WO2020124500A1

WO2020124500A1 - Nitrogen-containing heterocyclic compound, preparation method therefor, and application thereof

Info

Publication number: WO2020124500A1
Application number: PCT/CN2018/122419
Authority: WO
Inventors: 乐小勇; 蒋青; 朱健; 陈新; 胡代强
Original assignee: 江苏凯迪恩医药科技有限公司
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2020-06-25
Also published as: CN111601789A

Abstract

Disclosed in the present invention are a nitrogen-containing heterocyclic compound, a preparation method therefor, and an application thereof. The present invention provides a nitrogen-containing heterocyclic compound as represented by formula I, and a pharmaceutically acceptable salt, a hydrate, a solvate, a polymorph, a metabolite, a stereoisomer, a tautomer, or a prodrug thereof. The nitrogen-containing heterocyclic compound has an application of inducing and stimulating mesenchymal stem cells (MSCs) to differentiate into chondrocytes to repair cartilage. Also provided are a composition and method for treatment, prevention, or alleviation of arthritis or joint damage. The compound or composition provided by the present invention is applied to a joint, cartilage tissue or cartilage proximal tissue, or the whole body. The present invention also provides a composition and method for inducing MSCs to differentiate into chondrocytes.

Description

Nitrogen-containing heterocyclic compound, preparation method and application thereof

Technical field

The invention relates to a nitrogen-containing heterocyclic compound, its preparation method and application.

Background technique

In recent years, with the aging of the global entrance, more and more people are suffering from cartilage loss, especially in patients with osteoarthritis (Osteoarthritis, OA), which is caused by multiple factors Degenerative disease, the most common musculoskeletal disorder. It is usually characterized by degeneration of articular cartilage, reactive hyperplasia of the joint margin and subchondral bone, and the clinical manifestation is the slow development of symptoms such as joint pain, tenderness, joint swelling, restricted mobility, and joint deformity. Currently, about 237 million people worldwide suffer from symptoms and activity-restricted OA, which is the third largest disability-related disease worldwide. It is expected that by 2030, OA will become the first major disease causing disability. In the final stage of the disease, a total knee replacement is usually required.

Osteoarthritis (OA) is characterized by the progressive breakdown of articular cartilage and ultimately leads to functional disorders of synovial joints (Reginster, JY and NG. Khaltaev, 2002, Supp1: p.1-2), OA is caused by several types Mechanism-mediated, including enzymatic degradation of extracellular matrix, formation of defective new matrix, cell death and abnormal activation of cartilage and hypertrophic differentiation [Goldring, MB and SRGoldring Sci, 2010.1192(1): p.230-7] At present, OA is basically treated with pain intervention and surgical treatment.

Mesenchymal stem cells (MSCs) present in the bone marrow and most adult tissues can self-renew and differentiate into a variety of cell lineages, including chondrocytes, osteoblasts, and adipocytes (Pittenger, MF et al., Science, 1999.284 (5411 ):p.143-7), where mesenchymal stem cells include synovial mesenchymal stem cells, bone marrow mesenchymal stem cells, etc., studies have found that adult articular cartilage contains MSCs (about 3% of cells) capable of multi-lineage differentiation. In OA cartilage, these cells increase exponentially. These existing mesenchymal stem cells (MSC) have the ability to differentiate chondrocytes, so they retain the ability to repair damaged cartilage (Grogan, SP et al., Arthritis Res Ther, 2009.11(3): p.R85; Koelling, S . Et al. Cell Stem Cell, 2009.4(4): p.324-35).

However, there is still a lack of compounds for the treatment and/or amelioration of related diseases and disorders by inducing mesenchymal stem cells.

Summary of the invention

The problem to be solved by the present invention is the deficiency of the existing mesenchymal stem cell-induced compound deficiency, and provides a nitrogen-containing heterocyclic compound, intermediate, preparation method and application. The nitrogen-containing heterocyclic compound has the application of inducing the differentiation of mesenchymal stem cells (MSC) into chondrocytes and repairing cartilage. Also provided are compositions and methods for treating, preventing, or ameliorating arthritis or joint damage by applying the compounds or compositions of the present invention to joints, cartilage tissue, or proximal cartilage tissue, or the entire body. The invention also provides compositions and methods for inducing mesenchymal stem cells (MSC) to differentiate into chondrocytes.

The present invention solves the above technical problems through the following technical solutions.

The present invention provides a nitrogen-containing heterocyclic compound as shown in Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its Tautomer or its prodrug:

Among them, X is O or S;

Y is methylene or

Z is a single bond or C ₁ ～C ₄ alkylene;

Is unsubstituted or substituted with one or more R ^a is C ₆ ~ C ₁₀ aryl group, unsubstituted or substituted with one or more R ^B is 5 to 6-membered heteroaryl, unsubstituted or substituted with one or more R ^c- substituted C ₄ -C ₈ cycloalkenyl, or, unsubstituted or substituted with one or more ^Rd , C ₄ -C ₈ heterocycloalkenyl; in the 5-6 membered heteroaryl, the hetero atom One or more selected from N, O, and S, and the number of hetero atoms is 1 to 2; in the C ₄ to C ₈ heterocyclic alkenyl group, the hetero atom is selected from one of N, O, and S One or more kinds, the number of heteroatoms is 1 to 2; when there are multiple substituents ^Ra , ^Rb , ^Rc or ^Rd , the substituents are the same or different;

R ^a , R ^b , R ^c and R ^{d are} independently halogen, -OH, -CN, -COOH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl-O-, C ₁ -C ₆ alkane Group -C(=O)-, C ₁ -C ₆ alkyl-CO(=O)-, C ₁ -C ₆ alkyl-OC(=O)-, C ₁ -C ₆ alkyl-C(= O)NH-or

Is an unsubstituted or one or more R ¹ substituted C ₆ ～C ₁₀ aryl, or, unsubstituted or one or more R ² substituted 5-6 membered heteroaryl; the 5-6 membered In the heteroaryl group, the hetero atoms are selected from one or more of N, O and S, and the number of hetero atoms is 1 to 2; when there are multiple substituents R ¹ or R ² , the substituents are the same Or different

R ¹ and R ^{2 are} independently halogen, -OH, -CN, -NH ₂ , -COOH,

Unsubstituted or substituted with one or more of R ^1-7 is C ₁ ~ C ₆ alkyl, unsubstituted or substituted with one or more of R ^1-8 is C ₃ ~ C ₆ cycloalkyl, unsubstituted or substituted by a R ^1-9 is substituted with one or more C ₆ ~ C ₁₀ aryl group, unsubstituted or substituted with one or more R ^1-10 substituted 5- to 6-membered heteroaryl, unsubstituted or substituted with one or plural R ¹ s ^-11 substituted C ₁ ～C ₆ alkyl-O-, unsubstituted or substituted by one or more R ^1-12 C ₁ ～C ₆ alkyl-C(═O)-, unsubstituted or substituted by one or Multiple R ^1-13 substituted C ₁ ～C ₆ alkyl-C(═O)O-, unsubstituted or substituted by one or more R ^1-14 C ₁ ～C ₆ alkyl-OC(═O )-, or, unsubstituted or substituted by one or more R ^1-15 C ₁ ～C ₆ alkyl-C(=O)NH-; in the 5- to 6-membered heteroaryl, the hetero atom One or more selected from N, O and S, the number of hetero atoms is 1 to 3; when the substituents R ^1-7 , R ^1-8 , R ^1-9 , R ^1-10 , R ^{1- 11. When} there are a plurality of R ^1-12 , R ^1-13 , R ^1-14 or R ^1-15 , the substituents are the same or different;

Alternatively, when there are a plurality of substituents R ¹ or R ² , two adjacent R ¹ and two C directly connected to the C ₆ to C ₁₀ aryl group, or two adjacent R ² and 5 to Two Cs directly connected to the 6-membered heteroaryl group together form: C ₄ ～C ₆ cycloalkyl unsubstituted or substituted by one or more R ^1-5 , or unsubstituted or substituted by one or more C ₃ ˜C ₅ heterocycloalkyl substituted with R ^1-6 ; (that is, forming a fused ring with the C ₆ ˜C ₁₀ aryl group or 5-6 membered heteroaryl group); the C ₃ ˜ In the C ₅ heterocycloalkyl group, the hetero atoms are selected from one or more of N, O, and S, and the number of hetero atoms is 1 to 3; when the substituents R ^1-5 or R ^1-6 are plural , The substituents are the same or different;

R ^1-1 and R ^1-2 , R ^1-3 and R ^{1-4 are} independently H or C ₁ -C ₆ alkyl;

R ^1-5 , R ^1-6 , R ^1-7 , R ^1-8 , R ^1-9 , R ^1-10 , R ^1-11 , R ^1-12 , R ^1-13 , R ^1-14 and R ^{1-15 is} independently halogen, -OH, -CN, -NH ₂ , -COOH, -CONH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl -O-, C ₁ -C ₆ Alkyl-C(=O)-, C ₁ -C ₆ alkyl-CO(=O)-, or C ₁ -C ₆ alkyl-OC(=O)-.

In the present invention, the definitions of certain substituents in the nitrogen-containing heterocyclic compound as shown in Formula I may be as follows, and the definitions of unmentioned substituents are as described in any of the above schemes.

In a preferred embodiment of the present invention, the Z is methylene, ethylene (eg -CH ₂ CH ₂ -or -CH(CH ₃ )-) or isopropylene (eg -CH(CH ₃ ) CH ₂ -or -C(CH ₃ ) ₂ -), preferably methylene.

In a preferred embodiment of the present invention, the

, Said C ₆ ~ C ₁₀ aryl group is unsubstituted or substituted with one or more R ^a substituted phenyl.

In a preferred embodiment of the present invention, the

Is a bridged heterobicycloalkenyl group which is unsubstituted or substituted with one or more ^Rd , and the bridged heterobicycloalkenyl group is preferably 7-oxabicyclo[2.2.1]heptenyl (e.g.

).

In a preferred embodiment of the present invention, the numbers of ^Ra , ^Rb , ^Rc and ^Rd are independently 1 or 2.

In a preferred embodiment of the present invention, the ^Ra , ^Rb , ^Rc and ^{Rd are} independently located

Adjacent position, meta position or para position, preferably adjacent position.

In a preferred embodiment of the present invention, the ^Ra , ^Rb , ^Rc or ^Rd are fluorine, chlorine, bromine or iodine.

In a preferred embodiment of the present invention, said R ^a, R ^b, R ^c or R ^d is C ₁ ~ C ₆ alkyl group, C ₁ ~ C ₆ alkyl group -O-, C ₁ ~ C ₆ alkyl -C(=O)-, C ₁ -C ₆ alkyl-CO(=O)-, C ₁ -C ₆ alkyl-OC(=O)-, or, C ₁ -C ₆ alkyl-C( =O) NH-, the C ₁ -C ₆ alkyl (such as methyl, ethyl, propyl, butyl, pentyl or hexyl) is independently C ₁ -C ₄ alkyl, and for example Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

In a preferred embodiment of the present invention, the

In the above, the 5- to 6-membered heteroaryl group is linked to the

Connected.

In a preferred embodiment of the present invention, the

Is a phenyl group that is unsubstituted or substituted with one or more R ¹ .

In a preferred embodiment of the present invention, the

Is a 6-membered heteroaryl group that is unsubstituted or substituted by one or more R ² , in the 6-membered heteroaryl group, the hetero atoms are selected from N, the number of hetero atoms is 1 to 2, and the 6-membered hetero group Aryl is preferably pyridyl (e.g.

).

In a preferred embodiment of the present invention, the number of R ¹ and R ² is independently ¹ , ² , 3 or 4.

In a preferred embodiment of the present invention, the R ¹ or R ^{2 is} independently located

Adjacent position, meta position or para position, preferably meta position or para position.

In a preferred embodiment of the present invention, R ¹ or R ² is fluorine, chlorine, bromine or iodine, and for example, fluorine or chlorine.

In a preferred embodiment of the present invention, said R ¹ or R ² is unsubstituted or substituted with one or more R ^1-7 C ₁ -C ₆ alkyl, unsubstituted or substituted with one or more R ^{1 -11} substituted C ₁ ～C ₆ alkyl-O-, unsubstituted or substituted by one or more R ^1-12 C ₁ ～C ₆ alkyl-C(═O)-, unsubstituted or substituted by one or Multiple R ^1-13 substituted C ₁ ～C ₆ alkyl-C(═O)O-, unsubstituted or substituted by one or more R ^1-14 C ₁ ～C ₆ alkyl-OC(═O )-, or, unsubstituted or substituted by one or more R ^1-15 C ₁ ～C ₆ alkyl-C(═O)NH-, wherein the C ₁ ～C ₆ alkyl (for example Group, ethyl, propyl, butyl, pentyl, or hexyl) independently C ₁ -C ₄ alkyl, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl Radicals, sec-butyl or tert-butyl, again methyl.

