The indirect route: interacting with other transcription factors, etc., and performing anti-inflammatory and immune effects.
Glucocorticoid receptor agonists can be classified into endogenous or artificially synthesized ones according to sources. The synthetic glucocorticoid receptor agonists are a class of potent small molecule drugs used in the treatment of inflammatory diseases and immune diseases. Glucocorticoids that have been marketed include prednisone, prednisolone, betamethasone, dexamethasone, fluticasone propionate, budesonide, and the like. Glucocorticoid receptor agonists can be used for controlling inflammation and allergic diseases such as asthma, rheumatoid arthritis, obstructive airways disease, rhinitis, inflammatory bowel disease, psoriasis, eczema, and the like. However, if the medicine is taken for a long time at a supraphysiological dose, side effects such as muscle atrophy, osteoporosis and the like may appear, so that the dose and the duration of medication of the medicine for patients are limited, and the therapeutic potential of the medicine has not yet been effectively exploited. Therefore, there is a need to develop new glucocorticoid receptor agonists, which can treat more diseases and reduce toxic and side effects.
The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:
wherein
In some embodiments, R1 and R2 are each independently selected from H or F.
In some embodiments, ring A is selected from phenyl or 5-6 membered heteroaryl, wherein the phenyl or 5-6 membered heteroaryl is optionally substituted with R1a.
In some embodiments, ring A is selected from phenyl, wherein the phenyl is optionally substituted with R1a.
In some embodiments, R1a is selected from halogen, CN, ═O, OH, NH2, or C1-C6 alkyl.
In some embodiments, R1a is selected from NH2.
In some embodiments, ring A is selected from
In some embodiments, ring A is selected from
In some embodiments, X is selected from O, S, C1-C3 alkylene-O, C1-C3 alkylene-S, or C(R7)(R8).
In some embodiments, R7 and R8 are each independently selected from H, halogen, CN, OH, NH2, or C1-C3 alkyl, or R7 and R8, together with the atom linked thereto, form C3-C6 cycloalkyl, wherein the OH, NH2, C1-C3 alkyl, or C3-C6 cycloalkyl is optionally substituted with Rb.
In some embodiments, R7 and R8 are each independently selected from H, halogen, or C1-C3 alkyl, or R7 and R8, together with the atom linked thereto, form C3-C6 cycloalkyl.
In some embodiments, R7 and R8 are each independently selected from H, F, or methyl, or R7 and R8, together with the atom linked thereto, form cyclopropyl.
In some embodiments, X is selected from O, S, CH2O, CH2S, CH2, CF2, CHCH3, or
In some embodiments, X is selected from CH2.
In some embodiments, R10 is selected from OH, SH, O(C1-C3 alkyl), O—C(═O)—(C1-C3 alkyl), or
wherein the O(C1-C3 alkyl) or O—C(═O)—(C1-C3 alkyl) is optionally substituted with halogen. In some embodiments, the O(C1-C3 alkyl) is optionally substituted with halogen.
In some embodiments, R11 and R12 are each independently selected from H, methyl, or ethyl.
In some embodiments, R10 is selected from OH, SH, OCH2F, O(C═O)CH3,
In some embodiments, R10 is selected from OH or
In some embodiments, ring B is selected from C6-C10 aryl, 5-10 membered heteroaryl, or 4-14 membered heterocyclyl, wherein the C6-C10 aryl, 5-10 membered heteroaryl, or 4-14 membered heterocyclyl is optionally substituted with R2a.
In some embodiments, ring B is selected from 5-10 membered heteroaryl or 4-14 membered heterocyclyl, wherein the 5-10 membered heteroaryl or 4-14 membered heterocyclyl is optionally substituted with R2a.
In some embodiments, ring A is substituted with R1a and/or ring B is substituted with R2a.
In some embodiments, ring A is substituted with R1a or ring B is substituted with R2a. In some embodiments, R2a is selected from halogen, CN, ═O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclyl.
In some embodiments, R2a is selected from NH2 or ═O.
In some embodiments, R2a is selected from NH2. In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof is selected from a compound of formula (Ia) or a pharmaceutically acceptable salt thereof:
In some embodiments, R3 is selected from H, OH, or NHR9, and R4 and R5, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a, or R5 is selected from H, OH, or NHR9, and R3 and R4, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R9 is selected from H or C1-C6 alkyl.
In some embodiments, R9 is selected from H.
In some embodiments, R3 is selected from H, OH, or NH2, and R4 and R5, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R5 is selected from H, OH, or NH2, and R3 and R4, together with the atom linked thereto, form C5-C6 cycloalkenyl, 5-6 membered heterocyclyl, or 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, 5-6 membered heterocyclyl, or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R5 is selected from H, OH or NH2, and R3 and R4, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R3 is selected from H, and R4 and R5, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form C5-C6 cycloalkenyl, 5-6 membered heterocyclyl, or 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, 5-6 membered heterocyclyl, or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein the 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with R4a.
In some embodiments, R3 is selected from H, OH, or NH2, and R4 and R5, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R3 is selected from H, and R4 and R5, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H, OH, or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H, OH, or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H, OH, or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R5 is selected from H or NH2, and R3 and R4, together with the atom linked thereto, form
wherein the
is optionally substituted with R4a.
In some embodiments, R4a is selected from halogen, CN, ═O, OH, NH2, C1-C6 alkyl, or C3-C6 cycloalkyl.
In some embodiments, R4a is selected from halogen, CN, ═O, OH, NH2, or C1-C3 alkyl.
In some embodiments, R4a is selected from ═O or NH2.
In some embodiments, R3 is selected from H, and R4 and R5, together with the atom linked thereto, form
In some embodiments, R3 is selected from H, and R4 and R5, together with the atom linked thereto, form
In some embodiments, R5 is selected from NH2, and R3 and R4, together with the atom linked thereto, form
In some embodiments, R5 is selected from NH2, and R3 and R4, together with the atom linked thereto, form
In some embodiments, R13 and R14 are each independently selected from H or NH2.
In some embodiments, R13 is selected from H.
In some embodiments, R14 is selected from H or NH2.
In some embodiments, R14 is selected from H.
In some embodiments,
is selected from
In some embodiments,
is selected from
In some embodiments,
is selected from
In some embodiments,
is selected from
In some embodiments,
is selected from
In some embodiments,
is selected from
Without conflict, it should be understood that the embodiments described above may be combined arbitrarily, forming a technical solution that includes the features of the combined embodiments. Such combined technical solutions are within the scope of the present invention.
In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:
In another aspect, the present invention provides a pharmaceutical composition comprising the compound of formula (I) or the pharmaceutically acceptable salt thereof disclosed herein and a pharmaceutically acceptable excipient.
In another aspect, the present invention provides a method for treating a disease mediated by a glucocorticoid receptor in a subject (e.g., a mammal), comprising administering to a subject (e.g., a mammal, preferably a human) in need of such treatment a therapeutically effective amount of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof.
In another aspect, the present invention provides use of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof in preparing a medicament for use in preventing or treating a disease mediated by a glucocorticoid receptor.
In another aspect, the present invention provides use of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof in preventing or treating a disease mediated by a glucocorticoid receptor.
In another aspect, the present invention provides the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof for use in preventing or treating a disease mediated by a glucocorticoid receptor.
In another aspect, the present invention provides a method for treating a tumor, an inflammatory disease, or an autoimmune disease in a subject (e.g., a mammal), comprising administering to a subject (e.g., a mammal, preferably a human) in need of such treatment a therapeutically effective amount of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof.
In another aspect, the present invention provides use of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof in preparing a medicament for use in treating a tumor, an inflammatory disease, or an autoimmune disease.
In another aspect, the present invention provides use of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof in treating a tumor, an inflammatory disease, or an autoimmune disease.
In another aspect, the present invention provides the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof for use in treating a tumor, an inflammatory disease, or an autoimmune disease.
In some embodiments, the disease mediated by a glucocorticoid receptor is selected from a tumor, an inflammatory disease, or an autoimmune disease.
The compounds of the present invention have strong binding activity to glucocorticoid receptors and weak or inactive binding to other hormone receptors, and the strong selectivity of the compounds of the present invention is expected to reduce the side effects associated with acting on other hormone receptors. The compounds of the present invention also have good metabolic stability in liver microsomes. In addition, the compounds of the present invention have a low potential risk of drug-drug interaction (DDI) and have no obvious inhibitory effect on the hERG potassium channel, which suggests that the compounds of the present invention have a low risk of cardiotoxicity due to the inhibition of hERG potassium channel.
Unless otherwise stated, the terms used in the present invention have the following meanings, and the definitions of groups and terms described in the present invention, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions documented in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and incorporated with each other. A certain term, unless otherwise specifically defined, should not be considered uncertain or unclear, but construed according to its common meaning in the field. When referring to a trade name, it is intended to refer to its corresponding commercial product or its active ingredient. Herein,
represents a connection site.
The illustration of the pure compounds of the racemate or the enantiomer herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise stated, the absolute configuration of a stereogenic center is represented by a wedged bond and a wedged dashed bond ( and
), and the relative configuration of a stereogenic center (e.g., cis- or trans-configuration of alicyclic compounds) is represented by a black solid bond and a dashed bond (
and (
).
The term “tautomer” refers to functional isomers resulting from the rapid movement of an atom in a molecule between two positions. The compounds of the present invention may exhibit the tautomerism. Tautomeric compounds may exist in two or more interconvertible forms. Tautomers generally exist in an equilibrium form. Trying to separate a single tautomer usually leads to a mixture, the physicochemical properties of which are consistent with the mixture of the compound. The position of the equilibrium depends on the chemical properties of the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenol, the enol form predominates. In the present invention, all tautomeric forms of the compound are included.
The term “stereoisomer” refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, and diastereoisomers.
The compound of the present invention may have an asymmetric atom such as a carbon atom, a sulfur atom, a nitrogen atom, a phosphorus atom, or an asymmetric double bond, and thus the compound of the present invention may exist in the form of a particular geometric isomer or stereoisomer. The form of a particular geometric isomer or stereoisomer may be cis and trans isomers, E and Z geometric isomers, (−)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as an enantiomer or diastereoisomer enriched mixture, and all of the above isomers as well as mixtures thereof are encompassed within the definition scope of the compound of the present invention. An additional asymmetric carbon atom, asymmetric sulfur atom, asymmetric nitrogen atom, or asymmetric phosphorus atom may be present in substituents such as alkyl. All of these isomers and mixtures thereof referred to in the substituents are also encompassed within the definition scope of the compound of the present invention. The compound with asymmetric atoms disclosed herein can be separated in an optically active pure form or in a racemic form. The optically active pure form can be isolated from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.
The term “substituted” means that any one or more hydrogen atoms on a specific atom are substituted by substituents, as long as the valence of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (namely ═O), it means that two hydrogen atoms are substituted, and oxo is not available on an aromatic group.
