Tgr5-dpp4 dual activity compounds, methods of making, pharmaceutical compositions, and uses thereof

Abstract

The present application relates to TGR5-DPP4 dual activity compounds and preparation method, pharmaceutical composition and purposes thereof.The structural formula of the compound is shown as formula I.The present application proposes a kind of TGR5-DPP4 dual activity compound with intestinal local effect, and in vitro experiment confirms that it has good TGR5 agonist activity and / or DPP4 inhibitory activity, and in vivo experiment shows that it has good intestinal local effect and has good gallbladder safety.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a class of compounds with dual TGR5-DPP4 activity, their preparation methods, pharmaceutical compositions, and uses. Background Technology

[0002] TGR5, Takeda G protein receptor 5, also known as G protein-coupled bile acid receptor 1 (GPBAR1), is widely distributed in the gastrointestinal tract, gallbladder, brown adipose tissue, and muscle tissue. TGR5 mediates various physiological functions, such as glucose homeostasis, gastrointestinal homeostasis, and inflammatory responses. In the gastrointestinal tract, TGR5 activation promotes the release of gastrointestinal hormones such as GLP-1, GLP-2, and PYY. GLP-1 can promote insulin release and inhibit glucagon release in a glucose-dependent manner, thus participating in glucose homeostasis regulation; PYY is related to dietary regulation; and GLP-2 can play a role in intestinal repair by promoting the growth of intestinal villi and crypts. Simultaneously, studies have shown that bile acids can activate intestinal stem cells and promote intestinal epithelial cells through the TGR5 pathway, thereby promoting intestinal repair after injury. Furthermore, TGR5 participates in inflammatory responses by regulating the activation of immune cells. Therefore, TGR5 is considered a potential target for chronic metabolic diseases such as type 2 diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and gastrointestinal disorders such as inflammatory bowel disease. However, since TGR5 is highly expressed in the gallbladder, its activation can cause gallbladder distension side effects. Developing TGR5 agonists with local intestinal effects could improve gallbladder distension side effects.

[0003] TGR5 activation in intestinal L cells promotes the secretion of gastrointestinal hormones such as GLP-1 and GLP-2. However, GLP-1 and GLP-2 are rapidly hydrolyzed by dipeptidyl peptidase 4 (DPP4), with a half-life of only a few minutes. The intestine contains a large amount of DPP4 enzyme, which can hydrolyze more than half of GLP-1 and GLP-2. Therefore, inhibiting intestinal DPP4 enzyme activity is beneficial for TGR5 agonists to exert their pharmacological effects more effectively.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a compound that has dual TGR5-DPP4 activity.

[0006] The second objective of this invention is to provide a method for preparing the compound.

[0007] A third objective of this invention is to provide a pharmaceutical composition containing the compound.

[0008] A fourth objective of this invention is to provide the uses of the compound.

[0009] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0010] This invention proposes compounds of Formula I, their stereoisomers, or pharmaceutically acceptable salts.

[0011]

[0012] in,

[0013] A represents a thiophene ring or a pyridine ring;

[0014] R1, R2, R3, and R4 are each independently hydrogen and C. 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxyl, amino, nitro, or cyano groups; preferably each is independently hydrogen or C. 1-4 Alkyl, halogen or C 1-4 Alkoxy groups; more preferably, each is independently hydrogen or C. 1-2 Alkyl groups; preferably all are hydrogen atoms;

[0015] R5 is hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 1-6 alkoxy-substituted C 1-6 Alkyl, C 3-6 Heterocyclic group, C 1-6 alkyl carbonyl or C 1-6 Alkoxy carbonyl; preferably hydrogen, C 1-4 Alkyl, C 3-6 cycloalkyl or C 3-6 Heterocyclic group; most preferably hydrogen or cyclopropyl;

[0016] R6, R7, R8, and R9 are each independently hydrogen and C. 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxyl, amino, nitro, or cyano groups; preferably each is independently hydrogen or C. 1-4 Alkyl, halogen, C 1-4 Alkyl, hydroxyl, or amino; more preferably, R6 and R8 are halogens and R7 and R9 are hydrogen; or R7 and R9 are halogens and R6 and R8 are hydrogen.

[0017] W1 and W2 are each independently a group represented by IIa or IIb:

[0018]

[0019] m and n are each an independent integer from 1 to 7, preferably an integer from 1 to 3;

[0020] R 21 R 22Each independently is hydrogen, C 1-6 Alkyl groups, or C groups substituted with carboxyl groups 1-6 Alkyl groups, preferably each independently hydrogen or C 1-4 Alkyl groups or C1-C4 alkoxycarbonyl groups substituted with carboxyl or C4 groups 1-4 Alkyl groups, more preferably each being independently hydrogen or C2. 1-2 Alkyl groups or C1-C4 alkoxycarbonyl groups substituted with carboxyl or C4 groups 1-2 Alkyl groups are preferably each independently hydrogen, methyl, or carboxymethyl;

[0021] L represents a group indicated by IIIa or IIIb:

[0022]

[0023] Wherein, q is an integer from 1 to 20, preferably an integer from 1 to 10; r is an integer from 1 to 10, preferably an integer from 1 to 5;

[0024] X represents the group shown in IV:

[0025]

[0026] R is H, C1-C4 alkyl, phenyl-substituted C1-C4 alkyl, hydroxyl-substituted C1-C4 alkyl, C1-C6 alkoxy-substituted C1-C4 alkyl, preferably methyl, benzyl, hydroxyl-substituted methyl and tert-butoxy-substituted methyl; s is an integer from 1 to 3.

[0027] A method for preparing the above-mentioned compound, wherein the method is selected from at least one of the following methods 1 to 4:

[0028] Method 1: Prepare the compound represented by general formula IA, including at least the following steps:

[0029] S1, compound Ia was amide condensed with the linker precursor compound NH2-L-N3 to obtain compound Ib;

[0030] S2 and compound Ib are converted to compound Ic via a hydrogenation reduction reaction;

[0031] S3, compound Id reacts with benzyl bromoacetate via a nucleophilic substitution reaction to give Ie;

[0032] S4 and compound Ie undergo a hydrogenation-reduction reaction to debenzylate and yield If.

[0033] S5. Compound If and compound Ic undergo an amide condensation reaction to yield compound Ig;

[0034] S6, compound Ig undergoes deprotection reaction to give compound IA;

[0035] Method 2: Prepare the compound represented by general formula IB, including at least the following steps:

[0036] S1, Compound Ib reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IIa;

[0037] S2, Compound IIa is converted to Compound IIb via a hydrogenation reduction reaction;

[0038] S3, compound IIb and compound If undergo an amide condensation reaction to yield compound IIc;

[0039] S4, Compound IIc reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IId;

[0040] S5, compound IId is deprotected and hydrolyzed to give IB;

[0041] Method 3: Prepare the compound represented by general formula IC, including the following steps:

[0042] S1, compound If reacts with borane to give compound IIIa;

[0043] S2, Compound IIIa is oxidized with Dess-Martin reagent to give compound IIIb;

[0044] S3, compound IIIb and compound Ic react via a reductive amination reaction to yield compound IIIc;

[0045] S4, compound IIIc is deprotected to give compound IC;

[0046] Method 4: Prepare the compound represented by general formula ID, including the following steps:

[0047] S1, compound Ia is reduced with borane to give compound IVb;

[0048] S2, compound IVb was oxidized with Dess-Martin reagent to give compound IVc;

[0049] S3, compound IVc and the linker precursor compound NH2-L-N3 are reductively aminationly reacted with Boc anhydride to give compound IVd;

[0050] S4, Compound IVd was reduced with an azide group under hydrogen-palladium-carbon conditions to give compound IVe;

[0051] S5, compound IVe and compound If undergo amide condensation to yield compound IVf;

[0052] S6. Compound IVf is deprotected by the protecting group Boc to obtain compound ID.

[0053] The present invention also relates to the use of the compound, its stereoisomers, or pharmaceutically acceptable salts in the preparation of medicaments for treating diseases mediated by TGR5 and / or DPP4.

[0054] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the compounds, stereoisomers or pharmaceutically acceptable salts described above, and optionally, pharmaceutically acceptable excipients.

[0055] The present invention also relates to the use of the compound, its stereoisomers, or pharmaceutically acceptable salts in the preparation of medicaments as TGR5 agonists and / or DPP4 inhibitors.

[0056] Technical effect

[0057] This invention proposes a class of TGR5-DPP4 dual-activity compounds with local intestinal action. In vitro experiments have demonstrated good TGR5 agonist activity and DPP4 enzyme inhibitory activity. In vivo experiments show good local intestinal effects and therapeutic efficacy in a DSS-induced mouse colitis model. The advantages of this invention also lie in the good gallbladder safety of these compounds, making them suitable for the treatment of TGR5-mediated diseases such as type 2 diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and inflammatory bowel disease. Attached Figure Description

[0058] Figure 1-3 The figure shows the experimental results of the DSS-induced colitis experiment in mice;

[0059] Figure 4 The results of the drug concentration tests in plasma, gallbladder, and bile are shown in the figure. Detailed Implementation

[0060] Unless otherwise specified, the terms used in this invention have the following definitions:

[0061] In this invention, "substitution" means that a hydrogen atom on a group is replaced by one or more groups. When multiple groups are selected from the same series of candidate substituents, they may be the same or different. "Optionally" in this invention means that the defined group may be selected from a series of candidate groups, or may not be selected at all.

[0062] In this invention, "alkyl" refers to saturated straight-chain and branched alkyl groups with a specific number of atoms, specifically including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, etc. The "C"... 1-6"Alkyl" refers to a saturated straight-chain or branched alkyl group with 1 to 6 carbon atoms, specifically including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[0063] The "heterocyclic ring" described in this invention refers to a saturated monocyclic system having 4 to 7 cyclic atoms and containing 1 to 4 heteroatoms selected from N, O, and S. Representative examples include, but are not limited to, tetrahydrofuran, pyrrolidine, piperidine, piperazine, morpholine, and oxobutane. 3-6 Heterocyclic groups refer to the aforementioned heterocycles having 3 to 6 carbon atoms.

[0064] The "C" described in this invention 2-6 "Alkenyl" refers to a straight-chain or branched alkenyl group with 2 to 6 carbon atoms and a single double bond. Examples include vinyl, propenyl, butenyl, isobutenyl, pentenyl, and hexenyl.

[0065] The “C” mentioned in this invention 3-6 "Cycloalkyl" represents a non-aromatic, saturated, cyclic aliphatic hydrocarbon group having 3 to 6 cyclic atoms. Representative examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0066] The "C" described in this invention 1-6 "Alkoxy" refers to all straight-chain or branched alkoxy groups with 3 to 6 carbon atoms, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy.

[0067] The "C" described in this invention 1-6 "alkyl carbonyl" refers to all straight-chain or branched alkyl carbonyl groups with 1 to 6 carbon atoms, i.e., alkyl groups combined with... The groups formed by the linkage can be specifically listed as, but are not limited to, methyl carbonyl, ethyl carbonyl, n-propyl carbonyl, isopropyl carbonyl, n-butyl carbonyl, etc.

[0068] The "C" described in this invention 1-6 "Alkoxycarbonyl" refers to all straight-chain or branched alkoxycarbonyl groups with 1 to 6 carbon atoms, i.e., alkoxy groups with... The groups formed by the linkage can be specifically listed, but are not limited to, such as... wait.

[0069] In this invention, "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0070] A "pharmaceutically acceptable salt" is a salt prepared to increase the solubility or stability of a compound and suitable for pharmaceutical use. This pharmaceutically acceptable salt can be obtained directly during the preparation and purification of the compound, or indirectly through the reaction of the compound's free acid or free base with another suitable base or acid.

[0071] The inflammatory bowel disease described in this invention includes, but is not limited to, Crohn's disease and ulcerative colitis.

[0072] Method of use: When used for treatment, the compounds of this invention are typically administered in the form of a standard pharmaceutical composition comprising one or more compounds of general formula (I) in an effective therapeutic dose, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are pharmaceutically acceptable carriers, excipients, or sustained-release agents, etc.

[0073] The pharmaceutical compositions provided by this invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be contained in suitable solid or liquid carriers or diluents. The pharmaceutical compositions of this invention can also be stored in suitable sterile injection or infusion apparatus. The pharmaceutical compositions may also contain odorants, flavorings, etc.

[0074] In this invention, the pharmaceutical composition contains a safe and effective amount (e.g., 0.1 to 99.9 parts by weight, preferably 1 to 90 parts by weight) of a compound of Formula I or a pharmaceutically acceptable salt thereof; and the balance being a pharmaceutically acceptable excipient, wherein the total weight of the composition is 100 parts by weight. Alternatively, the pharmaceutical composition of this invention contains 0.1 to 99.9% by weight, preferably 1 to 90% by weight, of a compound of Formula I or a pharmaceutically acceptable salt thereof; and the balance being a pharmaceutically acceptable excipient, wherein the total weight of the composition is 100% by weight.

[0075] The preferred ratio of the compound represented by Formula I to a pharmaceutically acceptable carrier, excipient, or sustained-release agent is such that the compound represented by Formula I constitutes more than 60% of the total weight as the active ingredient, and the remaining portion constitutes 0 to 40% of the total weight, with the remaining portion preferably being 1 to 20%, and most preferably 1 to 10%.