In a preferred embodiment of the present invention, said R ¹ or R ² is phenyl which is unsubstituted or substituted by one or more R ^1-9 .

In a preferred embodiment of the present invention, two adjacent R ¹ and C ₆ to C ₁₀ aryl groups are directly connected to two Cs, or two adjacent R ² and 5 to 6 membered heteroaryl groups Two Cs directly connected to each other together form 1,3-dioxolane unsubstituted or substituted by one or more R ^1-6 (eg

among them

The carbon-to-carbon bond is shared with the C ₆ -C ₁₀ aryl group or the 5-6 membered heteroaryl group to form a paracyclic ring).

In a preferred embodiment of the present invention, in the R ^1-1 , R ^1-2 , R ^1-3 or R ^1-4 , the C ₁ -C ₆ alkyl group (for example, methyl, ethyl Group, propyl, butyl, pentyl or hexyl) independently C ₁ -C ₄ alkyl, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec Butyl or tert-butyl, for example isobutyl.

In a preferred embodiment of the present invention, the R ^1-5 , R ^1-6 , R ^1-7 , R ^1-8 , R ^1-9 , R ^1-10 , R ^1-11 , R ^{1- 12.} R ^1-13 , R ^1-14 or R ^1-15 are fluorine, chlorine, bromine or iodine, and for example, fluorine.

In a preferred embodiment of the present invention, the R ^1-5 , R ^1-6 , R ^1-7 , R ^1-8 , R ^1-9 , R ^1-10 , R ^1-11 , R ^{1- 12} , R ^1-13 , R ^1-14 or R ^1-15 are C ₁ ～C ₆ alkyl, C ₁ ～C ₆ alkyl-O-, C ₁ ～C ₆ alkyl-C(＝O)- , C ₁ -C ₆ alkyl-CO(=O)-, or C ₁ -C ₆ alkyl-OC(=O)-, wherein the C ₁ -C ₆ alkyl (eg methyl, ethyl Group, propyl, butyl, pentyl or hexyl) independently C ₁ -C ₄ alkyl, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec Butyl or tert-butyl.

In a preferred embodiment of the present invention, the

for

In a preferred embodiment of the present invention, the R ¹ or R ² is trifluoromethyl or hydroxymethyl (for example

).

In a preferred embodiment of the present invention, the R ¹ or R ² is

In a preferred embodiment of the present invention, the

for

In a preferred embodiment of the present invention, the

for

In a preferred embodiment of the present invention, when X is O, Y is methylene and Z is a single bond.

In a preferred embodiment of the present invention, when X is S, Y is

Z is a single bond or C ₁ -C ₄ alkylene group, preferably Z is a single bond.

In a preferred embodiment of the invention,

Is ^a C ₆ -C ₁₀ aryl group that is unsubstituted or substituted with one or more Ra, or a C ₄ -C ₈ heterocycloalkenyl group that is unsubstituted or substituted with one or more ^Rd ; preferably,

Is substituted with one R ^a C ₆ ~ C ₁₀ aryl group, or, R ^d is a substituted heterocyclyl C ₄ ~ C ₈ alkenyl group, a substituted located

Next door.

In a preferred embodiment of the invention,

It is ^a C ₆ -C ₁₀ aryl group which is unsubstituted or substituted with one or more Ra, preferably an unsubstituted C ₆ -C ₁₀ aryl group.

In a preferred embodiment of the present ^{^{invention, R a, R b, R}} c or R ^d is -COOH or

It is preferably -COOH.

In a preferred embodiment of the present invention, R ¹ and R ^{2 are} independently halogen, -OH, -CN, -NH ₂ , -COOH,

By one or more substituents R ^1-7 is C ₁ ~ C ₆ unsubstituted alkyl, unsubstituted or substituted with one or more of R ^1-9 is C ₆ ~ C ₁₀ aryl group, or unsubstituted or substituted C ₁ -C ₆ alkyl-O- substituted with one or more R ^1-11 ; or, when there are multiple substituents R ¹ or R ² , two adjacent R ¹ and C ₆ -C ₁₀ aromatic Two Cs directly connected to the base, or two adjacent R ² and two Cs directly connected to the 5-6 membered heteroaryl, together formed: unsubstituted or substituted by one or more R ^1-6 Substituted C ₃ -C ₅ heterocycloalkyl.

In a preferred embodiment of the present invention, X is O; Y is methylene; Z is a single bond;

It is substituted with one R ^a C ₆ ~ C ₁₀ aryl group, or substituted with one R ^d C ₄ ~ C ₈ alkenyl heterocyclyl group; the substituent is located

Adjacent to

R ^a or R ^d is COOH or

Is unsubstituted or substituted with one or more R ¹ phenyl, or, unsubstituted or substituted with one or more R ² 5-6 membered heteroaryl;

R ¹ and R ^{2 are} independently halogen, -OH, -CN, -NH ₂ , -COOH,

By one or more R substituents are not substituted with ^1-7 C ₁ ~ C ₆ alkyl, unsubstituted or substituted with one or more R ^1-9 is C ₆ ~ C ₁₀ aryl group, unsubstituted or substituted with one or C ₁ -C ₆ alkyl-O- substituted with multiple R ^1-11 ; or, when there are multiple substituents R ¹ or R ² , adjacent two R ¹ and C ₆ -C ₁₀ aryl Two directly connected Cs, or two adjacent R ² and two C directly connected to a 5-6 membered heteroaryl, together form an unsubstituted or substituted by one or more R ^1-6 C ₃ ~ C ₅ heterocycloalkyl;

Preferably, R ¹ and R ^{2 are} independently -CN, -COOH, unsubstituted or substituted with one or more R ^1-7 C ₁ -C ₆ alkyl, unsubstituted or substituted with one or more R ^{1 -9} substituted C ₆ -C ₁₀ aryl, unsubstituted or C ₁ -C ₆ alkyl-O- substituted with one or more R ^1-11 or, when there are multiple substituents R ¹ or R ² , Two adjacent R ¹ and two C directly connected to the C ₆ -C ₁₀ aryl group together form a C ₃ -C ₅ heterocycloalkyl group which is unsubstituted or substituted by one or more R ^1-6 .

In a preferred embodiment of the present invention, the definition of certain substituents in the nitrogen-containing heterocyclic compound as shown in Formula I can be as follows, and the definitions of unmentioned substituents are as described in any of the above schemes :

X is S, Y is

Z is a single bond or C ₁ ～C ₄ alkylene;

It is unsubstituted or substituted with one or more R ^a is C ₆ ~ C ₁₀ aryl group, preferably unsubstituted C ₆ ~ C ₁₀ aryl group;

R ¹ and R ^{2 are} independently halogen, -OH, -CN, -NH ₂ , -COOH,

By one or more R substituents are not substituted with ^1-7 C ₁ ~ C ₆ alkyl, unsubstituted or substituted with one or more R ^1-9 is C ₆ ~ C ₁₀ aryl group, unsubstituted or substituted with one or C ₁ -C ₆ alkyl-O- substituted with multiple R ^1-11 ; or, when there are multiple substituents R ¹ or R ² , adjacent two R ¹ and C ₆ -C ₁₀ aryl Two Cs directly connected, or two adjacent R ² and two Cs directly connected to the 5-6 membered heteroaryl, together formed: unsubstituted or substituted by one or more R ^1-6 C ₃ ～C ₅ heterocycloalkyl;

Preferably, R ¹ and R ^{2 are} independently halogen, OH, CN, COOH, or, unsubstituted or substituted with one or more R ^1-7 C ₁ -C ₆ alkyl.

In a preferred embodiment of the present invention, the nitrogen-containing heterocyclic compound represented by Formula I is preferably selected from any of the following compounds:

Thus, throughout the specification, those skilled in the art can refer to the nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, and its polycrystals Groups, their metabolites, their stereoisomers, their tautomers or their prodrugs, and their substituents are selected to provide a stable nitrogen-containing heterocyclic compound as shown in Formula I, Its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its tautomers or its prodrugs, including but not limited to the practice of the invention The compounds described in the examples.

The present invention also includes isotope-labeled nitrogen-containing heterocyclic compounds represented by Formula I, their pharmaceutically acceptable salts, their hydrates, their solvates, their polymorphs, their metabolites, and their A stereoisomer, its tautomer, or a prodrug thereof, in which one or more atoms are replaced by one or more atoms having a specific atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present invention include, but are not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, sulfur, and chlorine (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ¹⁸ F, ³⁵ S and ³⁶ Cl). Isotope-labeled compounds of the invention can be used for the determination of the tissue distribution of compounds and their prodrugs and metabolites; preferred isotopes for such determinations include ³ H and ¹⁴ C. In addition, in some cases, substitution with heavier isotopes (such as deuterium (2H or D)) can provide increased metabolic stability, which provides therapeutic advantages such as increased half-life in vivo or reduced dosage requirements.

The isotopically labeled compounds of the present invention can generally be prepared by replacing non-isotopically labeled reagents with isotopically labeled reagents according to the methods described herein.

The nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its mutual The variant isomers or their prodrugs can be synthesized by methods similar to those well known in the chemical arts. For the steps and conditions, refer to the steps and conditions of similar reactions in the art, especially according to the instructions herein. The starting materials are usually from commercial sources such as Aldrich or can be easily prepared using methods well known to those skilled in the art (available through SciFinder, Reaxys online database).

In the present invention, the nitrogen-containing heterocyclic compound as shown in Formula I can also be prepared through the preparation of the nitrogen-containing heterocyclic compound as shown in Formula I, using conventional methods in the art, and the peripheral modification Furthermore, the other nitrogen-containing heterocyclic compound represented by Formula I described above is obtained.

The present invention provides a method for preparing the nitrogen-containing heterocyclic compound as shown in Formula I, which is Scheme I, Scheme II and Scheme III; wherein Scheme I includes the following steps: In an organic solvent, The compound represented by Formula II and the compound represented by Formula III are subjected to the cyclization reaction shown below to obtain the nitrogen-containing heterocyclic compound represented by Formula I;

among them,

for

X is O; Y is methylene; Z and

Is defined as shown above;

Scheme two includes the following steps: In the phosphoric acid solution, the

for

The nitrogen-containing heterocyclic compound shown in Formula I undergoes an aromatization reaction to obtain

for

The nitrogen-containing heterocyclic compound shown in Formula I is sufficient; wherein, X is O; Y is methylene; Z and

Is defined as shown above;

Scheme three includes the following steps: in an organic solvent, the compound represented by Formula I'and the sulfurization reagent are subjected to a sulfurization reaction as shown below to obtain the nitrogen-containing heterocyclic compound represented by Formula I;

Among them, X'and Y'are O, Y is

X is S, Z,

The definition is shown above.

In scheme one, the organic solvent may be a conventional organic solvent in this type of reaction in the art, and preferably an aromatic hydrocarbon solvent (such as toluene) in the present invention.

In Scheme 1, the amount of the organic solvent may not be specifically limited so as not to affect the reaction; in the present invention, the mass volume of the compound represented by Formula II and the organic solvent is preferably 0.01 g/mL to 0.2g/mL (for example, 0.02g/mL to 0.03g/mL).

In Scheme 1, the molar ratio of the compound represented by Formula II to the compound represented by Formula III may be a conventional molar ratio in this type of reaction in the art, and the formula II described in the present invention The molar ratio of the compound shown to the compound shown by Formula III is preferably 1.2:1 to 1:1.2.

In the first scheme, the temperature of the cyclization reaction may be a conventional temperature in this type of reaction in the art, preferably 90°C to 120°C in the present invention.

In scheme one, the progress of the cyclization reaction can be monitored by conventional monitoring methods in the art (eg TLC or NMR), generally the end point of the reaction is when the compound of formula II disappears or no longer reacts .

In the second solution, the phosphoric acid solution may be a conventional phosphoric acid solution in this type of reaction in the art, and preferably 85% phosphoric acid aqueous solution in the present invention.

In scheme two,

for

The molar volume ratio of the nitrogen-containing heterocyclic compound shown in Formula I to the phosphoric acid solution may be a conventional molar volume ratio in this type of reaction in the art, preferably 0.05 mol/L in the present invention ~0.2mol/L (0.1mol/L).

In the second scheme, the temperature of the aromatization reaction may be a conventional temperature in this type of reaction in the art, preferably 90°C to 120°C in the present invention.

In scheme two, the progress of the aromatization reaction can be monitored by using conventional monitoring methods in the art (such as TLC or NMR), and the reaction is generally performed when the compound shown in Formula II disappears or no longer reacts end.

In scheme three, the organic solvent may be a conventional organic solvent in this type of reaction in the art, preferably an aromatic hydrocarbon solvent (such as toluene) in the present invention.

In scheme three, the sulfidation reagent may be a conventional sulfidation reagent in this type of reaction in the art, and it is preferably Lawson's reagent in the present invention.