The term “optional” or “optionally” means that the subsequently described event or circumstance may, but not necessarily, occur. The description includes instances where the event or circumstance occurs and instances where it does not. For example, an ethyl “optionally” substituted with halogen, means that the ethyl may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, or the like), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, or the like), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, or the like). It will be understood by those skilled in the art that for any group comprising one or more substituents, no substitution or substituting pattern that is spatially impossible and/or cannot be synthesized will be introduced.
When any variable (e.g., Ra or Rb) occurs more than once in the constitution or structure of a compound, the variable is independently defined in each case. For example, if a group is substituted with 2 Rb, the definition of each Rb is independent.
When the number of a linking group is 0, such as —(CH2)0—, it means that the linking group is a bond. When one of variables is selected from a chemical bond or is absent, it means that the two groups which it links are linked directly. For example, when L in A-L-Z represents a bond, it means that the structure is actually A-Z.
When the linking direction of the linking group referred to herein is not specified, the linking direction is arbitrary. For example, when X in the structural unit
is selected from “C1-C3 alkylene-O”, then X may link ring A and ring B in a direction from left to right to constitute “ring A-C1-C3 alkylene-O-ring B”, or link ring A and ring B in a direction from right to left to constitute “ring A-O-C1-C3 alkylene-ring B”.
When a bond of a substituent is cross-linked to two atoms on a ring, the substituent can be bonded to any atom on the ring. For example, the structural unit
represents that R5 may be substituted at any position on the benzene ring.
Cm-Cn used herein means that the portion has an integer number of carbon atoms in the range of m-n. For example, “C1-C10” means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.
The term “alkyl” refers to hydrocarbyl with a general formula of CnH2n+1. The alkyl may be linear or branched. The term “C1-C6 alkyl” may be understood to represent a linear or branched saturated hydrocarbyl having 1, 2, 3, 4, 5, or 6 carbon atoms. Specific examples of the alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like. The term “C1-C3 alkyl” may be understood to represent a linear or branched saturated alkyl group having 1-3 carbon atoms. The “C1-C6 alkyl” may further include “C1-C3 alkyl”.
The term “alkoxy” refers to a group derived from a linear or branched alcohol by loss of a hydrogen atom from hydroxyl, and may be understood as “alkyloxy” or “alkyl-O—”. The term “C1-C6 alkoxy” may be understood as “C1-C6 alkyloxy” or “C1-C6 alkyl-O—”. The “C1-C6 alkoxy” may further include “C1-C3 alkoxy”.
The term “cycloalkyl” refers to a fully saturated carbon ring group that exists in the form of a monocyclic ring, a fused ring, a bridged ring, a spiro ring, or the like. Unless otherwise specified, the carbon ring is generally a 3-10 membered ring (e.g., a 3, 4, 5, 6, 7, 8, 9, or 10 membered ring). The term “C3-C10 cycloalkyl” may be understood as a saturated monocyclic, fused, spiro, or bridged ring having 3-10 carbon atoms. Specific examples of the cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term “C3-C10 cycloalkyl” may include “C3-C6 cycloalkyl”, and the term “C3-C6 cycloalkyl” may be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3-6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like.
The term “cycloalkyloxy” may be understood as “cycloalkyl-O—”.
The term “cycloalkenyl” refers to a non-aromatic carbon ring group that is not fully saturated, has at least one carbon-carbon double bond, and exists in the form of a monocyclic ring, a fused ring, a bridged ring, a spiro ring, or the like. Unless otherwise specified, the carbon ring is generally a 5-8 membered ring. The term “C5-C6 cycloalkenyl” refers to cycloalkenyl with 5 or 6 ring carbon atoms. Specific examples include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
The term “heterocyclyl” refers to a fully saturated or partially saturated (non-aromatic heteroaromatic group on the whole) monocyclic, fused cyclic, spiro cyclic, or bridged cyclic group, and ring atoms of the group include 1-5 (e.g., 1-3 or 1-2) heteroatoms or heteroatom groups (i.e., heteroatom-containing atom groups). The “heteroatom or heteroatom group” includes, but is not limited to, a nitrogen atom (N), an oxygen atom (O), a sulfur atom (S), a phosphorus atom (P), a boron atom (B), —S(═O)2—, —S(═O)—, —P(═O)2—, —P(═O)—, —NH—, —S(═O)(═NH)—, —C(═O)NH—, -13 NHC(═O)NH—, and the like. The term “4-14 membered heterocyclyl” refers to a heterocyclyl group having a ring atom number of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and ring atoms of the heterocyclyl group include 1-5 heteroatoms or heteroatom groups independently selected from those described above. “4-14 membered heterocyclyl” includes “4-10 membered heterocyclyl”, “4-7 membered heterocyclyl”, or the like, wherein specific examples of 4-membered heterocyclyl include, but are not limited to, azetidinyl or oxetanyl; specific examples of 5-membered heterocyclyl include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5- dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; specific examples of 6-membered heterocyclyl include, but are not limited to, tetrahydropyranyl, piperidyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and specific examples of 7-membered heterocyclyl include, but are not limited to, diazepanyl. The heterocyclyl may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(1H)-yl; and specific examples of 5,6-membered bicyclic groups include, but are not limited to, hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, and 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclyl may be a benzo-fused ring group of the 4-7 membered heterocyclyl described above. Specific examples include, but are not limited to, dihydroisoquinolyl and the like. “4-10 membered heterocyclyl” may include “5-10 membered heterocyclyl”, “4-7 membered heterocyclyl”, “5-6 membered heterocyclyl”, “6-8 membered heterocyclyl”, “4-10 membered heterocycloalkyl”, “5-10 membered heterocycloalkyl”, “4-7 membered heterocycloalkyl”, “5-6 membered heterocycloalkyl”, “6-8 membered heterocycloalkyl”, and the like, and the “4-7 membered heterocyclyl” may further include “4-6 membered heterocyclyl”, “5-6 membered heterocyclyl”, “4-7 membered heterocycloalkyl”, “4-6 membered heterocycloalkyl”, “5-6 membered heterocycloalkyl”, and the like. Although some bicyclic heterocyclyl herein comprise, in part, a benzene ring or a heteroaromatic ring, the heterocyclyl is still non-aromatic on the whole.
The term “aryl” refers to an aromatic monocyclic or fused polycyclic group of carbon atoms with the conjugated pi-electron system. The term “C6-C10 aryl” may be understood as an aryl group having 6-10 carbon atoms, in particular a ring having 6 carbon atoms (“C6 aryl”), such as phenyl, a ring having 9 carbon atoms (“C9 aryl”), such as indanyl or indenyl, or a ring having 10 carbon atoms (“C10 aryl”), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl.
The term “aryloxy” may be understood as “aryl-O—”.
The term “heteroaryl” refers to an aromatic cyclic group having an aromatic monocyclic or fused polycyclic system in which at least one ring atom selected from N, O, and S is comprised, while the remaining ring atoms are C. The term “5-10 membered heteroaryl” may be understood to include an aromatic monocyclic or bicyclic ring system which has 5, 6, 7, 8, 9, or 10 ring atoms, in particular 5, 6, 9, or 10 ring atoms, and comprises 1-5, preferably 1-3, heteroatoms independently selected from N, O, and S. In particular, the heteroaryl is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, or the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, or the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, or the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, or the like; or azocinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, or the like. The term “5-6 membered heteroaryl” refers to an aromatic ring system which has 5 or 6 ring atoms, and comprises 1-3, preferably 1-2, heteroatoms independently selected from N, O, and S.
The term “heteroaryloxy” may be understood as “heteroaryl-O—”.
The term “halo” or “halogen” refers to fluorine, chlorine, bromine, or iodine.
The term “hydroxy” refers to —OH group.
The term “cyano” refers to —CN group.
The term “sulfydryl” refers to —SH group.
The term “amino” refers to —NH2 group.
The term “therapeutically effective amount” refers to an amount of the compound of the present invention for (i) treating a specific disease, condition or disorder; (ii) alleviating, ameliorating or eliminating one or more symptoms of a specific disease, condition or disorder, or (iii) delaying onset of one or more symptoms of a specific disease, condition or disorder described herein. The amount of the compound of the present invention constituting the “therapeutically effective amount” varies dependently on the compound, the disease state and its severity, the administration regimen, and the age of the mammal to be treated, but can be determined routinely by those skilled in the art in accordance with their knowledge and the present disclosure.
The term “prevent” or “prevention” means administering the compound or formulation described herein to prevent a disease or one or more symptoms associated with the disease, and includes: preventing the occurrence of the disease or disease state in an individual (e.g., a mammal), particularly when such an individual (e.g., a mammal) is predisposed to the disease state but has not yet been diagnosed with it.
The term “pharmaceutically acceptable” is used herein for those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit/risk ratio.
The term “pharmaceutically acceptable salt” refers to salts of pharmaceutically acceptable acids or bases, including salts formed from the compound and an inorganic or organic acid, and salts formed from the compound and an inorganic or organic base.
The term “pharmaceutical composition” refers to a mixture consisting of one or more of the compounds or the salts thereof of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition is intended to facilitate the administration of the compound of the present invention to an organism.
The term “pharmaceutically acceptable excipients” refers to those which do not have a significant irritating effect on an organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrate, wax, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oil, solvent, water, and the like.
The term “individual” includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock animals, such as cattle, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, guinea pigs, and the like. Examples of non-human mammals include, but are not limited to, birds, fish, and the like. In one embodiment associated with the methods and compositions provided herein, the mammal is a human.
The word “comprise” and variations thereof such as “comprises” or “comprising” may be understood in an open, non-exclusive sense, i.e., “including but not limited to”.
The phosphate ester compounds (for example, R10 is selected from a compound of
described herein are ester prodrugs, which are hydrolyzed by phosphatase in vivo to release compounds with biological activity. For example, the phosphate ester compound
is hydrolyzed by phosphatase in vivo to release a glucocorticoid receptor agonist
Thus, it will be understood or expected by those skilled in the art that if compound 009 has some biological activity (e.g., glucocorticoid receptor agonistic activity), then the corresponding ester compound 009-p also has the same or similar biological activity in vivo.
The present invention also comprises isotopically-labeled compounds which are identical to those documented herein but have one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I and 36Cl, respectively, and the like.
Certain isotopically-labeled compounds of the present invention (e.g., those labeled with 3H and 14C) can be used to analyze compounds and/or substrate tissue distribution. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as 15O, 13N, 11C and 18F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below while substituting a non-isotopically labeled reagent with an isotopically-labeled reagent.
The pharmaceutical composition of the present invention can be prepared by combining the compound of the present invention with a suitable pharmaceutically acceptable excipient, and can be formulated, for example, into a solid, semisolid, liquid, or gaseous formulation such as tablet, pill, capsule, powder, granule, ointment, emulsion, suspension, suppository, injection, inhalant, gel, microsphere, aerosol, and the like.