[0076] The compound represented by Formula I or a pharmaceutical composition comprising a compound represented by Formula I may be used clinically in mammals, including humans and animals, via routes of administration including oral, nasal inhalation, transdermal absorption, pulmonary administration, or gastrointestinal administration. Oral administration is preferred. A unit dosage form is preferred, with each dose containing 0.01 mg to 200 mg of the active ingredient, preferably 0.5 mg to 100 mg, taken once or in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a small dose is started, and the dose is gradually increased until the most suitable dose is found.

[0077] The pharmaceutical compositions of the present invention can be administered orally, as well as intravenously, intramuscularly, or subcutaneously. From the viewpoint of ease of preparation and administration, solid compositions are preferred, especially tablets and solid-filled or liquid-filled capsules. Oral administration of the pharmaceutical compositions is preferred.

[0078] Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include sterile water, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), provided they are suitable for the characteristics of the active ingredient and the desired specific route of administration. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, colorings, preservatives, and antioxidants like vitamin E, vitamin C, BHT, and BHA.

[0079] Injectable formulations include, but are not limited to, sterile, injectable, aqueous, oil-containing solutions, suspensions, emulsions, etc. These formulations can also be formulated with suitable parenteral diluents, dispersants, wetting agents, suspending agents, etc. Such injectable formulations can be sterilized by filtration through a bacteria-retaining filter. These formulations can also be formulated with bactericides dissolved or dispersed in an injectable medium or using other methods known in the art.

[0080] The first aspect of this invention provides a compound as shown in Formula I, its stereoisomers, or a pharmaceutically acceptable salt thereof.

[0081]

[0082] in,

[0083] A represents a thiophene ring or a pyridine ring;

[0084] R1, R2, R3, and R4 are each independently hydrogen and C. 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxyl, amino, nitro, or cyano groups; preferably each is independently hydrogen or C. 1-4 Alkyl, halogen or C 1-4 Alkoxy groups; more preferably, each is independently hydrogen or C. 1-2 Alkyl groups; preferably all are hydrogen atoms;

[0085] R5 is hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 1-6 alkoxy-substituted C 1-6 Alkyl, C 3-6 Heterocyclic group, C 1-6 alkyl carbonyl or C 1-6Alkoxy carbonyl; preferably hydrogen, C 1-4 Alkyl, C 3-6 cycloalkyl or C 3-6 Heterocyclic group; most preferably hydrogen or cyclopropyl;

[0086] R6, R7, R8, and R9 are each independently hydrogen and C. 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxyl, amino, nitro, or cyano groups; preferably each is independently hydrogen or C. 1-4 Alkyl, halogen, C 1-4 Alkyl, hydroxyl, or amino; more preferably, R6 and R8 are halogens and R7 and R9 are hydrogen; or R7 and R9 are halogens and R6 and R8 are hydrogen.

[0087] W1 and W2 are each independently a group represented by IIa or IIb:

[0088]

[0089] m and n are each independent integers from 1 to 7, such as 1, 2, 3, 4, 5, 6, etc., preferably integers from 1 to 3;

[0090] R 21 R 22 Each independently is hydrogen, C 1-6 Alkyl groups, or C groups substituted with carboxyl groups 1-6 Alkyl groups, preferably each independently hydrogen or C 1-4 Alkyl groups or C1-C4 alkoxycarbonyl groups substituted with carboxyl or C4 groups 1-4 Alkyl groups, more preferably each being independently hydrogen or C2. 1-2 Alkyl groups or C1-C4 alkoxycarbonyl groups substituted with carboxyl or C4 groups 1-2 Alkyl groups are preferably each independently hydrogen, methyl, or carboxymethyl;

[0091] L represents a group indicated by IIIa or IIIb:

[0092]

[0093] Where q is an integer from 1 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, etc., preferably an integer from 1 to 10; r is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9, etc., preferably 1 to 5;

[0094] X represents the group shown in IV:

[0095]

[0096] R is H, C1-C4 alkyl, phenyl-substituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C6 alkoxy-substituted C1-C4 alkyl, preferably methyl, benzyl, hydroxy-substituted methyl and tert-butoxy-substituted methyl; s is an integer from 1 to 3, preferably 2 or 3.

[0097] In this invention, a compound with TGR5 activating activity and a compound with DPP4 inhibitory activity are linked together via a linker chain to obtain the compound of Formula I. In Formula I, W1 and W2 represent linker groups, L represents the linker chain, and X represents the portion filling the S2 pocket in the non-peptide-like structure of the DPP4 inhibitor. During the research, this invention discovered that the specific selection of W1, W2, and L significantly affects the activity of the compound. By screening the groups, this invention simultaneously ensures the activity of both molecular components, obtaining a compound with dual activity, thus completing this invention.

[0098] In some embodiments, the compound represented by Formula I is selected from compounds represented by Formulas IA, IB, IC, and ID:

[0099]

[0100]

[0101] The definitions of A, R1, R2, R3, R4, R5, R6, R7, R8, R9, L, and X are as described in Equation I.

[0102] In particular, in some embodiments, in formulas I and IA, IB, IC and ID above,

[0103] R1, R2, R3, and R4 are each independently hydrogen, halogen, hydroxyl, amino, nitro, or cyano, preferably all of them being hydrogen;

[0104] R5 is hydrogen, C 1-3 Alkyl, C 3-6 cycloalkyl or C 3-6 Heterocyclic group, preferably hydrogen or cyclopropyl;

[0105] R6, R7, R8, and R9 are each independently hydrogen, halogen, and C. 1-3 Alkoxy, hydroxy, amino, nitro or cyano; preferably hydrogen or halogen.

[0106] In some embodiments, the compound of formula I is selected from compounds represented by the following structural formulas:

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] In some embodiments, the compounds of the present invention, their stereoisomers, or pharmaceutically acceptable salts may be present as crystalline hydrates or solvates. These crystalline hydrates or solvates are also included within the scope of the present invention.

[0113] Based on the known structure of the compounds of this invention, those skilled in the art can design and synthesize the compounds of this invention using reactions known in the art, or synthesize the compounds of this invention using methods similar to the preparation methods disclosed in this invention. Therefore, there are no particular limitations on the specific preparation methods for synthesizing the compounds of this invention, as long as the compounds of this invention can be obtained.

[0114] A second aspect of the present invention provides a method for preparing a portion of the compounds according to the present invention, wherein the compound is selected from at least one of the following methods 1 to 4.

[0115] Method 1: Prepare the compound represented by general formula IA by the following steps:

[0116] S1, compound Ia was amide condensed with the linker precursor compound NH2-L-N3 to obtain compound Ib;

[0117] S2 and compound Ib are converted to compound Ic via a hydrogenation reduction reaction;

[0118] S3, compound Id reacts with benzyl bromoacetate via a nucleophilic substitution reaction to give Ie;

[0119] S4 and compound Ie undergo a hydrogenation-reduction reaction to debenzylate and yield If.

[0120] S5. Compound If and compound Ic undergo an amide condensation reaction to yield compound Ig;

[0121] S6, compound Ig undergoes deprotection reaction to give compound IA;

[0122] The reaction formula is as follows:

[0123]

[0124] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, L, A, R, S, and X are as described above.

[0125] The reaction conditions will be explained in detail below:

[0126] (1) Ia undergoes an amide condensation reaction with different linker precursor compounds NH2-L-N3 to obtain Ib. The amide condensation reaction is carried out using conventional methods in the art. For example, compound Ia is dissolved in an organic solvent, a condensing agent and a basic reagent are added, and then the linker precursor compound NH2-L-N3 is added, and the reaction is carried out at room temperature to 100°C for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably N,N-dimethylformamide (DMF), dichloromethane (DCM), etc. The condensing agent is a commonly used condensing agent in the art, and includes, without limitation, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1,3-dicyclohexylcarbodiimide (DCC), diethyl azodicarbonate (DEAD) / triphenylphosphine, N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 1-hydroxybenzotriazole (HOBt), etc., with HATU being preferred. The alkaline conditions include, without limitation, organic bases such as triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP), as well as inorganic bases such as potassium carbonate, cesium carbonate, and sodium bicarbonate; with triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP) being preferred.

[0127] (2) Compound Ib is hydrogenated to yield compound Ic. The hydrogenation reaction can be a conventional method in the art. For example, compound Ib is dissolved in an organic solvent, a metal catalyst is added, and hydrogen is directly generated under a hydrogen atmosphere or by heating with ammonium formate. The reaction is carried out at room temperature to solvent reflux for 0.5–24 hours. Any solvent that does not adversely affect the reaction can be used, preferably methanol, ethanol, tetrahydrofuran, etc. The metal catalyst is not limited to palladium on carbon, rhodium on carbon, etc., but palladium on carbon is preferred.

[0128] (3) Compound Id reacts with benzyl bromoacetate via a nucleophilic substitution reaction to yield Ie. The nucleophilic substitution reaction can be a conventional method in the art. For example, compound Id is dissolved in an organic solvent, a basic reagent and benzyl bromoacetate are added, and the reaction is carried out at 0-60°C for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably tetrahydrofuran (THF), DMF, etc. The basic reagent is not limited to sodium hydride, sodium hydroxide, potassium hydroxide, etc., with sodium hydride being preferred.

[0129] (4) Compound Ie is debenzylated by hydrogenation to give If. The debenzylation reaction can be a conventional method in the art. For example, compound Ie is dissolved in an organic solvent, a metal catalyst is added, and hydrogen is directly generated under a hydrogen atmosphere or by heating with ammonium formate. The reaction is carried out at room temperature to solvent reflux for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably methanol, ethanol, tetrahydrofuran, etc. The metal catalyst is not limited to palladium on carbon, rhodium on carbon, etc., with palladium on carbon being preferred.

[0130] (5) If and Ic are reacted by amide condensation to yield Ig. The amide condensation reaction can be carried out using conventional methods in the art. For example, compound Ic is dissolved in an organic solvent, a condensing agent and a basic reagent are added, and then If is added, and the reaction is carried out at room temperature to 100°C for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably N,N-dimethylformamide (DMF), dichloromethane (DCM), etc. The condensing agent is a commonly used condensing agent in the art, and non-limiting examples include 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1,3-dicyclohexylcarbodiimide (DCC), diethyl azodicarbonate (DEAD) / triphenylphosphine, N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 1-hydroxybenzotriazole (HOBt), etc., with HATU being preferred. The alkaline conditions non-limitingly include organic bases such as triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP), as well as inorganic bases such as potassium carbonate, cesium carbonate, and sodium bicarbonate; preferred are triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP).

[0131] (6) Ig is deBoc protected to give IA. The deBoc protecting group removal reaction can be carried out using conventional methods in the art. For example, compound Ig is dissolved in an organic solvent, and hydrochloric acid / dioxane, hydrochloric acid / ethanol, hydrochloric acid / dichloromethane solution, or trifluoroacetic acid, etc., are added, and the reaction is carried out at room temperature for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably DCM, dioxane, methanol, etc.

[0132] Method 2: Prepare the compound represented by general formula IB, including at least the following steps:

[0133] S1, Compound Ib reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IIa;

[0134] S2, Compound IIa is converted to Compound IIb via a hydrogenation reduction reaction;

[0135] S3, compound IIb and compound If undergo an amide condensation reaction to yield compound IIc;

[0136] S4, Compound IIc reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IId;

[0137] S5, compound IId undergoes deprotection and hydrolysis to give IB; the reaction formula is as follows:

[0138]

[0139] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, L, A, and X are as described above.

[0140] The reaction conditions will be explained in detail below:

[0141] (1) Ib reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to yield IIa. The nucleophilic substitution reaction can be a conventional method in the art. For example, compound Ib is dissolved in an organic solvent, a basic reagent and tert-butyl bromoacetate are added, and the reaction is carried out at 0-60°C for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably tetrahydrofuran (THF), DMF, etc. The basic reagent is not limited to sodium hydride, sodium hydroxide, potassium hydroxide, etc., with sodium hydride being preferred.

[0142] (2) IIa is reduced by hydrogenation to obtain IIb. The hydrogenation reaction can be a conventional method in the art. For example, compound IIa is dissolved in an organic solvent, a metal catalyst is added, and hydrogen is directly generated under a hydrogen atmosphere or by heating with ammonium formate, and the reaction is carried out at room temperature to solvent reflux for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably methanol, ethanol, tetrahydrofuran, etc. The metal catalyst is not limited to palladium on carbon, rhodium on carbon, etc., with palladium on carbon being preferred.

[0143] (3) IIb and If undergo amide condensation to yield IIc. The amide condensation reaction can be carried out using conventional methods in the art. For example, compound If is dissolved in an organic solvent, a condensing agent and a basic reagent are added, and then IIb is added, and the reaction is carried out at room temperature to 100°C for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably N,N-dimethylformamide (DMF), dichloromethane (DCM), etc. The condensing agent is a commonly used condensing agent in the art, and non-limiting examples include 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1,3-dicyclohexylcarbodiimide (DCC), diethyl azodicarbonate (DEAD) / triphenylphosphine, N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 1-hydroxybenzotriazole (HOBt), etc., with HATU being preferred. The alkaline conditions non-limitingly include organic bases such as triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP), as well as inorganic bases such as potassium carbonate, cesium carbonate, and sodium bicarbonate; preferred are triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP).

[0144] (4) IIc reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to yield IId. The nucleophilic substitution reaction can be a conventional method in the art. For example, compound IIc is dissolved in an organic solvent, a basic reagent and tert-butyl bromoacetate are added, and the reaction is carried out at 0-60°C for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably tetrahydrofuran (THF), DMF, etc. The basic reagent is not limited to sodium hydride, sodium hydroxide, potassium hydroxide, etc., with sodium hydride being preferred.