In scheme three, the amount of the organic solvent may not be specifically limited so as not to affect the reaction; in the present invention, the mass of the compound represented by formula I'and the organic solvent are relatively good It is 0.01 g/mL to 0.2 g/mL (for example, 0.02 g/mL).

In scheme three, the molar ratio of the compound represented by formula I′ to the sulfurization reagent may be a conventional molar ratio in the art, and the compound represented by formula I” described in the present invention is The molar ratio of the vulcanizing agent is preferably 1:1 to 1:3 (for example, 1:2).

In scheme three, the temperature of the vulcanization reaction may be a conventional temperature in this type of reaction in the art, preferably 100°C to 120°C in the present invention.

In scheme three, the progress of the vulcanization reaction can be monitored by using conventional monitoring methods in the art (such as TLC or NMR). Generally, the end point of the reaction is when the compound of formula I′ disappears or no longer reacts .

The present invention also provides a pharmaceutical composition comprising the nitrogen-containing heterocyclic compound represented by Formula I, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a polymorphic form thereof, Metabolites, their stereoisomers, their tautomers or their prodrugs, and at least one pharmaceutical excipient. The pharmaceutical composition may also contain one or more additional active ingredients. For example, such a pharmaceutical composition may comprise one or more additional nitrogen-containing heterocycles represented by Formula I, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, polymorphs thereof, Its metabolites, its stereoisomers, its tautomers or its prodrugs. Alternatively or in addition, the pharmaceutical composition may, for example, include, in addition to the nitrogen-containing heterocycle shown in Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, and many others One or more active ingredients other than the crystalline form, its metabolite, its stereoisomer, its tautomer or its prodrug.

In a preferred embodiment of the present invention, the pharmaceutical composition further comprises a composite scaffold formed by one or more of collagen, chitosan and hyaluronic acid; it is used to induce mesenchymal stem cells to differentiate into chondrocytes.

In a preferred embodiment of the present invention, the pharmaceutical composition may further include components suitable for formulation for intra-articular delivery; the components for intra-articular delivery include but are not limited to: angiogenin-like 3 protein (ANGPTL3) or its cartilage-forming variant, oral salmon calcitonin, SD-6010 (iNOS inhibitor), vitamin D3 (choline calciferol), collagen hydrolysate, FGF18, BMP7, avocado soybean unsaponified One or more of ASU and hyaluronic acid. ANGPTL3 is described in more detail in WO/2011/008773 (incorporated in its entirety).

In the pharmaceutical composition, the nitrogen-containing heterocyclic compound as shown in Formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its The dosage of the stereoisomer, its tautomer or its prodrug may be a therapeutically effective amount.

The effective amount of the compound, pharmaceutical composition or drug of the present invention can be easily determined through routine experimentation, and the most effective and convenient route of administration and the most appropriate formulation can also be determined through routine experimentation.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or to promote the activity of the subject after the administration of the composition The ingredients are effectively absorbed. The pharmaceutical adjuvant may be an inert filler or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binder, suspending agent, emulsifier, diluent, filler, granulating agent, adhesive, disintegrant, lubricant, anti-adhesion Agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins, such as human serum proteins, buffer substances such as phosphate, glycine, sorbic acid, sorbic acid Potassium acid, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silicon, magnesium trisilicate, polyethylene Pyrrolidone, polyacrylate, wax, polyethylene-polyoxypropylene-blocking polymer, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl Sodium cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; auxiliary materials such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, Olive oil, corn oil and soybean oil; glycol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; seaweed Acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol, phosphate buffer solution, and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, Coating materials, sweeteners, flavors and spices, preservatives and antioxidants.

The pharmaceutical composition of the present invention can be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or lyophilizing processes.

The pharmaceutical dosage form of the compound of the present invention can be provided in the form of immediate release, controlled release, sustained release or target drug release system. For example, commonly used dosage forms include solutions and suspensions, (micro)emulsions, ointments, gels and patches, liposomes, tablets, sugar-coated pills, soft or hard shell capsules, suppositories, ovules, implants, amorphous Or crystalline powder, aerosol and lyophilized preparation. Depending on the route of administration used, special devices may be needed to administer or administer the drug, such as syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks. Pharmaceutical dosage forms often consist of drugs, excipients, and container/sealing systems. One or more excipients (also called inactive ingredients) can be added to the compounds of the present invention to improve or promote the manufacture, stability, administration and safety of the drug, and can provide the desired drug release Curve method. Therefore, the type of excipients added to the drug may depend on various factors, such as the physical and chemical properties of the drug, the route of administration, and the preparation steps. Pharmaceutical excipients exist in this field and include those listed in various pharmacopoeias. (See US Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP); US Food and Drug Administration (the US Food and Drug Administration) Administration, www.fda.gov) Center for Drug Evaluation and Research (CEDR) publications, such as "Inactive Components Guide" (Inactive, Guide, 1996); "Handbook of Drug Additives" by Ash and Ash "(Handbook of Pharmaceutical Additives, 2002, United Information Resources (Synapse Information, Inc., Endicott NY; etc.).

The pharmaceutical dosage form of the compound of the present invention can be manufactured by any method well known in the art, for example, by conventional mixing, sieving, dissolving, melting, granulating, manufacturing sugar-coated pills, tableting, suspending, squeezing, spray drying, Grinding, emulsification, (nano/micron level) encapsulation, wrapping or freeze-drying process. As described above, the composition of the present invention may include one or more physiologically acceptable inactive ingredients that will facilitate the processing of the active molecule into a formulation for medical use.

The pharmaceutical composition of the present invention can be administered in any form, including injection (joint cavity), mucosa, topical or parenteral (infusion, injection, implantation, subcutaneous, intramuscular) administration. The pharmaceutical composition of the present invention may also be a controlled-release or delayed-release dosage form (for example, liposomes or microspheres). Examples of topical preparations include, but are not limited to, creams, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, liquid dosage forms suitable for parenteral administration.

Liquid form preparations include solutions, suspensions and emulsions, for example water or water/propylene glycol solutions. For injection, liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

Suitable aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, stabilizers and thickeners as required. Suitable aqueous suspensions can be prepared by dispersing finely divided active components in water with viscous materials such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl Methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic, and dispersants or wetting agents, such as naturally occurring phospholipids (eg, lecithin), condensation products of alkylene oxides and fatty acids (eg , Polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (for example, heptadecyloxyethanol), ethylene oxide and partial esters derived from fatty acids and hexitol ( For example, condensation products of polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (for example, polyoxyethylene sorbitan monooleate) .

The aqueous suspension may also contain one or more preservatives, such as ethyl paraben or n-propyl paraben, one or more colorants, one or more flavoring agents and one or more Sweeteners such as sucrose, aspartame or saccharin. The osmotic pressure of the preparation can be adjusted.

The present invention also provides the nitrogen-containing heterocyclic compound as shown in Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, and its stereoisomers , The tautomer or its prodrug, or the pharmaceutical composition as described above in the preparation of proteoglycan (Aggrecan), type II collagen (Collagen II) and Sox9 one or more inducers. The "one or more inducers in proteoglycan (Aggrecan), type II collagen (Collagen II) and Sox9" can be passed through one of proteoglycan (Aggrecan), type II collagen (Collagen II) and Sox9 Or a variety of induced mesenchymal stem cells differentiate into chondrocytes.

In a preferred embodiment of the present invention, the inducer is a drug.

The present invention also provides a nitrogen-containing heterocyclic compound as shown in Formula I, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a polymorphic form thereof, a metabolite thereof, and a stereoisotope thereof Use of a conformer, its tautomer or its prodrug, or a pharmaceutical composition as described above in the preparation of a medicament.

In a preferred embodiment of the present invention, the drug refers to a drug that can be used to treat or prevent diseases related to the induction of differentiation of mesenchymal stem cells into chondrocytes; the diseases are preferably: arthritis or joint damage, such as Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis, osteoporosis, or osteoarthritis; the induction of differentiation of mesenchymal stem cells into chondrocytes means One or more of proteoglycan (Aggrecan), type II collagen (Collagen II) and Sox9 induce mesenchymal stem cells to differentiate into chondrocytes.

In a preferred embodiment of the present invention, the inducer is used to expand the chondrocyte population in culture in vitro; autologous or allogenic chondrocyte transplantation can be performed. The transplant can optionally be administered simultaneously with the treatment, which includes administration of the nitrogen-containing heterocyclic compound represented by Formula I, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, of the invention Polymorphs, their metabolites, their stereoisomers, their tautomers or their prodrugs, or pharmaceutical compositions as described above. In these methods, for example, chondrocytes can be harvested from an unimpaired small weight-bearing area of the damaged joint by arthroscopy, and can be described in the nitrogen-containing heterocyclic compound represented by Formula I of the present invention, its pharmacy In the presence of an acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, its tautomer or its prodrug, or a pharmaceutical composition as described above Culture to increase the number of cells before transplantation. The swollen culture is then combined with the nitrogen-containing heterocyclic compound of formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its The stereoisomers, their tautomers or their prodrugs, or the pharmaceutical composition as described above are mixed and placed in the joint space or directly into the defect.

The present invention also provides a method for improving arthritis or joint damage in mammals. The method comprises administering to the joints of mammals an effective dose of the nitrogen-containing heterocyclic compound represented by Formula I, which is pharmaceutically acceptable Accepted salts, their hydrates, their solvates, their polymorphs, their metabolites, their stereoisomers, their tautomers or their prodrugs or pharmaceutical compositions as described above. The disease is preferably arthritis or joint damage, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis, osteoporosis, or osteoarthritis.

Provided herein is a method for inducing mesenchymal stem cells to differentiate into chondrocytes, the method comprising contacting the mesenchymal stem cells with a sufficient amount of the nitrogen-containing heterocyclic compound shown in Formula I, which is pharmaceutically acceptable Salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, its tautomer or its prodrug or pharmaceutical composition as described above.

The chondrocytes produce and maintain a cartilage matrix composed of collagen and proteoglycan. Chondrocytes are derived from the differentiation of mesenchymal stem cells (MSC). MSCs are pluripotent stem cells, which can differentiate into several different types of cells, including but not limited to osteoblasts, chondrocytes, and adipocytes. Differentiation is a process of specialized cell types formed by less specialized cell types, such as chondrocytes from MSC.

The nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its mutual The variant isomer or its prodrug, or the pharmaceutical composition as described above can be directly injected into the synovial fluid of the joint, administered systemically (oral or intravenous) or directly into the cartilage defect, used alone or with appropriate The carrier is compounded to prolong the release of protein.

The nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its mutual The variant isomer or its prodrug, or the pharmaceutical composition as described above can be used in combination with a periosteum or periosteum graft containing cartilage that can form cartilage and/or contribute to transplantation Cells or their precursor cells are kept in place. The nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its Tautomers or their prodrugs, or pharmaceutical compositions as described above, can be used to repair cartilage damage together with joint lavage, bone marrow stimulation, abraded arthroplasty, subchondral drilling or microfragmentation of subchondral bone . In addition, due to the administration of the nitrogen-containing heterocyclic compound represented by Formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, and its stereotypes according to the present invention After the isomer, its tautomer or its prodrug, or a pharmaceutical composition as described above causes cartilage growth, additional surgical treatment may be required to properly contour the newly formed cartilage surface. In some embodiments, the pharmaceutical composition may be formulated for intra-articular delivery.

Definitions and general terms

Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated by reference in their entirety.

Unless otherwise stated, the following definitions used herein shall apply. For the purposes of the present invention, chemical elements are consistent with the CAS version of the periodic table of elements, and the Handbook of Chemistry and Physics, 75th edition, 1994. In addition, the general principles of organic chemistry can refer to the descriptions in "Organic Chemistry", Thomas Sorrell, University Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007 , The entire contents of which are incorporated herein by reference.

The term "comprising" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by specific substituents. Further, when the group is substituted with one or more substituents, the substituents are mutually independent, that is, the one or more substituents may be different from each other, or may be the same of. Unless otherwise indicated, a substituent group may be substituted at each substitutable position of the substituted group. When more than one position in the given structural formula can be substituted by one or more substituents selected from specific groups, the substituents may be substituted at the same positions or differently.

In various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for the group should be understood as the linking group. For example, if the structure requires a linking group and the Markush group definition for the variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents the linked group, respectively An alkylene group or an arylene group.

In the present invention, "halo" or "halogen" are each independently fluorine, chlorine, bromine, or iodine (for example, fluorine or chlorine).

The term "alkyl" is meant to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, C ₁ -C ₆ , as defined in “C ₁ -C ₆ alkyl”, includes groups with 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched structure . For example, "C ₁ -C ₆ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, isobutyl, pentyl, and hexyl, etc. .

In some specific structures, when the alkyl group is clearly represented as a linking group, then the alkyl group represents a linked alkylene group, for example, the group "halo-(C ₁ ～C ₆ The C ₁ -C ₆ alkyl group in the “alkyl group” should be understood as a C ₁ -C ₆ alkylene group.