Typical routes of administration of the compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof of the present invention include, but are not limited to, oral, rectal, local, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous and intravenous administration.
The pharmaceutical composition disclosed herein can be manufactured by methods well known in the art, such as by conventional mixing, dissolving, granulating, emulsifying, lyophilizing, and the like.
In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present invention to be formulated into tablets, pills, pastilles, dragees, capsules, liquids, gels, slurries, suspensions and the like for oral administration to a patient.
A solid oral composition can be prepared by conventional mixing, filling or tableting. For example, it can be obtained by the following method: mixing the active compounds with solid excipients, optionally grinding the resulting mixture, adding additional suitable excipients if desired, and processing the mixture into granules to get the core parts of tablets or dragees. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, or flavoring agents, and the like.
The pharmaceutical compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
In all of the administration methods of the compound described herein, the daily dose administered is from 0.001 mg/kg to 200 mg/kg body weight, preferably from 0.05 mg/kg to 50 mg/kg body weight, and more preferably from 0.1 mg/kg to 30 mg/kg body weight, given in individual or separated doses. The following abbreviations are used in the present invention:
The present invention is described in detail below by way of examples. However, this is by no means disadvantageously limiting the scope of the present invention. Although the present invention has been described in detail herein and specific examples have also been disclosed, it will be apparent to those skilled in the art that various modifications can be made to the specific examples without departing from the spirit and scope of the present invention. All reagents used in the present invention are commercially available and can be used without further purification.
Unless otherwise stated, the ratio represented by a mixed solvent is a volume mixing ratio.
Unless otherwise stated, % refers to wt %.
The eluent or the mobile phase may be a mixed eluent or mobile phase consisting of two or more solvents, and the ratio of the mixed eluent or mobile phase is the volume ratio of the solvents. For example, “0-10% methanol/dichloromethane” represents that the volume ratio of methanol to dichloromethane in the mixed eluent or mobile phase is 0:100-10:100.
Compounds are named either manually or by ChemDraw® software, and supplier's catalog names are given for commercially available compounds.
The structures of the compounds are determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts are given in 10−6 (ppm). Solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol or the like, and internal standard is tetramethylsilane (TMS); “IC50” refers to the half maximal inhibitory concentration, which is the concentration at which half of the maximal inhibitory effect is achieved; “EC50” refers to the half maximal effect concentration, a concentration that causes 50% of the maximal effect.
The synthetic route and the specific synthetic steps were as follows.
According to the method reported in the literature (WO2016169504 A1), 2,3-dihydrobenzofuran-7-amine (1.0 g, 7.40 mmol) was dissolved in dimethylformamide (20 mL), N-bromosuccinimide (1.48 g, 8.33 mmol) was added, and the mixture was stirred at 25° C. for 3 h. After the reaction was completed as detected by LCMS, water (20 mL) was added to dilute the mixture, the mixture was extracted with ethyl acetate (100 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (30 mL×2), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/4) to give 4-bromo-2,3-dihydrobenzofuran-7-amine 1-2 (1.0 g).
LC-MS: Rt: 0.444 min; MS m/z (ESI): 213.9 [M+H]+;
4-Bromo-2,3-dihydrobenzofuran-7-amine 1-2 (1.0 g, 4.67 mmol) was dissolved in dichloromethane (20 mL), di-tert-butyl dicarbonate (1.41 g, 6.46 mmol) and 4-dimethylaminopyridine (571 mg, 4.67 mmol) were added, and the mixture was stirred at 25° C. for 16 h. After the reaction was completed as detected by LCMS, water (20 mL) was added to dilute the mixture, the mixture was extracted with ethyl acetate (100 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (30 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/5) to give tert-butyl (4-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 1-3 (600 mg, 38.8%).
LC-MS: Rt: 1.756 min; MS m/z (ESI): 258.1 [M-56+H]+;
1H NMR (400 MHZ, CHLOROFORM-d) δ=7.71-7.54 (m, 1H), 6.87 (d, J=8.6 Hz, 1H), 6.44 (s, 1H), 4.55 (t, J=8.8 Hz, 2H), 3.15 (t, J=8.8 Hz, 2H), 1.43 (s, 9H).
tert-Butyl (4-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 1-3 (450 mg, 1.43 mmol) was dissolved in dioxane (10 mL), bis(pinacolato)diboron (909 mg, 3.58 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (105 mg, 0.14 mmol), and potassium acetate (422 mg, 4.30 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/6) to give compound 1-4 (410 mg, 79.2%).
LC-MS: Rt: 1.937 min; MS m/z (ESI): 306.2 [M-56+H]+.
The reactant 16-hydroxyprednisolone F1 (1 g, 2.66 mmol) and anhydrous magnesium sulfate (1.22 g, 10.1 mmol) were dissolved in acetonitrile (20 mL), and the mixture was stirred at 25° C. for 1 h. 4-(Bromomethyl)benzaldehyde (529 mg, 2.66 mmol) was dissolved in acetonitrile (2 mL) and added to the reaction mixture, the mixture was cooled to 0° C., trifluoromethanesulfonic acid (1.88 g, 12.5 mmol) was added dropwise, and the mixture was stirred at 25° C. for 2 h. After the reaction was completed as detected by LCMS, the reaction mixture was quenched with an aqueous sodium bicarbonate solution (20 mL), the mixture was extracted with ethyl acetate (100 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (50 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/2) to give a crude product which was separated and purified by SFC [column: DAICEL CHIRALCEL OD (250 mm×50 mm, 10 μm); mobile phase: A: a 0.1% solution of ammonia in methanol; B: CO2, 50%] to give intermediate 1-5 (1.2 g, 80.9%).
LC-MS: Rt: 1.551 min; MS m/z (ESI): 557.1 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.45 (s, 3H), 7.31 (d, J=10.1 Hz, 1H), 6.16 (dd, J=1.8, 10.1 Hz, 1H), 5.93 (s, 1H), 5.75 (s, 1H), 5.45 (s, 1H), 5.16-5.03 (m, 1H), 4.94 (d, J=4.9 Hz, 1H), 4.79 (d, J=3.0 Hz, 1H), 4.68 (s, 2H), 4.53 (d, J=19.5 Hz, 1H), 4.30 (s, 1H), 4.19 (d, J=19.4 Hz, 1H), 2.61-2.53 (m, 1H), 2.31-2.25 (m, 1H), 2.17-2.07 (m, 1H), 2.06-1.98 (m, 1H), 1.81-1.61 (m, 5H), 1.39 (s, 3H), 1.10-0.99 (m, 2H), 0.87 (s, 3H).
The absolute configuration of intermediate 1-5 was verified by two-dimensional nuclear magnetic resonance (see
Compound 1-5 (300 mg, 0.54 mmol) was dissolved in tetrahydrofuran (6 mL) and water (0.6 mL), tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate (194 mg, 0.54 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (78.8 mg, 0.11 mmol), and potassium carbonate (223 mg, 1.61 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/1) to give compound 1-6 (300 mg, 54.8%).
LC-MS: Rt: 0.584 min; MS m/z (ESI): 712.4 [M+H]+;
1-6 (280 mg, 0.39 mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at 25° C. for 1 h. After the reaction was completed as detected by LCMS, the reaction mixture was concentrated, and the crude product was separated and purified by preparative-HPLC [column: Phenomenex luna 30×30 mm×10 μm; YMC AQ 100×30×10 μm; mobile phase: A: water (HCl); B: ACN, B %, 30%-60%, 15 min] to give compound 001 (10 mg, 19.0%).
LC-MS: Rt: 1.511 min; MS m/z (ESI): 612.5 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=9.52-8.19 (m, 2H), 7.38 (d, J=8.1 Hz, 2H), 7.32 (d, J=10.1 Hz, 1H), 7.20 (d, J=8.0 Hz, 2H), 6.89 (d, J=8.0 Hz, 1H), 6.65 (d, J=8.0 Hz, 1H), 6.17 (dd, J=1.8, 10.1 Hz, 1H), 5.93 (s, 1H), 5.41 (s, 1H), 4.92 (d, J=5.0 Hz, 1H), 4.81 (br s, 1H), 4.60 (t, J=8.8 Hz, 2H), 4.50 (d, J=19.4 Hz, 1H), 4.29 (br s, 1H), 4.18 (d, J=19.4 Hz, 1H), 3.86 (s, 2H), 3.16-3.08 (m, 2H), 2.62-2.54 (m, 1H), 2.30 (m, 1H), 2.19-2.08 (m, 1H), 2.07-1.97 (m, 1H), 1.81-1.59 (m, 5H), 1.40 (s, 3H), 1.12-0.96 (m, 2H), 0.87 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
6-(4,4,5-Trimethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (1.00 g, 4.10 mmol), N,N-diisopropylethylamine (1.06 g, 8.19 mmol), 4-dimethylaminopyridine (50 mg, 0.41 mmol), and di-tert-butyl dicarbonate (894 mg, 4.10 mmol) were dissolved in anhydrous dichloromethane (20 mL), and the mixture was reacted at 25° C. for 6 h. After the reaction was completed as detected by LCMS, water (20 mL) was added to quench the reaction, the mixture was extracted with dichloromethane (50 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (30 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (ethyl acetate/petroleum ether=1/5) to give 2-2 (1.0 g, 69.5%).
LC-MS: Rt: 1.793 min; MS m/z (ESI): 345.3 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=8.50-8.24 (m, 2H), 8.00-7.75 (m, 2H), 1.70 (d, J=3.5 Hz, 9H), 1.36 (d, J=5.1 Hz, 12H).
2-2 (500 mg, 1.45 mmol), 4-(bromomethyl)benzaldehyde (376 mg, 1.89 mmol), potassium carbonate (602 mg, 4.36 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (106 mg, 0.14 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL), and the mixture was reacted at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (ethyl acetate/petroleum ether=1/5) to give 2-3 (380 mg, crude).
LC-MS: Rt: 1.563 min and 1.593; MS m/z (ESI): 337.3 [M+H]+;
16α-Hydroxyprednisolone F1 (230 mg, 0.61 mmol) was dissolved in anhydrous acetonitrile (1.5 mL), anhydrous magnesium sulfate (279 mg, 2.32 mmol) was added, and the mixture was stirred at 25° C. for 1 h. 2-3 (308 mg, 0.92 mmol) was dissolved in anhydrous acetonitrile (1.5 mL) and added to the reaction system, the mixture was cooled to 0° C., trifluoromethanesulfonic acid (431 mg, 2.87 mmol) was added dropwise, and the mixture was stirred at 25° C. for 2 h. After the reaction was completed as detected by LCMS, a saturated sodium bicarbonate solution (5 mL) was added to quench the reaction, the mixture was extracted with ethyl acetate (30 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (20 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was purified by Prep-HPLC [column: Phenomenex Luna 30×30 mm×10 μm; YMC AQ 100×30×10 μm; mobile phase: A: water (containing 0.225% formic acid); B: MeCN, B %, 15%-45%, 20 min] to give compound 002 (35.8 mg, 9.75%).
