CN109206423B - Thioamide substituted dihydropyrrolopyridine compounds as ROR gamma inhibitors - Google Patents

Abstract

The compound of formula (I) or pharmaceutically acceptable salt thereof provided by the invention has good ROR gamma inhibitory activity and is expected to be used for treating diseases mediated by ROR gamma receptors in mammals.

Description

Thioamide substituted dihydropyrrolopyridine compounds as ROR gamma inhibitors

Cross Reference to Related Applications

This application claims priority and benefit from the chinese patent application No. 201710552918.0 filed on 2017, month 07 to the chinese intellectual property office, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to a thioamide substituted dihydropyrrolopyridine compound serving as a ROR gamma inhibitor, belonging to the field of medical chemistry.

Background

Retinoic acid-related orphan nuclear receptors (RORs) are members of the nuclear receptor family, which are capable of modulating a variety of physiological and biochemical processes. The ROR family includes three types ROR α, ROR β, and ROR γ. Three different RORs can be expressed in different tissues and regulate different physiological processes. ROR α is distributed mainly in liver, skeletal muscle, skin, lung, adipose tissue, kidney, thymus and brain; ROR β acts primarily on the central nervous system; ROR γ can be expressed in many tissues, including liver, animal fat and skeletal muscle.

There are two subtypes of ROR γ: ROR γ 1 and ROR γ t (ROR γ 2). ROR γ 1 is found in many tissues, such as: thymus, muscle, kidney and liver, while ROR γ t is expressed only in immune cells. ROR γ T is thought to regulate T cell helper T17 (Th17) differentiation. Th17 is a type of T helper cell that produces interleukin 17(IL-17) and other cytokines. Th17 cells are implicated in the pathology of numerous autoimmune and inflammatory diseases including, but not limited to, psoriasis, multiple sclerosis, rheumatoid arthritis, crohn's disease, asthma, chronic obstructive pulmonary disease, Behcet's disease, and irritable bowel syndrome.

Patent applications of Vitae Pharmaceuticals Inc in the prior art, such as WO2014179564, WO2015116904, WO2016061160, etc.; and patent applications of the Puerarin Schke company, such as WO2013045431, WO2013160418, WO2013160419 and the like, all disclose a series of compounds which can be used as ROR gamma inhibitors. In view of the great potential value of ROR gamma inhibitors, it is necessary to further search for compounds having ROR gamma inhibitory function.

Disclosure of Invention

The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt thereof,

wherein the content of the first and second substances,

m is 0 or 1;

n is 0, 1 or 2;

R¹、R²、R⁴independently selected from hydrogen or C_1-6An alkyl group;

R³、R⁵independently selected from hydrogen or C_1-6An alkyl group; said C is_1-6Alkyl is optionally substituted by one or more hydroxy, amino, nitro, cyano, halogen, C_1-4Alkoxy radical, C_1-4Alkylamino or di (C)_1-4Alkyl) amino substitution;

Cy¹selected from the group consisting of optionally substituted by one or more R⁶Substituted 3-6 membered cycloalkyl, 5-8 membered cycloalkenyl, 3-10 membered heterocyclo, 6-10 membered aryl or 5-10 membered heteroaryl;

R⁶selected from hydroxy, amino, nitro, cyano, halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Alkoxy radical, C_1-4A haloalkoxy group;

Cy²selected from 6-10 membered aryl or 5-10 membered heteroaryl; said 6-10 membered aryl or 5-10 membered heteroaryl is optionally substituted with one or more hydroxy, amino, nitro, cyano or

Substitution;

R⁷presence or absence; when R is⁷When the water-soluble polymer is existed in the water,

is selected from

R⁸Is selected from C_1-6Alkyl or C_1-6An alkoxy group.

In some embodiments, m ═ 0; n is 0 or 1.

In some exemplary embodiments, m is 0 and n is 0.

In some embodiments, R¹、R²、R⁴Independently selected from hydrogen or C_1-4An alkyl group.

In some typical embodiments, R¹、R²、R⁴Independently selected from hydrogen, methyl, ethyl, n-propyl or isopropyl.

In some more typical embodiments, R¹、R²、R⁴Independently selected from hydrogen or isopropyl.

In some of the most typical embodiments, R¹、R²、R⁴At least one of which is isopropyl.

In some of the most typical embodiments, R¹And R⁴Is hydrogen, and R²Is isopropyl.

In some of the most typical embodiments, R¹And R⁴Is hydrogen, and R²Is composed of

In some embodiments, R³、R⁵Independently selected from hydrogen or C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted with one or more hydroxy, amino, nitro, cyano, fluoro, chloro, bromo, iodo, methoxy, ethoxy, methylamino or ethylamino groups.

In some typical embodiments, R³、R⁵Independently selected from hydrogen, methyl or ethyl, said methyl or ethyl being optionally substituted with one hydroxy, methoxy or ethoxy group.

In some more typical embodiments, R³Selected from hydrogen or methyl, said methyl being optionally substituted by hydroxyl; and R is⁵Selected from hydrogen or methyl, said methyl being optionally substituted by hydroxyl or methoxy.

In some of the most typical embodiments, R³Selected from hydrogen, methyl or hydroxymethyl; and R is⁵Selected from hydrogen, methyl, hydroxymethyl or-CH₂OCH₃。

In some embodiments, Cy is¹Selected from the group consisting of⁶Substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxiranyl, tetrahydrofuryl, dihydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1, 3-cyclohexadienyl, benzo [ d][1,3]Dioxolyl, phenyl, thienyl, furyl, thiazolyl, oxazolyl, isoxazolyl, pyrrolyl, pyridyl, pyrazinyl or pyrimidinyl.