The term "alkylene" means a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated linear or branched hydrocarbon group. Examples of alkylene groups include methylene (-CH ₂ -), ethylene (including -CH ₂ CH ₂ -or -CH(CH ₃ )-), isopropylene (including -CH(CH ₃ ) CH ₂ -or -C(CH ₃ ) ₂ -) and so on. Wherein, the alkylene group may be optionally substituted with one or more substituents described in the present invention.

The term "cycloalkyl" refers to a saturated monocyclic, polycyclic, or bridged carbocyclic substituent. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.; wherein, C ₃ ～C ₆ cycloalkyl Rings having 3 to 6 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term "cycloalkenyl" refers to a cyclic non-aromatic hydrocarbon group containing the specified number of carbon atoms and at least one carbon-carbon double bond. There is preferably one carbon-carbon double bond, and up to four non-aromatic carbon-carbon double bonds may be present.

The term "heterocycloalkyl" refers to a group of 3-10 membered saturated heterocyclic systems containing 1-4 heteroatoms selected from O, N and S. In a heterocycloalkyl group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as long as valency permits. Heterocycloalkyl groups may be either monocyclic ("monocyclic heterocycloalkyl") or fused, bridged or spiro ring systems (eg bicyclic systems ("bicyclic heterocycloalkyl ")) and is saturated. Heterocycloalkyl bicyclic ring systems can include one or more heteroatoms in one or two rings. The -CH ₂ -group in the ring may optionally be replaced by -C(=O)-. The sulfur atom of the ring can optionally be oxidized to an S-oxide. The nitrogen atom of the ring can optionally be oxidized to an N-oxide. Heterocycloalkyl within the scope of this definition includes, but is not limited to: azetidinyl, glycidyl, thietyl, tetrahydrofuranyl, pyrrolidinyl, dioxolyl, piperidinyl, Tetrahydropyranyl, sulfided cyclopentyl, piperazinyl, morpholinyl, azepanyl, oxepanyl, and thiepanyl. Examples of the -CH ₂ -group substituted by -C(=O)- in the heterocyclic group include, but are not limited to, 2-oxopyrrolidinyl, 2-piperidinone, 3-morpholinone, 3 -Thiomorpholinone and oxotetrahydropyrimidinyl etc.

Thus, "heterocyclenyl" refers to a 5-10 membered cyclic non-aromatic containing a specified number of carbon atoms and at least one carbon-carbon double bond, and containing 1-4 heteroatoms selected from O, N, and S Hydrocarbyl. A carbon-carbon double bond is preferably present; and includes bicyclic groups.

The term "aryl" refers to a 4n+2 aromatic having 6-14 ring atoms and zero heteroatoms provided in the aromatic ring system, monocyclic or polycyclic (eg, bicyclic or tricyclic) Groups of ring systems (eg, having 6, 10, or 14 shared p-electrons in a cyclic array) ("C ₆ -C ₁₄ aryl"). Examples of the above aryl unit include phenyl, naphthyl, phenanthrenyl, or anthracenyl.

The term "heteroaryl" refers to a 5-10 member having a ring carbon atom and 1-4 ring heteroatoms provided in the aromatic ring system (where each heteroatom is independently selected from nitrogen, oxygen, and sulfur) Groups of monocyclic or bicyclic 4n+2 aromatic ring systems (for example, having 6 or 10 shared p electrons in a cyclic array) ("5-10 membered heteroaryl"). Heteroaryl groups within the scope of this definition include, but are not limited to: acridinyl, carbazolyl, cinnoline, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furyl, thienyl , Benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, Tetrahydroquinoline.

The term "haloalkyl" means an alkyl group substituted with halogen at any position. Thus, "haloalkyl" includes the above definitions of halogen and alkyl.

Those skilled in the art can understand that according to the conventions used in the art, this application describes the structural formula used in the group

It means that the corresponding group is connected to other fragments and groups in the compound through this site.

In addition, it should be noted that, unless explicitly stated otherwise, the description method "...independently" used in the present invention should be understood in a broad sense, which means that the described individuals are independent of each other and can be independent Ground is the same or different specific group. In more detail, the description "...independently" may mean that in different groups, the specific options expressed between the same symbols do not affect each other; it may also mean that in the same group, the same symbol The specific options expressed between do not affect each other.

The compounds described herein may contain one or more asymmetric centers, and thus can exist in various isomer forms, for example, enantiomers and/or diastereomers. For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and containing one or more A mixture of stereoisomers. Isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be asymmetric Prepared synthetically. See, for example, Jacques et al., Enantiomers, Racemates and Solutions (Enantiomers, Racemates and Resolutions) (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33: 2725 (1977) ; Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Resolving Agents and Optical Resolution Tables (Tables of Resolving Agents and Optical Resolutions) p.268 (ELEliel Editor, University of Notre Dame Press (Univ. of Notre Dame Press), Notre Dame (Notre Dame, in 1972). In addition, the invention encompasses compounds that are single isomers (substantially free of other isomers) as described herein, and, alternatively, are mixtures of multiple isomers.

"Stereoisomer" refers to compounds that have the same chemical structure, but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans) isomers, atropisomers, etc. .

"Enantiomer" refers to two isomers of a compound that cannot overlap but are mirror images of each other.

"Diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties and reactivity. Diastereomer mixtures can be separated by high-resolution analytical operations such as electrophoresis and chromatography, such as HPLC.

The stereochemical definitions and rules used in the present invention generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemicals (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereo chemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

Any asymmetric atoms (for example, carbon, etc.) of the compounds disclosed in the present invention can exist in racemic or enantiomerically enriched forms, such as (R)-, (S)-, or (R,S)-configuration forms exist. In certain embodiments, each asymmetric atom has at least 0% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least in (R)- or (S)-configuration 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.

The resulting mixture of any stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography Method and/or step crystallization method.

The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted by a low energy barrier. If tautomerism is possible (as in solution), the chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropictautomers) include interconversion through proton migration, such as keto-enol isomerization and imine- Enamine isomerization. Valence tautomers (valencetautomer) include interconversion through the recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. A specific example of phenol-ketone tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "pharmaceutically acceptable" used in the present invention refers to those compounds, materials, compositions and/or dosage forms, which are within the scope of reliable medical judgment and are suitable for use with humans and animals Used in contact with tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" means a salt formed from a suitable non-toxic organic acid, inorganic acid, organic base, or inorganic base and Compound I, which retains the biological activity of Compound I. The organic acid can be various organic acids that can form salts in the art, preferably methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid , Propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, isethionic acid, naphthalene sulfonic acid and one or more of salicylic acid. The inorganic acid may be various conventional inorganic acids capable of forming salts in the art, preferably one or more of hydrochloric acid, sulfuric acid and phosphoric acid. The organic base may be a variety of conventional organic bases capable of forming salts, preferably one or more of pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines and anilines Species. The tertiary amine organic base is preferably triethylamine and/or N,N-diisopropylethylamine. The aniline organic base is preferably N,N-dimethylaniline. The pyridine organic base is preferably one or more of pyridine, picoline, 4-dimethylaminopyridine and 2-methyl-5-ethylpyridine. The inorganic base may be various inorganic bases conventionally capable of forming salts in the art, preferably alkali metal hydride, alkali metal hydroxide, alkali metal alkoxide, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate , One or more of potassium bicarbonate and sodium bicarbonate. The alkali metal hydride is preferably sodium hydride and/or potassium hydride. The alkali metal hydroxide is preferably one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide. The alkali metal alkoxide is preferably one or more of sodium methoxide, sodium ethoxide, potassium t-butoxide and sodium t-butoxide.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing acid or base groups by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

In addition to salt forms, the compounds provided by the invention also exist in prodrug forms. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to transform the compounds of the invention. Any compound that can be transformed in vivo to provide a biologically active substance (ie, a nitrogen-containing heterocyclic compound represented by Formula I) is a prodrug within the scope and spirit of the present invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester, which is used as a medicine by hydrolyzing in vivo to obtain the compound represented by Formula I itself. The prodrug is preferably administered orally, because hydrolysis occurs in many cases mainly under the influence of digestive enzymes. When the ester itself is active or hydrolysis occurs in the blood, parenteral administration can be used. In addition, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in the in vivo environment.

The compound of the present invention may contain unnatural proportions of atomic isotopes in one or more atoms constituting the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). The conversion of all isotopic compositions of the compounds of the present invention, whether radioactive or not, is included within the scope of the present invention.

The compounds of formula I and salts thereof of the present invention are intended to include all solid forms of compounds of formula I and salts thereof. Compounds of formula I and their salts are also intended to cover all solvated (eg hydrated) and unsolvated forms of compounds of formula I and their salts.

The term "metabolite" used in the present invention refers to a product obtained by metabolizing a specific compound or its salt in the body. The metabolite of a compound can be identified by techniques well known in the art, and its activity can be characterized by an experimental method as described in the present invention. Such products can be obtained by administering compounds through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage and the like. Accordingly, the present invention includes metabolites of the compound, including metabolites produced by fully contacting the compound of the present invention with a mammal for a period of time.

The "solvate" of the present invention refers to an association formed by one or more solvent molecules and the compound of the present invention. Solvent-forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed by the solvent molecule being water.

The "ester" in the present invention refers to an in vivo hydrolyzable ester formed by a compound containing a hydroxyl group or a carboxyl group. Such esters are, for example, pharmaceutically acceptable esters that are hydrolyzed in the human or animal body to produce the parent alcohol or acid. The compounds of formula I of the present invention contain carboxyl groups and can form in vivo hydrolyzable esters with appropriate groups. Such groups include, but are not limited to, alkyl groups, arylalkyl groups, and the like.

As used herein, "mammal" refers to any mammal classified as a mammal, including humans, domesticated animals and farm animals, as well as zoo, competitive or pet animals such as cattle (eg cows), horses, dogs, Sheep, pigs, rabbits, goats, cats, etc.

"Therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refers to a dose that produces a therapeutic effect. The exact dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Pharmaceutical, Science, and Technology) Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

"Treatment", "Treatment" plus "Improvement" and "Improvement" refer to any sign of success in treating or ameliorating injury, pathology, condition or symptom (eg pain), including any goals or subjective parameters such as relief; alleviation of symptoms ; Reduce the frequency or duration of symptoms or conditions; or, in some cases, prevent the onset of symptoms or conditions. The treatment or improvement of symptoms may be based on any objective or subjective parameters; including, for example, the results of physical examinations.

On the basis of not violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are commercially available.

The positive progress effect of the present invention is that the nitrogen-containing heterocyclic compound has a good expression-inducing activity for one or more of proteoglycan (Aggrecan), type II collagen (Collagen II) and Sox9; Mesenchymal stem cells (MSCs) differentiate into chondrocytes and repair cartilage.

detailed description

The present invention is further described by way of examples below, but it does not limit the present invention to the scope of the described examples. The experimental methods without specific conditions in the following examples are selected according to the conventional methods and conditions or according to the product description.

In the following examples, room temperature means 10-30°C; overnight means 8-15 hours, such as 12 hours; eq means equivalent; solvent ratio such as PE:EA means volume ratio; Lawessons reagent means Lawson's reagent.

Example 1

Synthesis of N-(furan-2-methyl)-[1,1'-biphenyl-4-amine]:

Take 4-iodobiphenyl (1.22g, 4.36mmol), furfuramine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30mmol), L-proline ( 100mg, 0.87mmol), dimethyl sulfoxide 10ml, in a 100ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Anhydrous sodium sulfate was dried, and the sample was passed through the column (PE:EA=5:1) to obtain 0.42g of yellow solid with a yield of 38%.

¹ H-NMR (400MHz, DMSO-d ₆ ) δ7.61-7.44(m, 3H), 7.47-7.32(m, 4H), 7.26-7.17(m, 1H), 6.77-6.68(m, 2H), 6.39 (dd, J = 3.2, 1.9 Hz, 1H), 6.34-6.25 (m, 2H), 4.28 (d, J = 6.0 Hz, 2H).

(3aR,6R)-2([1,1'-biphenyl-4])-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole Synthesis of -7-carboxylic acid:

Take N-(furan-2-methyl)-[1,1'-biphenyl-4-amine] (0.5g, 2.01mmol), maleic anhydride (0.2g, 2.01mmol), toluene 20ml in 100ml single mouth The flask was protected by argon and refluxed at 100 degrees Celsius for 12 hours. The reaction solution was filtered, washed with toluene, and dried to obtain a gray solid 0.38 g. The yield was 54.5%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.31 (s, 1H), 7.81-7.75 (m, 2H), 7.75-7.62 (m, 4H), 7.45 (dd, J=8.4, 7.0 Hz, 2H), 7.39-7.30 (m, 1H), 6.66 (d, J = 5.7Hz, 1H), 6.50 (dd, J = 5.7, 1.7Hz, 1H), 5.06 (d, J = 1.7Hz, 1H), 4.59 (d, J = 11.6 Hz, 1H), 4.13 (d, J = 11.6 Hz, 1H), 3.11 (d, J = 9.1 Hz, 1H), 2.61 (d, J = 9.1 Hz, 1H).