LC-MS: Rt: 1.390 min; MS m/z (ESI): 595.5 [M+H]; Chiral-HPLC: Rt: 2.211 min, chiral purity=97.56%.
1H NMR (400 MHz, DMSO-d6) δ=8.39 (br s, 1H), 7.52 (d, J=8.3 Hz, 1H), 7.44 (s, 1H), 7.40-7.35 (m, 2H), 7.33-7.26 (m, 3H), 7.12 (d, J=8.4 Hz, 1H), 6.16 (dd, J=1.6, 10.1 Hz, 1H), 5.93 (s, 1H), 5.40 (s, 1H), 5.08 (br s, 1H), 4.92 (d, J=4.9 Hz, 1H), 4.78 (d, J=3.0 Hz, 1H), 4.50 (br d, J=16.0 Hz, 1H), 4.28 (s, 1H), 4.17 (d, J=19.5 Hz, 1H), 4.05 (s, 2H), 2.30 (s, 1H), 2.15-1.98 (m, 2H), 1.81-1.60 (m, 5H), 1.39 (s, 3H), 1.10-0.97 (m, 2H), 0.86 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
Compound 1-5 (500 mg, 0.89 mmol) was dissolved in tetrahydrofuran (5 mL) and water (0.5 mL), 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole 3-1 (262 mg, 1.08 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (66 mg, 0.09 mmol), and potassium carbonate (372 mg, 2.69 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the mixture was diluted with ethyl acetate (100 mL), the organic phase was washed with a saturated aqueous sodium chloride solution (30 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/1) to give a crude product which was separated and purified by Prep-HPLC [column: Phenomenex luna 30×30 mm×10 μm+YMC AQ 100×30×10 μm; mobile phase: A: water (0.225% FA); B: MeCN, B %, 50%-80%, 20 min] to give compound 003 (60 mg 11.2%).
LC-MS: Rt: 2.098 min; MS m/z (ESI): 594.3 [M+H]+; Chiral-HPLC: Rt: 1.204 min, chiral purity=100%.
1H NMR (400 MHZ, DMSO-d6) δ=10.92 (s, 1H), 7.41 (d, J=8.1 Hz, 1H), 7.37 (d, J=8.0 Hz, 2H), 7.30 (d, J=10.1 Hz, 1H), 7.28-7.23 (m, 3H), 7.18 (s, 1H), 6.85 (dd, J=1.3, 8.1 Hz, 1H), 6.34 (s, 1H), 6.16 (dd, J=1.8, 10.1 Hz, 1H), 5.93 (s, 1H), 5.40 (s, 1H), 5.08 (t, J=5.9 Hz, 1H), 4.92 (d, J=5.0 Hz, 1H), 4.78 (d, J=3.1 Hz, 1H), 4.50 (dd, J=6.3, 19.4 Hz, 1H), 4.29 (s, 1H), 4.17 (dd, J=5.4, 19.4 Hz, 1H), 4.00 (s, 2H), 2.59-2.53 (m, 1H), 2.36-2.26 (m, 1H), 2.18-2.06 (m, 1H), 2.05-1.96 (m, 1H), 1.80-1.54 (m, 5H), 1.39 (s, 3H), 1.11-0.97 (m, 2H), 0.86 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
6-Bromoindoline (1.0 g, 5.05 mmol) was dissolved in dioxane (20 mL), bis(pinacolato)diboron (2.56 g, 10.1 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (369 mg, 504 μmol), and potassium acetate (1.49 g, 15.2 mmol) were added, and the mixture was reacted at 80° C. for 12 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/3) to give 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline 4-2 (1.2 g, 96.9%).
LC-MS: Rt: 1.509 min; MS m/z (ESI): 246.3 [M+H]+;
4-2 (500 mg, 2.04 mmol) was dissolved in dichloromethane (8 mL), di-tert-butyl dicarbonate (891 mg, 4.08 mmol), diisopropylethylamine (528 mg, 4.08 mmol), and 4-dimethylaminopyridine (49.8 mg, 408 μmol) were added, and the mixture was stirred at 25° C. for 6 h. After the reaction was completed as detected by LCMS, the mixture was diluted with dichloromethane (50 mL), the organic phase was washed with a saturated aqueous sodium chloride solution (15 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/5) to give 4-3 (260 mg, 36.9%).
LC-MS: Rt: 2.340 min; MS m/z (ESI): 290.3 [M-56+H];
tert-Butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-formate (260 mg, 753 μmol) was dissolved in tetrahydrofuran (5 mL), 4-(bromomethyl)benzaldehyde (299 mg, 1.51 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (165 mg, 226 μmol), and potassium carbonate (520 mg, 3.77 mmol) were added, and the mixture was stirred at 80° C. for 12 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/5) to give 4-4 (190 mg, 74.8%).
LC-MS: Rt: 1.810 min; MS m/z (ESI): 282.2 [M-56+H]+;
16α-Hydroxyprednisolone F1 (165 mg, 438 μmol) was dissolved in acetonitrile (3 mL), anhydrous magnesium sulfate (258 mg, 2.15 mmol) was added, and the mixture was stirred at 25° C. for 1 h. tert-Butyl 6-(4-formylbenzyl)indoline-1-formate (178 mg, 526 μmol) was dissolved in acetonitrile (1 mL) and added to the reaction mixture, the mixture was cooled to 0° C., trifluoromethanesulfonic acid (322 mg, 2.15 mmol) was added dropwise, and the mixture was stirred at 25° C. for 2 h. After the reaction was completed as detected by LCMS, a saturated aqueous sodium bicarbonate solution (10 mL) was added to quench the reaction, the mixture was extracted with ethyl acetate (30 mL×3), the organic phase was washed with a saturated aqueous sodium chloride solution (20 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by Prep-HPLC [column: Phenomenex luna 30×30 mm×10 μm+YMC AQ 100×30×10 μm; mobile phase: A: water (0.225% FA); B: MeCN, B %, 18%-48%, 20 min] to give compound 004 (42.9 mg, 6.45%).
LC-MS: Rt: 1.532 min; MS m/z (ESI): 596.3 [M+H]+; Chiral-HPLC: Rt: 2.502 min, chiral purity=100%.
1H NMR (400 MHZ, DMSO-d6) δ=7.37-7.33 (m, 2H), 7.30 (d, J=10.1 Hz, 1H), 7.20 (d, J=8.0 Hz, 2H), 6.88 (d, J=7.3 Hz, 1H), 6.37 (d, J=7.4 Hz, 1H), 6.28 (s, 1H), 6.18-6.13 (m, 1H), 5.95-5.91 (m, 1H), 5.39 (s, 2H), 5.12-5.06 (m, 1H), 4.91 (d, J=5.0 Hz, 1H), 4.80-4.75 (m, 1H), 4.54-4.45 (m, 1H), 4.32-4.25 (m, 1H), 4.21-4.12 (m, 1H), 3.75 (s, 2H), 3.38-3.34 (m, 2H), 2.84-2.77 (m, 2H), 2.55-2.47 (m 1H), 2.34-2.27 (m, 1H), 2.16-2.07 (m, 1H), 2.06-1.97 (m, 1H), 1.79-1.59 (m, 5H), 1.39 (s, 3H), 1.12-0.97 (m, 2H), 0.89-0.82 (m, 3H).
The synthetic route and the specific synthetic steps were as follows.
7-Bromo-3,4-dihydro-2H-benzo[b][1,4]oxazine (1.00 g, 4.67 mmol) 5-1 was dissolved in tetrahydrofuran (20 mL), bis(pinacolato)diboron (1.30 g, 5.14 mmol), potassium acetate (1.38 g, 14.0 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (341 mg, 467 μmol) were added, and the mixture was reacted at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (ethyl acetate/petroleum ether=1/5) to give 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine 5-2 (950 mg, 77.9%).
LC-MS: Rt: 1.454 min; MS m/z (ESI):262.3 [M+H]+;
5-2 (900 mg, 3.45 mmol) was dissolved in tetrahydrofuran (20 mL), 4-(bromomethyl)benzaldehyde (1.03 g, 5.17 mmol), potassium carbonate (1.43 g, 10.3 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (252 mg, 345 μmol) were added, and the mixture was reacted at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (ethyl acetate/petroleum =1/3) to give 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde 5-3 (397 mg).
LC-MS: Rt: 1.202 min; MS m/z (ESI):254.1 [M+H]+;
16α-Hydroxyprednisolone F1 (350 mg, 929 μmol) was dissolved in acetonitrile (15 mL), magnesium sulfate (425 mg, 3.53 mmol) was added, and the mixture was stirred at 25° C. for 1 h. 4-((3,4-Dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde (235 mg, 929 μmol) 5-3 was dissolved in acetonitrile (5 mL) and added to the reaction system, the mixture was cooled to 0° C., trifluoromethanesulfonic acid (655 mg, 4.37 mmol) was added dropwise, and the mixture was stirred at 25° C. for 2 h. After the reaction was completed as detected by LCMS, a saturated aqueous sodium bicarbonate solution (15 mL) was added to quench the reaction, the mixture was extracted with ethyl acetate (50 mL×3), the organic phase was washed with a saturated aqueous sodium chloride solution (20 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by prep-HPLC [column: Xtimate C18 150×40 mm×10 μm; mobile phase: A: water (10 mM NH4HCO3); B: MeCN, B %, 40%-70%, 10 min] to give compound 005 (20 mg, 21.1%).
LC-MS: Rt: 1.874 min; MS m/z (ESI): 612.3 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.41-7.27 (m, 3H), 7.19 (br d, J=7.9 Hz, 2H), 6.56-6.38 (m, 3H), 6.16 (dd, J=1.1, 10.0 Hz, 1H), 5.93 (s, 1H), 5.52 (br s, 1H), 5.39 (s, 1H), 5.05 (t, J=5.9 Hz, 1H), 4.92 (br d, J=5.0 Hz, 1H), 4.76 (br d, J=2.6 Hz, 1H), 4.49 (br dd, J=6.3, 19.4 Hz, 1H), 4.29 (br s, 1H), 4.17 (br dd, J=5.6, 19.4 Hz, 1H), 4.10-4.01 (m, 2H), 3.70 (s, 2H), 3.29-3.25 (m, 1H), 3.21 (br s, 2H), 2.37-2.25 (m, 1H), 2.18-1.96 (m, 2H), 1.84-1.56 (m, 5H), 1.39 (s, 3H), 1.14-0.97 (m, 2H), 0.86 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
The compound (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 6-1 (1.0 g, 2.21 mmol) was dissolved in a 30% aqueous fluoroboric acid solution (20 mL), and the mixture was stirred at room temperature for 48 h. After the reaction was completed as detected by LCMS, 40 mL of water was added, the mixture was stirred for 5 min and filtered, and the residue was washed with water (10 mL×3) and ethanol (10 mL×3) sequentially to give a crude product of (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2- hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one 6-2 (0.7 g).