[0145] (5) Deprotection of IId by Boc and hydrolysis yields IB. The deprotection of IId by Boc and hydrolysis can be carried out using conventional methods in the art. For example, compound IId is dissolved in an organic solvent, and hydrochloric acid / dioxane, hydrochloric acid / ethanol, hydrochloric acid / dichloromethane solution, or trifluoroacetic acid, etc., is added, and the reaction is carried out at room temperature for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably DCM, dioxane, methanol, etc.

[0146] Method 3: Prepare the compound represented by general formula IC, including the following steps:

[0147] S1, compound If reacts with borane to give compound IIIa;

[0148] S2, Compound IIIa is oxidized with Dess-Martin reagent to give compound IIIb;

[0149] S3, compound IIIb and compound Ic react via a reductive amination reaction to yield compound IIIc;

[0150] S4, compound IIIc is deprotected to give compound IC;

[0151] The reaction formula is as follows:

[0152]

[0153] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, L, A, and X are as described above.

[0154] The reaction conditions will be explained in detail below:

[0155] (1) If is reduced by borane to give IIIa. The reduction method can be a conventional method in the art. For example, the compound If is dissolved in an organic solvent, a borane / tetrahydrofuran solution is added, and the reaction is carried out at 0-50°C for 1-24 hours. The solvent can be any solvent that does not adversely affect the reaction, preferably tetrahydrofuran (THF) or the like.

[0156] (2) IIIa is oxidized by Dess-Martin to obtain IIIb. The Dess-Martin oxidation method is a conventional method in the art. For example, compound IIIa is dissolved in an organic solvent, a Dess-Martin oxidant is added, and the reaction is carried out at 0-50°C for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably dichloromethane (DCM), etc.

[0157] (3) IIIb and Ic are reacted by reductive amination to obtain IIIc. The reductive amination method can be a conventional method in the art. For example, compounds IIIb and Ic are dissolved in an organic solvent, activated molecular sieves are added, and the mixture is stirred for 1-24 hours at room temperature to the solvent boiling point. Sodium borohydride or sodium triacetoxyborohydride is then added, and the reaction is continued for 1-24 hours at room temperature to 50°C. The solvent can be any solvent that does not adversely affect the reaction, preferably methanol, dichloroethane, dichloromethane (DCM), etc.

[0158] (4) The deprotecting group of IIIc (Boc) is removed to give IIId. The deprotecting Boc reaction can be carried out using conventional methods in the art. For example, compound IIIc is dissolved in an organic solvent, and hydrochloric acid / dioxane, hydrochloric acid / ethanol, hydrochloric acid / dichloromethane solution, or trifluoroacetic acid, etc., are added, and the reaction is carried out at room temperature for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably DCM, dioxane, methanol, etc.

[0159] Method 4: Prepare the compound represented by general formula ID, including the following steps:

[0160] S1, compound Ia is reduced with borane to give compound IVb;

[0161] S2, compound IVb was oxidized with Dess-Martin reagent to give compound IVc;

[0162] S3, compound IVc and the linker precursor compound NH2-L-N3 are reductively aminationly reacted with Boc anhydride to give compound IVd;

[0163] S4, Compound IVd was reduced with an azide group under hydrogen-palladium-carbon conditions to give compound IVe;

[0164] S5, compound IVe and compound If undergo amide condensation to yield compound IVf;

[0165]

[0166] S6. Compound IVf is deprotected by the protecting group Boc to give compound ID;

[0167] The reaction formula is as follows:

[0168]

[0169] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, L, A, and X are as described above.

[0170] The reaction conditions will be explained in detail below:

[0171] (1) Ia is reduced by borane to give IVb. The reduction method can be a conventional method in the art. For example, compound Ia is dissolved in an organic solvent, a borane / tetrahydrofuran solution is added, and the reaction is carried out at 0-50°C for 1-24 hours. The solvent can be any solvent that does not adversely affect the reaction, preferably tetrahydrofuran (THF) or the like.

[0172] (2) IVb is oxidized with Dess-Martin reagent to obtain IVc. The oxidation method is a conventional method in the art. For example, compound IVb is dissolved in an organic solvent, Dess-Martin oxidant is added, and the reaction is carried out at 0-50°C for 1-24 hours. The solvent can be any solvent that does not adversely affect the reaction, preferably dichloromethane (DCM), etc.

[0173] (3) IVc reacts with the linker precursor compound NH2-L-N3 via a reductive amination reaction, followed by a reaction with Boc anhydride to yield IVd. The reductive amination method can be a conventional method in the art. For example, compound IVc and the linker precursor compound NH2-L-N3 are dissolved in an organic solvent, activated molecular sieves are added, and the mixture is stirred for 1-24 hours at room temperature to the solvent boiling point. Sodium borohydride or sodium triacetoxyborohydride is then added, and the reaction continues for 1-24 hours at room temperature to 50°C. The solvent can be any solvent that does not adversely affect the reaction, preferably methanol, dichloroethane, dichloromethane (DCM), etc. Finally, the reductive amination product reacts with Boc anhydride to yield IVd.

[0174] (4) IVd is reduced to IVe by the reduction of the azide group under hydrogen-palladium-carbon conditions. The hydrogenation reaction can be a conventional method in the art. For example, compound IVd is dissolved in an organic solvent, a metal catalyst is added, and hydrogen is directly generated under a hydrogen atmosphere or by heating with ammonium formate, and the reaction is carried out at room temperature to solvent reflux for 1-24 hours. Any solvent that does not adversely affect the reaction can be used, preferably methanol, ethanol, tetrahydrofuran, etc. The metal catalyst is not limited to palladium-carbon, rhodium-carbon, etc., and palladium-carbon is preferred.

[0175] (5) IVe and If undergo amide condensation to yield IVf. The amide condensation reaction can be carried out using conventional methods in the art. For example, compound If is dissolved in an organic solvent, a condensing agent and a basic reagent are added, followed by the addition of IVe, and the reaction is carried out at room temperature to 100°C for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably N,N-dimethylformamide (DMF), dichloromethane (DCM), etc. The condensing agent is a commonly used condensing agent in the art, and non-limiting examples include 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1,3-dicyclohexylcarbodiimide (DCC), diethyl azodicarbonate (DEAD) / triphenylphosphine, N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 1-hydroxybenzotriazole (HOBt), etc., with HATU being preferred. The alkaline conditions non-limitingly include organic bases such as triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP), as well as inorganic bases such as potassium carbonate, cesium carbonate, and sodium bicarbonate; preferred are triethylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP).

[0176] (6) Deprotection of IVf from Boc yields ID. The deprotection of Boc can be carried out using conventional methods in the art. For example, compound IVf is dissolved in an organic solvent, and hydrochloric acid / dioxane, hydrochloric acid / ethanol, hydrochloric acid / dichloromethane solution, or trifluoroacetic acid, etc., is added, and the reaction is carried out at room temperature for 1-24 hours. The organic solvent can be any solvent that does not adversely affect the reaction, preferably DCM, dioxane, methanol, etc.

[0177] A third aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount selected from one or more of the compounds, stereoisomers thereof, and pharmaceutically acceptable salts according to the present invention. The pharmaceutical composition may also comprise pharmaceutically acceptable excipients. Furthermore, the pharmaceutical composition may comprise one or more other drugs for treating diseases mediated by TGR5 and / or DPP4. Preferably, the other drugs are selected from one or more of hypoglycemic agents, weight-loss agents, non-alcoholic fatty liver disease (NAFLD) drugs, non-alcoholic steatohepatitis (NAHB) drugs, and inflammatory bowel disease (IBD) drugs. Hypoglycemic agents include, but are not limited to, sulfonylureas, biguanides, insulin sensitizers, α-glucosidase inhibitors, and DPP4 inhibitors; weight-loss agents include, but are not limited to, GLP-1 analogs; NAFLD / NAHB drugs include, but are not limited to, GLP-1 analogs and bile acid analogs; and IBD drugs include, but are not limited to, GLP-2 analogs.

[0178] The fourth aspect of the present invention proposes the use of one or more of the compounds, stereoisomers thereof or pharmaceutically acceptable salts selected from those described above according to the present invention in the preparation of medicaments as TGR5 agonists and / or DPP4 inhibitors.

[0179] A fifth aspect of this invention proposes the use of one or more compounds selected from those described above according to the invention, their stereoisomers, or pharmaceutically acceptable salts, in the preparation of a medicament for treating diseases mediated by TGR5 and / or DPP4. These diseases mediated by TGR5 and / or DPP4 include, but are not limited to, diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and inflammatory bowel disease.

[0180] The present invention will be further illustrated below with examples. It should be particularly noted that these examples are for illustrative purposes only and are not intended to limit the invention in any way. All parameters and other descriptions in the examples, unless otherwise stated, are based on quality. Unless otherwise specified, the packing material used for column chromatography separation is silica gel. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0181] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0182] Example 1

[0183] Intermediate A1-1;

[0184]

[0185] Except for the preparation of the seven-membered ring, which used a racemic precursor instead of a chiral precursor, the other preparation methods were based on the literature Bioorganic & Medicinal Chemistry Letters 17 (2007) 1903–1907, yielding Al-1. MS (ESI): m / z 444.2 [M+H] + 344.2[M-Boc+H] + . 1 H NMR(400MHz, CDCl3)δ7.07(m,1H),6.90(m,1H),6.64–6.33(m,1H),5.75–5 .42(m,1H),4.25–3.93(m,1H),3.82–3.60(m,1H),3.45–3.24(m,2H),3.24 –3.10(m,1H),2.97(q,J=7.0,5.1Hz,3H),2.83–2.66(m,1H),2.62(t,J=5. 9Hz,1H),2.05(m,1H),1.83(m,1H),1.51–1.39(3H),1.37(d,J=4.7Hz,9H).

[0186] Intermediate A1-2;

[0187]

[0188] Intermediate A1-1 (2.2 g, 5 mmol) was dissolved in tetrahydrofuran (THF) under an ice-water bath. Sodium hydride (dispersed in paraffin, 60% content, 400 mg, 10 mmol) was added, and the reaction was carried out under a nitrogen atmosphere and an ice bath for 0.5 h. Benzyl bromoacetate (2.3 g, 10 mmol) was added. The ice bath was removed, and the reaction was carried out at room temperature for 8 h. Thin-layer chromatography confirmed the completeness of the reaction. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, dried, and purified by column chromatography (eluent: dichloromethane:methanol = 20:1) to give 2.0 g of colorless oily product A1-2, yield 68%. MS (ESI): m / z 592.3 [M+H] + 492.3[M-Boc+H]+ .

[0189] Intermediate A1-3;

[0190]

[0191] Intermediate A1-2 (2 g, 3.38 mmol) was dissolved in methanol and tetrahydrofuran (1:1 v / v). 200 mg of palladium on carbon (10% palladium content) was added, and the mixture was purged with hydrogen three times. The reaction was carried out at room temperature for 3 hours, and thin-layer chromatography confirmed complete reaction. The palladium on carbon was removed by direct filtration, and the mixture was evaporated to dryness to obtain A1-3 as a white solid (1.37 g, 81% yield). No further purification was required, and it could be directly used in the next step. MS (ESI): m / z 502.3 [M+H] + 402.3[M-Boc+H] + .

[0192] Intermediate A1-4;

[0193]

[0194] The preparation of intermediate A1-4 was carried out according to the literature Sci Rep. 2016 Jun 24; 6:28676. MS (ESI): m / z 517.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ12.28 (s, 1H), 7.78 (d, J = 5.4Hz, 1H), 7.46 (s, 1H), 6.84 (td, J=7.7,7.1,1.5Hz,1H),6.78–6.72(m,1H),6.67(dd,J=10.1,6.3Hz,2H),6.43(td,J =7.6,1.4Hz,1H),6.00(s,1H),3.81(t,J=5.2Hz,2H),3.37(t,J=5.4Hz,2H),2.83(t ,J=7.6Hz,2H),2.52(t,J=5.4Hz,2H),2.23(m,1H),0.70(m,2H),0.35–0.25(m,2H).

[0195] Intermediate A1-5;

[0196]

[0197] 3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaloline-1-carbonyl)thiophene-3-yl)oxy)phenyl)propionic acid (Al-4, 103.4 mg, 0.2 mmol) and HATU (2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, 91.2 mg, 0.24 mmol) were dissolved in DMF (N,N-dimethylformamide, 10 mL), and DIPEA (diisopropylethylamine, 53 μL, 0.3 mmol) was added. After reacting at room temperature for 15 minutes, tert-butyl (2-(methylamino)ethyl)carbamate (35 mg, 0.2 mmol) was added. The reaction was continued at room temperature for 2 hours. After confirming the completeness of the reaction by thin-layer chromatography, saturated sodium chloride solution was added to the reaction solution, and the mixture was extracted twice with ethyl acetate, washed three times with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. After purification by column chromatography (dichloromethane:methanol = 50:1), 95 mg of a yellow oily substance A1-5 was obtained, which solidified into a yellow solid, with a yield of 71%. MS (ESI): m / z 673.2 [M+H] + 573.2[M-Boc+H] + .

[0198] Intermediate A1-6;

[0199]

[0200] Intermediate A1-5 (90 mg) was dissolved in dichloromethane, and 2 mL of hydrochloric acid / dioxane solution (molar concentration: 4N) was added. The mixture was reacted at room temperature for 2 hours. Thin-layer chromatography confirmed the complete reaction. The solution was directly evaporated to dryness to obtain 75 mg of yellow solid A1-6. No purification was required, and the mixture could be directly used in the next step. MS (ESI): m / z 573.2 [M+H] + .