Synthesis of 2([1,1'-biphenyl-4])-3-oxoisoindoline-4-carboxylic acid:

Take (3aR, 6R)-2([1,1'-biphenyl-4])-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindene Indole-7-carboxylic acid (0.2g, 0.576mmol) was dissolved in 10ml of phosphoric acid, and reacted at 100 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid was precipitated. The solid was then treated with EA:PE=1:1 The organic solvent was washed and dried to obtain 112 mg of a gray solid with a yield of 59.3%.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.11 (dd, J=7.7, 1.0 Hz, 1H), 8.00-7.93 (m, 3H), 7.91-7.68 (m, 6H), 7.49 (dd, J = 8.3, 6.9 Hz, 2H), 7.40 (d, J = 7.4 Hz, 1H), 5.27 (s, 2H).

Example 2

Synthesis of 4-((furan-2-methyl)amino)benzonitrile:

Take 4-iodobenzonitrile (1g, 4.36mmol), furfuramine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30mmol), L-proline (100mg , 0.87mmol), dimethyl sulfoxide 10ml, in a 100ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Anhydrous sodium sulfate was dried, and the sample was passed through the column (PE:EA=5:1) to obtain 0.6g of a yellow solid with a yield of 49.5%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.58 (dd, J=1.9, 0.9 Hz, 1H), 7.49-7.41 (m, 2H), 7.12 (t, J=6.0 Hz, 1H), 6.76 -6.68 (m, 2H), 6.39 (dd, J = 3.2, 1.9 Hz, 1H), 6.32 (dd, J = 3.2, 0.9 Hz, 1H), 4.32 (d, J = 5.8 Hz, 2H).

(3aR,6R)-2-(4-cyanophenyl)-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7-carboxy Acid synthesis:

Take 4-((furan-2-methyl)amino)benzonitrile (0.4g, 2.01mmol), 2.01mmol, maleic anhydride (0.2g, 2.01mmol), 20ml of toluene in a 100ml single-necked flask protected by argon, Reflux at 100 degrees Celsius for 12 hours. The reaction solution was filtered, washed with toluene, and dried to obtain a gray solid with a yield of 375 mg and a yield of 63%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.36 (s, 1H), 7.93-7.82 (m, 4H), 6.65 (d, J=5.7 Hz, 1H), 6.51 (dd, J=5.7, 1.7Hz, 1H), 5.06 (d, J = 1.6Hz, 1H), 4.56 (d, J = 11.6Hz, 1H), 4.19 (d, J = 11.6Hz, 1H), 3.15 (d, J = 9.1Hz , 1H), 2.63 (d, J=9.1Hz, 1H).

Synthesis of 2-(4-cyanophenyl)-3-oxoindoline-4-carboxylic acid:

Take (3aR,6R)-2-(4-cyanophenyl)-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7- Carboxylic acid (296 mg, 1 mmol) was dissolved in 10 ml of phosphoric acid and reacted at 100 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid precipitated. The solid was washed with an organic solvent of EA:PE=1:1 and dried. The yield of 163 mg gray solid was 58.6%.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 14.43 (s, 1H), 8.10 (d, J=8.5 Hz, 1H), 7.97 (tdd, J=19.5, 12.7, 7.1 Hz, 5H), 7.88- 7.82 (m, 1H), 5.22 (d, J = 14.1 Hz, 2H).

Example 3

Synthesis of N-(furan-2-methyl)benzo[d][1,3]dioxa-5-amine:

Take 4-bromo-1,2-methylenedioxybenzene (0.876g, 4.36mmol), furfurylamine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30 mmol), L-proline (100 mg, 0.87 mmol), 10 ml of dimethyl sulfoxide, in a 100 ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Dry over anhydrous sodium sulfate and mix the sample through the column (PE:EA=5:1) to obtain 0.36g of yellow solid with a yield of 38%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.56 (dd, J = 1.9, 0.9 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.40-6.31 (m, 2H), 6.27 (dd, J = 3.2, 0.9 Hz, 1H), 6.10-6.02 (m, 2H), 5.82 (s, 2H), 4.16 (d, J = 6.1 Hz, 2H).

(3aR,6R)-2-(benzo[d][1,3]dioxy-5-)-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6 -Synthesis of epoxyisoindole-7-carboxylic acid:

Take N-(furan-2-methyl)benzo[d][1,3]dioxa-5-amine (436mg, 2.01mmol), maleic anhydride (0.2g, 2.01mmol), toluene 20ml in 100ml The argon gas was protected in a single-necked flask and refluxed at 100 degrees Celsius for 12 hours. The reaction solution was filtered, washed with toluene and dried to obtain a gray solid with a yield of 430 mg of 68%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.27 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.00 (dd, J = 8.5, 2.2 Hz, 1H), 6.92 (d , J = 8.5 Hz, 1H), 6.62 (d, J = 5.7 Hz, 1H), 6.48 (dd, J = 5.6, 1.8 Hz, 1H), 6.02 (s, 2H), 5.04 (d, J = 1.7 Hz , 1H), 4.49 (d, J = 11.6 Hz, 1H), 4.00 (d, J = 11.6 Hz, 1H), 3.04 (d, J = 9.1 Hz, 1H), 2.57 (d, J = 9.1 Hz, 1H) ).

2–(Benzo[d][1,3]dioxy-5-)-3-oxoindole-4-carboxylic acid synthesis:

Take (3aR,6R)-2-(benzo[d][1,3]dioxy-5-)-1-oxo-1,2,3,6,7,7a-hexahydro-3a, 6-Epoxyisoindole-7-carboxylic acid (315mg, 1mmol) was dissolved in 10ml of phosphoric acid, and reacted at 100 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid precipitated. The solid was re-used with EA:PE= Washed with 1:1 organic solvent and dried to obtain 137mg gray solid in 46% yield.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 15.36 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.86 (t, J = 7.6Hz, 1H), 7.47 (d, J = 2.2Hz, 1H), 7.25 (dd, J = 8.4, 2.2Hz, 1H), 7.05 (d, J = 8.4Hz, 1H), 6.09 (s, 2H ), 5.15 (s, 2H).

Example 4

Synthesis of N-(furan-2-methyl)-4–(trifluoromethyl)aniline:

Take p-bromotrifluorotoluene (0.98g, 4.36mmol), furfurylamine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30mmol), L-proline ( 100mg, 0.87mmol), dimethyl sulfoxide 10ml, in a 100ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Anhydrous sodium sulfate was dried, and the sample was passed through the column (PE:EA=5:1) to obtain 0.54 g of yellow solid with a yield of 51.5%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.58 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 6.86 (t, J = 6.0 Hz, 1H), 6.75 (d, J = 8.5 Hz, 2H), 6.39 (dd, J = 3.2, 1.8 Hz, 1H), 6.32 (d, J = 3.1 Hz, 1H), 4.31 (d, J = 5.9 Hz, 2H).

(3aR,6R)-1-oxo-2-(4-(trifluoromethyl)phenyl)-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole Synthesis of -7-carboxylic acid:

Take N-(furan-2-methyl)-4–(trifluoromethyl)aniline (484mg, 2.01mmol), maleic anhydride 0.2g (2.01mmol), toluene 20ml in a 100ml single-necked flask protected by argon, 100 Reflux at 12 degrees Celsius for 12 hours. The reaction solution was filtered, washed with toluene, and dried to obtain a gray solid with a yield of 497 mg and a yield of 73%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.32 (s, 1H), 7.91 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 6.66 (d, J = 5.7Hz, 1H), 6.51 (dd, J = 5.7, 1.7Hz, 1H), 5.06 (d, J = 1.6Hz, 1H), 4.59 (d, J = 11.5Hz, 1H), 4.17 (d, J = 11.5 Hz, 1H), 3.14 (d, J=9.1 Hz, 1H), 2.63 (d, J=9.1 Hz, 1H).

Synthesis of 3-oxo-2-(4-(trifluoromethyl)phenyl)isoindoline-4-carboxylic acid:

Take (3aR,6R)-1-oxo-2-(4-(trifluoromethyl)phenyl)-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoind Indole-7-carboxylic acid (339mg, 1mmol) was dissolved in 10ml of concentrated hydrochloric acid and reacted at 80 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid was precipitated. The solid was then treated with EA:PE=1:1 organic The solvent was washed and dried to obtain 138 mg of gray solid with a yield of 43%.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 14.52 (s, 1H), 8.12 (d, J=8.6 Hz, 2H), 8.00-7.84 (m, 5H), 5.23 (s, 2H).

Example 5

Synthesis of 1-iodo-4-(methoxymethoxy)benzene:

4-Iodophenol (1g, 4.54mmol) was dissolved in tetrahydrofuran, sodium hydrogen (60%) (0.26g, 6.81mmol) was added under ice bath, and bromomethyl methyl ether (0.625g, 5mmol) was added dropwise with stirring. After the addition, the temperature was raised to room temperature and the reaction was continued for two hours. After the reaction was completed, a saturated solution of NH ₄ Cl was added to quench the reaction, extracted with DCM, the organic phase was dried over anhydrous Na ₂ SO ₄ , and passed through the column EA: PE = 1:20 to obtain 0.9 g of a light yellow oily substance with a yield of 75%.

¹ H NMR (400 MHz, Chloroform-d) δ 7.61-7.52 (m, 2H), 6.86-6.77 (m, 2H), 5.14 (s, 2H), 3.46 (s, 3H).

Synthesis of N-(furan-2-methyl)-4–(methoxymethoxy)aniline:

Take 1-iodo-4-(methoxymethoxy)benzene (1.15g, 4.36mmol), furfurylamine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30 mmol), L-proline (100 mg, 0.87 mmol), 10 ml of dimethyl sulfoxide, in a 100 ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Anhydrous sodium sulfate was dried, and the sample was passed through the column (PE:EA=5:1) to obtain 0.762 g of a yellow solid with a yield of 75%.

¹ H-NMR (400MHz, Chloroform-d) δ 7.36 (dd, J = 1.9, 0.9 Hz, 1H), 6.96-6.87 (m, 2H), 6.68-6.59 (m, 2H), 6.32 (dd, J = 3.2, 1.9 Hz, 1H), 6.26-6.19 (m, 1H), 5.08 (s, 2H), 4.27 (d, J = 0.9 Hz, 2H), 3.48 (s, 3H).

(3aR,6R)-1-oxo-2-(4-(methoxymethoxy)phenyl)-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoind Synthesis of indole-7-carboxylic acid:

Take N-(furan-2-methyl)-4–(methoxymethoxy)aniline (468mg, 2.01mmol), maleic anhydride 0.2g (2.01mmol), 20ml of toluene in a 100ml single-necked flask for argon protection, Reflux at 100 degrees Celsius for 12 hours. The reaction solution was filtered, washed with toluene, and dried to obtain a gray solid with a yield of 545 mg and a yield of 82%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.24 (s, 1H), 7.61-7.52 (m, 2H), 7.08-6.99 (m, 2H), 6.64 (d, J=5.7Hz, 1H) , 6.48 (dd, J = 5.7, 1.7 Hz, 1H), 5.17 (s, 2H), 5.04 (d, J = 1.7 Hz, 1H), 4.52 (d, J = 11.6 Hz, 1H), 4.02 (d, J = 11.5 Hz, 1H), 3.37 (s, 3H), 3.04 (d, J = 9.1 Hz, 1H), 2.57 (d, J = 9.1 Hz, 1H).

Synthesis of 2-(4-hydroxyphenyl)-3-oxoindoline-4-carboxylic acid:

Take (3aR,6R)-1-oxo-2-(4-((methoxymethoxy)phenyl)-1,2,3,6,7,7a-hexahydro-3a,6-epoxyiso Indole-7-carboxylic acid (331 mg, 1 mmol) was dissolved in 10 ml of concentrated hydrochloric acid, and reacted at 80 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid precipitated out. The solid was re-used with EA: PE=1:1 After washing with organic solvent and drying, 121 mg of gray solid was obtained with a yield of 45%.

Example 6

Synthesis of 4-((furan-2-ylmethyl)amino)benzaldehyde:

Take p-bromobenzaldehyde (0.807g, 4.36mmol), furfuramine (0.64g, 6.53mmol), cuprous iodide (83mg, 0.436mmol), potassium carbonate (180mg, 1.30mmol), L-proline (100mg , 0.87mmol), dimethyl sulfoxide 10ml, in a 100ml single-necked flask, protected by argon. Reflux at 100 degrees Celsius for 12 hours, cool to room temperature after the reaction is completed, dilute with 60 ml of ethyl acetate, and wash with saturated aqueous sodium chloride solution (45 ml×2). Anhydrous sodium sulfate was dried, and the sample was passed through the column (PE:EA=5:1) to obtain 0.33 g of a yellow solid with a yield of 38%.