LC-MS: Rt: 0.426 min; MS m/z (ESI): 413.1 [M+H]+;
6-2 (150 mg, 0.36 mmol) was dissolved in anhydrous acetonitrile (1.5 mL), magnesium sulfate (166 mg, 1.38 mmol) was added, and the mixture was stirred at 25° C. for 1 h. 4-((3,4-Dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde (138 mg, 0.55 mmol) was dissolved in anhydrous acetonitrile (1.5 mL) and added to the reaction system, the mixture was cooled to 0° C., trifluoromethanesulfonic acid (257 mg, 1.71 mmol) was added dropwise, and the mixture was reacted at 25° C. for 2 h. After the reaction was completed as detected by LCMS, a saturated aqueous sodium bicarbonate solution (10 mL) was added to quench the reaction, the mixture was extracted with ethyl acetate (50 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (20 mL), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by Prep-HPLC [column: Phenomenex Luna C18 100×30 mm×3 μm; mobile phase: A: water (0.225% FA); B: MeCN, B %: 40%-70%, 8 min] to give compound 006 (82.7 mg, 34.6%).
LC-MS: Rt: 1.553 min; MS m/z (ESI): 648.2 [M+H]+; Chiral-HPLC: Rt: 3.241 min, chiral purity=100%.
1H NMR (400 MHZ, DMSO-d6) δ=7.32 (d, J=8.1 Hz, 2H), 7.26 (d, J=10.1 Hz, 1H), 7.20 (d, J=8.1 Hz, 2H), 6.53-6.48 (m, 1H), 6.47-6.43 (m, 2H), 6.33-6.27 (m, 1H), 6.13 (s, 1H), 5.72-5.57 (m, 1H), 5.50 (d, J=3.0 Hz, 1H), 5.43 (s, 1H), 4.94 (d, J=4.9 Hz, 1H), 4.51 (d, J=19.5 Hz, 1H), 4.19-4.04 (m, 2H), 4.07-4.03 (m, 2H), 3.69 (s, 2H), 3.22-3.19 (m, 2H), 2.65-2.56 (m, 1H), 2.28-2.07 (m, 2H), 2.05-1.98 (m, 1H), 1.75-1.65 (m, 3H), 1.51-1.49 (m, 1H), 1.50 (s, 3H), 0.86 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
According to the method reported in the literature (CN110903258), benzo[d]oxazol-4-amine (1.0 g, 7.5 mmol) was dissolved in dimethylformamide (20 mL), NBS (1.3 g, 7.1 mmol) was added, and the mixture was stirred at 25° C. for 1 h. After the reaction was completed as detected by LCMS, water (20 mL) was added to dilute the reaction mixture, the mixture was extracted with ethyl acetate (100 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (30 mL×2), the organic phase was dried, filtered, and concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/2) to give 7-bromobenzo[d]oxazol-4-amine 7-2 (1.3 g, 64.5%).
LC-MS: Rt: 1.181 min; MS m/z (ESI): 214.9 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=8.59 (s, 1H), 7.24 (d, J=8.5 Hz, 1H), 6.52 (d, J=8.6 Hz, 1H), 5.86 (s, 2H).
7-Bromobenzo[d]oxazol-4-amine 7-2 (1.3 g, 6.1 mmol) and bis(pinacolato)diboron (3.1 g, 12.2 mmol) were dissolved in anhydrous dioxane (26 mL), potassium acetate (1.8 g, 18.3 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (447 mg, 0.6 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (tetrahydrofuran/petroleum ether=1/2) to give 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-4-amine 7-3 (1.4 g, 64.3%).
LC-MS: Rt: 1.368 min; MS m/z (ESI): 261.2 [M+H];
Compound 7-3 (112 mg, 0.4 mmol) and compound 1-5 (200 mg, 0.4 mmol) were dissolved in tetrahydrofuran (4 mL) and water (0.4 mL), potassium carbonate (149 mg, 1.1 mmol) and tetrakis(triphenylphosphine)palladium(0) (124 mg, 0.1 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered, the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/2), and the resulting crude product was separated and purified by preparative HPLC [column: Phenomenex C18 75×30 mm×3 μm; mobile phase: A: water (NH4HCO3); B: acetonitrile, B %, 34%-58%, 10 min] to give compound 007 (37.7 mg, 20.52%).
LC-MS: Rt: 1.832 min; MS m/z (ESI): 611.5 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=8.45 (s, 1H), 7.36 (d, J=8.1 Hz, 2H), 7.31 (d, J=10.1 Hz, 1H), 7.25 (d, J=8.1 Hz, 2H), 6.93 (d, J=8.0 Hz, 1H), 6.48 (d, J=8.0 Hz, 1H), 6.17 (dd, J=1.8, 10.1 Hz, 1H), 5.94 (s, 1H), 5.48 (s, 2H), 5.39 (s, 1H), 5.08 (s, 1H), 4.92 (d, J=5.0 Hz, 1H), 4.78 (d, J=2.9 Hz, 1H), 4.50 (dd, J=3.0, 19.3 Hz, 1H), 4.29 (s, 1H), 4.17 (d, J=18.4 Hz, 1H), 4.03 (s, 2H), 2.61 -2.53 (m, 1H), 2.37-2.29 (m, 1H), 2.16-1.97 (m, 2H), 1.82-1.59 (m, 5H), 1.40 (s, 3H), 1.13-1.00 (m, 2H), 0.86 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
The starting material benzo[d][1,3]dioxolan-4-amine 8-1 (2.00 g, 14.6 mmol) was dissolved in N,N-dimethylformamide (50 mL), N-bromosuccinimide (2.50 g, 13.9 mmol) was added at 0° C., and the mixture was stirred at 0° C. for 1 h. After the reaction was completed as detected by LCMS, ethyl acetate (200 mL) was added to dilute the reaction mixture, the mixture was washed with water (100 mL×2) and saturated brine (50 mL), the organic phase was dried, filtered, and concentrated, and the residue was purified by column chromatography (petroleum ether/tetrahydrofuran=2/1) to give 7-bromobenzo[d][1,3]dioxolan-4-amine 8-2 (1.30 g, 94.8%).
LC-MS: Rt: 1.311 min; MS m/z (ESI): 218.1 [M+H]+;
7-Bromo-benzo[d][1,3]dioxolan-4-amine 8-2 (1.00 g, 4.6 mmol) and di-tert-butyl dicarbonate (3.0 g, 13.9 mmol) were dissolved in 1,4-dioxane (20 mL), and the mixture was stirred at 100° C. for 16 h. After the reaction was completed as detected by LCMS, the reaction mixture was concentrated, and the residue was purified by column chromatography (petroleum ether/ethyl acetate=6/1) to give tert-butyl (7-bromobenzo[d][1,3]dioxolan-4-yl)carbamate 8-3 (820 mg, 43.5%).
LC-MS: Rt: 1.707 min; MS m/z (ESI): 262.1 [M+H-56]+;
1H NMR (400 MHZ, DMSO-d6) δ=8.95 (s, 1H), 6.98-6.89 (m, 2H), 6.10 (s, 2H), 1.44 (s, 9H).
tert-Butyl (7-bromobenzo[d][1,3]dioxolan-4-yl)carbamate 8-3 (820 mg, 2.60 mmol) and bis(pinacolato)diboron (1.30 g, 5.20 mmol) were dissolved in 1,4-dioxane (25 mL), potassium acetate (764 mg, 7.80 mmol) and [1,1′ bis(diphenylphosphino)ferrocene]palladium dichloride (190 mg, 0.30 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (petroleum ether/tetrahydrofuran=6/1) to give tert-butyl (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-4 (1.00 g, 83.8%).
LC-MS: Rt: 1.782 min; MS m/z (ESI): 308.3 [M+H-56]+;
tert-Butyl (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-4 (489 mg, 1.35 mmol) and (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 1-5 (500 mg, 0.9 mmol) were dissolved in a mixed solvent of tetrahydrofuran (20 mL) and water (2 mL), potassium carbonate (372 mg, 2.70 mmol) and tetrakis(triphenylphosphine)palladium(0) (311 mg, 0.3 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, water was added to dilute the reaction mixture, the mixture was extracted with ethyl acetate (100 mL), the organic phase was washed with saturated brine (30 mL), dried, filtered, and concentrated, and the residue was purified by column chromatography (petroleum ether/tetrahydrofuran=1/1) and then separated and purified by preparative high performance liquid chromatography [column: Gemini NX C18 5 μm×10 mm×150 mm; mobile phase: A: 0.1% aqueous formic acid solution; B: acetonitrile, 49%-79%, 10 min] to give intermediate tert-butyl (7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-5 (20 mg, 2.8%).
LC-MS: Rt: 1.742 min; MS m/z (ESI): 714.4 [M+H]+;
tert-Butyl (7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-5 (20 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at 25° C. for 0.5 h. After the reaction was completed as detected by LCMS, The reaction mixture was concentrated, and the residue was separated and purified by preparative high performance liquid chromatography [column: Xtimate C18 100×30 mm×10 μm; mobile phase: A: 0.1% aqueous ammonia solution; B: acetonitrile, B %, 20%-70%, 35 min] to give (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolan-4-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one (compound 008) (3.1 mg, 17.3%).
LC-MS: Rt: 1.681 min; MS m/z (ESI): 614.5 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.36 (d, J=8.0 Hz, 2H), 7.32 (d, J=10.1 Hz, 1H), 7.20 (d, J=7.9 Hz, 2H), 6.42 (d, J=8.3 Hz, 1H), 6.22-6.15 (m, 2H), 5.94 (s, 1H), 5.88 (s, 2H), 5.40 (s, 1H), 5.11 (t, J=5.9 Hz, 1H), 4.92 (d, J=5.0 Hz, 1H), 4.83-4.75 (m, 3H), 4.51 (dd, J=6.3, 19.4 Hz, 1H), 4.30 (s, 1H), 4.18 (dd, J=5.6, 19.4 Hz, 1H), 3.73 (s, 2H), 2.31 (d, J=2.3 Hz, 1H), 2.15-2.00 (m, 2H), 1.81-1.61 (m, 5H), 1.40 (s, 3H), 1.25 (s, 1H), 1.13-1.01 (m, 2H), 0.87 (s, 3H).
The synthetic route and the specific synthetic steps were as follows.