[0201] Intermediate A1-7;

[0202]

[0203] Intermediate A1-3 (50 mg, 0.1 mmol), HATU (46 mg, 0.12 mmol), and intermediate A1-6 (60 mg, 0.1 mmol) were dissolved in DMF (10 mL). DIPEA (35 μL, 0.2 mmol) was added, and the mixture was reacted at room temperature for 2 hours. After confirming the reaction was complete by thin-layer chromatography, saturated sodium chloride solution was added to the reaction solution. The mixture was extracted twice with ethyl acetate, washed three times with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. After purification by column chromatography (dichloromethane:methanol = 20:1), 67 mg of a yellow oily substance, A1-7, was obtained, with a yield of 64%. MS (ESI): 529 [M / 2+H] + ,479[(M﹣Boc) / 2+H]+ .

[0204] Compound 1

[0205]

[0206] Intermediate A1-7 (60 mg, 0.057 mmol) was dissolved in dichloromethane (5 mL), and 1 mL of hydrochloric acid / dioxane (molar concentration: 4N) was added. The mixture was reacted at room temperature for 5 hours. After confirming the completeness of the reaction by thin-layer chromatography, the solution was evaporated to dryness. Semi-preparative liquid chromatography purification yielded 32 mg (0.034 mmol, yield 59%) of a pale yellow oily final product (Example 1). MS (ESI): 479 [M / 2+H] + . 1 H NMR (400MHz, CDCl3) δ7.38(d,J=5.4Hz,1H),7.32-7.24(m,2H),7.18–6.98(m,2H),6.97–6.83(m,2H),6.80(d,J=7.9Hz,1H),6.51(t,J =7.5Hz,1H),6.46(d,J=5.4Hz,1H),6.17(s,1H),4.52–4.26(m,1H),4.17(d,J=16.4Hz,1H),3.96(s,2H),3.79–3.56(m,3H),3.48(t,J= 5.4Hz,2H),3.43(s,2H),3.21(d,J=15.9Hz,1H),3.02(s,3H),2.95(d,J=9.7Hz,2H),2.88–2.66(m,2H),2.58(t,J=7.9Hz,2H),2.42(m, 1H),2.32(m,1H),2.19-1.65(m,7H),1.56(dd,J=13.3,6.9Hz,1H),1.44(t,J=6.5Hz,2H),0.73(d,J=6.3Hz,2H),0.44(d,J=3.6Hz,2H).

[0207] Example 2

[0208] Intermediate A2-1:

[0209]

[0210] Intermediate A1-4 (104 mg, 0.2 mmol), HATU (92 mg, 0.24 mmol), and 1-amino-11-azido-3,6,9-trioxaundecan (CAS: 134179-38-7, 44 mg, 0.2 mmol) were dissolved in DMF (10 mL). DIPEA (53 μL, 0.3 mmol) was added, and the mixture was reacted at room temperature for 3 hours. After confirming the reaction was complete by thin-layer chromatography, saturated sodium chloride solution was added to the reaction solution. The mixture was extracted twice with ethyl acetate, washed three times with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. After purification by column chromatography (dichloromethane:methanol = 20:1), 107 mg of a yellow oily substance A2-1 was obtained, with a yield of 75%. MS (ESI): m / z 717 [M+H] + . 1 H NMR (500MHz, CDCl3) δ7.36 (d, J=5.5Hz, 1H), 7.22 (s, 1H), 6.88 (ddd, J=8.5, 7.1, 1.5Hz, 1H), 6.83 (dd, J=8.3, 1.5Hz, 1H), 6.7 7(d,J=7.9Hz,1H),6.52–6.45(m,1H),6.43(d,J=5.4Hz,1H),6.14(dd,J=11.0,5.3Hz,2H),3.93(t,J=5.4Hz,2H),3.71–3.63( m,8H),3.64–3.59(m,2H),3.55(dd,J=5.6,4.5Hz,2H),3.46(td,J=5.4,1.4Hz,4H),3.38(t,J=5.0Hz,2H),2.96(dd,J=8.7,6 .7Hz,2H),2.44(dd,J=8.7,6.8Hz,2H),2.29(tt,J=6.8,3.7Hz,1H),0.71(dd,J=6.6,1.9Hz,2H),0.40(dd,J=4.0,2.3Hz,2H).

[0211] Intermediate A2-2;

[0212]

[0213] Intermediate A2-1 (100 mg, 0.14 mmol) was dissolved in a methanol / tetrahydrofuran (V / V = 1:1) solution. After nitrogen purging, 15 mg of palladium on carbon (10% palladium content) was added. The mixture was then purged three times with hydrogen and reacted at room temperature for 45 minutes. The palladium on carbon was removed by filtration, and the product was directly evaporated to dryness to obtain 75 mg of a pale yellow oil, A2-2; yield 78%. MS (ESI): m / z 691.2 [M+H] + . 1H NMR(500MHz, CDCl3)8.27(s,3H),7.42(d,J=5.4Hz,1H),7.35(s,1H),7.28(s,1H),7.14–7.05(m,1H),6.96(dd ,J=8.1,1.4Hz,1H),6.89–6.82(m,1H),6.43(d,J=5.4Hz,1H),6.18(s,1H),4.11(t,J=5.9Hz,2H),3.86(t,J=4. 8Hz,2H),3.73–3.69(m,2H),3.69–3.60(m,8H),3.59(t,J=5.8Hz,2H),3.51(t,J=5.0Hz,2H),3.16(s,2H),3.05 (t,J=7.6Hz,2H),2.80(t,J=7.6Hz,2H),2.63(tt,J=7.0,3.7Hz,1H),0.95(d,J=4.1Hz,2H),0.80–0.73(m,2H).

[0214] Intermediate A2-3:

[0215]

[0216] A2-2 (68 mg, 0.1 mmol), HATU (46 mg, 0.12 mmol), and A1-3 (50 mg, 0.1 mmol) (44 mg, 0.2 mmol) were dissolved in DMF (10 mL), and DIPEA (35 μL, 0.2 mmol) was added. The mixture was reacted at room temperature for 3 hours. After confirming the completeness of the reaction by thin-layer chromatography, saturated sodium chloride solution was added to the reaction solution. The mixture was extracted twice with ethyl acetate, washed three times with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. After purification by column chromatography (dichloromethane:methanol = 20:1), 80 mg of a yellow oily substance, A2-3, was obtained, with a yield of 68%. MS (ESI): m / z 688 [M / 2+H] + ,638[(M﹣Boc) / 2+H] + .

[0217] Compound 2:

[0218]

[0219] Intermediate A2-3 (70 mg, 0.06 mmol) was dissolved in dichloromethane (5 mL), and 1 mL of hydrochloric acid / dioxane (molar concentration: 4N) was added. The mixture was reacted at room temperature for 5 hours. After confirming the completeness of the reaction by thin-layer chromatography, the solution was evaporated to dryness. Semi-preparative liquid chromatography purification yielded 43 mg (0.034 mmol, yield 67%) of a pale yellow oily compound 2. MS (ESI): m / z 638 [M / 2+H] +. 1 H NMR (400MHz, CDCl3) δ7.36 (d, J=5.4Hz, 1H), 7.22 (s, 1H), 7.07 (q, J=9.4, 9.0Hz, 1H) ,6.98–6.81(m,3H),6.77(d,J=7.3Hz,2H),6.56(s,1H),6.52–6.41(m,2H),6.14(s, 1H),5.19(s,1H),4.14(dd,J=15.5,5.7Hz,1H),3.94(t,J=5.4Hz,2H),3.86(dd,J=1 5.5,9.4Hz,1H),3.82–3.64(m,0H),3.62(d,J=4.2Hz,9H),3.55(q,J=4.9Hz,5H),3. 44(dt,J=10.1,5.3Hz,6H),3.28(t,J=24.6Hz,2H),2.96(t,J=7.8Hz,2H),2.83–2.7 3(m,1H),2.65(t,J=10.9Hz,1H),2.55(dd,J=16.2,3.4Hz,1H),2.44(t,J=7.7Hz,3H ),2.29(dt,J=6.4,2.7Hz,1H),2.22(t,J=7.6Hz,0H),2.16–1.96(m,1H),1.52(dd,J =12.9,7.0Hz,1H),1.38(t,J=6.4Hz,2H),0.70(t,J=6.0Hz,2H),0.45–0.36(m,2H).

[0220] Example 3

[0221]

[0222] Except for replacing 1-amino-11-azido-3,6,9,12,15-pentahepta-1-amine (CAS: 516493-93-9), 17-azido-3,6,9-trioxaundecan (CAS: 134179-38-7) was used; the rest of the preparation method was the same as in Example 2; MS (ESI): m / z 582 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.36(d,J=5.4Hz,1H),7.23(s,1H),7.15–7.01(m,2H),6.98–6.81(m,3H),6.81–6.65(m,2H),6.49(t,J=7.3Hz,1H), 6.44(d,J=5.4Hz,1H),6.13(s,1H),4.30–4.01(m,1H),3.94(t,J=5.4Hz,2H),3.72–3.57(m,20H),3.54(d,J=5.6Hz,4H),3.45(dd,J=11.4 ,6.0Hz,8H),3.22(d,J=15.1Hz,1H),3.01–2.92(m,2H),2.79(s,1H),2.74–2.62(m,1H),2.46(t,J=7.8Hz,2H),2.33–2.25(m,1H),2.22(t ,J=7.7Hz,1H),2.15–1.95(m,1H),1.70–1.57(m,1H),1.57–1.47(m,1H),1.38(t,J=6.3Hz,2H),0.71(q,J=5.9Hz,2H),0.45–0.35(m,2H).

[0223] Example 4

[0224]

[0225] Except for replacing 1-amino-11-azido-3,6,9,12,15,18-hexaoxaecoeicosane-1-amine (CAS: 957486-82-7) with 20-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7), the preparation method was the same as in Example 2; MS (ESI): m / z 604 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.36(d,J=5.5Hz,1H),7.25(s,2H),7.01–6.81(m,3H),6.77(d,J=7.9Hz,1H),6.49(t ,J=7.6Hz,1H),6.44(d,J=5.4Hz,1H),6.13(s,1H),3.94(t,J=5.4Hz,2H),3.81–3.52(m,28H),3.52–3.38(m ,6H),3.37–3.04(m,4H),2.96(t,J=7.9Hz,2H),2.50(t,J=8.0Hz,2H),2.36–2.25(m,1H),2.22(t,J=7.6Hz, 1H),2.13–1.94(m,2H),1.69–1.56(m,1H),1.52–1.37(m,3H),0.72(d,J=6.2Hz,2H),0.40(d,J=3.8Hz,2H).

[0226] Example 5

[0227]

[0228] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecan (CAS: 134179-38-7) with amino-heptaethylene glycol-azide (CAS: 1333154-77-0), the preparation method was the same as in Example 2; MS (ESI): m / z 626 [M / 2+H] + . 1HNMR(500MHz, CDCl3)δ7.36(d,J=5.3Hz,1H),7.34–7.28(m,1H),7.22(s,1H),6.92–6.80(m,3H),6.77(d,J=7.9Hz,1H),6.52–6.45( m,1H),6.43(d,J=5.4Hz,1H),6.32(t,J=5.5Hz,1H),6.13(s,1H),4.06(s,1H),3.93(t,J=5.4Hz,2H),3.88–3.72(m,1H),3.68–3.56( m,26H),3.54(t,J=5.1Hz,4H),3.45(q,J=5.5Hz,4H),3.38(s,2H),3.15(d,J=78.6Hz,3H),2.96(d,J=8.8Hz,2H),2.72(d,J=48.5Hz, 1H), 2.44(d,J=8.7Hz,2H), 2.29(tt,J=6.8,3.7Hz,1H), 2.08–1.87(m,2H), 1.67–1.58(m,1H), 1.43–1.29(m,3H), 0.75–0.66(m,2H).

[0229] Example 6

[0230]

[0231] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)heptapolyethylene glycol (CAS: 857891-82-8), the preparation method was the same as in Example 2; MS (ESI): m / z 648 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ8.24(d,J=45.3Hz,2H),7.37(d,J=5.2Hz,1H),6.99–6.81(m,3H),6.78(d,J=7.8H z,1H),6.49(t,J=7.4Hz,1H),6.44(d,J=5.2Hz,1H),6.12(s,1H),3.94(s,4H),3.65(s,36H),3.46(t,J =5.5Hz,6H),3.33(s,4H),2.97(s,2H),2.71(s,1H),2.50(s,2H),2.35–2.27(m,1H),2.23(t,J=7.6Hz, 1H), 2.10–1.95 (m, 1H), 1.69–1.56 (m, 1H), 1.54–1.38 (m, 3H), 0.72 (d, J = 6.3Hz, 2H), 0.47–0.37 (m, 2H).

[0232] Example 7

[0233]

[0234] Intermediate B1-1:

[0235]

[0236] In the synthesis of intermediate B1-1, except for the synthesis of the 7-membered ring which uses a racemic starting material instead of a chiral starting material, the other preparation methods can be found in the literature Bioorganic & Medicinal Chemistry Letters 17(2007)1903–1907. MS(ESI): m / z 420.2 [M+H] + .

