GLP-1 compound-containing pharmaceutical composition, preparation method therefor and use thereof

Abstract

Provided are a pharmaceutical composition and a use thereof for treating diseases. The pharmaceutical composition contains a GLP-1 compound and an absorption enhancer.

Description

A pharmaceutical composition containing a GLP-1 compound, its preparation method and application Technical Field

[0001] This invention belongs to the field of therapeutic peptide technology, specifically relating to a pharmaceutical composition containing a GLP-1 compound, its preparation method, and its application. Background Technology

[0002] Human GLP-1 and its analogues have low oral bioavailability. Both exposure and bioavailability of human GLP-1 and its analogues are very low after oral administration. If formulated in specific amounts with certain absorption enhancers, human GLP-1 and its analogues can only be detected in plasma after oral administration.

[0003] Currently, several patent documents have reported pharmaceutical compositions of oral GLP-1 peptides, such as CN201180060463.1, WO2010 / 020978, WO2019149880, WO2013 / 189988, and WO2013 / 139694.

[0004] However, existing oral GLP-1 receptor agonist drugs must be administered once daily, which is a relatively high frequency of administration and does not improve patient convenience and compliance. Therefore, there is still a need to develop oral GLP-1 peptide drugs that can be administered at a frequency of less than once daily. Summary of the Invention

[0005] In view of the problems existing in the prior art, this application provides a pharmaceutical composition containing a GLP-1 compound, a method for preparing the composition and its application, which can improve bioavailability and achieve lower frequency of administration.

[0006] Specifically, the present invention is achieved through the following technical solutions:

[0007] In a first aspect, this application provides a pharmaceutical composition comprising: a compound or a salt thereof and an absorption enhancer.

[0008] As a preferred embodiment of the present invention, the compound is selected from:

[0009] As a preferred embodiment of the present invention, the compound is selected from:

[0010] As a preferred embodiment of the present invention, the compound is selected from:

[0011] As a preferred embodiment of the present invention, the compound is selected from:

[0012] As a preferred embodiment of the present invention, the content of the compound is 0.1-50%, preferably 1%-50%, and more preferably 3%-40%.

[0013] Wherein, each unit of the compound or its salt, calculated as a compound, is 5mg-1000mg, preferably 5mg-500mg, more preferably 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 350mg, 400mg, 450mg, or 500mg.

[0014] As a preferred embodiment of the present invention, the content of the absorption promoter is 10%-95%, preferably 15%-90%, more preferably 30%-90%, and even more preferably 50%-80%.

[0015] As a preferred embodiment of the present invention, the absorption promoter is selected from at least one of SNAC, decanoic acid, sodium decanoate, sodium dodecyl sulfate, polyoxyethylene ether, sodium octanoate, and Labrasol.

[0016] SNAC is sodium 8-(2-hydroxybenzoylamino)octanoate, with the following structure:

[0017] As a preferred embodiment of the present invention, the absorption promoter is SNAC or sodium decanoate.

[0018] As a preferred embodiment of the present invention, the absorption promoter is SNAC, and the content of SNAC is 20%-75%, preferably 30%-60%, and more preferably 35%-55%.

[0019] As a preferred embodiment of the present invention, the absorption enhancer is SNAC. The dosage of SNAC is 100mg-400mg, preferably 150mg, 200mg, 250mg, 275mg, 300mg, 325mg, or 350mg, and more preferably 300mg.

[0020] In a preferred embodiment of the present invention, the absorption promoter is SNAC and sodium decanoate. Preferably, the mass ratio of SNAC to sodium decanoate is in the range of 300:90-300:300, further comprising 300:130-300:160, or the mass ratio of SNAC to sodium decanoate is in the range of 300:200-300:300, preferably 300:250.

[0021] As a preferred embodiment of the present invention, the absorption promoter is SNAC and Labrasol, wherein the mass ratio of SNAC to Labrasol is in the range of 300:90-300:300, further comprising 300:130-300:160, or the mass ratio of SNAC to Labrasol is in the range of 300:200-300:300, preferably 300:250.

[0022] As a preferred embodiment of the present invention, the pharmaceutical composition further includes a lubricant, wherein the lubricant content is 0.1-10%, preferably 0.5-5%, more preferably 0.5%-3%, more preferably 0.5%-1%, and even more preferably 1%-3%.

[0023] As a preferred embodiment of the present invention, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium stearate, and calcium stearate, with magnesium stearate being preferred.

[0024] The lubricant can be added in the required amount at once and mixed with other excipients to prepare tablets of the target size, or a portion of the lubricant can be added first, mixed with some excipients, and then another portion of the lubricant can be added and mixed to prepare tablets of the target size.

[0025] As a preferred embodiment of the present invention, the content of magnesium stearate is 0.1-10%, preferably 0.2-5%, and more preferably 0.3-3%.

[0026] As a preferred embodiment of the present invention, when the magnesium stearate is added internally and externally, the content of the magnesium stearate when added internally is 1-10%, preferably 1-5%, more preferably 1-3%; and the content of the magnesium stearate when added externally is 0.1-3%, preferably 0.1-1%.

[0027] Among them, internal addition refers to mixing the excipients with a portion of magnesium stearate first; external addition refers to adding a portion of magnesium stearate to the mixed excipients again and mixing to prepare the target particles. It should be noted that the method of adding magnesium stearate has little impact on the performance of the prepared tablets.

[0028] As a preferred embodiment of the present invention, the pharmaceutical composition further includes a water-soluble growth promoter, wherein the content of the water-soluble growth promoter is 5%-50%, 5%-30%, more preferably 10%-30%, more preferably 15-30%, and even more preferably 10%-20%.

[0029] As a preferred embodiment of the present invention, the water-soluble growth promoter is selected from at least one of resorcinol, catechol, pyrogallol, gentian acid xylene sulfonate, p-toluene sulfonate, nicotinamide, dimethylbenzamide, diethylbenzamide, 1-methylnicotinamide, salicylic acid and p-hydroxybenzoic acid, preferably nicotinamide.

[0030] As a preferred embodiment of the present invention, the pharmaceutical composition further includes: a water-soluble growth promoter, preferably, the water-soluble growth promoter is selected from nicotinamide, and the absorption promoter is selected from SNAC and / or sodium decanoate.

[0031] As a preferred embodiment of the present invention, the water-soluble growth promoter is nicotinamide, and the nicotinamide content is 10-50%, preferably 10-30%, more preferably 12-25%, and even more preferably 10%-20%.

[0032] As a preferred embodiment of the present invention, the mass ratio of SNAC to nicotinamide ranges from 300:90 to 300:300, and further includes 300:90 to 300:110.

