Polypeptide Compounds Containing Lactam Modifications

Abstract

Polypeptide compounds containing lactam modifications and their use in the manufacture of medicaments for treating associated diseases are disclosed.

Description

Detailed Description of the Invention

[0001] This application claims priority to: CN202011402979.7, application date: December 2, 2020; CN202011409947.X, filing date: December 2, 2020; CN202110432060.0, application date: April 21, 2021; CN202110587056.1, application date: May 27, 2021.

[0002] [Technical field] The present invention relates to polypeptide compounds containing lactam modifications and their use in the manufacture of medicaments for treating associated diseases.

[0003] [Background technology] There are approximately 110 million diabetes patients in China, accounting for 24% of the world's diabetes patients. With economic development and lifestyle changes, the incidence of diabetes in China has risen to 12.8% (as high as 19.9% ​​in some provinces and cities), and is associated with obesity or cardiovascular disease (CVD). Recent studies have demonstrated that glucose-dependent insulinotropic polypeptide (GIP) / glucagon-like peptide-1 (GLP-1) dual agonists can be used to treat diabetes.

[0004] GLP-1 protects islet β cells in the pancreatic islets and stimulates islet β cells to release insulin in a glucose-dependent manner, effectively controlling postprandial blood glucose. Its unique mechanism of action significantly reduces the risk of hypoglycemia. Although GLP-1R agonists have shown excellent hypoglycemic effects in clinical practice, many type 2 diabetes patients still fail to achieve the goals of blood glucose reduction and weight loss. Therefore, combining GLP-1R agonists with other hypoglycemic targets, such as GIP, for the treatment of type 2 diabetes is an urgent and promising demand. GIP is a polypeptide secreted by neuroendocrine K cells in the small intestine, and its physiological effects are mediated by GIPR, mainly including non-glucose-dependent stimulation of insulin secretion, enhancement of glucagon secretion, and enhancement of lipid metabolism. Although the beneficial effects of GIPR agonists appear to be weakened in hyperglycemic symptoms in type 2 diabetes patients, studies have shown that the reduced insulin secretion-promoting effect of GIP can be fully restored after a period of normalization of plasma glucose levels. This suggests that GLP-1R / GIPR coagonism can exert synergistic hypoglycemic effects. Dual agonism of GIP and GLP-1 may have synergistic effects on regulating glucose / lipid metabolism, resulting in better therapeutic effects on lowering blood glucose levels, reducing body weight, and alleviating liver fat.

[0005] [Summary of the Invention] The present invention provides the following compound or a pharma- ceutically acceptable salt thereof: SEQ ID NO: 1: YAibEGT FTSDY SIAibLD KK 1 AZ0K0Z1FZ2E0W LZ3AGG PSSGA PPPS0 SEQ ID NO: 2: YAibEGT FTSDY SIAibLD KE0AQK0AFVK 1 W LIAGG PSSGA PPPS0 SEQ ID NO: 3: YAibEGT FTSDY SIE0LD KK0AQK 1 AFVQW LIAGG PSSGA PPPS0 SEQ ID NO: 4: YAibEGT FTSDY SIE0LD K0IAQK 1 AFVQW LIAGG PSSGA PPPS0 however, The structure of Aib is

[0006] [ka] and

[0007] S0 is

[0008] [ka] is selected from

[0009] Z0 is selected from glutamine (Q) and asparagine (N); Z1 is selected from alanine (A) and glutamic acid (E); Z2 is selected from valine (V) and isoleucine (I); Z3 is selected from isoleucine (I) and leucine (L); E0 and K0 represent the carboxyl on the side chain of glutamic acid and the amino on the side chain of lysine together forming a lactam, and the structure of K0Z1FZ2E0 is

[0010] [ka] and the structure of E0AQK0 is

[0011] [ka] and the structure of E0LDKK0 is

[0012] [ka] and the structure of E0LDK0 is

[0013] [ka] and

[0014] K 1 represents the linkage between the amino acid on the side chain of lysine and -X-X1-X2, and the structure is

[0015] [ka] and

[0016] X is

[0017] [ka] is selected from

[0018] R1 and R2 are each independently selected from H and CH3; X1 is,

[0019] [ka] is selected from

[0020] X2 is

[0021] [ka] is selected from

[0022] m, n and p are each independently selected from 2 and 3; s is selected from 2 and 3; q is selected from 15, 16, 17, 18 and 19.