[0237] Intermediate B1-2:

[0238]

[0239] Intermediate B1-1 (2.6 g, 5 mmol) was dissolved in tetrahydrofuran under an ice-water bath. Sodium hydride (dispersed in paraffin, 60% content, 400 mg, 10 mmol) was added, and the reaction was carried out under a nitrogen atmosphere and an ice bath for 0.5 h. Benzyl bromoacetate (2.3 g, 10 mmol) was then added. The ice bath was removed, and the reaction was carried out at room temperature for 8 h. Thin-layer chromatography confirmed the completeness of the reaction. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, dried, and purified by column chromatography to give 2.3 g of colorless oily substance B1-2, yield 70%. MS (ESI): m / z 668 [M+H] + ,568[M–Boc+H] + .

[0240] Intermediate B1-3:

[0241]

[0242] Intermediate B1-2 (2.2 g, 3.3 mmol) was dissolved in methanol and tetrahydrofuran (1:1 v / v). 220 mg of palladium on carbon (10% palladium content) was added, and the mixture was purged with hydrogen three times. The reaction was carried out at room temperature for 3 hours, and thin-layer chromatography confirmed complete reaction. The palladium on carbon was removed by direct filtration, and the mixture was evaporated to dryness to give white solid B1-3 (1.5 g, 80% yield). No further purification was required, and the mixture could be directly added to the next step. MS (ESI): m / z 578, [M+H] + ,478[M–Boc+H] + .

[0243] Intermediate B1-4:

[0244]

[0245] Intermediate B1-3 (60 mg, 0.1 mmol), HATU (46 mg, 0.12 mmol), and intermediate A2-2 (70 mg, 0.1 mmol) were dissolved in DMF (10 mL). DIPEA (35 μL, 0.2 mmol) was added, and the mixture was reacted at room temperature for 3 hours. After confirming the reaction was complete by thin-layer chromatography, saturated sodium chloride solution was added to the reaction solution. The mixture was extracted twice with ethyl acetate, washed three times with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. After purification by column chromatography (dichloromethane:methanol = 20:1), 94 mg of a yellow oily substance, B1-4, was obtained, with a yield of 75%. MS (ESI): m / z 626 [M / 2+H] + ,601[M / 2-Boc+H] + .

[0246] Compound 7

[0247]

[0248] Intermediate B1-4 (75 mg, 0.06 mmol) was dissolved in dichloromethane (5 mL), and 1 mL of hydrochloric acid / dioxane (molar concentration: 4N) was added. The mixture was reacted at room temperature for 5 hours. After confirming the completeness of the reaction by thin-layer chromatography, the solution was evaporated to dryness. Semi-preparative liquid chromatography purification yielded 40 mg (0.035 mmol, yield 58%) of a pale yellow oily compound 7. MS (ESI): m / z 576 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.35(dd,J=5.5,2.2Hz,1H),7.26–7.20(m,5H),7.20–7.09(m,2H),7.09–6.97(m,1H),6.97–6.80(m,3H),6.80–6.63(m,2H), 6.48(t,J=7.6Hz,1H),6.42(dd,J=5.6,2.2Hz,1H),6.12(s,1H),4.17–4. 04(m,1H),3.93(t,J=5.4Hz,2H),3.86(dd,J=15.5,8.1Hz,1H),3.63(d,J= 2.4Hz,8H),3.55(t,J=4.9Hz,4H),3.44(dt,J=11.5,5.1Hz,8H),3.36–3. 15(m,3H),3.01–2.91(m,2H),2.72(dd,J=13.7,5.6Hz,1H),2.65–2.55(m ,1H),2.54–2.39(m,3H),2.33–2.25(m,1H),2.25–2.16(m,1H),2.08–1.9 3(m,2H),1.78(s,2H),1.31(s,2H),0.75–0.65(m,2H),0.44–0.36(m,2H).

[0249] Example 8

[0250]

[0251] Except for replacing intermediate A1-3 with intermediate B1-3, the preparation method was the same as in Example 3; MS (ESI): m / z 620 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.36(d,J=5.4Hz,1H),7.24(q,J=6.6Hz,5H),7.16(td,J =7.5,6.5,1.7Hz,1H),7.10–7.00(m,1H),7.00–6.80(m,4H),6.77(d,J=7.9Hz ,1H),6.61(s,1H),6.48(t,J=7.3Hz,1H),6.42(d,J=5.4Hz,1H),6.12(s,1H), 4.26–4.04(m,1H),3.94(t,J=5.4Hz,2H),3.87(dd,J=15.7,7.9Hz,1H),3.72– 3.57(m,16H),3.54(t,J=4.9Hz,4H),3.46(d,J=9.9Hz,10H),3.37–3.28(m,1H ),3.22(d,J=14.3Hz,2H),2.96(t,J=7.8Hz,2H),2.73(dd,J=13.8,5.7Hz,1H) ,2.61(dd,J=13.7,7.8Hz,1H),2.55–2.40(m,3H),2.34–2.19(m,2H),2.04–1. 94(m,2H),1.37–1.23(m,1H),0.71(dd,J=6.6,1.8Hz,2H),0.46–0.32(m,2H).

[0252] Example 9

[0253]

[0254] Except for replacing intermediate A1-3 with intermediate B1-3, the preparation method was the same as in Example 4; MS (ESI): m / z 642 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.36 (d, J = 5.4Hz, 1H), 7.27–7.21 (m, 4H), 7.19–7.11 ( m,1H),7.10–6.99(m,1H),6.98–6.72(m,5H),6.52–6.46(m,2H),6.43(d,J= 5.4Hz,1H),6.12(s,1H),4.25–4.01(m,1H),3.94(t,J=5.4Hz,2H),3.86(dd ,J=15.6,5.9Hz,1H),3.72–3.58(m,20H),3.54(t,J=5.1Hz,4H),3.45(dt,J= 9.9,5.2Hz,7H),3.33(dd,J=13.8,6.7Hz,1H),3.30–3.15(m,2H),2.98(q,J =9.7,7.9Hz,3H),2.72(dd,J=13.7,5.6Hz,1H),2.60(dd,J=13.7,7.8Hz,1H) ,2.54–2.40(m,3H),2.29(dq,J=6.6,3.3Hz,1H),2.27–2.17(m,1H),1.79(s ,1H),1.37–1.23(m,2H),0.71(dd,J=6.6,1.9Hz,2H),0.40(p,J=4.6Hz,2H).

[0255] Example 10

[0256]

[0257] Except for replacing intermediate A1-3 with intermediate B1-3, the preparation method was the same as in Example 5; MS (ESI): m / z 664 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.36(d,J=5.4Hz,1H),7.27–7.19(m,5H),7.19–7.13(m,1H) ,7.10–6.98(m,1H),6.98–6.80(m,3H),6.77(d,J=7.7Hz,2H),6.52–6.46(m,1H), 6.44(d,J=5.4Hz,1H),6.11(s,1H),4.22–4.02(m,1H),3.93(q,J=9.4,7.4Hz,2H) ,3.87–3.70(m,1H),3.63(q,J=4.9,3.8Hz,24H),3.55(t,J=5.1Hz,4H),3.45(tt, J=7.7,4.1Hz,7H),3.33(dd,J=13.9,6.6Hz,1H),3.23(d,J=14.3Hz,2H),3.15–3. 01(m,1H),2.96(t,J=7.8Hz,2H),2.74(dd,J=13.9,5.8Hz,1H),2.63(dd,J=13.8, 7.6Hz,1H),2.56–2.40(m,3H),2.28(td,J=6.7,3.3Hz,2H),1.98(s,2H),1.85–1. 56(m,1H),1.26(d,J=5.0Hz,2H),0.71(dd,J=6.6,1.9Hz,2H),0.44–0.34(m,2H).

[0258] Example 11

[0259]

[0260] Except for replacing intermediate A1-3 with intermediate B1-3, the preparation method was the same as in Example 6; MS (ESI): m / z 686 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.38(d,J=5.4Hz,1H),7.28–7.20(m,4H),7.17(t,J=6.7Hz,1H),7. 11–7.00(m,1H),6.98–6.81(m,3H),6.81–6.69(m,2H),6.53–6.47(m,1H),6.45(d,J=5.4 Hz,1H),6.39(d,J=5.4Hz,1H),6.12(s,1H),4.24–4.02(m,1H),3.95(t,J=5.4Hz,2H),3. 87(dd,J=15.6,4.8Hz,1H),3.71–3.60(m,28H),3.55(d,J=5.2Hz,4H),3.46(qd,J=8.0,6. 7,3.1Hz,7H),3.35(dd,J=13.8,6.7Hz,1H),3.31–3.17(m,2H),3.14–3.00(m,1H),2.97( dd,J=8.9,6.8Hz,2H),2.74(dd,J=13.7,5.7Hz,1H),2.61(dd,J=13.6,7.8Hz,1H),2.45( dd,J=8.8,6.7Hz,3H),2.30(tt,J=7.7,4.2Hz,1H),2.27–2.19(m,1H),2.00(s,2H),1.72 (d,J=62.9Hz,1H),1.27(d,J=5.1Hz,1H),0.72(dd,J=6.6,1.9Hz,2H),0.46–0.36(m,2H).

[0261] Example 12

[0262]

[0263] Intermediate B3-1: N-(26-azido-3,6,9,12,15,18,21,24-octaoxohexyl)-3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalo-1-carbonyl)thiophene-3-yl)oxy)phenylpropionamide

[0264]

[0265] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)-heptaethylene glycol (CAS: 857891-82-8), the preparation process is the same as A2-1. MS (ESI): m / z 937.3 [M+H] + .1 H NMR (500MHz, CDCl3) δ7.36(d,J=5.4Hz,1H),7.22(s,1H),6.88(ddd,J=8.5,7.1,1.5Hz,1H),6.83(dd,J=8.3,1.5Hz,1H),6.77(d ,J=8.1Hz,1H),6.49(ddd,J=8.3,7.2,1.5Hz,1H),6.44(d,J=5.4Hz,1H),6.27(t,J=5.6Hz,1H),6.13(s,1H),3.94(t,J=5.4Hz,2H ),3.70–3.58(m,24H),3.55(dd,J=5.6,4.5Hz,2H),3.46(td,J=5.4,1.7Hz,4H),3.39(t,J=5.1Hz,2H),2.96(d,J=4.0Hz,4H),2. 88(s,2H),2.80(s,2H),2.44(dd,J=8.7,6.8Hz,2H),2.29(dt,J=6.6,3.1Hz,1H),0.75–0.68(m,2H),0.41(dd,J=4.0,2.3Hz,2H).

[0266] Intermediate B3-2: tert-butyl-29-azido-3-(3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalin-1-carbonyl)thiophene-3-yl)oxy)phenyl)propionyl)-6,9,12,15,18,21,24,27-octaoxa-3-azanaphthoic acid

[0267]

[0268] Intermediate B3-1 (280 mg, 0.3 mmol) was dissolved in tetrahydrofuran under an ice-water bath. Sodium hydride (60% concentration, 24 mg, 0.6 mmol) was added, and the reaction was carried out under a nitrogen atmosphere and an ice bath for 0.5 hours. Tert-butyl bromoacetate (117 mg, 0.6 mmol) was then added. The ice bath was removed, and the reaction was carried out at room temperature for 8 hours. Thin-layer chromatography confirmed the completeness of the reaction. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, dried, and purified by column chromatography to obtain 205 mg of yellow oily substance B3-2, with a yield of 65%. MS (ESI): m / z 526.5 [M / 2+H] + .

[0269] Intermediate B3-3: tert-butyl-29-amino-3-(3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalin-1-carbonyl)thiophene-3-yl)oxy)phenyl)propionyl)-6,9,12,15,18,21,24,27-octaoxa-3-azanaphthoic acid

[0270]

[0271] The preparation process was the same as for A2-2, except that intermediate B3-2 was used instead of intermediate A2-1; MS (ESI): m / z 513.3 [M / 2+H] + .

[0272] Intermediate B3-4: di-tert-butyl3-(3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalin-1-carbonyl)thiophene-3-yl)oxy)phenyl)propionyl)-6,9,12,15,18,21,24,27-octaoxazole-3,30-diazadecanoic acid

[0273]

[0274] Intermediate B3-3 (300 mg, 0.29 mmol), potassium bicarbonate (30 mg, 0.3 mmol), and tert-butyl bromoacetate (48 μL, 0.3 mmol) were dissolved in acetonitrile and reacted at room temperature for 2-3 hours. The reaction was terminated when a significant amount of the target product was detected by thin-layer chromatography. The reaction solution was filtered to remove solids, and the filtrate was concentrated and purified by preparative TLC to obtain 125 mg of intermediate B3-4, with a yield of approximately 38%. MS (ESI): m / z 526.5 [M / 2+H] + .

[0275] Intermediate B3-5: di-tert-butyl3-(2-(3-benzyl-4-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butyryl)-2-oxo-1,4-diaza-1-yl)acetyl)-30-(3-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalin-1-carbonyl)thiophene-3-yl)oxy)phenyl)propionyl)-6,9,12,15,18,21,24,27-octaoxa-3,30-diazadecanoic acid)

[0276]

[0277] Except for replacing intermediate A2-2 with intermediate B3-4, the rest of the preparation process is the same as that of intermediate A2-3.