In some typical embodiments, Cy¹Selected from the group consisting of⁶Substituted cyclohexyl, cyclohexenyl, benzo [ d ]][1,3]Dioxolyl, phenyl, pyridyl or pyrimidinyl.

In some more typical embodiments, Cy¹Selected from the group consisting of⁶Substituted by

In some more typical embodiments, Cy¹Is selected from

In some embodiments, R⁶Selected from the group consisting of fluorine, chlorine, bromine, iodine, methyl, fluoromethyl, chloromethyl, bromomethyl, iodomethyl, ethyl, fluoroethyl, chloroethyl, bromoethyl, iodoethyl, methoxy, fluoromethoxy, chloromethoxy, bromomethoxy, iodomethoxy, ethoxy, fluoroethoxy, chloroethoxy, bromoethoxy, or iodoethoxy.

In some typical embodiments, R⁶Selected from fluoro, methyl, one or more fluoro substituted methyl, methoxy or one or more fluoro substituted methoxy.

In some more typical embodiments, R⁶Selected from fluorine, -CF₃or-OCF₃。

In some embodiments, Cy is²Selected from phenyl, thienyl, furyl, thiazolyl, oxazolyl, isoxazolyl, pyrrolyl, pyridyl, pyrazinyl or pyrimidinyl; said groups being optionally substituted by one or more hydroxy, amino, nitro, cyano,

Substitution; wherein R is⁸Is selected from C_1-3An alkyl group.

In some typical embodiments, Cy²Selected from phenyl or pyridyl; said phenyl or pyridyl being optionally substituted by one

Substitution; wherein R is⁸Selected from methyl or ethyl.

In some more typical embodiments, Cy²Is selected from

Said group being optionally substituted by one

And (4) substitution.

In some of the most typical embodiments, Cy²Is selected from

In some embodiments, the aforementioned compound of formula (I) has the structure shown in formula (II),

wherein the content of the first and second substances,

x is CH or N;

R³、R⁵、R⁷、Cy¹as defined above for the compounds of formula (I).

In some embodiments, the compound of formula (I) of the present application is selected from the following compounds, or pharmaceutically acceptable salts thereof:

in another aspect, the present application relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as described herein. In some embodiments, the pharmaceutical compositions of the present application further comprise a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.

Typical routes of administration of a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical compositions of the present application can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These adjuvants enable the compounds of the present application to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, optionally the mixture obtained is milled, if desired with further suitable adjuvants, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

In another aspect, the present application relates to a method of treating a disease mediated by the ROR γ receptor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In some embodiments, the dosage administered daily in all methods of administration of the compounds of formula (I) described herein is from 0.01 to 300mg/kg body weight, preferably from 10 to 300mg/kg body weight, more preferably from 25 to 200mg/kg body weight, in single or divided doses.

In another aspect, the present application relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for the prevention or treatment of a ROR γ receptor mediated disease.

Definition of

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═ O), meaning that two hydrogen atoms are substituted, oxo does not occur on the aryl.

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with one or more fluoro or chloro, meaning that the ethyl group may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F、CHClCH₃) Polysubstituted (e.g. CHFCH)₂F、CHClCHF₂、CH₂CHF₂Etc.) or are taken entirelyGeneration (CCl)₂CF₃、CF₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

Herein C_m-nIt is the moiety that has an integer number of carbon atoms in the given range. E.g. "C_1-6By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 2R, then there are separate options for each R.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "hydroxy" refers to an-OH group.

The term "cyano" refers to the group — CN.

The term "amino" refers to the group-NH₂A group.

The term "nitro" means-NO₂A group.

The term "hydroxyalkyl" refers to-C_nH_2nAnd (5) OH. For example, hydroxymethyl means-CH₂OH, 2-hydroxyethyl means-CH₂CH₂OH。

The term "alkyl" refers to a group of formula C_nH_2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "C_1-6Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., 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). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio groups have the same definitions as above.

The term "alkoxy" refers to-O-alkyl.

The term "alkylamino" refers to-NH-alkyl.

The term "dialkylamino" refers to-N (alkyl)₂。

The term "cycloalkyl" refers to a carbon ring that is fully saturated and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the carbocycle is typically a 3 to 10 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and may exist as a monocyclic, bicyclic, or spiro ring. Unless otherwise indicated, the carbocycle is typically a 5 to 8 membered ring. Non-limiting examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and the like.

The term "heterocycloaliphatic" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated heteroaromatic) and may exist as a monocyclic, bicyclic, or spiro ring. Unless otherwise indicated, the aliphatic heterocyclic ring is generally a 3 to 6 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. Non-limiting examples of lipoheterocyclyl groups include, but are not limited to, oxiranyl, tetrahydrofuryl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, and the like.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated pi-electron system. For example, the aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and 1,2,3, 4-tetrahydronaphthalene, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryls have a single 4-to 8-membered ring, especially a 5-to 8-membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition;

(ii) inhibiting the disease or disease state, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "therapeutically effective amount" means an amount of a compound of the present application that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art with their own knowledge and this disclosure.

The term "pharmaceutically acceptable" is intended to refer to 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 problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" includes, but is not limited to, acid addition salts of compounds of formula (i) with inorganic acids, acid addition salts of compounds of formula (i) with organic acids, or addition salts of compounds of formula (i) with acidic amino acids, and the like. The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

The words "comprise" or "comprise" and variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "including but not limited to.