[0023] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following:

[0024] [ka]

[0025] however, Aib, Z0, Z1, Z2, Z3 and K 1 is as defined in the present invention. In some embodiments of the present invention, X is selected from -C(=O)-CH2-, and all other variables are as defined herein.

[0026] In some embodiments of the invention, m, n, and p are each independently selected from 2, and all other variables are as defined herein. In some embodiments of the present invention, s is selected from 2, and all other variables are as defined herein.

[0027] In some embodiments of the invention, q is selected from 15 and 17, and all other variables are as defined herein. In some embodiments of the present invention, X1 is

[0028] [ka] and the other variables are as defined herein.

[0029] In some embodiments of the invention, X2 is

[0030] [ka] and the other variables are as defined herein.

[0031] In some embodiments of the present invention, -X-X1-X2 is

[0032] [ka] and the other variables are as defined herein. The present invention provides a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0033] YAibEGT FTSDY SIAibLD KK 1 AQK0AFVE0W LIAGG PSSGA PPPS0 YAibEGT FTSDY SIAibLD KK 1 ANK0AFVE0W LIAGG PSSGA PPPS0 YAibEGT FTSDY SIAibLD KK 1 AQK0EFVE0W LIAGG PSSGA PPPS0 YAibEGT FTSDY SIAibLD KK 1 AQK0AFIE0W LIAGG PSSGA PPPS0 YAibEGT FTSDY SIAibLD KK 1 AQK0AFVE0W LLAGG PSSGA PPPS0 however, The structure of Aib is

[0034] [ka] and

[0035] S0 is

[0036] [ka] is selected from

[0037] K0 and E0 represent the amino on the side chain of lysine and the carboxyl on the side chain of glutamic acid that together form a lactam, the structure of which is

[0038] [ka] and

[0039] K 1 represents the linkage between the amino acid on the side chain of lysine and -X-X1-X2, and the structure is

[0040] [ka] and

[0041] X is

[0042] [ka] is selected from

[0043] R1 and R2 are each independently selected from H; X1 is,

[0044] [ka] is selected from

[0045] X2 is

[0046] [ka] is selected from

[0047] m and n are each independently selected from 2 and 3; s is selected from 2 and 3; q is selected from 15, 16, 17, 18 and 19.

[0048] In some embodiments of the present invention, X is selected from -C(=O)-CH2-, and all other variables are as defined herein. In some embodiments of the invention, m and n are each independently selected from 2, and all other variables are as defined herein.

[0049] In some embodiments of the present invention, s is selected from 2, and all other variables are as defined herein. In some embodiments of the invention, q is selected from 15 and 17, and all other variables are as defined herein.

[0050] In some embodiments of the present invention, X1 is

[0051] [ka] and the other variables are as defined herein.

[0052] In some embodiments of the invention, X2 is

[0053] [ka] and the other variables are as defined herein.

[0054] In some embodiments of the present invention, -X-X1-X2 is

[0055] [ka] and the other variables are as defined herein. The present invention provides a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0056] YAibEGT FTSDY SIAibLD KK 1 AZ0K0Z1FZ2E0W LZ3AGG PSSGA PPPS0 YAibEGT FTSDY SIAibLD KE0AQK0AFVK 1 W LIAGG PSSGA PPPS0 YAibEGT FTSDY SIE0LD KK0AQK 1 AFVQW LIAGG PSSGA PPPS0 however, The structure of Aib is

[0057] [ka] and

[0058] S0 is

[0059] [ka] is selected from

[0060] Z0 is selected from glutamine (Q) and asparagine (N); Z1 is selected from alanine (A) and glutamic acid (E); Z2 is selected from valine (V) and isoleucine (I); Z3 is selected from isoleucine (I) and leucine (L); E0 and K0 represent the carboxyl on the side chain of glutamic acid and the amino on the side chain of lysine together forming a lactam, and the structure of K0Z1FZ2E0 is

[0061] [ka] and the structure of E0AQK0 is

[0062] [ka] and the structure of E0LDKK0 is

[0063] [ka] and

[0064] K 1 represents the linkage between the amino acid on the side chain of lysine and -X-X1-X2, and the structure is

[0065] [ka] and

[0066] X is

[0067] [ka] is selected from

[0068] R1 and R2 are each independently selected from H; X1 is,

[0069] [ka] is selected from

[0070] X2 is

[0071] [ka] is selected from

[0072] m, n and p are each independently selected from 2 and 3; s is independently selected from 2 and 3; q is independently selected from 15, 16, 17, 18, and 19.