[0033] As a preferred embodiment of the present invention, the pharmaceutical composition further includes: a lubricant and a water-soluble growth promoter, wherein the lubricant is selected from magnesium stearate, the water-soluble growth promoter is selected from nicotinamide, and the absorption promoter is selected from SNAC and / or sodium decanoate.

[0034] Preferably, the mass ratio of SNAC to sodium decanoate is in the range of 300:130-300:160, and the mass ratio of SNAC to nicotinamide is in the range of 300:90-300:110.

[0035] When the product contains two absorption promoters but no water-soluble growth promoter, the preferred mass ratio of SNAC to other absorption promoters is 300:200-300:300; when the product contains both absorption promoters and water-soluble growth promoters, the preferred mass ratio of SNAC to water-soluble growth promoters (+ other absorption promoters, if any) is 300:200-300:300.

[0036] As a preferred embodiment of the present invention, the amount of magnesium stearate is 0.1%-5%, preferably 0.5%-5%, more preferably 0.5%-3%; the amount of nicotinamide is 5%-30%, preferably 10%-25%, more preferably 15%-20%; the amount of SNAC is 40%-60%, preferably 45%-55%, more preferably 50%-55%; and the amount of sodium decanoate is 10%-40%, preferably 15%-35%, more preferably 20%-30%.

[0037] As a preferred embodiment of the present invention, sodium decanoate and / or nicotinamide are adjusted according to the dosage of SNAC in a certain ratio. Specifically, when the SNAC is 300mg, the dosage of sodium decanoate is 130mg-160mg, specifically including: 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, etc.; when the SNAC is 300mg, the dosage of nicotinamide is 90mg-110mg, specifically including: 92mg, 94mg, 96mg, 98mg, 100mg, 102mg, 104mg, 106mg, 108mg, 110mg, etc.

[0038] As a preferred embodiment of the present invention, the pharmaceutical composition further includes: a water-soluble growth promoter selected from nicotinamide, and an absorption enhancer selected from SNAC. The mass ratio of SNAC to nicotinamide ranges from 300:100 to 300:300, preferably 300:150 to 300:300, and more preferably 300:200 to 300:300.

[0039] As a preferred embodiment of the present invention, the pharmaceutical composition further includes a lubricant and a water-soluble growth promoter, wherein the lubricant is selected from magnesium stearate and the water-soluble growth promoter is selected from nicotinamide.

[0040] As a preferred embodiment of the present invention, the pharmaceutical composition further includes a filler, wherein the filler content is 1-20%, preferably 5-15%, and more preferably 10%-15%.

[0041] The filler is selected from at least one of microcrystalline cellulose, starch, lactose, mannitol, sorbitol, and silicified microcrystalline cellulose, preferably microcrystalline cellulose. The content of microcrystalline cellulose is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0042] As a preferred embodiment of the present invention, when the pharmaceutical composition contains SNAC, sodium decanoate, and nicotinamide, the ratio of SNAC, sodium decanoate, and nicotinamide is approximately 2:1:1; when the pharmaceutical composition contains SNAC and sodium decanoate, the ratio of SNAC and sodium decanoate is 1.2:1-2:1; when the pharmaceutical composition contains SNAC and nicotinamide, the ratio of SNAC and nicotinamide is 1.2:1-2:1; when the pharmaceutical composition contains SNAC and Labrasol, the ratio of SNAC and Labrasol is 1.2:1-2:1.

[0043] The “content” mentioned above refers to the percentage of a certain substance in the total weight of the pharmaceutical composition (such as tablets, capsules, etc.), where the total weight is the sum of the weights of all excipients and active ingredients; the “dosage” mentioned above refers to the percentage of a certain substance in the total weight of the excipients, where the total weight of the excipients does not include the weight of the active ingredient.

[0044] As a preferred embodiment of the present invention, the pharmaceutical composition comprises the prescription shown in Tables 1-11.

[0045] Secondly, the present invention also provides a method for preparing the pharmaceutical composition as described above, comprising:

[0046] The compound is mixed with excipients and lubricant; lubricant may be added again or not; tableting is performed to obtain the target tablet.

[0047] As a preferred technical solution, before or after mixing the compound and excipients, one or more of the compound and excipients can be granulated, or one or more of the excipients can be granulated and then mixed with the compound, and then other excipients except lubricants can be added, and granulation can be performed again to prepare mixed particles of the compound and excipients.

[0048] The granulation methods include one or two of dry granulation, wet granulation, and melt granulation;

[0049] Mix the granules and lubricant; add lubricant again or not; compress to obtain the target tablet.

[0050] Thirdly, the present invention also provides the use of the pharmaceutical composition described above in the preparation of a medicament for treating a disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, cardiovascular disease, neurodegenerative diseases (including AD), chronic kidney disease, diabetic nephropathy, peripheral artery disease, and / or heart failure, heart failure including HFpEF, HFrEF, HFmEF, etc.

[0051] For clarity, this article defines the general terminology used in the description of compounds.

[0052] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0053] The term "pharmaceutically acceptable salt" refers to a salt of the deuterated compound of the present invention, prepared by a deuterated compound with specific substituents discovered in the present invention and a pharmaceutically acceptable acid or base.

[0054] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids linked together by amide (or peptide) bonds. The term polypeptide is used interchangeably with the terms "peptide" and "protein".

[0055] As used herein, the term "GLP-1 compound" refers to a polypeptide capable of binding to and / or activating the GLP-1 receptor. In other words, a GLP-1 compound is a polypeptide with GLP-1 activity. In other words, GLP-1 is a GLP-1 receptor agonist. GLP-1 can bind to and / or activate other types of receptors; that is, a compound qualifies as a GLP-1 compound simply by binding to and / or activating the GLP-1 receptor, regardless of any other receptor interactions that may be involved. In addition to the amino acid residues responsible for GLP-1 receptor interactions, GLP-1 compounds may also contain other amino acid residues that do not participate in GLP-1 receptor interactions.

[0056] In some embodiments, the term "GLP-1 analog" or "GLP-1 analogue" as used herein refers to a peptide, or a compound, that is a variant of human glucagon-like peptide-1 (GLP-1(7-37)). GLP-1(7-37) has the sequence HAEGTFTSDV SSYLEGQAAKEFIAWLVKGRG (SEQ ID No:1). In some embodiments, the term "variant" refers to a compound that comprises one or more amino acid substitutions, deletions, additions, and / or insertions.

[0057] For pharmaceuticals or pharmacologically active agents, the term "effective amount" or "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. For the oral dosage forms of this invention, the "effective amount" of one active substance in the composition refers to the quantity required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. A suitable effective amount in any given case can be determined by a person skilled in the art through routine testing.