Unless otherwise indicated, the abbreviations used in the present application have the following meanings

EDCI: 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride;

HOBt: 1-hydroxybenzotriazole;

TEA: triethylamine;

TLC: thin layer chromatography;

PE: petroleum ether;

EtOAc: ethyl acetate;

MeOH: methanol;

1M：1mol/L；

CDI: n, N' -carbonyldiimidazole;

DABCO: 1, 4-diazabicyclo [2.2.2] octane;

NH₄OAc: acetic acid amine;

DMF: n, N-dimethylformamide;

n-BuOH: n-butanol;

KOH: potassium hydroxide;

HATU: 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate;

DIPEA: n, N-diisopropylethylamine;

MsCl: methanesulfonyl chloride;

Pd(OAc)₂: lead acetate;

DMSO, DMSO: dimethyl sulfoxide;

THF: tetrahydrofuran;

Pre-HPLC: preparing a liquid phase with high efficiency;

SFC: supercritical fluid chromatography;

LC-MS: liquid chromatography-mass spectrometry.

The intermediates and compounds of the present application may also exist in different tautomeric forms, and all such forms are included within the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, wherein the proton can migrate between two ring nitrogens. Valence tautomers include interconversion by recombination of some of the bonding electrons. Exemplary enol tautomers are shown below, but are not limited thereto.

The present application also includes isotopically-labeled compounds of the present application, which are identical to those recited herein, but for the fact that one or more atoms are 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 compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as respectively²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、¹²³I、¹²⁵I and³⁶cl, and the like.

Certain isotopically-labelled compounds of the present application (e.g. with³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation)³H) And carbon-14 (i.e.¹⁴C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as¹⁵O、¹³N、¹¹C and¹⁸f can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

In addition, heavier isotopes are used (such as deuterium (i.e., deuterium)²H) Substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances where deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is substituted with at least one deuterium. Exemplary deuterated compounds are shown below, but are not limited thereto.

The compounds of the present application may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the present application containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents. Non-limiting examples of stereoisomers include, but are not limited to:

the compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

An important consideration in the art of synthetic route planning is the selection of suitable protecting Groups for reactive functional Groups (such as amino Groups in the present application), for example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed.) Hoboken, New Jersey: John Wiley & Sons, Inc. all references cited herein are incorporated herein in their entirety.

In some embodiments of the present application, the compounds of formula (II) are prepared by the synthetic routes shown below:

X、R3、Cy1、

as defined for the compounds of formula (II).

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Example 1

Step a: synthesis of Compound 1-2

To a solution of compound 1-1(25g) and N-methoxymethylamine hydrochloride (14.9g) in 500mL of dichloromethane were added TEA (51.6g,71.0mL), EDCI (36.7g) and HOBt (25.8 g). The reaction solution is reacted for 16 hours at the temperature of 10-20 ℃. TLC (PE: EtOAc ═ 1:1) showed the reaction was complete. The reaction mixture was dispersed in 500mL of methylene chloride and 500mL of water. The organic phase was washed once with 500mL of saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude product. The crude product was passed through a flash column (220 g)

Silica gel flash column, eluent: ethyl acetate, petroleum ether, gradient elution of eluent: 0-100% of petroleum ether/ethyl acetate (V/V); flow rate: 100mL/min) to obtain the compound 1-2.

¹H NMR(400MHz,CHLOROFORM-d)δ3.71(s,3H),3.19(s,3H),2.62-2.74(m,1H),1.99-2.07(m,3H),1.92(br d,J＝13.6Hz,2H),1.48-1.61(m,2H),1.32-1.44(m,2H)；

¹⁹F NMR(400MHz,CHLOROFORM-d)δ＝-73.8。

Synthesis of Compounds 1-3

A solution of compound 1-2(8.0g) in 100mL of tetrahydrofuran was added dropwise to a 50mL tetrahydrofuran suspension of lithium aluminum hydride (1.5g) at-65 ℃. After the dropwise addition is finished, the reaction solution reacts for 5 hours at the temperature of between 70 ℃ below zero and 65 ℃ below zero. TLC (PE: EtOAc ═ 5:1) showed complete reaction of compound 1-2. The reaction was quenched with MeOH (5.14g) at-65 ℃. When the temperature is increased to-10 ℃, 1M citric acid aqueous solution is dripped, the pH value is adjusted to be about 4, and the temperature is controlled to be-10 to-5 ℃ in the process. The resulting mixture was extracted with ethyl acetate (300mL x 3). The organic phases were combined, washed with 300mL of saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude compound 1-3, which was used in the next step without further purification.

¹HNMR(400MHz,CHLOROFORM-d)δ9.65(s,1H),2.09-2.16(m,1H),2.03-2.05(m,4H),1.92-1.99(m 1H),1.23-1.32(m,4H)。

Synthesis of Compounds 1-5

Compound 1-4(50.0g) was dissolved in N-methylpyrrolidone (500mL), and potassium carbonate (49.1g) and sodium ethylmercaptide (31.0g) were added to the reaction solution, and the mixture was stirred at 20 ℃ for 12 hours. When TLC (PE: EtOAc ═ 5:1) showed complete reaction of starting material, the reaction was poured into 1500mL of water and a solid precipitated, filtered and the filter cake was dried in vacuo to give compound 1-5.

MS:m/z 165.1[M+H]⁺。

Synthesis of Compounds 1-6

Compound 1-5(58.0g) was dissolved in methanol (600mL), a suspension of oxone complex salt (434.2g) in 900mL of water was added dropwise to the reaction solution at 0 ℃ and the reaction solution was stirred at 0-20 ℃ for 16 hours. When TLC (PE/EtOAc ═ 3:1) showed complete reaction of starting material. The reaction was filtered and excess oxone complex salt in the reaction was quenched with sodium thiosulfate. After removing methanol under reduced pressure, extracting with ethyl acetate (500 mL. times.2), combining the organic phases, washing with saturated brine (300mL), drying over anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain crude compounds 1-6. And purifying the crude product by column chromatography (silica gel of 100-200 meshes, eluent comprising petroleum ether and ethyl acetate, and elution gradient comprising petroleum ether and ethyl acetate in a ratio of 10: 1-2: 1) to obtain a compound 1-6.