¹ H-NMR (400MHz, Chloroform-d) δ 7.36 (dd, J = 1.9, 0.8 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 6.70-6.62 (m, 2H), 6.32 ( dd, J = 3.2, 1.9 Hz, 1H), 6.23 (d, J = 3.2 Hz, 1H), 4.54 (s, 2H), 4.32 (s, 2H).

(3aR,6R)-2-(4-formylphenyl)-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7-carboxy Acid synthesis:

Take 4-((furan-2-ylmethyl)amino)benzaldehyde (404mg, 2.01mmol), maleic anhydride (0.2g, 2.01mmol), 20ml of toluene in a 100ml single-necked flask protected by argon, reflux at 100 degrees Celsius 12 In hour, the reaction solution was filtered, washed with toluene and dried to obtain a gray solid with a yield of 216 mg and a yield of 36%.

Synthesis of 2-(4-formylphenyl)-3-oxoindoline-4-carboxylic acid:

Take (3aR,6R)-2-(4-formylphenyl)-1-oxo-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7- Carboxylic acid (299 mg, 1 mmol) was dissolved in 10 ml of concentrated hydrochloric acid, and reacted at 80 degrees Celsius for 1.5 hours. After the reaction was completed, the reaction solution was poured into water, and a solid precipitated. The solid was washed with an organic solvent of EA:PE=1:1 and dried. The yield of 135 mg gray solid was 46%.

Synthesis of 2-(4-(hydroxymethyl)phenyl)-3-oxoindole-4-carboxylic acid:

Take 2-(4-formylphenyl)-3-oxoindoline-4-carboxylic acid (100mg, 0.36mmol), dissolve with tetrahydrofuran, add NaHB ₄ (68.4mg, 1.8mmol) under ice bath, add After completion, the temperature was raised to room temperature, the reaction was carried out overnight, then it was diluted with ethyl acetate, washed with saturated brine, and the organic phase was filtered to obtain a solid yield of 64 mg of 63%.

Example 7

Synthesis of 2-(4-hydroxyphenyl)isoindoline-1,3-dione:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, p-aminophenol (1.62g, 14.86mmol), put it in a 250ml single-necked bottle and protect it at 120°C under reflux for 3 hours. After the reaction, cool to room temperature and add 1000ml of water was diluted with solid precipitation. The solid was filtered and washed with water to obtain 1.36g of light yellow solid. The yield was 46.4%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.73 (s, 1H), 7.98-7.83 (m, 4H), 7.25-7.17 (m, 2H), 6.91-6.84 (m, 2H).

Synthesis of 2-(4-hydroxyphenyl)isoindoline-1,3-dithione:

Take 2-(4-hydroxyphenyl)isoindoline-1,3-dione (1g 3.69mmol), Lawson's reagent (2.98g, 7.38mmol), 50ml of toluene, and protect with argon in a 250ml single-necked bottle Reflux at 110 degrees Celsius for 24 hours. The reaction solution was cooled to room temperature and diluted with 300 ml of ethyl acetate. It was washed with saturated aqueous sodium chloride solution (300 ml×2), dried over anhydrous sodium sulfate, and passed through the column with sample preparation. EA: PE=1:4. The yield of 160 mg brown solid was 14.1%.

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 9.98-9.72 (m, 1H), 7.91 (s, 4H), 7.19-7.12 (m, 2H), 6.91-6.84 (m, 2H).

Example 8

Synthesis of 5-(1,3-dioxoisoindolin-2-yl)-2-fluorobenzonitrile:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, 5-amino-2-fluorobenzonitrile (2.03g, 14.86mmol), put it in a 250ml single-necked bottle and protect it by refluxing at 120°C for 3 hours, the reaction is over After cooling to room temperature, solids were diluted by adding 1000 ml of water. The solids were filtered and washed with water to obtain 1.96 g of light yellow solids. The yield was 60%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.06 (dd, J = 5.8, 2.6 Hz, 1H), 8.00 (dt, J = 7.3, 3.7 Hz, 2H), 7.92 (ddd, J = 7.5, 5.2, 2.8 Hz, 3H), 7.74 (t, J=9.1 Hz, 1H).

Synthesis of 5-(1,3-dithioisoindolin-2-yl)-2-fluorobenzonitrile:

Take 5-(1,3-dioxoisoindolin-2-yl)-2-fluorobenzonitrile (0.98g, 3.69mmol), Lawson reagent (2.98g, 7.38mmol), toluene 50ml, in 250ml Argon gas in a single-necked bottle was refluxed at 110 degrees Celsius for 24 hours. The reaction solution was cooled to room temperature and diluted with 300 ml of ethyl acetate, washed with a saturated sodium chloride aqueous solution (300 ml×2), dried over anhydrous sodium sulfate, and mixed with the sample, EA: PE=1:4, and finally a yield of 180 mg of brown solid was 16.3%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.21 (dd, J=5.8, 2.5 Hz, 1H), 8.02-7.91 (m, 5H), 7.77 (t, J=9.0 Hz, 1H).

Example 9

Synthesis of 6-(1,3-dioxoisoindolin-2-yl)cyanopyridine:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, 6-aminonicotinic acid nitrile (1.77g, 14.86mmol), put it in a 250ml single-necked bottle under argon protection at 120°C and reflux for 3 hours. After the reaction, cool to room temperature. Then, 1000 ml of water was added to dilute the solid, and the solid was filtered and washed with water to obtain 2.43 g of light yellow solid. The yield was 72%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.12 (d, J = 2.3 Hz, 1H), 8.56 (dd, J = 8.3, 2.3 Hz, 1H), 8.02 (dt, J = 7.3, 3.7 Hz , 2H), 7.95 (dd, J=5.5, 3.1 Hz, 2H), 7.79 (d, J=8.4 Hz, 1H).

Synthesis of 6-(1,3-dithioisoindolin-2-yl)cyanopyridine:

Take 5-(1,3-dioxoisoindolin-2-yl)-2-fluorobenzonitrile (0.92g, 3.69mmol), Lawson's reagent (2.98g, 7.38mmol), toluene 50ml, in 250ml Argon gas in a single-necked bottle was refluxed at 110 degrees Celsius for 24 hours. The reaction solution was cooled to room temperature and diluted with 300 ml of ethyl acetate, washed with a saturated sodium chloride aqueous solution (300 ml×2), dried over anhydrous sodium sulfate, and mixed with the sample, EA: PE=1:4, and finally the yield of 208 mg yellow solid was 20%.

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 9.25-9.15 (m, 1H), 8.65 (dd, J=8.2, 2.1 Hz, 1H), 7.98 (s, 5H).

Example 10

Synthesis of 2-(4-(trifluoromethyl)phenyl)isoindole-1,3-dione:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, 4-trifluoromethylaniline (2.39g, 14.86mmol), place in a 250ml single-necked bottle under argon protection at 120 °C and reflux for 3 hours, the reaction is cooled to At room temperature, after adding 1L of water to dilute the solid precipitate, the solid was filtered and washed with water to obtain 3.59g yellow solid. The yield was 83%.

Synthesis of 2-(4-(trifluoromethyl)phenyl)isoindolin-1,3-dithione:

Take 2-(4-(trifluoromethyl)phenyl)isoindole-1,3-dione (1.08g, 3.69mmol), Lawson's reagent (2.98g, 7.38mmol), toluene 50ml, 250ml single mouth Argon in the bottle was protected by refluxing at 110°C for 24 hours. The reaction solution was cooled to room temperature and diluted with 300ml of ethyl acetate. The solution was washed with saturated aqueous sodium chloride solution (300ml×2), dried over anhydrous sodium sulfate, and mixed with the sample through the column. EA: PE = 1:4, and finally the yield of 358 mg of yellow solid was 30%.

Example 11

Synthesis of 4-(1,3-dioxoisoindolin-2-yl)benzonitrile:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, p-aminobenzonitrile (1.76g, 14.86mmol), put it in a 250ml single-necked bottle under argon protection at 120°C and reflux for 3 hours. After the reaction, cool to room temperature. After adding 1L of water to dilute a solid precipitate, the solid was filtered, washed with water and dried to give 2.31g white solid. The yield was 69%.

Synthesis of 4-(1,3-dithioisoindolin-2-yl)benzonitrile:

Take 2-(4-(trifluoromethyl)phenyl)isoindole-1,3-dione (0.92g, 3.69mmol), Lawson's reagent (2.98g 7.38mmol), 50ml of toluene in a 250ml single-necked bottle Reflux under medium argon at 110°C for 24 hours. The reaction solution was cooled to room temperature and diluted with 300ml of ethyl acetate. It was washed with saturated aqueous sodium chloride solution (300ml×2), dried over anhydrous sodium sulfate, and passed through the column with mixed samples. EA:PE= 1:4, the final yield of 320mg yellow solid is 30%.

Example 12

Synthesis of 4-(1,3-dioxoisoindolin-2-yl)benzoic acid:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, p-aminobenzoic acid (2.04g, 14.86mmol), put it in a 250ml single-necked bottle, protect it by refluxing at 120°C for 3 hours under argon, and cool to room temperature at the end of the reaction. After adding 1L of water to dilute, solid precipitated out, the solid was filtered, washed and dried to obtain 3.21g, and the yield of white solid was 89%.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.07 (d, J = 8.3 Hz, 2H), 7.99-7.85 (m, 4H), 7.59 (d, J = 8.3 Hz, 2H).

Synthesis of 4-(1,3-dithioisoindolin-2-yl)benzoic acid:

Take 4-(1,3-dioxoisoindolin-2-yl)benzoic acid (0.99g, 3.69mmol), Lawson's reagent (2.98g, 7.38mmol), 50ml of toluene in a 250ml single-necked bottle, Argon gas was refluxed at 110°C for 24 hours. The reaction solution was cooled to room temperature and diluted with 300 ml of ethyl acetate, washed with saturated sodium chloride aqueous solution (300 ml × 2), dried over anhydrous sodium sulfate, and mixed with the sample through the column. EA:PE=1 : 4, the final yield of 397mg yellow solid is 36%.

Example 13

Synthesis of 2-(5-methylpyridin-3-yl)isoindoline-1,3-dione:

Take phthalic anhydride (2g, 13.5mmol), acetic acid 100ml, 3-amino-5-methylpyridine (1.60g, 14.86mmol), put it in a 250ml single-necked bottle under argon protection at 120°C, and reflux for 3 hours to react After cooling to room temperature, add 1L of water to dilute, solid precipitates out, filter to obtain solid, wash and dry to obtain 1.42g white solid. The yield was 44%.

Synthesis of 2-(5-methylpyridin-3-yl)isoindoline-1,3-dithione:

Take 2-(5-methylpyridin-3-yl)isoindoline-1,3-dione (0.88g, 3.69mmol), Lawson's reagent (2.98g, 7.38mmol), toluene 50ml, 250ml single mouth Argon in the bottle was protected by refluxing at 110°C for 24 hours. The reaction solution was cooled to room temperature and diluted with 300ml of ethyl acetate. The solution was washed with saturated aqueous sodium chloride solution (300ml×2), dried over anhydrous sodium sulfate, and mixed with the sample through the column. EA: PE = 1:4, and finally the yield of 269 mg of yellow solid was 27%.

Example 14

Specific steps

1. Dissolve 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 8.77g of m-cyanoaniline and stir at room temperature for 30min. The product is completely precipitated and filtered directly. After washing the filter cake with dichloromethane, 9.56 g of product was obtained. The yield is 51%.

2. Take 2g of raw material, 2.3g of phosphorus pentasulfide into a single-necked bottle, add 5ml of triethylamine, 50ml of toluene, warm to reflux and stir the reaction, after 30min thin layer monitoring, (PE:EA=3:1), The raw material Rf value is about 0.1, and the product Rf value is about 0.3. Column chromatography (PE:EA=5:1) gave 0.24 g of product. The yield was 11%.

HRMS (ESI ⁺ ): C ₁₅ H ₈ N ₂ S ₂ [MH] ⁺ 279.3596; ¹ H NMR {300 MHz, DMSO (d ₆ ), d (ppm)}: 7.74-8.03 (m, 8H, ArH).

Example 15

Specific steps

1. Dissolve 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 11.97g of 3-trifluoromethylaniline and stir at room temperature for 30min, the product is completely precipitated. Direct suction filtration. After washing the filter cake with methylene chloride, 10.11 g of product was obtained. The yield was 46%.

2. Take 5g of raw material, 3.6g of phosphorus pentasulfide into a single-necked bottle, add 10ml of triethylamine, 80ml of toluene, warm to reflux and stir the reaction, after 30min thin layer monitoring, (PE:EA=3:1), The raw material Rf value is about 0.2, and the product Rf value is about 0.5. Column chromatography (PE:EA=5:1) gave 0.22 g of product. The yield is 5%.

HRMS (ESI ⁺ ): C ₁₅ H ₈ F ₃ NS ₂ [MH] ⁺ 322.3481; ¹ H NMR {300 MHz, DMSO (D ₆ ), d (ppm)}: 7.91-8.00 (m, 8H, ArH).