[0058] "Optional" or "optionally" means that the event or condition described below may occur but is not required to occur, and the description includes both the scenario in which said event or condition occurs and the scenario in which said event or condition does not occur.

[0059] The compositions of the present invention can be solid compositions and administered orally. The compositions can be administered in several dosage forms, such as tablets, coated tablets, chewing gum, capsules (e.g., hard or soft gelatin capsules), or powders. The compositions can be further formulated into a drug carrier or drug delivery system, for example, to improve stability and / or solubility, or to further improve bioavailability. The compositions can be freeze-dried or spray-dried compositions.

[0060] In some embodiments, the composition may be granulated before compression. The composition may comprise an inner portion and an outer portion, wherein the inner portion is granulated and the outer portion is added after granulation. The inner portion may comprise a GLP-1 agonist, a delivery agent, and a binder. In some embodiments, the inner portion comprises povidone. The outer portion may comprise a filler, a lubricant, and / or a flow aid. In some embodiments, the outer portion comprises microcrystalline cellulose, such as Avicel, for example, Avicel PH120 or Avicel PH200. In some embodiments, the outer portion comprises magnesium stearate.

[0061] A tableting machine can be used to compress tableting materials into solid oral dosage forms, such as tablets. In a tableting machine, the tableting material is first filled (e.g., by forced feeding or gravity feeding) into a mold cavity. The tableting material is then compressed under pressure by a punch. Next, the resulting compressed material or tablet is ejected from the tableting machine. The above tableting process will be referred to herein as the "tableting process". Suitable tableting machines include, but are not limited to, rotary tableting machines and eccentric tableting machines. Detailed Implementation

[0062] The present application will be described in further detail below with reference to the embodiments, but the implementation of the present application is not limited thereto.

[0063] The synthetic route of compound 1 in Example 1 is as follows:

[0064] Compound 1

[0065] Step 1: F1 Synthesis

[0066] Synthesis process description: Compound F1 was obtained by solid-state synthesis.

[0067] (1) Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0068] (2) After washing, methanol and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0069] (3) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0070] (4) Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add them to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0071] (5) Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD) and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0072] (6) Repeat step (3) to remove the Fmoc protecting group.

[0073] (7) Weigh out Fmoc-Asp-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0074] (8) Weigh out tert-butyl hexadecanoate and repeat step (7) to carry out the condensation reaction.

[0075] (9) After mixing mercaptoethanol, DBU and DMF respectively, add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0076] (10) Weigh out Boc-Gly-OH and repeat step (7) to carry out the condensation reaction.

[0077] (11) The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0078] (12) The resin was cut with 20% TFE / DCM and the concentrated solution was filtered to obtain compound F1.

[0079] Step 2: Synthesis of F2 (Smigratide fully protected peptide resin)

[0080] The synthetic route of smegglutinin is referenced from the synthetic routes in patent applications CN200680006674.6 and WO2022096636A1.

[0081] His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-octadecanedioic acid monotert-butyl ester)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin

[0082] F2

[0083] Synthesis steps:

[0084] (1) Wang resin was used as a carrier. It was first swollen with DMF. After the swelling was completed, the resin was washed with DMF.

[0085] (2) Weigh out Fmoc-Gly-OH, HOBt and 4-dimethylaminopyridine (DMAP), dissolve them in DMF, add DIC and mix well. Then add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0086] (3) After washing, acetic anhydride and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0087] (4) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0088] (5) Weigh Fmoc-Arg(Pbf)-OH and HOBt, add DMF to mix and dissolve, then add DIC to activate for 3-5 min. After activation, add to the reactor to start the coupling reaction; the amino acid coupling reaction takes 1.0-3.0 h, and the reaction endpoint is monitored with ninhydrin throughout the coupling process. After coupling, wash the resin with DMF.

[0089] (6) Repeat step (5) to sequentially couple amino acids according to the peptide sequence to obtain F2 (smeglucopyrein fully protected peptide resin).

[0090] (7) After the reaction is complete, wash the peptide resin with dichloromethane and methanol and air dry at room temperature.

[0091] Step 3: Preparation of the target compound (F2+F1)

[0092] (1) First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling, wash the resin with DMF.

[0093] (2) Weigh out F1 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane and methanol respectively and air dry.

[0094] (3) Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0095] (4) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0096] (5) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0097] (6) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0098] (7) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0099] After lyophilization, the samples were aliquoted and stored as required. Samples were taken and tested to obtain the target compounds. The measured molecular weights of compound 1 were 4654.3, M / 3 = 1551.4, M / 4 = 1163.9, and M / 5 = 931.3.

[0100] The synthetic route of compound 2 in Example 2 is as follows:

[0101] Compound 2

[0102] Step 1: Synthesis of F3

[0103] Synthesis process description: Compound F3 was obtained by solid-state synthesis.

[0104] (1) Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0105] (2) After washing, methanol and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0106] (3) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0107] (4) Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add them to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0108] (5) Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD) and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0109] (6) Repeat step (3) to remove the Fmoc protecting group.

[0110] (7) Weigh out Fmoc-Asp-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0111] (8) Weigh out tert-butyl hexadecanoate and repeat step (7) to carry out the condensation reaction.

[0112] (9) After mixing mercaptoethanol, DBU and DMF respectively, add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0113] (10) Weigh out Boc-Gly-OH and repeat step (7) to carry out the condensation reaction.

[0114] (11) The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0115] (12) The resin was cut with 20% TFE / DCM and the concentrate was filtered to obtain compound F3.

[0116] Step 2: Preparation of the target compound (F2+F3)

[0117] (1) First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling, wash the resin with DMF.

[0118] (2) Weigh out F3 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane and methanol respectively and air dry.

[0119] (3) Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0120] (4) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0121] (5) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0122] (6) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0123] (7) Concentration and freeze-drying: The qualified fraction is concentrated, and the concentrated sample is filtered, divided into trays, and freeze-dried according to the process specifications. The freeze-dried sample is packaged and stored as required. Samples are taken for testing, and the target compound is obtained. The measured molecular weight of compound 2 is 4698.4, M / 3 = 1566.4, M / 4 = 1174.5, and M / 5 = 940.1.

[0124] Example 3 Preparation of Compound 3

[0125] Step 1: Synthesis of F4

[0126] Synthesis process description: Compound F4 was obtained by solid-state synthesis.

[0127] (1) Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0128] (2) After washing, methanol and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0129] (3) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0130] (4) Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add them to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0131] (5) Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD) and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0132] (6) Repeat step (3) to remove the Fmoc protecting group.