MS:m/z 197.0[M+H]⁺；

¹H NMR(400MHz,CDCl₃-d)δ9.13(d,J＝1.6Hz,1H),8.38(dd,J＝8.4,2.4Hz,1H),7.94(d,J＝8.4Hz,1H),3.21(Q,J＝7.2Hz,2H),1.35(d,J＝7.6Hz,3H)。

Synthesis of Compounds 1-7

Compounds 1-6(10g) were dissolved in dry methanol (100.00mL), and dry palladium on carbon (10%, 0.58g) was added under nitrogen, and the mixture was replaced with nitrogen 3 times and then with hydrogen 3 times. Reaction solution in H₂(50Psi) was stirred at 25 ℃ for 3 hours. When the LC-MS showed the reaction was complete, the reaction solution was filtered through Celite, and the filtrate was concentrated under reduced pressure to give compounds 1-17. MS M/z 201.0[ M + H ]]⁺；

¹H NMR(400MHz,CDCl₃)δ9.04(d,J＝2.0Hz,1H),8.15(dd,J＝2.4,8.4Hz,1H),7.56(d,J＝8.4Hz,1H),4.12(s,2H),3.16(q,J＝7.6Hz,2H),1.32(t,J＝7.6Hz,3H).

Synthesis of Compounds 1-9

Compound 1-8(400.0g) was dissolved in anhydrous THF (1.5L), CDI (400.0g) was added at 15 ℃ and the reaction mixture was reacted at 15 ℃ for 1 hour. Anhydrous magnesium chloride (180.0g) and potassium salt of monoethyl malonate (400.0g) were then added. The mixture was stirred at 50 ℃ for 15 hours and LC-MS indicated that compounds 1-8 were completely reacted. To the reaction solution, 1.5L of water was added, extracted with ethyl acetate (1000 mL. times.2), and the organic phases were combined and washed once with 1L of saturated saline. Adding anhydrous sodium sulfate, drying, filtering, and concentrating the filtrate under reduced pressure to obtain residue. To the residue was added 1L of dichloromethane, stirred to give a suspension, filtered and the filter cake washed with dichloromethane (250 mL. times.3). The filtrates were combined and concentrated to give crude compounds 1-9, which were used directly in the next step without further purification.

MS:m/z 188.0[M_De-Boc+H]⁺；

¹H NMR(400MHz,DMSO)δ5.11(m,1H),4.34(dd,J＝8.8,4.0Hz,1H),4.23-4.19(m,2H),3.53(s,1H),2.5(m,1H),1.44(s,9H),1.27(m,4H),1.02(d,J＝6.8,3H),0.83(m,3H)。

Synthesis of Compounds 1-10

Compound 1-9(600.0g) was dissolved in tetrahydrofuran (2500mL), potassium tert-butoxide (225.0g) was added at 0 ℃ and the reaction mixture was reacted at 15 ℃ for 60 minutes. DABCO (210.0g) and Compound A (870.0g) were then added sequentially. The reaction solution was stirred at 15 ℃ for 1 hour, and NH was added to the reaction solution₄OAc ((300.0 g.) the reaction was then stirred at 15 ℃ for 13 hours LC-MS showed the starting material reacted completely, 4.0L of water was added to the reaction, extracted with ethyl acetate (1000mL x 3), the organic phases combined, washed once with 1L of saturated saline, dried over anhydrous sodium sulfate added, filtered, the filtrate concentrated under reduced pressure to give a residue, which was purified by column chromatography (silica, eluent gradient elution: petroleum ether: ethyl acetate: 100: 0-5: 1) to give compounds 1-10.

MS:m/z 357.1[M+H]⁺；

¹H NMR(400MHz,CDCl3)δ8.61(d,J＝5.6Hz,1H),8.19(d,J＝5.6Hz,1H),5.73(d,J＝9.6Hz,1H),5.61(dd,J＝9.6,5.2Hz,1H),4.47-4.38(m,2H),2.08-2.00(dt,J＝17.2,6.8Hz,1H),1.40-1.44(m,12Hz),0.94(d,J＝6.8Hz,3H),0.84(d,J＝9.2Hz,3H)。

Synthesis of Compounds 1-11

Compounds 1-10(60.0g) were dissolved in absolute ethanol (500.0mL) and NaBH was added portionwise at 0 deg.C₄(12.7g) and CaCl2(18.7g), and maintaining the temperature of the reaction solution at 0-5 ℃. After the completion of the dropwise addition, the reaction solution was stirred at 0 ℃ for 90 minutes. LC-MS showed the starting material reaction was complete. The reaction was quenched by the addition of 1000mL of saturated ammonium chloride solution at 0 ℃. Then, ethyl acetate was added for extraction (500 mL. times.3), the organic phases were combined and washed with 1L of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compounds 1-11.