[0278] Compound 12

[0279]

[0280] Except for replacing intermediate A2-3 with intermediate B3-5, the rest of the preparation process was the same as in Example 2 for the final product. MS(ESI): m / z 744 [M / 2+H] + . 1 H NMR(500MHz,DMSO-d6)δ7.76(dd,J=8.0,5.4Hz,1H),7.48(dd,J=13.0,2.0Hz,3H),7.31–7.14(m,4H),7.12(dd,J=7.1,2.1Hz,1H),6.9 0–6.81(m,1H),6.77(t,J=8.5Hz,1H),6.72–6.61(m,2H),6.43(q,J=7.5Hz,1H),6.03(d,J=18.2Hz,1H),4.33(d,J=24.1Hz,1H),4.18–4 .00(m,1H),4.01–3.73(m,8H),3.67–3.39(m,34H),3.36(d,J=5.1Hz,4H),3.33–2.98(m,5H),2.80(q,J=8.3Hz,4H),2.70–2.62(m,1H) ,2.47(d,J=7.7Hz,2H),2.24(dh,J=10.1,3.5Hz,1H),1.78(d,J=21.0Hz,2H),0.69(d,J=6.5Hz,2H),0.30(dq,J=10.8,4.8,3.8Hz,2H).

[0281] Example 13

[0282]

[0283] Intermediate C1-1:

[0284]

[0285] C1-1 synthesis was performed according to Bioorganic & Medicinal Chemistry Letters 21(2011)3809–3812. MS (ESI): m / z 502.2 [M+H] + 402.2[M-Boc+H] + .

[0286] Intermediate C1-2:

[0287]

[0288] The preparation process was the same as for A1-2, except that intermediate C1-1 was used instead of intermediate A1-1; MS (ESI): m / z 650.2 [M+H] + 550.2[M-Boc+H] + .

[0289] Intermediate C1-3:

[0290]

[0291] The preparation process was the same as for A1-3, except that intermediate C1-2 was used instead of intermediate A1-2; MS (ESI): m / z 560.3 [M+H] + 460.3[M-Boc+H] + .

[0292] Intermediate C1-4:

[0293]

[0294] The preparation process was the same as for A2-3, except that intermediate C1-3 was used instead of intermediate A1-3; MS (ESI): m / z 617 [M / 2+H] + ,567[(M–Boc) / 2+H] + .

[0295] Compound 13

[0296]

[0297] Except for replacing intermediate A2-3 with intermediate C2-1, the preparation process was the same as in Example 2; MS (ESI): m / z 567 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.36(d,J=5.4Hz,1H),7.22(s,1H),7.14–7.04(m,1H),6 .98–6.81(m,3H),6.81–6.70(m,1H),6.67–6.60(m,1H),6.53–6.46(m,1H),6.4 4(d,J=5.4Hz,1H),6.40–6.31(m,1H),6.15(d,J=7.1Hz,1H),4.95(s,1H),4.71 (dd,J=13.0,4.1Hz,1H),4.46(d,J=6.8Hz,1H),4.21(s,1H),4.01(dd,J=9.5,2 .7Hz,1H),3.94(t,J=5.4Hz,2H),3.87–3.78(m,1H),3.78–3.70(m,2H),3.69– 3.59(m,10H),3.55(q,J=5.2Hz,5H),3.45(t,J=5.4Hz,5H),3.40–3.25(m,1H), 2.96(t,J=8.8Hz,2H),2.83–2.61(m,2H),2.54–2.41(m,3H),2.34–2.25(m,1H) ,1.15(s,4H),1.11(d,J=2.2Hz,5H),0.70(t,J=6.1Hz,2H),0.46–0.37(m,2H).

[0298] Example 14

[0299]

[0300] Intermediate C1-4 (90 mg) was dissolved in dichloromethane / methanol (V / V = 5:1, 3 mL), and 3 mL of hydrochloric acid / dioxane (molar concentration: 4N) was added. The mixture was reacted at room temperature for 10 hours. The solution was directly evaporated to dryness, and purified by semi-preparative HPLC to give 40 mg of a yellow oily compound 14. MS (ESI): m / z 540 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.43(s,1H),7.38–7.33(m,1H),7.23(s,1H),7.11–7.01(m,1H),6.98–6.82(m,3H),6.78(d,J=7. 9Hz,1H),6.65(s,1H),6.53–6.44(m,2H),6.18(s,1H),4.90(d,J=10.9Hz,1H),4.63(d,J=15.8Hz,1H),4.28–4.19(m,1 H),4.05–3.89(m,4H),3.78–3.68(m,1H),3.68–3.30(m,23H),2.96(t,J=7.7Hz,2H),2.79(dd,J=13.7,6.2Hz,1H),2.7 2–2.62(m,1H),2.45(td,J=13.8,6.6Hz,3H),2.30(dd,J=7.1,3.7Hz,1H),0.71(t,J=6.1Hz,2H),0.42(d,J=3.7Hz,2H).

[0301] Example 15

[0302]

[0303] Except for replacing 1-amino-11-azido-3,6,9,12,15-pentahepta-1-amine (CAS: 516493-93-9), 17-azido-3,6,9-trioxaundecan (CAS: 134179-38-7) was used; the rest of the preparation method was the same as in Example 13; MS (ESI): m / z 611 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.36 (d, J=5.4Hz, 1H), 7.23 (s, 1H), 7.15–7.03 (m, 1H ),6.98–6.81(m,4H),6.78(d,J=7.9Hz,1H),6.59–6.45(m,2H),6.44(d,J= 5.4Hz,1H),6.15(d,J=5.7Hz,1H),4.95(d,J=2.8Hz,1H),4.75–4.65(m,1H ),4.52–4.43(m,1H),4.25(dd,J=15.7,3.5Hz,1H),4.01(h,J=5.9Hz,1H),3 .93(q,J=7.9,6.5Hz,2H),3.87–3.70(m,2H),3.69–3.38(m,29H),3.36–3. 24(m,1H),2.97(dd,J=8.9,6.7Hz,2H),2.79(dd,J=14.0,5.9Hz,1H),2.69( td,J=14.1,13.0,6.6Hz,1H),2.54–2.41(m,3H),2.29(dq,J=6.8,3.4Hz,1 H),1.13(d,J=13.5Hz,9H),0.71(dt,J=6.6,3.3Hz,2H),0.46–0.36(m,2H).

[0304] Example 16

[0305]

[0306] Except for replacing 1-amino-11-azido-3,6,9,12,15-pentahepta-1-amine (CAS: 516493-93-9), 17-azido-3,6,9-trioxaundecan (CAS: 134179-38-7) was used, the preparation method was the same as in Example 14. MS (ESI): m / z 583 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.51(d,J=12.5Hz,1H),7.36(d,J=5.4Hz,1H),7.23(s,1H),7.10(d,J=13.9Hz,1H),6.98–6.81(m,3H),6.78( d,J=7.9Hz,1H),6.57–6.46(m,1H),6.44(d,J=5.4Hz,1H),6.16(s,1H),4.93(s,1H),4.85(d,J=16.4Hz,1H),4.69–4.59(m,1H),4.4 6(s,0H),4.26–4.16(m,1H),4.06–3.88(m,4H),3.79–3.69(m,1H),3.68–3.36(m,29H),2.96(dd,J=8.8,6.8Hz,2H),2.79(dt,J=13 .9,7.0Hz,1H),2.69(dd,J=13.7,7.6Hz,1H),2.57–2.34(m,4H),2.29(td,J=6.6,3.4Hz,1H),0.75–0.67(m,2H),0.45–0.34(m,2H).

[0307] Example 17

[0308]

[0309] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)heptapolyethylene glycol (CAS: 857891-82-8), the preparation method was the same as in Example 13. MS (ESI): m / z 677 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ7.36(d,J=5.4Hz,1H),7.23(s,1H),7.09(td,J=9.5,8.9 ,7.0Hz,1H),6.99–6.80(m,3H),6.77(d,J=7.8Hz,2H),6.53–6.46(m,1H),6.4 4(d,J=5.4Hz,1H),6.38(d,J=6.0Hz,1H),6.13(s,1H),4.96(d,J=2.7Hz,1H), 4.70(dd,J=13.1,4.3Hz,1H),4.26(t,J=15.7Hz,1H),4.02(dp,J=11.0,3.7Hz ,1H),3.94(t,J=5.4Hz,2H),3.87–3.70(m,2H),3.69–3.38(m,41H),3.30(qd, J=8.5,6.7,4.5Hz,1H),2.96(dd,J=8.8,6.7Hz,2H),2.80(dd,J=13.8,5.6Hz, 1H),2.69(td,J=13.6,12.1,6.7Hz,1H),2.57–2.34(m,4H),2.29(tt,J=6.8,3 .7Hz,1H),1.19–1.05(m,9H),0.70(dt,J=6.6,3.3Hz,2H),0.45–0.34(m,2H).

[0310] Example 18

[0311]

[0312] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)heptapolyethylene glycol (CAS: 857891-82-8), the preparation method was the same as in Example 14. MS (ESI): m / z 649 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.55(d,J=6.0Hz,1H),7.36(d,J=5.4Hz,1H),7.23(s,1H),7.10(tt,J=9.9,5.2Hz,1H),6.98–6.81(m,3H),6.77(d,J= 7.9Hz,1H),6.61(d,J=5.3Hz,1H),6.53–6.46(m,1H),6.44(d,J=5.4Hz,1H),6.13(s,1H),4.95–4.82(m,1H),4.62(dd,J=14.7,11.2Hz,1H), 4.21(dt,J=11.0,3.8Hz,1H),4.07–3.89(m,4H),3.78–3.36(m,43H),2.96(dd,J=8.9,6.7Hz,2H),2.80(td,J=12.5,11.6,6.1Hz,1H),2.70( dd,J=13.7,7.5Hz,1H),2.56(dd,J=16.2,3.5Hz,1H),2.50–2.35(m,3H),2.29(tt,J=6.8,3.7Hz,1H),0.75–0.68(m,2H),0.44–0.37(m,2H).

[0313] Example 19

[0314]

[0315] Intermediate D1-1:

[0316]

[0317] The synthesis of intermediate D1-1 is described in J. Med. Chem. 2015, 58, 3315-3328.

[0318] Compound 19

[0319]

[0320] Except for replacing intermediate A1-3 with intermediate D1-1, the preparation method was the same as in Example 13; MS (ESI): m / z 664 [M / 2+H] + . 1H NMR(400MHz, CDCl3)δ8.80(s,1H),8.41(d,J=5.8Hz,1H),7.34(s,1H),7.16–6.99(m,2H),7.00–6.86(m,1H),6.80(s,1H),6.71(s,1H), 6.49(d,J=8.7Hz,2H),6.38(d,J=7.3Hz,1H),6.30(d,J=5.7Hz,1H),5.77(s,1H),4.95(d,J=2.6Hz,1H),4.71(dd,J=12.9,4.2Hz,0H),4 .47(dd,J=7.5,2.9Hz,0H),4.22(dd,J=15.6,10.0Hz,1H),4.09–3.95(m,1H),3.97–3.69(m,3H),3.69–3.27(m,22H),3.02(t,J=7.7Hz, 2H), 2.74(dtd,J=32.1,13.6,6.5Hz,2H),2.59–2.34(m,4H),2.25(d,J=14.0Hz,1H),1.13(d,J=15.6Hz,9H),0.66(s,3H),-0.22(s,1H).

[0321] Example 20

[0322]

[0323] Except for replacing intermediate A1-4 with intermediate D1-1, the preparation method was the same as in Example 14; MS (ESI): m / z 536 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ8.78(s,1H),8.41(d,J=5.7Hz,1H),7.44(s,1H),7.34(s,1H),7.06(dt,J=17.7,8.5Hz,2H),6.98–6.86 (m,1H),6.71(s,1H),6.49(d,J=7.7Hz,1H),6.45–6.28(m,2H),5.79(s,1H),4.96–4.78(m,1H),4.65(t,J=13.6Hz,1H),4.26 (d,J=9.8Hz,1H),3.99(t,J=9.6Hz,2H),3.74(d,J=13.2Hz,0H),3.70–3.29(m,26H),3.02(t,J=7.6Hz,2H),2.79(dd,J=13.9 ,6.5Hz,1H),2.67(dd,J=13.7,7.7Hz,1H),2.51(d,J=8.1Hz,3H),2.32–2.24(m,1H),0.76–0.53(m,3H),-0.11–-0.31(m,1H).

[0324] Example 21

[0325] 2-((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butyryl)-3-(tert-butoxymethyl)-2-oxoperazin-1-yl)-N-(15-(2,5-dichloro-4-((2-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxalin-1-carbonyl)thiophene-3-yl)oxy)phenyl)-3,6,9-trioxa-12-azapentadecanyl)acetamide

[0326]

[0327] Intermediate E1-1:

[0328]

[0329] Under ice-water bath conditions, A1-4 (517 mg, 1 mmol) was added to a tetrahydrofuran solution, followed by 4 mL of borane-tetrahydrofuran solution (molar concentration: 1N). After half an hour, the ice-water bath was removed, and the reaction was continued at room temperature for 4 hours. Thin-layer chromatography confirmed the completeness of the reaction. The reaction was quenched by slow dropwise addition of methanol, evaporated to dryness, and separated by column chromatography to obtain 420 mg of a pale yellow solid, E1-1, with a yield of 84%. MS (ESI): m / z 503 [M+H] + . 1H NMR (400MHz, CDCl3) δ7.35 (d, J=5.4Hz, 1H), 7.18 (s, 1H), 6.88 (ddd, J=8.6, 7.1, 1.5Hz, 1H), 6.81 (d d,J=8.3,1.5Hz,1H),6.76(d,J=7.9Hz,1H),6.49(ddd,J=8.4,7.2,1.5Hz,1H),6.43(d,J=5.4Hz,1H ),6.08(s,1H),3.94(t,J=5.4Hz,2H),3.69(t,J=6.3Hz,2H),3.47(t,J=5.4Hz,2H),2.79–2.66(m,2 H),2.28(tt,J=6.8,3.7Hz,1H),1.91–1.78(m,2H),0.69(dt,J=6.6,3.3Hz,2H),0.44–0.34(m,2H).