[0133] (7) Weigh out Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0134] (8) Weigh out tert-butyl hexadecanoate and repeat step (7) to carry out the condensation reaction.

[0135] (9) After mixing mercaptoethanol, DBU and DMF respectively, add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0136] (10) Weigh out Boc-Pro-OH and repeat step (7) to carry out the condensation reaction.

[0137] (11) The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0138] (12) The resin was cut with 20% TFE / DCM and the concentrate was filtered to obtain compound F4.

[0139] Step 2: Preparation of the target compound (F2+F4)

[0140] (1) First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling, wash the resin with DMF.

[0141] (2) Weigh out F4 and HOBt, dissolve them in DMF, add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane and methanol respectively and air dry.

[0142] (3) Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0143] (4) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0144] (5) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0145] (6) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0146] (7) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0147] After lyophilization, the samples were aliquoted and stored as required. Samples were taken for testing, and the target compounds were obtained. The measured molecular weights of compound 3 were 4708.4, M / 3 = 1569.5, M / 4 = 1177.4, and M / 5 = 942.2.

[0148] The synthetic route for compound 4 in Example 4 is as follows:

[0149] Compound 4

[0150] Step 1: Synthesis of F5:

[0151] Synthesis process description: Compound F5 was obtained by solid-state synthesis.

[0152] a: Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0153] b: After washing, methanol and DIEA are added separately for end-capping. After end-capping, the resin is washed several times with DMF.

[0154] c: Deprotect the resin twice with a 20% piperidine / DMF mixed solution, 10 minutes each time. After the deprotection is complete, wash the resin with DMF.

[0155] d: 2-Nitrobenzenesulfonyl chloride (NsCl) and DIEA are mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction is completed, the resin is washed with DMF.

[0156] e: Fmoc-aminocyclobutane methanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and then added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0157] f: Repeat step c to remove the Fmoc protecting group.

[0158] g: Weigh out Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. Add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0159] h: Weigh out tert-butyl hexadecanoate and repeat step g to carry out the condensation reaction.

[0160] i: Mercaptoethanol, dicycloamidine (DBU) and DMF were mixed and added to the resin to remove Ns protecting groups. After the reaction was completed, the resin was washed with DMF.

[0161] j: Weigh out Boc-Gly-OH and repeat step g to carry out the condensation reaction.

[0162] k: The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin. The resin was cut with 20% TFE / DCM, and the concentrated resin was obtained by filtration to obtain compound F5.

[0163] Step 2: Preparation of the target compound (F5+F2)

[0164] a. First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling is complete, wash the resin with DMF.

[0165] b. Weigh out F5 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane, and methanol respectively, and then air dry.

[0166] c. Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0167] d. Dissolve the crude product in acetonitrile / water solution by sonication, then filter through a 0.45 μm filter membrane. Transfer the filtered solution to a purification tank for crude HPLC analysis.

[0168] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0169] (2) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0170] (3) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0171] (4) The freeze-dried sample was packaged and stored according to the requirements. The sample was sampled and tested, and the target compound 4 was obtained. The measured molecular weight of compound 4 was 4708.4, M / 3 = 1570.2, M / 4 = 1177.9, and M / 5 = 942.5.

[0172] Example 5: Preparation of Compound 5

[0173] Compound 5

[0174] Step 1: F6 Synthesis:

[0175] Synthesis process description: Compound F6 was obtained by solid-state synthesis.

[0176] a. Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0177] b. After washing, methanol and DIEA are added separately for end-capping. After end-capping, the resin is washed several times with DMF.

[0178] c. Perform Fmoc removal twice with a 20% piperidine / DMF mixed solution, 10 minutes each time. After the deprotection is completed, wash the resin with DMF.

[0179] d. Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add the mixture to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0180] e. Fmoc-(1S,3R)-3-aminocyclohexanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and then added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0181] f. Repeat step c to remove the Fmoc protecting group.

[0182] g. Weigh out Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. Add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0183] h. Weigh out tert-butyl hexadecanoate and repeat step g to carry out the condensation reaction.

[0184] i. Mix mercaptoethanol, dicycloamidine (DBU) and DMF separately, and add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0185] j. Weigh out Boc-Gly-OH and repeat step g to carry out the condensation reaction.

[0186] k. The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0187] l. Cut the resin with 20% TFE / DCM, filter and concentrate to obtain compound F6.

[0188] Step 2: Preparation of the target compound (F6+F2)

[0189] a. First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling is complete, wash the resin with DMF.

[0190] b. Weigh out F6 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane, and methanol respectively, and then air dry.

[0191] c. Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0192] d. Dissolve the crude product in acetonitrile / water solution by sonication, then filter through a 0.45 μm filter membrane. Transfer the filtered solution to a purification tank for crude HPLC analysis.

[0193] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0194] (2) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0195] (3) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0196] (4) The freeze-dried sample was packaged and stored according to the requirements. The target compound 5 was obtained by sampling and testing. The measured molecular weight of compound 5 was 4722.4, M / 3 = 1574.8, M / 4 = 1181.4, and M / 5 = 945.3.

[0197] The synthetic route for compound 6 in Example 6 is as follows:

[0198] Compound 6

[0199] Step 1: F7 Synthesis:

[0200] Synthesis process description: Compound F7 was obtained by solid-state synthesis.

[0201] a. Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0202] b. After washing, methanol and DIEA are added separately for end-capping. After end-capping, the resin is washed several times with DMF.

[0203] c. Perform Fmoc removal twice with a 20% piperidine / DMF mixed solution, 10 minutes each time. After the deprotection is completed, wash the resin with DMF.

[0204] d. Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add the mixture to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0205] e. Fmoc-(1R,3R)-3-aminocyclohexanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and then added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0206] f. Repeat step c to remove the Fmoc protecting group.

[0207] g. Weigh out Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. Add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0208] h. Weigh out tert-butyl hexadecanoate and repeat step g to carry out the condensation reaction.

[0209] i. Mix mercaptoethanol, dicycloamidine (DBU) and DMF separately, and add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0210] j. Weigh out Boc-Gly-OH and repeat step g to carry out the condensation reaction.

[0211] k. The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0212] l. Cut the resin with 20% TFE / DCM, filter and concentrate to obtain compound F7.

[0213] Step 2: Preparation of the target compound (F7+F2)

[0214] a. First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling is complete, wash the resin with DMF.

[0215] b. Weigh out F7 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane, and methanol respectively, and then air dry.

[0216] c. Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0217] d. Dissolve the crude product in acetonitrile / water solution by sonication, then filter through a 0.45 μm filter membrane. Transfer the filtered solution to a purification tank for crude HPLC analysis.