Example 16

Specific steps

1. Dissolve 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 8.25g of 4-fluoroaniline and stir at room temperature for 30min. The product is completely precipitated and filtered directly. After washing the filter cake with methylene chloride, 7.25 g of product was obtained. The yield is 40%.

2. Take 5 grams of raw materials, 3.16 grams of phosphorus pentasulfide into a single-necked bottle, add 10ml of triethylamine, 80ml of toluene, warm to reflux and stir the reaction, thin layer monitoring after 30min, (PE:EA=3:1), The raw material Rf value is about 0.2, and the product Rf value is about 0.6. Column chromatography (PE:EA=5:1) gave 0.25 g of product. The yield is 5%.

HRMS (ESI+): C14H8FNS2 [MH] + 272.3406; ¹ H NMR {300 MHz, DMSO (D6), d (ppm)}: 7.35-7.98 (m, 8H, ArH).

Example 17

Specific steps

1. Dissolve 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 8.25g of 4-fluoroaniline and stir at room temperature for 30min. The product is completely precipitated and filtered directly. After washing the filter cake with methylene chloride, 8.33 g of product was obtained. The yield was 46%.

2. Take 5 grams of raw materials, 3.16 grams of phosphorus pentasulfide into a single-necked bottle, add 10ml of triethylamine, 80ml of toluene, warm to reflux and stir the reaction, thin layer monitoring after 30min, (PE:EA=3:1), The raw material Rf value is about 0.2, and the product Rf value is about 0.5. Column chromatography (PE:EA=5:1) gave 0.30 g of product. The yield is 6%.

HRMS (ESI ⁺ ): C ₁₄ H ₈ FNS ₂ [MH] ⁺ 272.34062; ¹ H NMR {300 MHz, DMSO (D ₆ ), d (ppm)}: 6.81-8.08 (m, 8H, ArH).

Example 18

Specific steps

1. Dissolve 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 11.5g of p-aminophthalic acid and stir at room temperature for 30min. The product is completely precipitated and directly After suction filtration, the filter cake was washed with dichloromethane to obtain 7.35 g of product, yield 34%

2. Take 5 grams of raw materials, 3.48 grams of phosphorus pentasulfide into a single-necked bottle, add 10ml of triethylamine, 80ml of toluene, warm to reflux and stir the reaction, thin layer monitoring after 30min, (PE:EA=3:1), The raw material Rf value is about 0.1, and the product Rf value is about 0.5. Column chromatography (PE:EA=5:1) gave 0.25 g of product in 5% yield.

HRMS(ESI+): C ₁₅ H ₈ ClNO ₂ S ₂ [MH] ⁺ 332.8047;

¹ H NMR {300 MHz, DMSO (D ₆ ), d (ppm)}: 7.54-8.17 (m, 7H, ArH). 8.17 (s, 1H, -COOH).

Example 19

Specific steps

1. Take 10 grams of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 12.57 grams of p-cyano-trifluoromethylaniline and stir at room temperature for 30min. The product is complete Precipitated and filtered directly with suction. After washing the filter cake with methylene chloride, 12.22 g of product was obtained with a yield of 54%

2. Take 3g of raw material, 3.33g of phosphorus pentasulfide into a single-necked bottle, add 5ml of triethylamine, 50ml of toluene, warm to reflux and stir the reaction, thin layer monitoring after 30min, (PE:EA=3:1), The raw material Rf value is about 0.2, and the product Rf value is about 0.5. Column chromatography (PE:EA=5:1) gave 0.2 g of product in 6% yield.

HRMS(ESI ⁺ ): C ₁₆ H ₇ F ₃ N ₂ S ₂ [MH] ⁺ 347.3576; ¹ H NMR{300MHz, DMSO(D ₆ ), d(ppm)}: 7.95-8.09(m,7H,ArH) .

Example 20

Specific steps

1. Take 10g of raw material phthalic anhydride in 100ml of acetone and stir at room temperature. After the phthalic anhydride is completely dissolved, add 11.2g of p-amino-o-chlorobenzoic acid and stir at room temperature for 30min. The product is completely precipitated and directly After suction filtration, the filter cake was washed with dichloromethane to obtain 9.66 g of product, yield 46%

2. Take 3 grams of raw materials, 2.23 grams of phosphorus pentasulfide into a single-necked bottle, add 5ml of triethylamine, 50ml of toluene, warm to reflux and stir the reaction, thin layer monitoring after 30min, (PE:EA=3:1), The raw material Rf value is about 0.1, and the product Rf value is about 0.6. Column chromatography (PE:EA=5:1) gave 0.36 g of product in 11% yield.

HRMS (ESI ⁺ ): C ₁₆ H ₁₁ NO ₂ S ₂ [MH] ⁺ 312.3862; ¹ H NMR {300MHz, DMSO (D ₆ ), d (ppm)}: 4.68 (s, 2H, -CH ₂ ), 7.26 -8.13 (m, 8H, ArH). 11.02 (s, 1H, -COOH).

Biological Example 1: In vitro cartilage induction and differentiation experiment of human-derived synovial stem cells

1. Experimental materials

1.1 Experimental sample

Human-derived synovial stem cells: The experimental samples were provided by Nanjing Gulou Hospital.

1.2 Experimental reagents

DMEM/F-12 medium, gibco

FBS, Sciencell

Penicillin-Streptomycin Liquid, gibco

Collagenase Type I, gibco

0.25% trypsin EDTA, gibco

Reverse transcription kit (RR047A), Takara Corporation

QPCR kit (Q411-02), Nanjing Nuoweizan Biological Technology Co., Ltd.

1.3 Experimental instruments

PCR Amplifier, Biometra

Real-time fluorescence quantitative PCR instrument (LC480II), ROCHE company

1.4 Preparation of main working fluid

1.4.1 Preparation of PBS solution (1L)

The balance weighs NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ · 12H ₂ O 3.58g, KH ₂ PO ₄ 0.24g, ddH ₂ O to 800ml, add appropriate amount of NaOH to adjust the pH to 7.2 and then ddH ₂ O to 1L, used after autoclaving.

1.4.2 Preparation of 0.4% type I collagen degrading enzyme

Weigh 0.08g type I collagen degrading enzyme, add 20ml DMEM/F-12 medium, mix well, filter sterilize with 0.22μm filter.

1.4.3 Preparation of drugs

n(mmol)=(Wt(mg)/Mol·Wt)×HPLC(%), the drug is dissolved in DMSO.

2. Experimental method

2.1 Human synovial mesenchymal stem cell isolation and culture

Day 1: Take samples. Remove the synovial tissue on the ultra-clean workbench, rinse with PBS, and remove the fat part. The synovial tissue was shredded, PBS was aspirated, DMEM/F-12 medium containing type I collagen degrading enzyme was added, 1% double antibody was added, and digested overnight at 37°C and 5% CO ₂ cell incubator.

The next day: After filtering the filter, the liquid was centrifuged at 1500 rpm, 5 min, and 20°C. After centrifugation, aspirate the supernatant, add 2ml of 10% FBS DMEM/F-12 medium to pipette the cells evenly. Add 8ml of DMEM/F-12 medium containing 10% FBS to a 10cm Petri dish, then add 2ml of cell suspension, shake gently, add 1% penicillin streptomycin (double antibody), 37℃, 5% CO ₂ Culture in a cell incubator.

After the cells adhere to the wall, change the fluid every 2-3 days. When the cell fusion degree is about 90%, trypsin EDTA digestion and centrifugation and subculture seed plate.

2.2 QPCR detection and result analysis

Human-derived synovial mesenchymal stem cells (SMSCs) were seeded into each well of a six-well plate, 5% CO ₂ , and cultured at 37°C until the cell density per well reached 95%. Compounds (in DMSO solution) were added to the cells at a final concentration of 10 μM, and the cells were cultured at 37° C. for 21 days in 5% CO ₂ . Trizol method extracts RNA and reverse transcribes it into cDNA. The relative expression levels of Aggrecan, Collagen II and Sox9 in cells were detected. The relative expression level was detected using the LightCycler 480II system (Roche). The carrier (DMSO) was used as a control to determine the basal level of chondrocyte differentiation. The target genes to be tested in this experiment were Aggrecan, Collagen II, sox9, and the internal reference gene was GAPDH. Among them, the three genes Aggrecan, Collagen II, and sox9 are chondrogenic genes. QPCR analysis showed that if the expression levels of Aggrecan, Collagen II, and sox9 genes increased, it proved that the mesangial mesenchymal stem cells induced by drugs had a tendency to differentiate into chondrocytes. Select proteoglycan (Aggrecan), type Ⅱ collagen (Collagen II), Sox9 expression higher than the vehicle control compound for the next induction experiment.

EXCEL statistical results. Calculate △Ct=Ct (target gene)-Ct (internal reference gene) to get ΔCt for each target gene in each group. Calculate △△Ct=△Ct (administration group)-△Ct (control group), and take the opposite result to get -△△Ct. Finally, perform a power of 2 on ^-△△Ct , that is, 2 ^-△△Ct , to get the relative change of the target gene expression. The specific results are shown in the table:

Table: Different compounds induce human-derived synovial stem cells to differentiate into cartilage in vitro

Remarks: A～D represent different activity levels, see the table below for details:

活性级别Activity level	mAGGRECANmAGGRECAN	mCOL2A1mCOL2A1	SOX9SOX9
AA	0-0.50-0.5	0-1.00-1.0	0-1.00-1.0
BB	0.5-1.00.5-1.0	1.0-5.01.0-5.0	1.0-10.01.0-10.0

CC	1.0-5.01.0-5.0	5.0-20.05.0-20.0	10.0-100.010.0-100.0
DD	>5.0>5.0	>20.0>20.0	>100>100

The specific structure of Kartogenin is:

Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these embodiments without departing from the principle and essence of the present invention. modify. Therefore, the protection scope of the present invention is defined by the appended claims.

Claims

A nitrogen-containing heterocyclic compound represented by formula I, its pharmaceutically acceptable salts, its hydrates, its solvates, its polymorphs, its metabolites, its stereoisomers, its tautomerism Body or its prodrug:

Among them, X is O or S;

Y is methylene or

Z is a single bond or C 1 ～C 4 alkylene;

Is unsubstituted or substituted with one or more R a is C 6 ~ C 10 aryl group, unsubstituted or substituted with one or more R B is 5 to 6-membered heteroaryl, unsubstituted or substituted with one or more R c- substituted C 4 -C 8 cycloalkenyl, or, unsubstituted or substituted with one or more Rd , C 4 -C 8 heterocycloalkenyl; in the 5-6 membered heteroaryl, the hetero atom One or more selected from N, O, and S, and the number of hetero atoms is 1 to 2; in the C 4 to C 8 heterocyclic alkenyl group, the hetero atom is selected from one of N, O, and S One or more kinds, the number of heteroatoms is 1 to 2; when there are multiple substituents Ra , Rb , Rc or Rd , the substituents are the same or different;

R a , R b , R c and R d are independently halogen, -OH, -CN, -COOH, C 1 -C 6 alkyl, C 1 -C 6 alkyl-O-, C 1 -C 6 alkane Group -C(=O)-, C 1 -C 6 alkyl-CO(=O)-, C 1 -C 6 alkyl-OC(=O)-, C 1 -C 6 alkyl-C(= O)NH-or

Is an unsubstituted or one or more R 1 substituted C 6 ～C 10 aryl, or, unsubstituted or one or more R 2 substituted 5-6 membered heteroaryl; the 5-6 membered In the heteroaryl group, the hetero atoms are selected from one or more of N, O and S, and the number of hetero atoms is 1 to 2; when there are multiple substituents R 1 or R 2 , the substituents are the same Or different

R 1 and R 2 are independently halogen, -OH, -CN, -NH 2 , -COOH,
Unsubstituted or substituted with one or more of R 1-7 is C 1 ~ C 6 alkyl, unsubstituted or substituted with one or more of R 1-8 is C 3 ~ C 6 cycloalkyl, unsubstituted or substituted by a R 1-9 is substituted with one or more C 6 ~ C 10 aryl group, unsubstituted or substituted with one or more R 1-10 substituted 5- to 6-membered heteroaryl, unsubstituted or substituted with one or plural R 1 s -11 substituted C 1 ～C 6 alkyl-O-, unsubstituted or substituted by one or more R 1-12 C 1 ～C 6 alkyl-C(═O)-, unsubstituted or substituted by one or Multiple R 1-13 substituted C 1 ～C 6 alkyl-C(═O)O-, unsubstituted or substituted by one or more R 1-14 C 1 ～C 6 alkyl-OC(═O )-, or, unsubstituted or substituted by one or more R 1-15 C 1 ～C 6 alkyl-C(=O)NH-; in the 5- to 6-membered heteroaryl, the hetero atom One or more selected from N, O and S, the number of hetero atoms is 1 to 3; when the substituents R 1-7 , R 1-8 , R 1-9 , R 1-10 , R 1- 11. When there are a plurality of R 1-12 , R 1-13 , R 1-14 or R 1-15 , the substituents are the same or different;