MS:m/z 315.0[M+H]⁺；

¹H NMR (400MHz, CDCl3) δ 8.47(d, J ═ 2.4Hz,1H),7.68(d, J ═ 2.4Hz,1H),5.39-5.46(m,1H),5.00(d, J ═ 12.0Hz,1H),4.55(t, J ═ 9.2Hz,1H),4.35-4.47(m,2H),2.10-2.20(m,1H),1.37(s,9H),1.10(d, J ═ 6.8Hz,3H),0.70(d, J ═ 6.8Hz, 3H). Synthesis of Compounds 1-12, 1-13

Compounds 1-11(53g) and TEA (25.6g,35.0mL) were dissolved in 500mL of dichloromethane. To this solution was added MsCl (24.9g,16.8mL) dropwise at 0 ℃. After the dropwise addition, the reaction solution is stirred for 16 hours at 15-25 ℃. LC-MS showed the starting material reaction was complete. The reaction was quenched by the addition of 500mL of saturated sodium bicarbonate solution and extracted with ethyl acetate (500 mL. times.2). The organic phases were combined, washed with 500mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of a mixture of compounds 1-12 and compounds 1-13 (molar ratio about 1: 1). The crude product was used directly in the next step without further purification.

Compounds 1-12, MS: M/z 276.9[ M_de-56+H]⁺；

Compounds 1-13, MS M/z 337.0[ M ]_de-56+H]⁺。

Synthesis of Compounds 1-14

A mixture of compounds 1-12 and compounds 1-13 (62g, molar ratio about 1:1) was dissolved in 500mL of anhydrous THF. NaH (24.4g, 60% pure) was added to the solution in portions at 0 ℃ and the temperature was maintained at 0-5 ℃. After the addition is finished, the reaction solution is stirred for 16 hours at 15-25 ℃. LC-MS showed the maintenance of the starting material and NaI (15g) was added to the mixture. And after the addition is finished, continuously stirring the reaction solution for 16 hours at the temperature of 15-25 ℃. LC-MS shows disappearance of the raw material and generation of the product. The reaction mixture was quenched with 500mL of saturated ammonium chloride solution at 25 ℃ and extracted with aqueous ethyl acetate (500 mL. times.2). The combined organic phases were washed with 500mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude product. The crude product was passed through a flash column (120 g)

Silica gel flash column, gradient elution of eluent: 0-28% of ethyl acetate/petroleum ether (V/V); flow rate: 85mL/min, and purifying to obtain the compounds 1-14.

MS:m/z 297.1[M+H]⁺；

¹H NMR(400MHz,CHLOROFORM-d)δ8.43(s,1H),7.48-7.59(m,1H),4.69-4.98(m,2H),4.45-4.55(m,1H),2.33-2.59(m,1H),1.51(s,9H),0.98-1.10(m,3H),0.69-0.81(m,3H)。

Synthesis of Compounds 1-15

To a solution of compounds 1-14(50.0g) in 250mL anhydrous DMF and 250mL n-BuOH (250mL) at 25 deg.C was added Pd (OAc)₂(1.89g), Compound B (10.3g) and K₂CO₃(34.9 g). The reaction solution was evacuated and carbon monoxide gas-displaced 3 times. Then stirring for 16 hours at 100 ℃ under the atmosphere of carbon monoxide gas. LC-MS showed disappearance of starting material. The solvent was removed under vacuum, and 250mL of aqueous KOH (1M) and 250mL of ethanol were added to the residue and stirred for 2 hours. LC-MS showed the formation of compounds 1-15. The mixed solution was concentrated to a volume of 250 mL. The pH value is adjusted to 3-5 by 1M hydrochloric acid, and ethyl acetate (500mL x 2) is added for extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude product. The crude product was used directly in the next step without further purification.

MS:m/z 307.1[M+H]⁺；

¹H NMR(400MHz,CHLOROFORM-d)δ9.21(s,1H),9.05(s,1H),8.19-8.29(m,1H),4.76-5.13(m,2H),4.57(d,J＝15.6Hz,1H),2.38-2.63(m,1H),1.53(s,9H),0.96-1.09(m,3H),0.74-0.85(m,3H)。

Synthesis of Compounds 1-16

Compounds 1-15(8.0g, ca.) were dissolved in anhydrous DMF (100mL), to which HATU (14.9g, ca.), compounds 1-7(5.8g) and DIPEA (10.1g,13.6mL) were added in that order, and the reaction was stirred at 20 ℃ for 2 hours. When the LC-MS showed the reaction was complete, the reaction was concentrated under reduced pressure to remove DMF, diluted with 120mL of water and extracted with dichloromethane (200mL x 3). The combined organic phases were washed with 150mL of saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product is subjected to flash column chromatography (40 g)

Silica gel flash column, gradient elution of eluent: 0-100% of petroleum ether/ethyl acetate (V/V); flow rate: 35mL/min) to yield compounds 1-16.

MS:m/z 489.2[M+H]⁺；

¹H NMR(400MHz,CDCl₃)δ8.98(d,J＝2.0Hz,1H),8.91(s,1H),8.12(dd,J＝8.0,2.4Hz,1H),7.96-8.03(m,1H),7.55(br s,1H),7.50(d,J＝8.0Hz,1H),4.88(br d,J＝11.2Hz,1H),4.85-4.85(m,1H),4.83(d,J＝5.2Hz,1H),4.50(br t,J＝14.4Hz,1H),3.10(q,J＝7.2Hz,2H),2.66-2.94(m,1H),2.23-2.59(m,1H),1.45(s,9H),1.26(t,J＝7.2Hz,3H),0.89-1.05(m,3H),0.62-0.76(m,3H)。

Synthesis of Compounds 1-17

Xylene (50.00mL) was preheated to 80 ℃ and then compounds 1-16(3.1g) were slowly added to xylene to dissolve them sufficiently, and Lawson's reagent (1.00g) was added to the reaction solution at 80 ℃. The reaction mixture was stirred at 80 ℃ for 1 hour under nitrogen. When LC-MS showed the reaction was complete, the reaction was concentrated under reduced pressure to remove xylene, diluted with 80mL of water and extracted with dichloromethane (200mL x 2), the combined organic phases were washed with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product which was purified by column chromatography (80 g of column chromatography)

Silica gel flash column, eluent: petroleum ether, ethyl acetate; elution gradient is 0-80%; flow rate: 35mL/min) to yield compounds 1-17.