[0218] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0219] (2) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0220] (3) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0221] (4) The freeze-dried sample was packaged and stored according to the requirements. The target compound 6 was obtained by sampling and testing. The measured molecular weight of compound 6 was 4722.4, M / 3 = 1574.8, M / 4 = 1181.4, and M / 5 = 945.3.

[0222] The excipients used in preparing the formulation of this invention are commercially available.

[0223] Preparation of tablets in Examples 7-12

[0224] Table 1 shows the composition of the tablets.

[0225] The tablet preparation process in Example 7 is as follows:

[0226] Step 1: Preprocessing

[0227] Sieve compound 1 and set aside.

[0228] Step 2: Granulation

[0229] (1) Wet granulation – Preparation of excipient granules (sodium decanoate)

[0230] Add an appropriate amount of purified water to the prescribed amount of sodium decanoate for wet granulation, sieve the granules, dry them in an oven to obtain dry granules, and then sieve them again to obtain excipient granules (sodium decanoate).

[0231] (2) Melt granulation – Preparation of excipient granules (SNAC + nicotinamide)

[0232] The excipient mixture (SNAC + nicotinamide) is placed in a container and heated until it melts at a temperature of 170-180°C. After cooling and solidification, it is ground into granules and sieved to obtain excipient granules (SNAC + nicotinamide).

[0233] Step 3: Mix

[0234] Compound 1, excipient granules (SNAC + nicotinamide), and excipient granules (sodium decanoate) are added to the prescription amount, mixed, then sieved, and mixed again. Magnesium stearate is added to the prescription amount and mixed to obtain mixed granules.

[0235] Step 4: Tableting

[0236] A rotary tablet press is used to compress the mixed granules from step 3 into tablets of the target size.

[0237] The tablets in Examples 8-12 were prepared using the same process as in Example 7.

[0238] Preparation of tablets in Examples 13-18

[0239] Table 2 shows the composition of the tablets.

[0240] The tablets in Examples 13-18 were prepared in accordance with the preparation process in Example 7.

[0241] Preparation of tablets in Examples 19-24

[0242] Table 3 shows the composition of the tablets.

[0243] The tablets in Examples 19-24 were prepared in accordance with the preparation process in Example 7.

[0244] Preparation of tablets in Examples 25-30

[0245] Table 4 Tablet Composition

[0246] The preparation process of the tablet in Example 25 is as follows:

[0247] Step 1: Preprocessing

[0248] Sieve compound 1 and set aside.

[0249] Step 2: Granulation

[0250] 2.1.1 Dry Granulation – Preparation of Excipient Granules (Sodium Decanoate)

[0251] Sodium decanoate was prepared according to the prescription in Table 4 and granulated using a dry granulator. The granules were then sieved and sized to obtain excipient granules (sodium decanoate).

[0252] 2.1.2 Dry Granulation – Preparation of Granules (Sodium Decanoate + Compound 1)

[0253] According to the prescription in Table 4, the excipient granules (sodium decanoate) prepared in step 2.1.1 are mixed with the compound, sieved, and then mixed again to obtain a granular mixture (sodium decanoate + compound). Then, the granular mixture (sodium decanoate + compound 1) is dry granulated using a dry granulator and sieved to obtain mixed granules (sodium decanoate + compound 1).

[0254] 2.2 Dry Granulation – Preparation of Excipient Granules (SNAC + Nicotinamide + Added Magnesium Stearate)

[0255] The prescribed amount of excipient granules (SNAC + nicotinamide + internally added magnesium stearate) was prepared using the method described in 2.1.2.

[0256] Step 3: Mix

[0257] Add the prescribed amount of excipient granules (SNAC + nicotinamide + internally added magnesium stearate) and mixed granules (sodium decanoate + compound 1) to a mixer, mix them, sieve them and mix them again; finally add magnesium stearate (external) and mix to obtain total mixed granules, compress the granules to obtain tablets of the target size.

[0258] Another preparation process for the tablets of Example 25 is as follows:

[0259] Step 1: Preprocessing

[0260] Sieve compound 1 and set aside.

[0261] Step 2: Granulation

[0262] 2.1 Wet Granulation – Preparation of Excipient Granules (Sodium Decanoate)

[0263] Add an appropriate amount of purified water to the prescribed amount of sodium decanoate for wet granulation, sieve, and then dry in an oven to obtain dry granules. After sieving, obtain excipient granules (sodium decanoate).

[0264] 2.2 Dry Granulation – Preparation of Excipient Granules (SNAC + Nicotinamide)

[0265] Weigh SNAC and nicotinamide according to the prescription amounts in Table 4 of the prescription composition, add them to the mixing tank, mix, sieve, and then mix again to obtain the excipient mixture (SNAC + nicotinamide); then, use a dry granulator to dry granulate the excipient mixture (SNAC + nicotinamide), sieve and granulate to obtain excipient granules (SNAC + nicotinamide).

[0266] Step 3: Mix

[0267] Add the excipient granules (SNAC + nicotinamide) and the prescribed amount of magnesium stearate (added internally) to the mixing tank, and mix to obtain a premixed powder (SNAC + nicotinamide + magnesium stearate); then, add the prescribed amount of compound 1 and excipient granules (sodium decanoate) to the premixed powder and mix using a multi-directional motion mixer, sieve and mix again; finally, add magnesium stearate (added externally) and mix to obtain mixed granules.

[0268] Step 4: Tableting

[0269] A rotary tablet press is used to compress the mixed granules from step 3 into tablets of the target size.

[0270] The tablets in Examples 26-30 were prepared using the same process as in Example 25.

[0271] Preparation of tablets in Examples 31-36

[0272] Table 5 Tablet Composition

[0273] The preparation process of the tablets in Example 31 is as follows:

[0274] Step 1: Preprocessing

[0275] Sieve compound 1 and set aside.

[0276] Step 2: Granulation

[0277] 2.1 Dry Granulation – Preparation of Granules (Sodium Decanoate + Compound 1)

[0278] According to the prescription in Table 6, the prescribed amounts of sodium decanoate and compound 1 are mixed, sieved, and then mixed again to obtain a granular mixture (sodium decanoate + compound 1). Then, the granular mixture (sodium decanoate + compound 1) is granulated by dry granulation using a dry granulator and sieved to obtain mixed granules (sodium decanoate + compound 1).

[0279] 2.2 Dry Granulation – Preparation of Excipient Granules (SNAC + Nicotinamide + Added Magnesium Stearate)

[0280] The prescribed amount of excipient granules (SNAC + nicotinamide + internally added magnesium stearate) was prepared using method 2.1.