Alternatively, when there are a plurality of substituents R 1 or R 2 , two adjacent R 1 and two C directly connected to the C 6 to C 10 aryl group, or two adjacent R 2 and 5 to Two Cs directly connected to a 6-membered heteroaryl group together form an unsubstituted or substituted C 1-4 C 6 cycloalkyl group by one or more R 1-5 , or, unsubstituted or substituted by one or more R 1-6 substituted C 3 -C 5 heterocycloalkyl; in the C 3 -C 5 heterocycloalkyl, the hetero atoms are selected from one or more of N, O and S, and the number of hetero atoms is 1 to 3; when there are multiple substituents R 1-5 or R 1-6 , the substituents are the same or different;

R 1-1 and R 1-2 , R 1-3 and R 1-4 are independently H or C 1 -C 6 alkyl;

R 1-5 , R 1-6 , R 1-7 , R 1-8 , R 1-9 , R 1-10 , R 1-11 , R 1-12 , R 1-13 , R 1-14 and R 1-15 is independently halogen, -OH, -CN, -NH 2 , -COOH, -CONH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkyl -O-, C 1 -C 6 Alkyl-C(=O)-, C 1 -C 6 alkyl-CO(=O)-, or C 1 -C 6 alkyl-OC(=O)-.
The nitrogen-containing heterocyclic compound represented by formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, Its tautomer or its prodrug: the Z is a single bond, methylene, ethylene or isopropylene;

And/or
Is unsubstituted or substituted with one or more R a phenyl groups, unsubstituted or substituted with one or more R b 5-6 membered heteroaryl groups, unsubstituted or substituted with one or more R c C 4 ~C 8 cycloalkenyl, or, unsubstituted or bridged heterobicycloalkenyl substituted with one or more Rd ; the bridged heterobicycloalkenyl is preferably 7-oxadienedi Ring [2.2.1] heptenyl, more preferably

And / or, said R a, R b, R c or R d is a fluorine, chlorine, bromine, iodine, -OH, -CN, -COOH, C 1 ~ C 6 alkyl group, C 1 ~ C 6 alkyl Group -O-, C 1 -C 6 alkyl-C(=O)-, C 1 -C 6 alkyl-CO(=O)-, C 1 -C 6 alkyl-OC(=O)-, Or, C 1 ～C 6 alkyl-C(＝O)NH- or
R 1-1 or R 1-2 is H or C 1 -C 6 alkyl; wherein, the C 1 -C 6 alkyl is independently C 1 -C 4 alkyl;

And/or
In the above, the 5- to 6-membered heteroaryl group is linked to the
Connected

And/or
Is unsubstituted or substituted with one or more R 1 phenyl, or, unsubstituted or substituted with one or more R 2 6-membered heteroaryl, in the 6-membered heteroaryl, the heteroatom is selected from N, the number of heteroatoms is 1 to 2; the 6-membered heteroaryl group is preferably unsubstituted or substituted by one or more R 2 pyridyl;

And/or R 1 or R 2 is fluorine, chlorine, bromine, iodine, -OH, -CN, -NH 2 , -COOH,
Unsubstituted or substituted with one or more of R 1-7 is C 1 ~ C 6 alkyl, unsubstituted or substituted with one or more of R 1-8 is C 3 ~ C 6 cycloalkyl, unsubstituted or substituted by a Or more than one R 1-9 substituted phenyl, unsubstituted or substituted with one or more R 1-10 5-6 membered heteroaryl, unsubstituted or substituted with one or more R 1-11 C 1 to C 6 alkyl-O-, unsubstituted or substituted by one or more R 1-12 C 1 to C 6 alkyl-C(=O)-, unsubstituted or substituted by one or more R 1- 13- substituted C 1 -C 6 alkyl-C(=O)O-, unsubstituted or substituted by one or more R 1-14 C 1 -C 6 alkyl-OC(=O)-, or, C 1 -C 6 alkyl-C(=O)NH- which is unsubstituted or substituted by one or more R 1-15 ; wherein, the C 1 -C 6 alkyl is independently C 1 -C 4 Alkyl, preferably independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl;

Alternatively, two adjacent R 1 and two C directly connected to the C 6 to C 10 aryl group, or two adjacent two R 2 and two C directly connected to the 5 to 6 membered heteroaryl group , Together forming 1,3-dioxolane that is unsubstituted or substituted by one or more R 1-6 ;

And/or, said R 1-3 or R 1-4 is H or C 1 ˜C 4 alkyl;

And/or, said R 1-5 , R 1-6 , R 1-7 , R 1-8 , R 1-9 , R 1-10 , R 1-11 , R 1-12 , R 1- 13 , R 1-14 or R 1-15 are fluorine, chlorine, bromine, iodine, -OH, -CN, -NH 2 , -COOH, -CONH 2 , C 1 ～C 6 alkyl, C 1 ～C 6 Alkyl-O-, C 1 ～C 6 alkyl-C(═O)-, C 1 ～C 6 alkyl-CO(═O)-, or, C 1 ～C 6 alkyl-OC(═O )-, wherein the C 1 -C 6 alkyl group is independently a C 1 -C 4 alkyl group.
The nitrogen-containing heterocyclic compound represented by formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, Its tautomer or its prodrug: the Z is a single bond or methylene;

And/or, the number of said Ra , Rb , Rc and Rd is independently 1 or 2;

And/or, said Ra , Rb , Rc and Rd are independently located
Adjacent, interposition or counterposition;

And / or, said R a, R b, R c or R d is a fluorine, chlorine, bromine, iodine, -OH, -CN, -COOH, C 1 ~ C 6 alkyl group, C 1 ~ C 6 alkyl Group -O-, C 1 -C 6 alkyl-C(=O)-, C 1 -C 6 alkyl-CO(=O)-, C 1 -C 6 alkyl-OC(=O)-, Or, C 1 ～C 6 alkyl-C(＝O)NH- or
R 1-1 or R 1-2 is H or C 1 -C 6 alkyl; wherein, the C 1 -C 6 alkyl is independently methyl, ethyl, n-propyl, isopropyl, N-butyl, isobutyl, sec-butyl or tert-butyl;

And/or, the number of R 1 and R 2 is independently 1 , 2 , 3 or 4;

And/or R 1 or R 2 are independently located
Adjacent, interposition or counterposition;

And/or, said R 1-3 or R 1-4 are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl ;

And/or, said R 1-5 , R 1-6 , R 1-7 , R 1-8 , R 1-9 , R 1-10 , R 1-11 , R 1-12 , R 1- 13 , R 1-14 or R 1-15 are fluorine, chlorine, bromine, iodine, -OH, -CN, -NH 2 , -COOH, -CONH 2 , C 1 ～C 6 alkyl, C 1 ～C 6 Alkyl-O-, C 1 ～C 6 alkyl-C(═O)-, C 1 ～C 6 alkyl-CO(═O)-, or, C 1 ～C 6 alkyl-OC(═O )-, wherein the C 1 -C 6 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
The nitrogen-containing heterocyclic compound represented by the formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorphic form, its metabolite, its stereoisomer according to claim 3, Its tautomer or its prodrug: said Ra , Rb , Rc or Rd is -COOH, or

The R 1 or R 2 is fluorine, chlorine, -OH, -CN, -COOH, -CONH 2 , trifluoromethyl, hydroxymethyl, phenyl, or
Alternatively, two adjacent R 1 and two C directly connected to the C 6 to C 10 aryl group, or two adjacent two R 2 and two C directly connected to the 5 to 6 membered heteroaryl group , Formed together
The nitrogen-containing heterocyclic compound represented by the formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer according to claim 4; Its tautomer or its prodrug: when X is O, Y is methylene and Z is a single bond; or, when X is S, Y is
Z is a single bond or C 1 ～C 4 alkylene;

And/or
for

And/or
for
The nitrogen-containing heterocyclic compound represented by formula I, its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, Its tautomer or its prodrug: wherein the nitrogen-containing heterocyclic compound represented by Formula I is selected from any of the following compounds:
A method for preparing a nitrogen-containing heterocyclic compound as shown in formula I according to any one of claims 1 to 6, characterized in that it is scheme 1, scheme 2 and scheme 3; wherein scheme 1 includes the following Step: In an organic solvent, the compound represented by Formula II and the compound represented by Formula III are subjected to a cyclization reaction as shown below to obtain the nitrogen-containing heterocyclic compound represented by Formula I;

among them,
for
X is O; Y is methylene; Z and
Is defined in any one of claims 1 to 6;

Scheme two includes the following steps: In the phosphoric acid solution, the
for
The nitrogen-containing heterocyclic compound shown in Formula I undergoes an aromatization reaction to obtain
for
The nitrogen-containing heterocyclic compound shown in Formula I is sufficient; wherein, X is O; Y is methylene; Z and
Is defined in any one of claims 1 to 6;

Scheme three includes the following steps: in an organic solvent, the compound represented by Formula I'and the sulfurization reagent are subjected to a sulfurization reaction as shown below to obtain the nitrogen-containing heterocyclic compound represented by Formula I;

Among them, X'and Y'are O, Y is
X is S, Z,
Is defined in any one of claims 1 to 6.
The preparation method according to claim 7, wherein in the first solution, the organic solvent is an aromatic hydrocarbon solvent;

And/or, in scheme 1, the mass volume ratio of the compound represented by formula II to the organic solvent is 0.01 g/mL to 0.2 g/mL;

And/or, in scheme 1, the molar ratio of the compound represented by formula II to the compound represented by formula III is 1.2:1 to 1:1.2;

And/or, in the first scheme, the temperature of the cyclization reaction is 90°C to 120°C;

And/or, in the second solution, the phosphoric acid solution is an 85% phosphoric acid aqueous solution;

And/or, in Option 2,
for
The molar volume ratio of the nitrogen-containing heterocyclic compound shown in Formula I to the phosphoric acid solution is 0.05mol/L to 0.2mol/L;

And/or, in the second scheme, the temperature of the aromatization reaction is 90°C to 120°C;

And/or, in scheme three, the organic solvent is an aromatic hydrocarbon solvent;

And/or, in the third scheme, the vulcanization reagent is Lawson's reagent;

And/or, in scheme three, the mass volume ratio of the compound represented by formula I'to the organic solvent is 0.01 g/mL to 0.2 g/mL;

And/or, in scheme three, the molar ratio of the compound represented by formula I'to the sulfurization reagent is 1:1 to 1:3;

And/or, in scheme three, the temperature of the vulcanization reaction is 100°C to 120°C.
A pharmaceutical composition comprising the nitrogen-containing heterocyclic compound represented by Formula I, the pharmaceutically acceptable salt thereof, the hydrate thereof, the solvate thereof and the plurality thereof according to any one of claims 1 to 6. The crystal form, its metabolite, its stereoisomer, its tautomer or its prodrug, and at least one pharmaceutical excipient.
The pharmaceutical composition according to claim 9, further comprising a composite scaffold formed by one or more of collagen, chitosan, and hyaluronic acid;

And/or, further includes components for intra-articular delivery; the components for intra-articular delivery preferably include: angiopoietin-like 3 protein or cartilage-forming variants, oral salmon calcitonin, One or more of SD-6010, vitamin D3, collagen hydrolysate, FGF18, BMP7, avocado soybean unsaponifiable and hyaluronic acid.
A nitrogen-containing heterocyclic compound represented by formula I according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a polymorphic form thereof, and a metabolite thereof , Its stereoisomer, its tautomer or its prodrug, or one or more inducers in the preparation of proteoglycan, type II collagen and Sox9 in the pharmaceutical composition according to claim 9 or 10 Application.
The use according to claim 11, wherein the inducer is a drug;

Alternatively, the inducer is used to expand the chondrocyte population in culture in vitro.
The use according to claim 12, wherein the medicament refers to a medicament for treating or preventing diseases related to the induction of differentiation of mesenchymal stem cells into chondrocytes; The diseases preferably include: arthritis or joint damage, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis, osteoporosis, or osteoarthritis.
A method for improving arthritis or joint damage in a mammal, the method comprising administering an effective dose of a nitrogen-containing heterocyclic compound represented by formula I according to any one of claims 1 to 6 to a joint of a mammal, Its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, its tautomer or its prodrug, or as claimed in claim 9 or 10 The pharmaceutical composition described.
A method for inducing mesenchymal stem cells to differentiate into chondrocytes, the method comprising contacting the mesenchymal stem cells with a sufficient amount of the nitrogen-containing heterocyclic compound represented by formula I according to any one of claims 1 to 6, Its pharmaceutically acceptable salt, its hydrate, its solvate, its polymorph, its metabolite, its stereoisomer, its tautomer or its prodrug, or as claimed in claim 9 or 10 The pharmaceutical composition described.