[0330] Intermediate E1-2:

[0331]

[0332] Intermediate E1-1 (400 mg, 0.8 mmol) was dissolved in dichloromethane (15 mL), and Dess-Martin oxidant (424 mg, 1 mmol) was added. The mixture was reacted at room temperature for 2 hours, evaporated to dryness, and separated by column chromatography to obtain 330 mg of pale yellow solid E1-2, with a yield of 83%. MS (ESI): m / z 501 [M+H] + .

[0333] Intermediate E1-3:

[0334]

[0335] Intermediate E1-2 (300 mg, 0.6 mmol) and 1-amino-11-azido-3,6,9-trioxaundecan (130 mg, 0.6 mmol) were dissolved in anhydrous methanol. After stirring at room temperature for 10 hours, sodium borohydride (38 mg, 1 mmol) was added, and the reaction was continued at room temperature for 2 hours. Thin-layer chromatography confirmed the complete reaction. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, washed once with water, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was then redissolved in tetrahydrofuran / aqueous solution (V / V = 1:1), and sodium bicarbonate (84 mg, 1 mmol) and Boc anhydride (220 mg, 1 mmol) were added. The reaction was carried out at room temperature for 10 hours, and thin-layer chromatography confirmed the complete reaction. After extraction twice with ethyl acetate, drying over anhydrous magnesium sulfate, and evaporation to dryness, column chromatography yielded 216 mg of a pale yellow oil, E1-3, in 45% yield. MS (ESI): m / z 803 [M+H] +,703[M-Boc+H] + .

[0336] Intermediate E1-4:

[0337]

[0338] Except for replacing A2-1 with E1-3, the preparation process is the same as that for A2-2; MS (ESI): m / z 777 [M+H] + .

[0339] Intermediate E1-5:

[0340]

[0341] Except for replacing intermediate A2-2 with intermediate E1-4 and intermediate A1-3 with intermediate C1-3, the preparation process is the same as that for preparing A2-3; MS (ESI): m / z 560 [(M-2Boc) / 2+H] + .

[0342] Compound 21

[0343]

[0344] The preparation process was the same as for compound 1, except that intermediate E1-5 was used instead of intermediate A1-3. MS (ESI): m / z 560 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.36(d,J=5.4Hz,1H),7.18(d,J=2.7Hz,1H),7.10(dt,J=10.4 ,7.7Hz,1H),6.98–6.80(m,3H),6.77(d,J=7.9Hz,1H),6.53–6.46(m,1H),6.43(dd,J =5.4,1.1Hz,1H),6.11(s,1H),5.34(td,J=5.6,3.3Hz,1H),4.95(d,J=2.8Hz,1H),4. 70(dd,J=13.2,4.3Hz,1H),4.18(dd,J=21.2,15.7Hz,1H),4.07–3.97(m,1H),3.93(q ,J=7.1,6.2Hz,2H),3.88–3.70(m,2H),3.64(d,J=10.6Hz,10H),3.55(dt,J=8.2,5. 1Hz, 2H), 3.45 (dt, J=7.5, 4.6Hz, 4H), 3.30 (td, J=8.1, 4.2Hz, 1H), 2.89 (q, J=5.2Hz, 2H),2.84–2.62(m,6H),2.29(tt,J=6.8,3.7Hz,1H),1.85(p,J=7.6Hz,2H),1.63(q,J =7.3Hz,2H),1.13(d,J=14.0Hz,9H),0.70(td,J=6.7,4.8Hz,2H),0.47–0.35(m,2H).

[0345] Example 22

[0346]

[0347] Except for replacing intermediate C1-4 with intermediate E1-5, the preparation process was the same as in Preparation Example 14. MS(ESI): m / z 534 [M / 2+H] + . 1H NMR (400MHz, CDCl3) δ7.75 (s, 1H), 7.36 (d, J = 5.4Hz, 1H), 7.19 (s, 1H), 7.09 (ddd, J = 10.4, 8.6, 6.7Hz, 1H), 6. 98–6.81(m,3H),6.78(d,J=7.9Hz,1H),6.53–6.46(m,1H),6.45(d,J=5.4Hz,1H),6.13(s,1H),4.92(s,1H),4 .26–4.13(m,1H),4.09–3.88(m,4H),3.72–3.37(m,22H),2.90(q,J=5.4,5.0Hz,2H),2.77(dq,J=14.0,7.3,6 .8Hz,3H),2.68(q,J=7.6Hz,3H),2.59–2.36(m,2H),2.34–2.26(m,1H),0.76–0.66(m,2H),0.45–0.35(m,2H).

[0348] Example 23

[0349]

[0350] Intermediate F1-1

[0351]

[0352] Except for replacing A1-4 with intermediate C1-3, the preparation process was the same as that for E1-1; MS (ESI): m / z 546 [M+H] + ,446[M–Boc+H] + .

[0353] Intermediate F1-2

[0354]

[0355] Except for replacing E1-1 with F1-1, the preparation process is the same as that for E1-2; MS (ESI): m / z 544 [M+H] + ,444[M–Boc+H] + .

[0356] Intermediate F1-3:

[0357]

[0358] Intermediate F1-2 (110 mg, 0.2 mmol) and intermediate A2-2 (140 mg, 0.2 mmol) were dissolved in anhydrous methanol. After stirring at room temperature for 10 hours, sodium borohydride (16 mg, 0.4 mmol) was added, and the reaction was continued at room temperature for 2 hours. Thin-layer chromatography confirmed the completeness of the reaction. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, washed once with water, dried over anhydrous magnesium sulfate, evaporated to dryness, and separated by column chromatography to obtain 97 mg of a pale yellow oil, F1-3, in 40% yield. MS (ESI): m / z 610 [M / 2+H] + ,560[(M–Boc) / 2+H] + .

[0359] Compound 23

[0360]

[0361] Except for replacing A2-3 with F1-3, the preparation process is the same as that for A2-4; MS (ESI): m / z 560 [M / 2+H] + . 1 H NMR(500MHz,DMSO-d6)δ8.74(s,1H),8.07(t,J=5.7Hz,3H),8.03(t,J=5.7Hz,1H ),7.79(d,J=5.4Hz,1H),7.63–7.46(m,2H),7.41(s,1H),6.85(ddd,J=8.6,7.2, 1.6Hz,1H),6.75(d,J=8.2Hz,1H),6.66(t,J=6.3Hz,2H),6.44(td,J=7.6,1.4Hz ,1H),4.62(dt,J=19.1,3.0Hz,1H),4.43(td,J=11.5,9.6,6.3Hz,1H),3.91–3.6 3(m,7H),3.63–3.42(m,11H),3.42–3.33(m,5H),3.25–3.04(m,7H),2.96(ddq,J =20.8,14.4,6.2Hz,2H),2.81(dd,J=8.7,6.7Hz,2H),2.74(dd,J=9.2,5.8Hz,2H ),2.36(dd,J=8.7,6.8Hz,2H),2.25(tt,J=6.8,3.7Hz,1H),1.10(s,3H),1.07(s ,2H),1.04(s,2H),0.96(s,2H),0.70(dd,J=6.6,2.0Hz,2H),0.35–0.25(m,2H).

[0362] Example 24

[0363]

[0364] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)heptapolyethylene glycol (CAS: 857891-82-8), the preparation process was the same as in Preparation Example 23; MS (ESI): m / z 670 [M / 2+H] + . 1 H NMR (500MHz, Methanol-d4) δ7.64(d,J=5.3Hz,1H),7.42(s,1H),7.35(s,1H),7.28–7.19(m,1H),6.95–6.89(m,1H),6.87(d,J=8.1Hz,1 H),6.75–6.70(m,1H),6.50(t,J=7.0Hz,2H),6.05(s,1H),4.86(d,J=23.7Hz,1H),4.64(d,J=13.1Hz,0H),4.54(s,0H),3.94(dt,J=26.1 ,6.3Hz,6H),3.84–3.73(m,4H),3.72–3.57(m,30H),3.51(dt,J=11.1,5.3Hz,4H),3.41–3.33(m,4H),3.09(s,2H),2.96(t,J=7.5Hz,2H) ,2.92–2.80(m,1H),2.52(t,J=7.5Hz,2H),2.33(dp,J=6.7,3.5Hz,1H),1.18(s,4H),1.11(s,5H),0.79–0.66(m,2H),0.43–0.31(m,2H).

[0365] Example 25

[0366]

[0367] Except for replacing 1-amino-11-azido-3,6,9-trioxaundecanane (CAS: 134179-38-7) with O-(2-aminoethyl)-O′-(2-azidoethyl)-heptapolyethylene glycol (CAS: 857891-82-8), the preparation process was the same as in Example 22. MS (ESI): m / z 642 [M / 2+H] + . 1H NMR(500MHz, Methanol-d4)δ7.62(d,J=5.4Hz,1H),7.33(s,1H),7.31–7.24(m,1H),7. 15(dtd,J=14.7,8.2,7.5,3.9Hz,1H),6.91(ddd,J=8.6,7.2,1.5Hz,1H),6.84(dd,J=8 .3,1.4Hz,1H),6.70(d,J=7.8Hz,1H),6.49(d,J=5.4Hz,1H),6.46(td,J=7.6,1.4Hz,1 H),6.02(s,1H),5.34(ddd,J=5.6,4.4,1.1Hz,1H),4.76(dt,J=6.4,3.4Hz,1H),4.61( d,J=14.4Hz,1H),4.43(dd,J=7.4,3.5Hz,1H),4.08(ddd,J=11.3,3.6,1.6Hz,1H),4.0 4–3.79(m,4H),3.71–3.53(m,34H),3.53–3.43(m,6H),2.94(dd,J=8.3,6.9Hz,2H),2. 91–2.76(m,4H),2.66–2.55(m,1H),2.48(dd,J=8.3,6.9Hz,2H),2.29(tt,J=6.8,3.7H z,1H),2.08–1.96(m,2H),1.60(t,J=7.3Hz,1H),0.74–0.68(m,2H),0.38–0.31(m,2H).

[0368] Experimental Example 1: In vitro test of TGR5 agonist activity and DPP4 inhibitory activity

[0369] 1.1 TGR5 agonist activity assay method

[0370] Stable hTGR5 / CRE / HEK293 or mTGR5 / CRE / HEK293 cell lines were obtained by transfecting HEK293 cells with expression plasmids carrying the human or mouse TGR5 gene (hTGR5-pcDNA3.1 and mTGR5-pcDNA3.1, respectively) and CRE-driven fluorescent reporter gene plasmids (pGL4.29, Promega, Madison, WI, USA). These cells were used to test the in vitro TGR5 agonistic activity of compounds.

[0371] Before the test, cells were seeded in 96-well plates using DMEM containing 10% FBS and cultured overnight at 37°C with 5% CO2. The next day, cells were incubated for 5.5 hours with fresh culture medium containing various concentrations of the compounds. A positive control was performed using 20 μM INT-777. After incubation, the activity of firefly luciferase was measured using the Steady-Glo Luciferase Assay System (Promega) according to protocol, thus reflecting the agonistic effect of the compounds on TGR5. EC 50 The value was calculated using GraphPad Prism software.

[0372] 1.2 DPP4 inhibitory activity test method:

[0373] Enzyme activity was detected by in vitro incubation with mouse serum and a fluorescent substrate (Gly-Pro-7-amido-4-methylcoumarin hydrobromide, Gly-Pro-7-AMC).

[0374] 35 μL / well of serum enzyme activity reaction buffer (25 mM HEPES, 140 mM NaCl, pH adjusted to 7.8, MgCl2 added to a final concentration of 80 mM) and 5 μL / well of C57 mouse serum were added to each well of a 96-well plate. 10 μL of the compound diluted to the specified concentration with serum enzyme activity reaction buffer was added to each sample well, and 0.5% DMSO (containing the same concentration as the compound group) was added to each negative control well. No serum was added to the blank control wells; only an equal volume of serum enzyme activity reaction buffer and solvent control (0.5% DMSO) were added. 50 μL / well of the substrate Gly-Pro-7-AMC was diluted to 10 μM with serum enzyme activity reaction buffer and added to each well of the 96-well plate. The emission intensity at 460 nM under 380 nM excitation light was measured using a multi-mode microplate reader. Dynamic monitoring was performed for 18 minutes, with measurements taken every 3 minutes for a total of 7 measurements, and the reaction rate was recorded.

[0375] The inhibition rate (%) is calculated using the formula: [1 - (SB) / (NB)] × 100%

[0376] Wherein, S: sample; B: blank control; N: negative control.

[0377] IC 50 The value was calculated using GraphPad Prism software.

[0378] 1.3 Results of in vitro activity tests of some compounds

[0379] Methods of expressing activity range: A: 0-10 nM; B: 10-100 nM; C: 100-500 nM; D: >500 nM.

[0380] The experimental results are shown in Table 1:

[0381] Table 1

[0382] compound <![CDATA[hTGR5(EC 50 )]]> <![CDATA[mTGR5(EC 50 )]]> <![CDATA[mDPP4(IC 50 ) <!-- 42 -->]]> 1 B A C 2 A A C 3 A A B 4 A A B 5 A A B 6 A A B 7 A A C 8 A A C 9 A A C 10 A A C 11 A A C 12 B A B 13 A A C 14 A A C 15 A A C 16 A A B 17 A A C 18 A A B 19 C C B 20 C C C 21 C C C 22 C C B 23 A A C 24 A A B 25 A A C OL3 86nM 17nM 69.98μM

[0383] The structural formula of OL3 is as follows. The preparation method is referenced in OL3, a novel low-absorbed TGR5 agonist with reduced side effects, lowered blood glucose via dual actions on TGR5 activation and DPP-4 inhibition (Acta Pharmacologica Sinica (2016) 37:1359-1369).