5-Bromo-2,3-dihydrobenzofuran-7-carboxylic acid 9-1 (1.00 g, 4.11 mmol) was dissolved in toluene (10 mL) and tert-butanol (10 mL), 4A molecular sieves (200 mg) and triethylamine (1.25 g, 12.3 mmol) were added at room temperature, and the mixture was stirred at 110° C. for 0.5 h and cooled to room temperature. Diphenylphosphoryl azide (1.70 g, 6.17 mmol) was added dropwise, and the mixture was warmed to 110° C. and stirred for 10 h. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and diluted with ethyl acetate (150 mL), the organic phase was washed with saturated brine (30 mL), dried, filtered, and concentrated, and the residue was separated and purified by column chromatography (0-40% ethyl acetate/petroleum ether) to give intermediate tert-butyl (5-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 9-2 (1.03 g, 79.7%).
LC-MS: Rt: 1.065 min; MS m/z (ESI): 259.7 [M+H-56]+;
1H NMR (400 MHZ, DMSO-d6) δ=8.59 (s, 1H), 7.50 (br s, 1H), 7.13-7.11 (m, 1H), 4.61-4.50 (m, 2H), 3.24-3.17 (m, 2H), 1.48-1.41 (m, 9H).
tert-Butyl (5-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 9-2 (600 mg, 1.91 mmol), bis(pinacolato)diboron (533 mg, 2.10 mmol), potassium acetate (562 mg, 5.73 mmol), and 1,1′-bis(diphenylphosphino)ferrocene palladium chloride (139 mg, 190 μmol) were dissolved in anhydrous 1,4-dioxane (10 mL), and the mixture was reacted at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (0-10% tetrahydrofuran/petroleum ether) to give intermediate tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3 (990 mg, 71.8%).
LC-MS: Rt: 4.417 min; MS m/z (ESI): 306.2 [M+H-56]+;
1H NMR (400 MHZ, CHLOROFORM-d) δ=8.17-8.27 (m, 1 H) 7.38 (s, 1 H) 7.25-7.31 (m, 1 H) 4.59-4.66 (m, 2 H) 3.20-3.29 (m, 2 H) 1.33 (s, 12 H) 1.31 (s, 9 H)
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(Bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 1-5 (300 mg, 538 μmol), tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3 (300 mg, 830 μmol), tetrakis(triphenylphosphine)palladium(0) (62.2 mg, 53.8 μmol), and potassium carbonate (148 mg, 1.08 mmol) were dissolved in anhydrous tetrahydrofuran (5 mL) and water (1 mL), and the mixture was stirred at 80° C. for 2 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was extracted with ethyl acetate (50 mL), the organic phase was washed with saturated brine (10 mL), dried, filtered, and concentrated, and the residue was separated and purified by column chromatography (0-50% tetrahydrofuran/petroleum ether) to give intermediate (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)-2,3-dihydrobenzofuran-7- yl)carbamate 9-4 (300 mg, 78.3%).
LC-MS: Rt: 2.156 min; MS m/z (ESI): 712.3 [M+H]+;
The intermediate tert-butyl (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4- oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)-2,3- dihydrobenzofuran-7-yl)carbamate 9-4 (220 mg, 309 μmol) was dissolved in anhydrous dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at 25° C. for 40 min. After the reaction was completed as detected by LCMS, The reaction mixture was blow-dried with nitrogen, and the resulting residue was separated and purified by preparative high performance liquid chromatography [column: YMC-Pack CN 150×30 mm×5 μm; mobile phase: A: 0.1% aqueous formic acid solution; B: acetonitrile, B %, 40%-60%, 12 min] to give the compound (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 009 (2.00 mg, 1.06%).
LC-MS: Rt: 2.576 min; MS m/z (ESI): 612.3 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.33-7.38 (m, 2 H) 7.31 (d, J=10.13 Hz, 1 H) 7.19 (d, J=8.13 Hz, 2 H) 6.29 (d, J=17.26 Hz, 2 H) 6.17 (dd, J=10.13, 1.88 Hz, 1 H) 5.94 (s, 1 H) 5.39 (s, 1 H) 5.08 (s, 1 H) 4.90-4.94 (m, 1 H) 4.77-4.81 (m, 1 H) 4.45-4.73 (m, 3 H) 4.42 (t, J=8.63 Hz, 2 H) 4.29 (d, J=2.88 Hz, 1 H) 4.13-4.23 (m, 1 H) 3.71 (s, 2 H) 3.04 (t, J=8.69 Hz, 2 H) 2.30 (s, 1 H) 2.09-2.17 (m, 1 H) 1.98-2.06 (m, 1 H) 1.56-1.84 (m, 6 H) 1.40 (s, 3 H) 0.97-1.11 (m, 2 H) 0.86 (s, 3 H).
The synthetic route and the specific synthetic steps were as follows.
The starting material 6-alpha,9-alpha-difluoro-11-beta, 16-alpha, 17-alpha,21-tetrahydroxypregna-1,4-diene-3,20-dione SM-1 (1.00 g, 2.42 mmol) was dissolved in anhydrous acetonitrile (10 mL), anhydrous magnesium sulfate (1.46 g, 12.1 mmol) was added, and the mixture was stirred at 25° C. for 1 h. A solution of 4-bromomethylbenzaldehyde (579 mg, 2.91 mmol) in acetonitrile (2 mL) was added at 0° C., a solution of trifluoromethanesulfonic acid (1.82 g, 12.12 mmol) in acetonitrile (2 mL) was slowly added dropwise, and the mixture was stirred at 0° C. for 5 min. After the reaction was completed as detected by LCMS, the reaction was quenched with a saturated aqueous sodium bicarbonate solution (20 mL), the mixture was extracted with ethyl acetate (50 mL×2), the organic phase was washed with a saturated aqueous sodium chloride solution (20 mL), dried, filtered, and concentrated, and the residue was separated and purified by Prep-HPLC [column: YMC-Pack CN 150×30 mm×5 μm; mobile phase: A, 0.1% aqueous formic acid solution; B, acetonitrile, B %, 53%-63%, 12 min] to give intermediate (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 010-3 (640 mg, 42%).
LC-MS: Rt: 2.845 min; MS m/z (ESI): 595.1 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.62-7.39 (m, 4H), 7.27 (d, J=10.3 Hz, 1H), 6.30 (dd, J=1.8, 10.0 Hz, 1H), 6.13 (s, 1H), 5.77-5.45 (m, 3H), 5.01-4.95 (m, 1H), 4.68 (s, 2H), 4.55 (br d, J=19.3 Hz, 1H), 4.22 (br d, J=19.3 Hz, 2H), 2.74-2.56 (m, 1H), 2.37-2.12 (m, 3H), 2.09-1.95 (m, 1H), 1.77-1.64 (m, 3H), 1.56-1.42 (m, 4H), 0.87 (s, 3H);
19F NMR (376 MHz, DMSO-d6) δ=−164.99 (s, 1F), −186.39 (s, 1F).
4-Bromo-2,3-dihydrobenzofuran-7-amine 1-3 (7.30 g, 34.1 mmol) was dissolved in 1,4-dioxane (150 mL), bis(pinacolato)diboron (20.0 g, 78.8 mmol), 1,1′-bis(diphenylphosphino)ferrocene palladium chloride (2.50 g, 3.4 mmol), and potassium acetate (10.0 g, 102.3 mmol) were added, and the mixture was stirred at 80° C. for 16 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the residue was separated and purified by column chromatography (tetrahydrofuran/petroleum ether=1/6) to give intermediate 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine 010-2 (9.50 g, 96.0%).
LC-MS: Rt: 1.470 min; MS m/z (ESI): 262.0 [M+H];
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 010-3 (150 mg, 253 μmol) was dissolved in tetrahydrofuran (2 mL) and water (0.5 mL), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine (79 mg, 303 μmol), potassium carbonate (79 mg, 303 μmol), and tetrakis(triphenylphosphine)palladium(0) (29 mg, 25.3 μmol) were added sequentially, and the mixture was reacted at 80° C. for 2 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was extracted with ethyl acetate (10 mL×3), the organic phase was washed with a saturated aqueous sodium chloride solution (5 mL), dried, filtered, and concentrated, and the residue was separated and purified by preparative high performance liquid chromatography [column: Phenomenex C18 75×30 mm×3 μm; mobile phase: A: 0.1% aqueous ammonia solution; B: acetonitrile, B %, 30%-70%, 9 min] to give the compound (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 010 (20.0 mg).
LC-MS: Rt: 2.092 min; MS m/z (ESI): 648.3 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.33 (d, J=8.0 Hz, 2H), 7.27 (d, J=9.8 Hz, 1H), 7.18 (d, J=8.0 Hz, 2H), 6.42-6.37 (m, 2H), 6.30 (dd, J=1.8, 10.3 Hz, 1H), 6.13 (s, 1H), 5.75-5.56 (m, 1H), 5.52 (d, J=2.8 Hz, 1H), 5.45 (s, 1H), 5.11 (t, J=6.0 Hz, 1H), 4.95 (d, J=4.8 Hz, 1H), 4.56-4.38 (m, 5H), 4.25-4.15 (m, 2H), 3.73 (s, 2H), 2.97 (t, J=8.7 Hz, 2H), 2.44 (br s, 1H), 2.32-2.19 (m, 2H), 2.10-2.00 (m, 1H), 1.78-1.64 (m, 3H), 1.50 (s, 4H), 0.86 (s, 3H);
19F NMR (376 MHz, DMSO-d6) δ=−164.96 (s, 1F), −186.39 (s, 1F).
The synthetic route and the specific synthetic steps were as follows.
2,3-Dihydro-1H-inden-4-amine (2.00 g, 15.0 mmol) was dissolved in N,N-dimethylformamide (20 mL), N-bromosuccinimide (NBS) (2.67 g, 15.0 mmol) was added at 0° C., and the mixture was stirred at 0° C. for 1 h. After the reaction was completed as detected by LCMS, the mixture was quenched with a saturated sodium bicarbonate solution (50 mL) at 0° C. and extracted with ethyl acetate (100 mL×2), the organic phase was washed with water (50 mL×3) and saturated brine (50 mL), dried, filtered, and concentrated, and the resulting residue was separated and purified by column chromatography (0-15% ethyl acetate/petroleum ether) to give 7-bromo-2,3-dihydro-1H-inden-4-amine 11-2 (1.70 g, 53.4%).
LC-MS: Rt: 0.800 min; MS m/z (ESI): 213.8 [M+H]+;
7-Bromo-2,3-dihydro-1H-inden-4-amine 11-2 (100 mg, 471 μmol) and bis(pinacolato)diboron (239 mg, 943 μmol) were dissolved in 1,4-dioxane (5 mL), 1,1′-bis(diphenylphosphino)ferrocene palladium chloride (34.5 mg, 47.2 μmol) and potassium acetate (92.5 mg, 943 μmol) were added, and the mixture was stirred at 80° C. for 12 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the reaction mixture was filtered and concentrated, and the resulting residue was separated and purified by column chromatography (0-60% ethyl acetate/petroleum ether) to give intermediate 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-4-amine 11-3 (77.0 mg, 63.0%).