[0073] In some embodiments of the present invention, X is selected from -C(=O)-CH2-, and all other variables are as defined herein. In some embodiments of the invention, m, n, and p are each independently selected from 2, and all other variables are as defined herein.

[0074] In some embodiments of the present invention, s is independently selected from 2, and other variables are as defined herein. In some embodiments of the present invention, q is independently selected from 15 and 17, and all other variables are as defined herein.

[0075] In some embodiments of the present invention, q is independently selected from 17, and all other variables are as defined herein. In some embodiments of the present invention, X1 is

[0076] [ka] and the other variables are as defined herein.

[0077] In some embodiments of the invention, X2 is

[0078] [ka] and the other variables are as defined herein.

[0079] In some embodiments of the present invention, -X-X1-X2 is

[0080] [ka] and the other variables are as defined herein. The present invention provides a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0081] YAibEGT FTSDY SIAibLD KK 1 AQK0AFVE0W LIAGG PSSGA PPPS0 however, The structure of Aib is

[0082] [ka] and

[0083] S0 is

[0084] [ka] is selected from

[0085] K0 and E0 represent the amino on the side chain of lysine and the carboxyl on the side chain of glutamic acid that together form a lactam, the structure of which is

[0086] [ka] and

[0087] K 1 represents the linkage between the amino acid on the side chain of lysine and -X-X1-X2, and the structure is

[0088] [ka] and

[0089] X is

[0090] [ka] is selected from

[0091] R1 and R2 are each independently selected from H; X1 is,

[0092] [ka] is selected from

[0093] X2 is

[0094] [ka] is selected from

[0095] m, n and p are each independently selected from 2 and 3; q is independently selected from 15, 16, 17, 18, and 19. In some embodiments of the present invention, X is selected from -C(=O)-CH2-, and all other variables are as defined herein.

[0096] In some embodiments of the invention, m, n, and p are each independently selected from 2, and all other variables are as defined herein. In some embodiments of the present invention, q is independently selected from 17, and all other variables are as defined herein.

[0097] In some embodiments of the present invention, X1 is

[0098] [ka] and the other variables are as defined herein.

[0099] In some embodiments of the invention, X2 is

[0100] [ka] and the other variables are as defined herein.

[0101] In some embodiments of the present invention, -X-X1-X2 is

[0102] [ka] and the other variables are as defined herein. The present invention provides a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0103] YAibEGT FTSDY SIE0LD K0IAQK 1 AFVQW LIAGG PSSGA PPPS0 however, The structure of Aib is

[0104] [ka] and

[0105] S0 is

[0106] [ka] is selected from

[0107] E0 and K0 represent the carboxyl on the side chain of glutamic acid and the amino on the side chain of lysine together forming a lactam, the structure of which is

[0108] [ka] and

[0109] K 1 represents the linkage between the amino acid on the side chain of lysine and -X-X1-X2, and the structure is

[0110] [ka] and

[0111] X is

[0112] [ka] is selected from

[0113] R1 and R2 are each independently selected from H; X1 is,

[0114] [ka] is selected from

[0115] X2 is

[0116] [ka] is selected from

[0117] m, n and p are each independently selected from 2 and 3; q is independently selected from 15, 16, 17, 18, and 19. In some embodiments of the present invention, X is selected from -C(=O)-CH2-, and all other variables are as defined herein.

[0118] In some embodiments of the invention, m, n, and p are each independently selected from 2, and all other variables are as defined herein. In some embodiments of the present invention, q is independently selected from 17, and all other variables are as defined herein.

[0119] In some embodiments of the present invention, X1 is

[0120] [ka] and the other variables are as defined herein.

[0121] In some embodiments of the invention, X2 is

[0122] [ka] and the other variables are as defined herein.

[0123] In some embodiments of the present invention, -X-X1-X2 is

[0124] [ka] and the other variables are as defined herein. Some embodiments of the present invention are further formed by any combination of the variables described above.

[0125] The present invention further provides a compound represented by the formula: or a pharma- ceutically acceptable salt thereof.

[0126] [ka] TIFF0007597934000058.tif213168TIFF0007597934000059.tif243170

[0127] The present invention further provides a compound represented by the formula: or a pharma- ceutically acceptable salt thereof.

[0128] [ka] TIFF0007597934000061.tif243169TIFF0007597934000062.tif149170

[0129] In some embodiments of the invention is the use of the above compound or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating disorders related to obesity and diabetes. The present invention further provides the following test method.