[0384]

[0385] The above data indicate that the compound in this patent has good TGR5 agonist activity and DPP4 inhibitory activity.

[0386] Experiment Example 2: Caco-2 Cell Permeability Experiment

[0387] Experimental Methods: Caco-2 cells were derived from ATCC (Cat#HTB-37) and cultured in DMEM medium containing 10% FBS. The culture environment was 37°C, 5% CO2, and 90% humidity. Cells were passaged every seven days at a ratio of 1:10. Cells from passages 30 to 40 were used for the experiment. After 21 days of culture, the integrity of the cell monolayer was confirmed by measuring transmembrane resistance.

[0388] The monolayer cell has two ends: the apical side and the basolateral side. Compound transport from the apical to the basolateral side is abbreviated as AB, while compound transport from the basolateral to the apical side is abbreviated as BA; both are measured using similar methods. Propranolol and atenolol were used as high-permeability and low-permeability controls, respectively. Digoxin was used as a positive control for Pgp efflux substrates. The experimental procedure was roughly as follows: the monolayer cell membrane was washed three times with HBSS, the compounds were diluted and added to the corresponding chambers (pH 6.8 at the apical side and pH 7.4 at the basolateral side). Incubation was performed at 37°C for 95 min. Samples were collected at 5 and 95 min on the side where the compounds were added, and at 35 and 95 min on the side receiving the compounds. The concentration of the compounds in the samples was determined by LC-MS / MS.

[0389] P app The calculation formula is as follows:

[0390] P app =(V A / (SA×T))×([drug] acceptor / [drug] initial donor )

[0391] Among them, V A This is the volume of the receiving chamber, SA is the surface area of ​​the membrane, T is the total transport time, [drug] acceptor This refers to the concentration of the compound on the receiving side, [drug]. initial donor It is the concentration on the side where the compound was added at 0 min.

[0392] The results of cell permeability experiments for some compounds, such as Caco-2, are shown in Table 2.

[0393] Table 2

[0394] compound <![CDATA[Papp A-B(1×10 -6 cm / s)]]> <![CDATA[Papp B-A(1×10 -6 cm / s)]]> 5 <0.01 14.4 12 0.01 0.01

[0395] AB: from the apical side to the basal side; BA: from the basal side to the apical side.

[0396] The above data shows that the compound in this invention patent has extremely low cell permeability, which is beneficial for achieving local intestinal effects.

[0397] Experimental Example 3: Intestinal Enzyme Activity and Plasma Enzyme Activity Experiment

[0398] Experimental Methods: ICR mice were randomly grouped according to body weight. A single oral dose of either the compound or a solvent was administered as a control. Serum was collected 8 hours after administration, and approximately 1 cm of ileal tissue was collected, its contents removed, and the tissue was weighed. The samples were rinsed with ice-cold saline, blotted dry with filter paper, and placed into 2 mL grinding tubes. Both serum and tissue samples were stored in an ultra-low temperature freezer for later use.

[0399] On the day of testing, 5 μL of serum sample was added to 45 μL of serum enzyme activity reaction buffer (25 mM HEPES, 140 mM NaCl, pH adjusted to 7.8, MgCl2 added to a final concentration of 80 mM) and then added to a 96-well microplate. Only 50 μL of serum enzyme activity reaction buffer was added to the blank control wells. The substrate Gly-Pro-7-AMC was diluted to 10 μM with serum enzyme activity reaction buffer and added to the microplate at 50 μL / well. The emission intensity at 460 nm under 380 nm excitation light was dynamically detected using a multi-mode microplate reader, measured every 3 minutes until the 18th minute, for a total of 7 measurements, and the reaction rate was recorded. The percentage of relative enzyme activity in the serum of mice in each treatment group was calculated with the pre-drug serum enzyme activity as 100%.

[0400] Ileal samples were added to an appropriate amount of tissue enzyme activity reaction buffer (50mM Tris buffer, adjusted to pH 7.5) at a concentration of 50 mg / ml and vortexed at 50 Hz for 120 s to disrupt the tissue. The disrupted ileal tissue samples were centrifuged at 4℃ and 10000 rpm for 5 min, and the supernatant was used for enzyme activity assay. In a 96-well microplate, 30 μL of tissue enzyme activity reaction buffer was added to each well, followed by 20 μL of the above tissue sample per well, and the mixture was stirred. The substrate Gly-Pro-7-AMC was diluted to 10 μM with tissue enzyme activity reaction buffer and added to the microplate at 50 μL per well. The emission intensity at 460 nm under 380 nm excitation light was dynamically detected using a multi-mode microplate reader, measured every 1 minute until the 10th minute, for a total of 11 measurements, and the reaction rate was recorded. The relative percentage of enzyme activity in the ileum of mice in each treatment group was calculated with the enzyme activity of the blank control group as 100%.

[0401] The results of the ileal DPP4 enzyme activity assay are shown in Table 3, and the results of the plasma DPP4 enzyme activity assay are shown in Table 4.

[0402] Table 3

[0403]

[0404] Table 4

[0405]

[0406] The above experiments show that the compound in this patent can significantly inhibit the activity of intestinal DPP4 enzyme, while having only a weak inhibitory effect on the activity of systemic DPP4 enzyme, thus exhibiting good local intestinal effects.

[0407] Experiment Example 4: DSS-induced colitis test in mice

[0408] Experimental Methods: BALB / c mice were randomly divided into groups according to body weight. The normal control group was given standard drinking water. The model mice were given free access to 3% DSS aqueous solution to induce ulcerative colitis. From the date of induction, each group of model mice was administered either the solvent control or a corresponding dose of compound 12 via gavage. During the experiment, mouse body weight, fecal characteristics, and fecal blood were recorded daily. The Disease Index (DAI) was calculated based on various scores. The experimental results are shown below. Figures 1-3 As shown.

[0409] Figure 1 The results, which showed that the administered compound 12 significantly slowed down weight loss compared to the solvent control group, were displayed as a curve of weight change.

[0410] Figure 2 The symptom scoring curve is displayed. The symptom scoring criteria are: stool characteristics (0 points: normal; 2 points: loose stool; 4 points: watery diarrhea) and fecal occult blood (0 points: no blood in stool; 2 points: minor blood in stool; 4 points: severe blood in stool). From Figure 2 The results showed that compound 12 significantly improved the symptoms of enteritis in mice compared with the solvent control group.

[0411] Figure 3 The display shows the Disease Activity Index (DAI) scoring curve. The DAI is obtained by combining weight and symptom scores. The scoring criteria cover weight loss (0 points: normal; 1 point: weight loss 0%-5%; 2 points: weight loss 5%-10%; 3 points: weight loss 10%-15%; 4 points: weight loss >15%), stool characteristics (0 points: normal; 2 points: loose stool; 4 points: watery diarrhea), and fecal occult blood (0 points: no blood in stool; 2 points: small amount of blood in stool; 4 points: severe blood in stool).

[0412] Depend on Figure 3 The results showed that compound 12 significantly slowed the disease progression of DSS-induced colitis in mice compared to the solvent control group. Figures 1-3 It is known that the compounds of this invention can slow down the disease progression in a DSS-induced mouse colitis model.

[0413] Experiment Example 5: Drug Concentration Testing Experiment in Plasma, Ileum, Gallbladder, and Bile

[0414] Experimental Methods: ICR mice were randomly divided into groups of three based on body weight. They were fasted overnight before the experiment and administered a single oral dose of 30 mg / kg of the compound from Example 12. Blood samples were collected at 1 h, 2 h, 4 h, and 8 h. The mice were then sacrificed, and their ileum, gallbladder, and bile were collected for drug concentration testing. Quantitative analysis was performed using LC-MS / MS. The experimental results are as follows: Figure 4 As shown.

[0415] Depend on Figure 4 It is known that the compounds of the present invention are mainly distributed in intestinal tissues, with low systemic exposure.

[0416] Experiment Example 6: Gallbladder Experiment

[0417] Experimental Methods: ICR mice were randomly divided into groups of 7-8 mice each according to their body weight. They were fasted overnight before the experiment and administered a single oral dose of the compound and a blank control. Feeding began 0.5 hours after the last administration. Mice were sacrificed 4 hours after administration, dissected, and the gallbladder volume and bile weight were measured. The gallbladder volume was equal to length × width × height × 3.14 / 6. The bile weight was measured using an analytical balance. The experimental results are shown in Table 5.

[0418] Table 5

[0419]

[0420] Among them, MN6 is a positive control, whose full chemical name is 1-[4-(2,5-dichlorophenoxy)pyridine-3-formyl]-4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline, see WO2011089099 for details.

[0421] The above data indicate that the compound in this patent has good gallbladder safety compared to systemic TGR5 agonists.

Claims

1. A compound as shown in Formula I, its stereoisomer, or a pharmaceutically acceptable salt, in, A represents a thiophene ring or a pyridine ring; R1, R2, R3, and R4 are all hydrogen; R5 is cyclopropyl; R6 and R8 are halogens, and R7 and R9 are hydrogen; or R7 and R9 are halogens, and R6 and R8 are hydrogen. W1 and W2 are each independently a group represented by IIa or IIb: ； ； m is 1 or 2; n is 2 or 3; R 21 It is hydrogen, methyl, or carboxymethyl; R 22 It is hydrogen; L represents a group indicated by IIIa or IIIb: ； ； Where q is an integer from 3 to 8; r is 2; X represents the group shown in IV: R represents H, benzyl, hydroxy-substituted methyl groups, and tert-butoxy-substituted methyl groups; s represents an integer from 1 to 3.

2. The compound of formula I according to claim 1, its stereoisomers, or pharmaceutically acceptable salts, characterized in that, The compound represented by Formula I is selected from compounds represented by Formulas IA, IB, IC, and ID: The definitions of A, R1, R2, R3, R4, R5, R6, R7, R8, R9, L, and X are as described in claim 1.

3. The compound of formula I according to claim 1 or 2, its stereoisomers, or pharmaceutically acceptable salts, characterized in that, The compound represented by Formula I is selected from the compounds shown in the following structural formulas: 。 4. A method for preparing the compound according to any one of claims 1 to 3, characterized in that, The method is selected from at least one of the following methods 1 to 4: Method 1: Prepare the compound represented by general formula IA, including at least the following steps: S1, compound Ia was amide condensed with the linker precursor compound NH2-L-N3 to obtain compound Ib; S2 and compound Ib are then subjected to a hydrogenation reduction reaction to obtain compound Ic; S3, compound Id reacts with benzyl bromoacetate via a nucleophilic substitution reaction to give Ie; S4 and compound Ie undergo a hydrogenation-reduction reaction to debenzylate and yield If. S5. Compound If and compound Ic undergo an amide condensation reaction to yield compound Ig; S6, compound Ig undergoes deprotection reaction to give compound IA; The reaction formula is as follows: Method 2: Prepare the compound represented by general formula IB, including at least the following steps: S1, Compound Ib reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IIa; S2, Compound IIa is converted to Compound IIb via a hydrogenation-reduction reaction; S3, compound IIb and compound If undergo an amide condensation reaction to yield compound IIc; S4. Compound IIc reacts with tert-butyl bromoacetate via a nucleophilic substitution reaction to give compound IId. S5, compound IId is deprotected and hydrolyzed to give IB; The reaction formula is as follows: Method 3: Prepare the compound represented by general formula IC, including the following steps: S1, compound If reacts with borane to give compound IIIa; S2, Compound IIIa is oxidized with Dess-Martin reagent to give compound IIIb; S3, compound IIIb and compound Ic react via a reductive amination reaction to yield compound IIIc; S4, compound IIIc is deprotected to give compound IC; The reaction formula is as follows: Method 4: Prepare the compound represented by general formula ID, including the following steps: S1, compound Ia is reduced with borane to give compound IVb; S2, compound IVb was oxidized with Dess-Martin reagent to give compound IVc; S3, compound IVc and the linker precursor compound NH2-L-N3 undergo a reductive amination reaction, followed by reaction with Boc anhydride to yield compound IVd; S4, Compound IVd was reduced with an azide group under hydrogen-palladium-carbon conditions to give compound IVe; S5, compound IVe and compound If undergo amide condensation to yield compound IVf; S6. Compound IVf is deprotected by the protecting group Boc to give compound ID; The reaction formula is as follows: Where X1 is selected from ; The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, L, A, R, S, and X are the same as those in the corresponding claims.

5. The use of any compound, stereoisomer, or pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of a medicament for treating diseases mediated by TGR5 and / or DPP4, said diseases being selected from type 2 diabetes and inflammatory bowel disease.

6. A pharmaceutical composition comprising a therapeutically effective amount of one or more compounds, stereoisomers, and pharmaceutically acceptable salts selected from any one of claims 1-3, and optionally, pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further comprises other drugs selected from one or more of hypoglycemic drugs, weight-loss drugs, non-alcoholic fatty liver disease drugs, and inflammatory bowel disease drugs.

8. The pharmaceutical composition according to claim 7, characterized in that, The non-alcoholic fatty liver disease mentioned above is non-alcoholic steatohepatitis.

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