LC-MS: Rt: 0.914 min; MS m/z (ESI): 260.3 [M+H]+;
7-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-4-amine 11-3 (77 mg, 295.98 μmol) and (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a- dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one 1-5 (110 mg, 197 μmol) were dissolved in tetrahydrofuran (5 mL) and water (1 mL), 1,1′-bis(diphenylphosphino)ferrocene palladium chloride (14.4 mg, 19.7 μmol) and potassium carbonate (54.5 mg, 394 μmol) were added, and the mixture was stirred for reaction at 80° C. for 2 h under nitrogen atmosphere. After the reaction was completed as detected by LCMS, the mixture was diluted with water (20 mL) and then extracted with ethyl acetate (50 mL×2), the organic phase was washed with saturated brine (20 mL), dried, filtered, and concentrated, and the resulting residue was separated and purified by preparative high performance liquid chromatography [column: Boston Prime C18 150×30 mm×5 μm; mobile phase: A: 0.1% aqueous formic acid to give solution; B: ACN, 29%-49%, 12 min] to give (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydro-1H-inden-4-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-4(2H)-one (55.0 mg, 45.7%).
LC-MS: Rt: 0.856 min; MS m/z (ESI): 610.4 [M+H]+;
1H NMR (400 MHZ, DMSO-d6) δ=7.37-7.28 (m, 3H), 7.16-7.11 (m, 1H), 7.14 (d, J=7.8 Hz, 1H), 6.66 (d, J=7.8 Hz, 1H), 6.33 (d, J=8.0 Hz, 1H), 6.16 (dd, J=1.6, 10.2 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 5.08 (s, 1H), 4.91 (d, J=5.3 Hz, 1H), 4.80-4.75 (m, 1H), 4.75-4.55 (m, 2H), 4.50 (d, J=17.8 Hz, 1H), 4.29 (s, 1H), 4.17 (d, J=18.1 Hz, 1H), 3.73 (s, 2H), 2.66 (t, J=7.5 Hz, 2H), 2.63-2.57 (m, 2H), 2.57-2.53 (m, 1H), 2.30 (s, 1H), 2.16-1.98 (m, 2H), 1.91 (quin, J=7.4 Hz, 2H), 1.81-1.60 (m, 5H), 1.40 (s, 3H), 1.11-0.97 (m, 2H), 0.90-0.81 (m, 3H).
Compound 012 was prepared by referring to Example 5 and Example 9 and using compound 12-1 (CAS 1677706-25-0) as the starting material.
LC-MS: MS m/z (ESI): 612.3 [M+H]+.
Compound 013 was prepared by referring to Example 7 and using compound
(CAS 1267216-18-1) as the starting material.
LC-MS: MS m/z (ESI): 611.5 [M+H]+.
The synthetic route and the specific synthetic steps were as follows.
Intermediate 9-4 (80.0 mg, 112 μmol) was dissolved in N,N-dimethylformamide (4 mL), di-tert-butyl N,N-diethylphosphoramidite (336 mg, 1.35 mmol) and tetrazole (78.7 mg, 1.12 mmol) were added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed as monitored by TLC, the mixture was cooled to 0° C., hydrogen peroxide (580 mg, 5.12 mmol) was added to the reaction mixture, and the mixture was returned to room temperature and stirred for 1 h. After the reaction was completed as detected by LCMS, the reaction was quenched with an aqueous sodium sulfite solution (10 mL) at 0° C., the mixture was extracted with ethyl acetate (30 mL), the organic phase was washed with saturated brine (10 mL), dried, filtered, and concentrated, and the resulting residue was separated and purified by column chromatography (0-60% ethyl acetate/petroleum ether) to give intermediate 009-p-1 (50.0 mg, 49.2%).
LC-MS: Rt: 1.216 min; MS m/z (ESI): 904.6 [M+H]+;
Intermediate 009-p-1 (50.0 mg, 55.3 μmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (630 mg, 5.53 mmol) was added, and the mixture was stirred at room temperature for reaction for 30 min. After the reaction was completed as detected by LCMS, the reaction mixture was blow-dried with nitrogen, and the resulting residue was separated and purified by preparative high performance liquid chromatography [column: Boston Prime C18 150×30 mm×5 μm; mobile phase: A, 0.1% aqueous formic acid solution; B, acetonitrile, B %, 38%-58%, 12 min) to give compound 009-p (10.0 mg, 26.1%).
LC-MS: Rt: 2.752 min; MS m/z (ESI): 692.3 [M+H];
1H NMR (400 MHZ, DMSO-d6) δ=7.37-7.24 (m, 3H), 7.14 (d, J=6.8 Hz, 2H), 6.27 (s, 2H), 6.16 (dd, J=1.6, 10.0 Hz, 1H), 5.93 (s, 1H), 5.47 (s, 1H), 4.94-4.84 (m, 2H), 4.66-4.56 (m, 1H), 4.40 (t, J=8.6 Hz, 2H), 4.28 (s, 1H), 3.68 (s, 2H), 3.00 (t, J=8.7 Hz, 2H), 2.29 (d, J=2.9 Hz, 1H), 2.16-1.94 (m, 3H), 1.83-1.60 (m, 5H), 1.39 (s, 3H), 1.04-0.83 (m, 5H).
Phosphate ester compounds 001-p, 002-p, 003-p, 004-p, 005-p, 006-p, 007-p, 008-p, 010-p, 011-p, 012-p, and 013-p can be prepared by referring to the phosphorylation procedure described in Example 14 and using compounds 001, 002, 003, 004, 005, 006, 007, 008, 010, 011, 012, 013, or N-Boc intermediates of the compounds described above (e.g., intermediate 1-6 in Example 1) as the starting material.
Compounds other than the compounds synthesized in Examples 1-15 were synthesized by referring to the synthetic route and source material in Examples 1-15.
The compounds in the following test examples were prepared according to the methods in the above examples disclosed herein.
To generate the parental K562-GRE cell line, K562 cells were seeded at 5×105 cells/well onto a 6-well plate (brand: Costar, Cat. No: 3516) containing 2 mL of complete medium (RPM1640, 10% FBS, penicillin-streptomycin) and cultured at 37° C. with 5% CO2 for 24 h. The next day, 3 μg of pNL2.2[NLucP/MMTV/Hygro-NANO] (Promega) and 3 μL of PLUS reagent (brand: Invitrogen, Cat. No: 11514-015) were diluted in 150 μL of Opti-MEM (brand: Gibco, Cat. No: 11058021) and incubated at room temperature for 5 min. The pNL2.2[NLucP/MMTV/Hygro-NANO] vector contains MMTV LTR (mouse mammary tumor virus long terminal repeat), which drives transcription of the luciferase reporter gene NanoLuc in response to activation of several nuclear receptors (e.g. glucocorticoid receptor and androgen receptor). After incubation, the diluted DNA solution was mixed with Lipofectamine LTX solution (brand: Invitrogen, Cat. No: 15338-100) (6 μL of Lipofectamine LTX+144 μL of Opti-MEM) in a 1:1 ratio, pre-incubated, and incubated at room temperature for 15 min to form a DNA-Lipofectamine LTX complex. After incubation, 300 μL of the DNA-Lipofectamine complex was added directly to the cell wells. The K562 cells were transfected at 37° C. with 5% CO2 for 24 h. After transfection, the cells were washed with 3 mL of PBS and selectively grown for two weeks with complete growth medium containing 125 μg/mL hygromycin B (brand: Invitrogen, Cat. No: 10687010). “K562-GRE (pNL2.2[NLucP/MMTV/Hygro-NANO])” cells were generated.
The K562-GRE (pNL2.2[NLucP/MMTV/Hygro-NANO) cells were seeded at 50000 cells per well in 50 μL assay medium (RPMI 1640 medium, 1% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acid and 1% penicillin-streptomycin) on a 96-well tissue culture treated white plate (brand: Costar, Cat. No: 3917), and the cells were treated with 50 μL (2×) of compounds in 5-fold serial dilution with the assay medium, each compound at a total of 8 concentrations with a final concentration of 0.0000128 μM-1 μM in the system. After mixing, the cells were cultured in an incubator at 37° C. for 24 h. After 24 h of incubation, an equal volume of 100 μL of Nano-Glo luciferase assay system (brand: Promega, Cat. No: N1120) was added to the cells, the mixture was reacted in a shaker at 500 g for 10 min, and the chemiluminescence values were detected using the PHERAstar instrument. The antibody binding curves were plotted using the logarithmic value of the antibody concentration as the abscissa and the corresponding chemiluminescence reading as the ordinate, and the EC50 value was calculated by four-parameter fitting (GraphPad Prism9). The results are shown in Table 1.
The binding activity of the compound to GR was determined using the Polarscreen Glucocorticoid Receptor Assay Kit, Red (brand: Thermo, Cat. No: A15898). The test compound was diluted 10-fold with DMSO in a 96-well V-bottom plate (brand: Nunc, Cat. No: 249944). The highest concentration was 100 μM, 8 concentrations in total. The compound was then further diluted 50-fold with the detection buffer Complete GR Screening buffer provided in the kit, 10 μL of the diluted compound was transferred to a 384 microplate (brand: Corning, Cat. No: 4514), 5 μL of Fluormone GS Red (4× concentration) was added to the test compound, then a mixture of 5 μL of GR Full length (4× concentration) was added, and the experiment was performed in duplicate. The 384 microplate were incubated at room temperature in the dark for 2 h, and the fluorescence polarization (mP) was detected using the En Vision multifunctional microplate reader (manufacturer: Perkinelmer). With the log value of the final concentration of the compound as the X axis and the mP value as the Y axis, data was input into the processing software Graphpad Prism 9, and the EC50 value was calculated by four-parameter fitting
The binding activity of the compound to ER was determined using the LanthaScreen® TR-FRET ER Alpha Coactivator Assay kit (brand: Thermo, Cat. No: A15885). The ER receptor agonist Estradiol (brand: Sigma, Cat. No: E8875-25) was diluted 10-fold with DMSO in a 96-well V-bottom plate. The highest concentration was 100 μM, 8 concentrations in total. The test compound was diluted 10-fold with DMSO. The highest concentration was 3000 μM, 8 concentrations in total. The compound was then further diluted 50-fold with the detection buffer Nuclear Receptor Buffer E (containing 5 mM DTT) provided in the kit, 10 μL of the diluted compound was transferred to a 96-well half-area microplate (brand: Corning, Cat. No: 3694), 5 μL of ER-LBD protein (4× concentration) was added to the test compound, then a mixture of 5 μL of fluorescein-coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added, and the experiment was performed in duplicate. The plate was incubated at room temperature for 2 h, the fluorescence values (Excitation 337, Emission 520/495 nm) were detected using the PHERAstar instrument, and the 520:495 ratio was calculated. With the log value of the final concentration of the compound as the X axis and the 520/495 ratio as the Y axis, data was input into the software Graphpad Prism 9, and the EC50 value was calculated by four-parameter fitting. The results are shown in Table 1. The test compounds have relatively weak binding activity to estrogen receptors.