[0281] Step 3: Mix

[0282] Add the prescribed amount of excipient granules (SNAC + nicotinamide + internally added magnesium stearate) and mixed granules (sodium decanoate + compound 1) to a mixer, mix them, sieve them and mix them again; finally add magnesium stearate (external) and mix to obtain total mixed granules, compress the granules to obtain tablets of the target size.

[0283] The tablets in Examples 32-36 were prepared using the same process as in Example 31.

[0284] Preparation of tablets in Examples 37-42

[0285] Table 6 Tablet Composition

[0286] The tablets of Examples 37-42 were prepared using the same process as in Example 31.

[0287] Preparation of tablets in Examples 43-48

[0288] Table 7 Tablet Composition

[0289] The tablets of Examples 43-48 were prepared using the same preparation processes as in Examples 25 and 31, or they can be prepared using the following method: Step 1: Premixing

[0290] Add the prescribed amount of compound, each excipient, and added raw materials to a mixing tank and mix using a multi-directional motion mixer for 5-10 minutes at 10-20 rpm. Transfer the mixture to a pulverizer and granulator, sieve it, and then mix for another 10-20 minutes at 10-20 rpm to obtain a premixed powder.

[0291] Step 2: Dry granulation

[0292] The premixed powder is dry granulated to obtain flakes, which are then sieved and granulated using a granulator to obtain inner granules.

[0293] Step 3: Total Mixing

[0294] Mix the internal particles using a multi-directional motion mixer for 5-10 minutes at 10-20 rpm, then transfer them to a pulverizer and granulator for sieving, and mix again for 5-10 minutes at 10-20 rpm.

[0295] Finally, add magnesium stearate (added externally) and mix for 5-10 minutes at 10-20 rpm to obtain 50 mg granules.

[0296] Step 4: Tableting

[0297] The ZPS016 rotary tablet press was used, with a 17*7mm capsule-shaped die. The tablets were pressed according to the theoretical tablet weight, and the production speed was set to 15-30 rpm.

[0298] Tablet preparation in Examples 49-54

[0299] Table 8 Tablet Composition

[0300] The tablets of Examples 49-54 were prepared using the same preparation processes as those of Examples 25 and 31.

[0301] Examples 55-60 Tablet Preparation

[0302] Table 9 Tablet Composition

[0303] The tablets of Examples 55-60 were prepared using the same preparation processes as those of Examples 25 and 31.

[0304] Examples 61-63

[0305] Table 10 Tablet Composition

[0306] The preparation processes of the raw material mixtures in Examples 61 and 62 are as follows:

[0307] Step 1: Mix

[0308] Mix the prescribed amount of raw and auxiliary materials for 5 minutes, pass through a 30-mesh sieve, and continue mixing for 10 minutes to obtain the raw and auxiliary material mixture.

[0309] The preparation process of the raw material mixture in Example 63 is as follows:

[0310] Step 1: Mix

[0311] Mix the raw and excipient materials (excluding octanoic acid and caprylic acid glycerol) in the prescribed amounts for 5 minutes, pass them through a 30-mesh sieve, and continue mixing for 10 minutes to obtain mixture A;

[0312] Add the prescribed amount of caprylic / capric acid glyceride (PEG-Glyceryl ester) to mixture A and mix thoroughly to obtain the raw material mixture.

[0313] Examples 64-66

[0314] Table 11 Tablet Composition

[0315] (1) Dry granulation – preparation of drug-containing granules

[0316] 1.1 Premix

[0317] Add the prescribed amounts of compound 3, sodium decanoate, and microcrystalline cellulose to a mixing tank and mix using a multi-directional motion mixer. Transfer the mixture to a pulverizer and granulator for sieving, and then mix for 5-10 minutes to obtain a drug-containing premixed powder.

[0318] 1.2 Granulation

[0319] The drug-containing premixed powder is dry-granulated to obtain flakes, which are then sieved and granulated using a granulator to obtain drug-containing granules.

[0320] (2) Dry granulation – preparation of excipient granules

[0321] 1.1. Premix

[0322] Add SNAC and nicotinamide to the mixing tank and mix using a multi-directional motion mixer. Then transfer to a pulverizer and granulator for sieving and mix for 5-10 minutes to obtain powder A. Add powder A and magnesium stearate (added internally) to the mixing tank and mix to obtain the auxiliary premix powder.

[0323] 1.2. Granulation

[0324] The premixed excipient powder is dry granulated to obtain flakes, which are then sieved and granulated using a granulator to obtain excipient granules.

[0325] (3) Total Mixing

[0326] Add the prescribed amount of excipient granules and drug-containing granules, mix using a multi-directional motion mixer for 5-10 minutes, transfer to a pulverizer and granulator for sieving, and mix again for 5-10 minutes. Finally, add magnesium stearate (added externally) and mix to obtain 50mg granules.

[0327] (4) Tableting

[0328] The ZPS016 rotary tablet press was used with a 17*7mm capsule-shaped die. The tablets were compressed according to the theoretical tablet weight, and the production speed was set to 15-30 rpm to obtain the target tablets.

[0329] Example 67: Evaluation of Dissolution Effect

[0330] Take the tablets from Examples 7-66 and, according to the "Chinese Pharmacopoeia 2020 Edition General Chapter 0931 Determination of Dissolution and Release Rate", the dissolution medium volume is 500 ml, the dissolution medium temperature is 37±0.2℃, and the paddle method is used at 50 rpm. Take 5 mL of solution at regular intervals. Finally, increase the speed to 200 rpm and run for 60 min. Take 5 mL of solution and immediately add the same volume of dissolution medium (pH 6.8 phosphate buffer) to the cup. Specific dissolution data for some examples are shown in Table 12.

[0331] Table 12 shows the tablet dissolution data.

[0332] As shown in Table 12, the tablets of the present invention can dissolve more than 70% in the dissolution medium pH 6.8 phosphate buffer within 15 minutes, preferably more than 80%.

[0333] Example 68 Pharmacokinetic Experiment

[0334] 1. Experimental materials

[0335] Crab-eating macaques: male, 6-9 kg, conducted at Guangdong Ruigu Biotechnology Co., Ltd.

[0336] Reagents: EDTA-K2, DMSO, acetonitrile, formic acid, and propranolol (internal standard) are all commercially available.

[0337] Instruments: Thermo Fisher LC-MS (Vanquish UPLC, TSQAltis Plus triple quadrupole mass spectrometer).