MS:m/z 505.1[M+H]⁺；

¹H NMR(400MHz,CDCl₃)δ9.49(br s,1H),8.99(br s,2H),8.16(br d,J＝8.0Hz,2H),7.56(brd,J＝7.6Hz,1H),5.16(br s,2H),4.39-4.59(m,1H),3.66-3.85(m,2H),3.10(q,J＝7.6Hz,2H),2.25-2.58(m,1H),1.45(s,9H),1.25(t,J＝7.6Hz,3H),0.84-1.07(m,3H),0.63-0.84(m,3H)。

Synthesis of Compounds 1-18

Compounds 1-17(3.1g) were dissolved in hydrochloric acid-dioxane (30.00mL) and stirred at 20 ℃ for 1 hour. When the reaction was complete as shown by LC-MS, the reaction was concentrated under reduced pressure to remove dioxane, and the residue was dissolved in dichloromethane (30.00mL) and water (20.00mL), adjusted to pH 8 by the addition of saturated sodium bicarbonate solution, and then extracted with dichloromethane (25mL x 2). The combined organic phases were washed with 30.00mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude compounds 1-18. The crude product was used directly in the next step without further purification.

MS:m/z 405.1[M+H]⁺。

Synthesis of the Compound of example 1

Compounds 1-18(2.5g) and compounds 1-3(2.2g) were dissolved in methanol (30mL) and acetic acid (371.1mg) was added to adjust the pH to 5, then sodium cyanoborohydride (1.6g) was added slowly and the reaction stirred at 70 ℃ for 1 hour. When the LC-MS showed the reaction was complete, the reaction was quenched with saturated sodium bicarbonate solution, concentrated under reduced pressure to remove methanol, diluted with water (15mL), and extracted with dichloromethane (30mL × 3). The combined organic phases were washed with brine (20mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude Compound 1. The crude product was separated by pre-HPLC (formic acid system) and SFC (column: Chiralpak AD-350 x 4.6mm I.D.,3um mobile phase: A: CO2, B: isopropanol (0.05% DEA); gradient: maintaining B fraction 5% for 0.2 min, then 1.4 min the B fraction content was ramped from 5% to 40% and maintaining B fraction content 40% for 1.05 min, then the B fraction was ramped to 5% for 0.35 min; flow rate: 4mL/min column temperature: 40 ℃ C.) to give the compound of example 1 in free form.

The free compound was dissolved in acetonitrile and water, and 2.29mL of 1M hydrochloric acid was added to the solution at 0 ℃ and lyophilized to give the compound of example 1.

MS:m/z 569.1[M+H]⁺；

¹H NMR(400MHz,CDCl₃)δ9.20-9.45(m,1H),9.08(s,1H),8.90(br s,1H),8.20-8.31(m,1H),8.10(br s,1H),7.60(d,J＝8.28Hz,1H),5.22(d,J＝4.27Hz,2H),4.89(br s,1H),4.38(br s,1H),3.20(q,J＝7.36Hz,2H),2.62(br s,1H),2.13-2.34(m,1H),2.02(br d,J＝8.53Hz,3H),1.24-1.45(m,8H),1.22(br d,J＝6.02Hz,1H),0.70-1.17(m,8H)。

Example 2

The compound of example 2 was prepared by substituting 3-fluoro-4-trifluoromethoxybenzaldehyde for the compounds of formulae 1-3, according to the preparation of the compound of example 1 in free form.

MS:m/z 597.1[M+H]⁺；

¹HNMR(400MHz,METHANOL-d4)δ8.82-8.96(m,1H),8.76(s,1H),8.15(dd,J＝8.4,2.4Hz,1H),7.94(s,1H),7.56(d,J＝8.4Hz,1H),7.21-7.36(m,3H),5.09-5.16(m,2H),4.05-4.20(m,2H),3.94-4.04(m,1H),3.72(d,J＝14.8Hz,1H),3.56-3.67(m,1H),3.15-3.19(m,2H),2.20(td,J＝7.2,2.8Hz,1H),1.15(br t,J＝7.6Hz,3H),0.95(br d,J＝7.2Hz,3H),0.87-0.93(m,3H)。

Example 3

Example 3 was prepared by substituting 2, 2-difluoro-5-formylbenzodioxole for the compound of formula 1-3, according to the method for preparation of the compound of example 1 in free form.

MS:m/z 575.1[M+H]⁺；

¹H NMR(400MHz,METHANOL-d4)δ8.89(br s,1H),8.76(s,1H),8.13-8.20(m,1H),7.94(s,1H),7.56(br d,J＝8.0Hz,1H),7.22(s,1H),7.14(br d,J＝8.0Hz,1H),7.05-7.10(m,1H),5.11(s,2H),4.07-4.17(m,2H),3.99(br s,1H),3.61-3.74(m,2H),3.15-3.19(m,2H),2.15-2.26(m,1H),1.15(br t,J＝7.2Hz,3H),0.93(br dd,J＝12.8,6.80Hz,6H)。

Example 4

The compound of example 4 was prepared by following the procedure for the preparation of example 1 in free form, replacing the compound of formula 1-3 with 3, 5-difluoro-4-trifluoromethoxybenzaldehyde.