The binding activity of the compound to AR was determined using the LanthaScreen® TR-FRET Androgen Receptor Coactivator Assay kit (brand: Thermo, Cat. No: A15878). The AR receptor agonist dihydrotestosterone (DHT) (brand: Sigma, Cat. No: D-073) was diluted 10-fold with DMSO in a 96-well V-bottom plate. The highest concentration was 100 μM, 8 concentrations in total. The test compound was diluted 10-fold with DMSO. The highest concentration was 3000 μM, 8 concentrations in total. The compound was further diluted 50-fold with the detection buffer Nuclear Receptor Buffer A (containing 5 mM DTT) provided in the kit, 10 μL of the diluted compound was transferred to a 96-well half-area microplate, 5 μL of AR-LBD protein (4× concentration) was added to the test compound, then a mixture of 5 μL of fluorescein-coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added, and the experiment was performed in duplicate. The plate was incubated at room temperature for 2 h, the fluorescence values (Excitation 337, Emission 520/495 nm) were detected using the PHERAstar instrument, and the 520:495 ratio was calculated. With the log value of the final concentration of the compound as the X axis and the 520/495 ratio as the Y axis, data was input into the processing software Graphpad Prism 9, and the EC50 value was calculated by four-parameter fitting. The results are shown in Table 1. The test compounds have relatively weak binding activity to androgen receptors.
The binding activity of the compound to PR was determined using the LanthaScreen® TR-FRET Progesterone Receptor Coactivator Assay kit (brand: Thermo, Cat. No: A15903). The PR receptor agonist Progesterone (brand: Sigma, Cat. No: P0130) was diluted 10-fold with DMSO in a 96-well V-bottom plate. The highest concentration was 100 μM, 8 concentrations in total. The test compound was diluted 10-fold with DMSO. The highest concentration was 3000 μM, 8 concentrations in total. The compound was then further diluted 50-fold with the detection buffer Nuclear Receptor Buffer F (containing 5 mM DTT) provided in the kit, 10 μL of the diluted compound was transferred to a 96-well half-area microplate, 5 μL of PR-LBD protein (4× concentration) was added to the test compound, then a mixture of 5 μL of fluorescein-coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added, and the experiment was performed in duplicate. The plate was incubated at room temperature for 2 h, the fluorescence values (Excitation 337, Emission 520/495 nm) were detected using the PHERAstar instrument, and the 520:495 ratio was calculated. With the log value of the final concentration of the compound as the X axis and the 520/495 ratio as the Y axis, data was input into the processing software Graphpad Prism 9, and the EC50 value was calculated by four-parameter fitting. The results are shown in Table 1. The test compounds have a certain binding activity to progesterone receptors.
The test results show that the test compounds have strong GR binding activity to GR, weak binding activity to PR, and no ER and AR binding, and are expected to reduce the side effects associated with acting on other hormone receptors.
Construction of CHS mouse model: 6- to 8-week-old female C57BL/6N mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected, and all mice were shaved off abdominal hair using a small animal hair clipper. 400 μL of the sensitizer was pipetted with a micropipette and applied evenly to the abdomen of the mice for epidermal sensitization. After application, the mice were held for 3-5 s and the solvent was allowed to evaporate from the skin as clean as possible. The specific preparation method for the sensitizer was: 0.5 g of FITC (fluorescein isothiocyanate; sigma-Aldrich) powder was weighed and fully dissolved in a solvent of 50 mL of acetone (SinoPharm reagent) and 50 mL of DBP (dibutyl phthalate; sigma-Aldrich) in equal proportion to give the sensitizer with the FITC content of 0.5%. On the 6th day after sensitization, the thickness of the right ear of the mice was measured with a dial thickness gauge as a baseline value, then a freshly prepared sensitizer was pipetted with a micropipette and applied evenly inside and outside of the right ear of the mice respectively for epidermal stimulation, and 10 μL of the sensitizer was applied on each side. 24 h after stimulation, the thickness of the right ear of the mice was measured again with a dial thickness gauge to obtain the thickness change of the right ear of the mice, A thickness of right ear=(thickness of right ear 24 h after stimulation)−(baseline value of thickness of right ear).
Therapeutic regimen for CHS model mouse: mice were divided into a blank group, a negative control group, a positive control group, and an experimental group. The experimental group was given compound 001 and compound 009 at a dose of 3 μg/mouse, the positive control group was given compound a (synthesized according to the method reported in patent WO2017210471 for compound 41) at a dose of 3 μg/mouse, and the negative control group was given blank vehicle (0.5% DMSO/PBS). The drug was administered by one-time intraperitoneal injection 1 h before the right ear epidermal stimulation.
The results are shown in
The metabolic stability of the compound of the present invention in liver microsomes was determined using the following method.
All data were calculated by Microsoft Excel software. The peak area was detected through an extracted ion chromatogram. The in vitro half-life (t1/2) of the compound was determined by linear fitting of the natural logarithm of compound elimination percentage over time.
In vitro half-life (t1/2) was calculated by the slope k:
The in vitro intrinsic clearance (in μL/min/mg protein) was calculated using the following formula:
CLint was intrinsic clearance; k was an elimination rate constant; volume of incubation was the incubation volume (μL); amount of proteins was the amount of protein (mg)
The corresponding liver microsome stability of the compound of the present invention is shown in Table 2.
The membrane permeability and transport properties of the compound of the present invention were determined using the following method.
The resistance of the monolayer cell membrane <230 Ωcm2 indicates that a monolayer cell membrane is poorly dense and can not be used for experiment.
Iacceptor referred to the fluorescence density of the receiving end (0.3 mL), and Idonor referred to the fluorescence density of the administration end (0.1 mL). LY>1.0% indicates that a monolayer cell membrane is poorly dense and the corresponding results will be excluded from the evaluation.
The peak areas of the compound on the administration end and the receiving end were measured, and the apparent permeability coefficient (Papp, in cm/s) and the Efflux ratio (efflux ratio) of the compound were calculated:
VA was the volume of the receiving end solution (A→B was 0.3 mL, and B→A was 0.1 mL). Area was the area of the membrane of the Transwell 96-well plate (0.143 cm2). Incubation time was the time for incubation (in s). [drug]acceptor was the concentration of the drug at the receiving end. [drug]initial donor was the initial concentration of the drug at the administration end.
Papp (B-A) represented the apparent permeability coefficient from the basal end to the apical end; Papp (A-B) represented the apparent permeability coefficient from the apical end to the basal end.
The protein binding rates of the compound of the present invention in plasma of 5 species (humans, monkeys, dogs, rats, and mice) were determined using the following method.
The peak areas of the compound on the buffer side and the plasma side were measured. The plasma protein binding rate of the compound was calculated by the following formula:
The inhibition of CYP2C9 and CYP2D6 activity by the compound of the present invention was determined using the following method.
3.2 μL of 20 mg/mL liver microsome solution, 1 μL of substrate working solution, 1 μL of compound working solution, and 176 μL of PBS buffer were taken, mixed well, and placed in a 37° C. water bath for pre-incubation for 15 min. 1 μL of sulfaphenazole or quinidine working solution was added to the positive control group instead of the compound working solution. At the same time, 10 mM NADPH solution was placed together in the 37° C. water bath for pre-incubation for 15 min. After 15 min, 20 μL of NADPH was added to each well to initiate the reaction. The mixture was incubated at 37° C. for 5 min (CYP2C9) or 20 min (CYP2D6). All incubated samples were in duplicate. After incubation for the corresponding period of time, 400 μL of icy methanol containing internal standard was added to all samples to stop the reaction. The mixture was vortexed, mixed well, and centrifuged for 40 min at 4° C. at 3220 g. 100 μL of the supernatant was transferred to a feeding plate after the centrifugation was completed, and 100 μL ultrapure water was added. The mixture was well mixed for LC-MS/MS analysis.
The reduction of metabolite production in the treatment groups versus the control group was compared by the peak area ratios of the sample to the internal standard, and the IC50 value was calculated using Excel XLfit 5.3.1.3.
Percentages of remaining activity were calculated according to the following formula:
Drug-drug interaction (DDI) refers to the physical or chemical changes caused by no less than 2 drugs, as well as changes in efficacy due to such changes. Interpretation of the drug-drug interaction can provide better pharmaceutical services for patients, promote reasonable medication, and avoid adverse reactions to the maximum extent. The drug-drug interaction is dominated by metabolic interactions which are primarily associated with CYP450 enzymes involved in drug metabolism. The results in Table 5 show that the compound of the present invention has a weak inhibitory effect on CYP450, indicating that the compound of the present invention has a lower potential risk of developing DDI.
The inhibition of hERG activity by the compound of the present invention was determined using the following method.
HEK293 cells (Cat. No: K1236) stably expressing hERG ion channel were purchased from Invitrogen. The cells were cultured in a culture medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U/mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg/mL blasticidin, and 400 μg/mL geneticin. When the cell density increased to 40-80% of the bottom area of the culture dish, the cells were digested with trypsin and subcultured thrice every week. Before the experiment, the cells were cultured at a density of 5×105 in 6-cm culture dishes, induced for 48 h with 1 μg/mL doxycycline, and then digested and inoculated on a slide for the subsequent manual patch-clamp experiment.
Assay of the test compound for inhibition of hERG current: The hERG current measured in the extracellular fluid containing 0.1% DMSO was used as the baseline of the assay. The solution containing the test compound was perfused around the cells sequentially in an ascending order of concentration after the hERG current remained stable for at least 5 minutes. After the completion of each perfusion, a suspension of about 5 minutes was set to allow the compound to act sufficiently on the cells and the hERG current was recorded simultaneously. The last 5 hERG current values were recorded after the recorded current tended to stabilize, and the average value thereof was taken as the final current value at the specific concentration. After the compound was tested, 150 nM dofetilide was added to the same cell to completely inhibit the current as a positive control for the cell. Meanwhile, the positive compound dofetilide was synchronously detected by the same patch-clamp system before and after the test compound experiment was finished, so that the reliability and the sensitivity of the whole detection system were ensured.
The data was output by PatchMaster software and analyzed as follows:
The compound of the present invention has no obvious inhibitory effect on the hERG potassium channel, which suggests that the compound has a low risk of cardiotoxicity due to the inhibition of hERG potassium channel. The hERG potassium channel inhibitory activity of the test compounds is shown in Table 6.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110706921.X | Jun 2021 | CN | national |
| 202210101639.3 | Jan 2022 | CN | national |
| 202210340109.4 | Apr 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/100881 | 6/23/2022 | WO |