[0338] 2. Experimental Methods

[0339] Crab-eating macaques were fasted for at least 12 hours before administration. One tablet of the finished formulation was administered orally with 10 mL of water. Approximately 400 μL of venous blood was collected in EDTA-K2 anticoagulant tubes before administration and at 1, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours after administration. The blood was centrifuged at 12,000 rpm for 2 min, and the plasma was stored at -80°C for later analysis. A precise amount of the test sample was dissolved in ultrapure water to a concentration of 2 mg / mL to prepare a stock solution. An appropriate amount of the stock solution was accurately pipetted and diluted with 50% acetonitrile aqueous solution (V:V) to prepare a series of standard solutions. Accurately pipette 20 μL of each of the above standard series solutions and add 180 μL of blank plasma. Vortex to mix, preparing plasma samples equivalent to plasma concentrations of 1, 3, 10, 30, 100, 300, 500, 1000, 3000, and 5000 ng / mL. Perform dual-sample analysis for each concentration to establish a standard curve. QC samples are prepared in the same manner, with concentrations of 20, 400, and 4000 ng / mL. Take 20 μL of plasma and add 200 μL of acetonitrile solution containing propranolol (5 ng / mL) as internal standard. Vortex to mix and centrifuge at 4000 rpm for 5 min. Collect the supernatant for LC / MS / MS analysis. The LC / MS / MS detection conditions are as follows:

[0340] Chromatographic column: YMC-Triact C8 UPLC column, 50×2.1 mm l.DS-3μm.

[0341] Mobile phase: Water (0.1% formic acid) - methanol, gradient elution performed according to the table below.

[0342] 3. Data Processing

[0343] After LC-MS detection of blood drug concentration, pharmacokinetic parameters were calculated using WinNonlin 6.1 software and a non-compartmental model method. Results of some examples are shown in Table 13.

[0344] Table 13

[0345] As shown in Table 13, the oral administration of the prescription of this embodiment results in a high level of in vivo exposure.

[0346] Example 70: Pharmacokinetic Study in Rats

[0347] (1) Experimental materials

[0348] SD rats: male, 180-250g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0349] Reagents: DMSO (dimethyl sulfoxide), acetonitrile, methanol, formic acid, ammonium formate, and propranolol (internal standard) are all commercially available.

[0350] Instrument: LC-MS / MS (Thermo TSQAltis triple quadrupole mass spectrometer).

[0351] (2) Experimental methods

[0352] The prescription formulations listed in Tables 1-11 were weighed and mixed with ultrapure water to obtain the drug solution. The concentration of different prescription formulations was uniformly prepared to 10 mg / mL. After each formulation was administered to four rats by gavage, 200 μL of venous blood was collected at 15 min, 30 min, 1 h, 2 h, 5 h, 7 h, and 24 h into EDTA-K2 anticoagulant EP tubes. The tubes were centrifuged at 12000 rpm for 2 min, and the plasma was stored at -80℃ for later analysis. A certain amount of SAL0150 was accurately weighed and dissolved in DMSO to 2 mg / mL as a stock solution. An appropriate amount of the compound stock solution was accurately pipetted and diluted with 50% acetonitrile aqueous solution to prepare a series of standard solutions. 20 μL of each of the above standard solutions was accurately pipetted and added to 180 μL of blank plasma. The mixture was vortexed and mixed to prepare plasma samples equivalent to plasma concentrations of 1, 3, 10, 30, 100, 300, 1000, 3000, and 10000 ng / mL. Two samples were analyzed for each concentration to establish a standard curve. Take 30 μL of plasma, add 200 μL of acetonitrile methanol solution containing propranolol (100 μg / mL) as internal standard, vortex to mix, centrifuge at 4000 rpm for 10 min, take the supernatant, add 150 μL of purified water, vortex to mix again, and analyze the concentration of SAL0150 by LC-MS / MS.

[0353] 3. Data Processing

[0354] After detecting the blood concentration of the compound by LC-MS / MS, the pharmacokinetic parameters were calculated using WinNonlin software and a non-compartmental model method.

[0355] 4. The experimental results are shown in Table 14.

[0356] Table 14. Comparison of exposure levels in rats after oral administration of different formulations

[0357] As shown in Table 14, the formulation of the present invention has a good in vivo exposure level.

[0358] Example 71 GLP-1 Activity Assay

[0359] The agonistic effect of the test substance on GLP-1R was determined using U2OS-GLP-1R stable cell lines and HTRF assay.

[0360] Experimental methods:

[0361] 1. Following the experimental procedures of the cAMP-Gs Dynamic HTRF kit, dilute the 5x Stimulation buffer (SB) provided with the kit to 1xSB using ddH2O. Add IBMX to the 1xSB solution to a final concentration of 500 μM to prevent cAMP degradation. Then, use the 1xSB to prepare the working solution of the test substance and perform a series of serial dilutions.

[0362] 2. U2OS-GLP-R cells were digested and collected, resuspended, counted, and then diluted to a density of 2 x 10^6 cells using 1xSB solution. 6 Cells / mL, and then seeded into a 384-well cell plate at a rate of 5 μL per well, so that the number of cells per well is 10,000.

[0363] 3. Add 5 μL of the test substance to each well and incubate at 37°C for 30 mins.

[0364] 4. Prepare cAMP standard solutions of different concentrations according to the steps provided in the cAMP-Gs Dynamic HTRF kit and add them to 384-well cell plates.

[0365] 5. Dilute the cAMP d2 reagent and Eu Cryptate antibody provided in the kit to 1x using lysis & Detection Buffer. Add 5 μL of d2 and Eu to each well of a 384-well plate. Incubate at room temperature for 2 hours and then detect the results using the HTRF module (665 / 620 nm) of a microplate reader. Collect the experimental data.

[0366] 6. By plotting the signal values ​​against the compound concentration, curve fitting and EC50 calculation were performed using the nonlinear regression method in GraphPadPrism software. The test results show that the preferred compound of this invention exhibits excellent GLP-1 activity, with EC50 < 100 nM. The results are shown in Table 15.

[0367] Table 15 GLP-1 activity of compounds

[0368] Example 72 Pharmacokinetic Experiment

[0369] 1. Reagents and Instruments

[0370] Duchenne phosphate-buffered saline (DPBS) (batch number: F2326001, Shanghai Aladdin Biochemical Technology Co., Ltd.). LC-MS instrument (Thermo TSQ Altis Plus).

[0371] 2. Laboratory animals

[0372] SD rats: male, 180-250g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0373] 3. Formulation preparation

[0374] Accurately weigh the sample powder, ensuring it is free of Ca. 2+ / Mg 2+ The Duchenne phosphate buffer solution was completely dissolved and mixed to a concentration of 0.1 mg / mL. Administer intravenously at a rate of 2 mL / kg.