MS:m/z 615.1[M+H]⁺；

¹H NMR(400MHz,CHLOROFORM-d)δ9.28(br s,1H),9.07(d,J＝2.0Hz,1H),8.89(s,1H),8.24(dd,J＝8.4,2.4Hz,1H),8.04(s,1H),7.59(d,J＝8.4Hz,1H),7.46-7.53(m,1H),7.05(td,J＝8.8,1.6Hz,1H),5.21(d,J＝4.4Hz,2H),4.26(br d,J＝13.6Hz,1H),4.04-4.14(m,2H),3.93(d,J＝14.4Hz,1H),3.73(br d,J＝14.4Hz,1H),3.19(q,J＝7.2Hz,2H),2.32(qd,J＝6.8,4.0Hz,1H),1.34(t,J＝7.2Hz,3H),1.08(d,J＝6.8Hz,3H),1.02(d,J＝6.8Hz,3H)。

Biological activity experiment 1: in vitro ROR γ inhibitory Activity assay

The test method comprises the following steps:

a reporter cell: reporter cells expressing chimeric ROR gamma receptors were used in this experiment. The N-terminal DNA binding domain of the chimeric ROR γ receptor, i.e. the native ROR γ protein, was replaced with the DNA binding domain of the yeast Gal4 protein. The reporter luciferase is located downstream of the Gal4 activation sequence (UAS). As shown in the following table:

step 1: reporter cells were prepared as cell suspensions using INDIGO cell recovery medium (CRM, containing 5% charcoal treated fetal bovine serum). The cell suspension was dispensed into a white 96-well plate at 100. mu.l/well.

Step 2: 8 concentration gradients were set for each test compound, and each concentration was tested 2 times. Prior to the experiment, master stocks of test compounds were serially diluted in DMSO to prepare "1000X-concentration" solutions for each final test concentration. Subsequently, the compounds were further diluted with cell recovery medium (CRM, containing 5% charcoal treated fetal bovine serum) to prepare a "2X-concentration" of test working solution. Test solutions were added to test wells pre-loaded with reporter cells at a volume of 100 μ l/well to obtain the desired final test concentration. The residual concentration of DMSO in all test wells was 0.1%. The test plate was incubated for 24 hours in a cell incubator, which set the conditions: the temperature is 37 ℃, the CO2 is 5 percent, and the humidity is 85 percent.

And step 3: after 24 hours of incubation, the plate was discarded, 100 microliters of luciferase assay was added to each test well, and the fluorescence intensity in each well was read (Relative Luminescence Units, RLUs).

And (3) testing:

the test uses the reference compound ursolic acid as an internal standard of a positive compound to confirm the inhibition effect of the test compound on ROR gamma in a specific batch of report cells. The test of the reference compound and the test compound is performed simultaneously and is therefore exposed to the same test reagents and environment. The reference compound group containing 0.1% DMSO as solvent was used as an internal positive compound standard to determine the effect of DMSO as solvent on the test results and to calculate the percent ROR γ activity reduction.

Data processing:

the test data was managed and archived by microsoft Excel and the mean +/-Standard Deviation (SD), fold reduction, inhibition, coefficient of variation (% CV) and Z factor of RLU were calculated, where:

coefficient of variation (% CV): 100 [ (SD/ave. rlu) ];

fold reduction in inverse agonists: RLU [ Ave^Solvent/Ave.RLU^{Test compounds}]；

Percent reduction in inverse agonist: 100 (1- [ Ave^{Test compounds}/Ave.RLU^Solvent])；

The theoretical minimum reduction (0% reduction) is for the solvent control without compound;

factor Z: 1- [ (3. multidot. SD)^Solvent+SD^{Test compounds}])/(RLU^Solvent_RLU^{Test compounds})]。

The graphic data processing method comprises the following steps:

dose Response Curves (DRC) for ROR γ tests for reference and test compounds were obtained by nonlinear fitting of the log values of ROR γ activity inhibition and compound concentration by GraphPad Prism software.

And (3) testing results:

results of in vitro screening assays for the compounds of the examples are detailed in Table 1

TABLE 1 in vitro screening test results for the compounds of the invention

Definition of biological activity: a: EC (EC)₅₀≤100nM；B:100nM＜EC₅₀≤500nM；C:500nM＜EC₅₀≤1000nM；D:1000nM＜EC₅₀≤5000nM；

And (4) experimental conclusion: in the in vitro ROR γ activity test, the compounds of the examples all showed strong inhibitory activity against ROR γ.

Biological activity experiment 2: pharmacokinetic evaluation

Balb/c mice (female, 15-30g, 7-9 weeks old, Shanghai Ling Chang) were used to test the in vivo pharmacokinetics of the compounds, with the experimental method as follows:

rodent pharmacokinetic characteristics after intravenous injection and oral administration of the compound are tested by a standard scheme, and the candidate compound is prepared into a clear solution in an experiment and is administered to mice by single intravenous injection and single oral administration. The Intravenous (IV) vehicle was a mixture of 5% DMSO and 95% 10% Cremophor EL, and the oral (PO) vehicle was a mixture of 1% tween80, 9% PEG400, and 90% water. Collecting a whole blood sample within 48 hours, centrifuging for 15 minutes at 3000g at 4 ℃, separating supernatant to obtain a plasma sample, adding 20 times of acetonitrile solution containing an internal standard for precipitating protein, centrifuging to obtain supernatant, adding equal times of water into the supernatant, centrifuging again, taking supernatant for sampling, quantitatively analyzing blood concentration by an LC-MS/MS analysis method, and calculating drug substitution parameters such as peak reaching concentration, peak reaching time, clearance rate, half-life period, area under a drug time curve, bioavailability and the like. The test results are shown in Table 2.

The term "10% Cremophor EL" refers to a 10% Cremophor EL deionized water solution, for example, taking 10ml Cremophor EL as an example of 100ml preparation, adding deionized water, stirring uniformly, and adding deionized water to make the total volume 100 ml. "a mixed solvent of 5% DMSO and 95% 10% Cremophor EL" refers to a mixed solvent of DMSO and 10% Cremophor EL, wherein DMSO 10% Cremophor EL and 10% Cremophor EL account for 95% of the volume of the mixed solvent.

"internal standards" include, but are not limited to, 100ng/mL Labetalol, 100ng/mL dexamethasone, 100ng/mL Tolbutamide, 100ng/mL Verapamide, 100ng/mL Glyburide, or 100ng/mL Celecoxib in ACN.

Table 2 PK parameters in plasma of compounds of examples

Biological activity experiment 3: hERG potassium channel inhibition assay

1. Purpose of the experiment:

the influence of the embodiment 1 to be tested on the hERG potassium channel is detected by a full-automatic patch clamp method.

2. Experimental methods

2.1. Cell culture

The experiment used stable expression of hERG potassium channel cells from Aviva Biosciences CHO-hERE, CHO-hERG cultured in 5% CO₂And at 37 ℃. The CHO hERG culture broth is shown in Table 3.

TABLE 3 CHO hERG culture broth

Reagent	Supplier	Catalog Number	Volume(mL)
				F12 Hams	Invitrogen	31765-092	500
FBS	Invitrogen	10099-141	50
				G418/Geneticin	Invitrogen	10131-027	1
Hygromycin B	Invitrogen	10687-010	1

2.2. Pre-preparation of cells

The CHO-hERG cells prepared for the experiment were cultured for at least two days or more and the cell density reached 75% or more. Before the experiment began, cells were digested with TrypLE and then resuspended with extracellular fluid to collect cells.

2.3. Preparation of intracellular and extracellular fluids

The extracellular fluid needs to be prepared once a month. The intracellular fluid should be frozen at-20 deg.C. The intracellular and extracellular fluid compositions are shown in Table 4.

TABLE 4 intracellular and extracellular fluid composition

Composition of matter	Extracellular fluid (mM)	Intracellular fluid (mM)
			NaCl	145	‐
KCl	4	120
			KOH	‐	31.25
CaCl2	2	5.374
			MgCl2	1	1.75
Glucose	10	‐
			Na2ATP	‐	4
HEPES	10	10
			EGTA	‐	10
pH	7.4 with NaOH	7.2 with KOH
			Osmotic pressure	295mOsm	285mOsm

2.4. Preparation of the Compounds

Dissolving a compound to be detected and positive control Amitriptyline into stock solution with a certain concentration by DMSO, then diluting according to different gradients, and finally adding the stock solution into extracellular fluid according to a certain proportion to dilute the stock solution into the concentration to be detected. The presence or absence of the precipitate was checked visually before the start of the experiment. Finally, the concentration of DMSO in the solution to be tested and the positive control Amitriptyline cannot exceed 0.3 percent at most.

2.5. Voltage stimulation protocol

Keeping the clamping potential at-80 mv, firstly giving a voltage stimulus of-50 mv, lasting for 80ms to record the cell leakage current value, then depolarizing to +20mv, maintaining for 4800ms, opening the channel of hERG, then repolarizing to-50 mv and maintaining for 5000ms, leading out the tail current of hERG and recording, and finally, restoring the voltage to the clamping potential of-80 mv, and maintaining for 3100 ms. The above voltage stimulation was repeated every 15000 ms.

2.6.QPatch^HTXWhole cell patch clamp recordings

hERG QPatch^HTXThe experiments were performed at room temperature. Whole cell protocols, voltage stimulation protocols and compound detection protocols were established on QPatch Assay Software 5.2 (pH Bioscience) Software.

The voltage stimulation was first repeated 30 times, this segment being the baseline region for subsequent analysis, followed by the addition of 5 μ l of extracellular fluid, repeated three times. The effect concentration of each compound was added sequentially, again in a volume of 5 μ l three times. Cells were incubated at least 5mins per concentration tested. In the whole recording process, each index needs to reach the data analysis receiving standard, if the index does not reach the standard, the cell is not counted in the analysis range, the compound is tested again, and the recording process is automatically operated by Qpatch analysis software. Each compound was tested at a concentration of 0.24. mu.M, 1.20. mu.M, 6.00. mu.M, 30.00. mu.M in sequence, at least two cells were repeated at each concentration.

2.7. Data analysis

In each complete current record, the percent inhibition of the effect concentration of each compound was calculated based on the percentage of peak current in the negative control. Fitting by using a standard equation of Hill form to obtain a dose-effect relationship curve, wherein the specific equation is as follows:

I_(C)＝I_b+(I_fr-I_b)*cⁿ/(IC₅₀ ⁿ+cⁿ)

c is the test concentration of the compound and n is the slope

The curve fitting and the inhibition rate calculation are both completed by the analysis of Qpatch analysis software, and if the inhibition rate at the lowest concentration exceeds half inhibition or the inhibition rate at the highest concentration does not reach half inhibition, the corresponding IC of the compound₅₀Below minimum concentration or IC₅₀The value is greater than the maximum concentration.

2.8. Test results

The results of the hERG IC50 values for the example compounds are shown in table 5.

TABLE 5 results for hERG IC50 values for the compounds of the examples

Claims (3)

1. The following compounds or pharmaceutically acceptable salts thereof:

2. a pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, for the preparation of a medicament for preventing or treating a ROR γ receptor mediated disease.