[0375] 4. Blood sample collection

[0376] After intravenous administration to rats, 200 μL of venous blood was collected before administration and at 0, 0.5 h, 2 h, 5 h, 24 h, 48 h, and 72 h after administration into EP tubes containing EDTA-K2 anticoagulant. The tubes were centrifuged at 12,000 rpm for 2 min, and the plasma was frozen at -20 °C for later analysis.

[0377] 5. Biological Analysis

[0378] Accurately weigh a certain amount of the test sample and dissolve it in DMSO to a concentration of 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the stock solution and dilute it with acetonitrile aqueous solution (ACN:H2O = 1:1) to prepare a series of standard solutions. Accurately pipette 4 μL of each of the above standard solutions and add 36 μL of blank plasma. Vortex to mix, preparing plasma samples equivalent to plasma concentrations of 0.3, 1, 3, 10, 30, 100, 300, 500, 1000, 3000, 5000, and 10000 ng / mL, and establish a standard curve. Take 30 μL of plasma and add 150 μL of an acetonitrile / methanol (1:1) solution containing the internal standard propranolol (5 ng / mL). Vortex to mix, centrifuge at 4000 rpm for 10 min, collect 100 μL of the supernatant, add 100 μL of ultrapure water, and perform LC-MS analysis. The LC-MS detection conditions are as follows:

[0379] Chromatographic column: YMC-Triart C18, 33*2.1mm, 5μm.

[0380] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution as shown in the table below:

[0381] 6. Data Processing

[0382] After detecting blood drug concentrations by LC-MS, the pharmacokinetic parameters of rats after drug administration were calculated using WinNonlin 6.1 software and a non-compartmental model. The results are shown in Table 16.

[0383] Table 16 shows the exposure levels converted to smegglutinin.

[0384] As shown in Table 16, the compounds of the present invention have higher exposure levels and are superior to control compound 1.

[0385] Example 73 In vivo efficacy

[0386] The experiment used 16-week-old DIO (diet-induced obesity) mice (purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.) induced by high-fat diets, with wild-type mice of the same age serving as normal controls. Before the experiment, the DIO mice were randomly divided into three groups of five mice each, based on their body weight. Wild-type mice served as a blank control group, totaling five mice. Both DIO and wild-type mice were administered the drug subcutaneously every three days. During the experiment, animal behavior, coat color, water intake, and urination were observed. Animals were weighed every other day, and any abnormalities were recorded. The experiment lasted for 15 days.

[0387] Table 17 Grouping of mice under drug administration

[0388] The results showed that there were no abnormalities in mice in each administration group. After the last administration, the weight loss rate of mice in administration group 4 was 35.4% compared with that before administration, while the weight loss rate of mice in the control group was 21.7% compared with that before administration. The results indicate that the compound of the present invention has a better weight loss effect on DIO mice than the control compound 1.

[0389] Reference compound 1

[0390] The synthetic route of control compound 1 can be obtained by referring to the preparation method of Example 1 in the patent document with application number CN202180075068.4.

[0391] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, include: One of the following compounds or its salt and an absorption enhancer, 2. The pharmaceutical composition according to claim 1, characterized in that, The compound is selected from:

3. The pharmaceutical composition according to claim 1, characterized in that, The compound is selected from:

4. The pharmaceutical composition according to claim 1, characterized in that, The compound is selected from:

5. The pharmaceutical composition according to claim 1, characterized in that, The compound is selected from:

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The content of the compound is 0.1%-50%, preferably 1%-50%, and more preferably 3%-40%.

7. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The absorption enhancer content is 10%-95%, preferably 15%-90%, more preferably 30%-90%, and even more preferably 50%-80%.

8. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The absorption enhancer is selected from at least one of SNAC, decanoic acid, sodium decanoate, sodium dodecyl sulfate, polyoxyethylene ether, sodium octanoate, and Labrasol.

9. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The absorption enhancer is SNAC or sodium decanoate.

10. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The absorption enhancer is SNAC and sodium decanoate.

11. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The absorption enhancers are SNAC and Labrasol.

12. The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical composition further comprises a lubricant, said lubricant having a content of 0.1%-10%, preferably 0.5%-5%, more preferably 0.5%-3%, more preferably 0.5%-1%, and even more preferably 1%-3%.

13. The pharmaceutical composition according to claim 12, characterized in that, The lubricant is selected from at least one of magnesium stearate, stearic acid, sodium stearate, and calcium stearate, with magnesium stearate being preferred.

14. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The pharmaceutical composition further includes a water-soluble growth promoter, wherein the content of the water-soluble growth promoter is 5%-50%, preferably 5%-30%, more preferably 10%-30%, more preferably 15-30%, and even more preferably 10%-20%.

15. The pharmaceutical composition according to claim 14, characterized in that, The water-soluble growth promoter is selected from at least one of resorcinol, catechol, pyrogallol, gentian acid xylenesulfonate, p-toluenesulfonate, nicotinamide, dimethylbenzamide, diethylbenzamide, 1-methylnicotinamide, salicylic acid and p-hydroxybenzoic acid, preferably nicotinamide.

16. The pharmaceutical composition according to any one of claims 1-5, characterized in that, Also includes: A water-soluble growth promoter, wherein the water-soluble growth promoter is selected from nicotinamide, and the absorption promoter is selected from SNAC and / or sodium decanoate.

17. The pharmaceutical composition according to any one of claims 1-5, characterized in that, Also includes: The lubricant and water-soluble growth promoter are selected from magnesium stearate, the water-soluble growth promoter is selected from nicotinamide, and the absorption promoter is selected from SNAC and / or sodium decanoate.

18. The pharmaceutical composition according to claim 16, characterized in that, The amount of magnesium stearate is 0.1%-5%, preferably 0.5%-5%, more preferably 0.5%-3%; the amount of nicotinamide is 5%-30%, preferably 10%-25%, more preferably 15%-20%; the amount of SNAC is 40%-60%, preferably 45%-55%, more preferably 50%-55%; and the amount of sodium decanoate is 10%-40%, preferably 15%-35%, more preferably 20%-30%.

19. Use of the pharmaceutical composition according to any one of claims 1-18 in the preparation of a medicament for treating a disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, cardiovascular disease, neurodegenerative diseases, chronic kidney disease, diabetic nephropathy, peripheral artery disease, and / or heart failure.

20. A method for preparing a pharmaceutical composition according to any one of claims 1-18, characterized in that, include: The compound is mixed with excipients and lubricants; Add or remove lubricant; The tablets are compressed to obtain the target tablets.

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