[0130] Experimental example: hERG potassium channel inhibition test A. Main Ingredients CHO-hERG cell line (Chinese hamster ovary cells stably expressing hERG channel), constructed in Shanghai Institute of Materia Medica, Chinese Academy of Sciences; patch clamp amplifier Axopatch 200B, Taser International Inc; B. Method a) Cell culture CHO cells stably expressing hERG were placed in a 35 mm diameter cell culture dish and cultured in a 37°C, 5% CO2 incubator, subcultured at a ratio of 1:5 every 48 hours, and the medium components were: 90% F12 (Invitrogen), 10% fetal bovine serum (Gibco), 100 μg / mL G418 (Invitrogen) and 100 μg / mL Hygromycin B (Invitrogen). On the day of the test, the cell medium was aspirated and washed once with extracellular solution, after which 0.25% Trypsin-EDTA (Invitrogen) solution was added and digested at room temperature for 3 to 5 minutes. After aspirating the digestion solution and resuspending in extracellular solution, the cells were transferred to a dish for electrophysiological recording.

[0131] b) Preparation of intracellular and extracellular solutions Extracellular fluid should be prepared monthly. Intracellular fluid should be aliquoted and stored frozen at -20°C.

[0132] Extracellular solution (mM): 145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 Glucose, and 10 HEPES, pH was adjusted to 7.4 with NaOH, and osmolarity was 295 mOsm.

[0133] Intracellular solution (mM): 120 KCl, 31.25 KOH, 5.374 CaCl2, 1.75 MgCl2, 4 Na2 ATP, 10 HEPES, and 10 EGTA, pH was adjusted to 7.2 with KOH, and osmolarity was 285 mOsm.

[0134] c) Preparation of Compounds Compounds are dissolved in 20mM mother solution with DMSO, and on the day of testing, compound mother solution is serially diluted 3 times with DMSO, that is, 10μL compound mother solution is added to 20μL DMSO to obtain 6 compound intermediate concentrations serially diluted with DMSO, which are 20, 6.66, 2.22, 0.74, 0.24 and 0.082mM respectively. Then, 10μL compound intermediate concentration is taken and added to 4990μL extracellular solution, which is diluted 500 times to obtain the final concentration to be tested, the highest test concentration is 40μM, which are 40, 13.3, 4.44, 1.48, 0.49 and 0.16μM respectively. The DMSO content of the final test concentration does not exceed 0.2%, and this concentration of DMSO does not affect the hERG potassium channel.

[0135] d) Electrophysiological recording process CHO (Chinese Hamster Ovary) cells stably expressing the hERG potassium channel were used to record hERG potassium channel currents at room temperature using the whole-cell patch clamp technique. Glass microelectrodes were made by pulling glass electrode blanks (BF150-86-10, Sutter) with a stretching machine. The tip resistance after filling with the electrode solution was approximately 2-5 MΩ, and the glass microelectrode was inserted into the amplifier probe to connect to an Axopatch 200B (Molecular Devices) patch clamp amplifier. Clamp voltage and data recording were controlled and recorded by a computer using pClamp 10 software, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at -80 mV, and the step voltage to elicit the hERG potassium current (I hERG) was a depolarizing voltage of -80 mV to +20 mV for 2 s, followed by repolarization to -50 mV and back to -80 mV after 1 s. The voltage stimulation was applied every 10 seconds, and the administration process was started after confirming that the hERG potassium current was stable (1 min). Successive administrations were started from the lowest test concentration of the compound, and each test concentration was administered for at least 1 min. Each concentration of the compound was tested in at least three cells (n≧3), and each concentration of the positive compound was tested in at least two cells (n≧2).

[0136] e) Data analysis For each complete current record, the inhibition rate of each compound's effective concentration can be calculated based on the ratio of the peak current of the negative control. The dose-effect relationship curve is obtained by fitting with the standard Hill equation, and the specific formula is as follows:

[0137] I (C) =I b +(I fr -I b )*c n / (I C 50 n +c n ) c is the compound test concentration and n is the slope. Both curve fitting and inhibition calculation were performed by Qpatch analysis software. If the inhibition rate at the lowest concentration was greater than half inhibition or the inhibition rate at the highest concentration was less than half inhibition, the corresponding IC 50 is lower than the lowest concentration or IC 50 The value is greater than the maximum concentration.

[0138] C. Test Results and Conclusions The compounds of the present invention have no hERG-related cytochrome P450 isozyme inhibition. A. Purpose of the experiment The objective of the present invention is to measure the inhibitory effect of test compounds on the activity of human hepatic microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4).

[0139] B. Experimental Procedure First, the test compound (10 mM) was gradient-diluted to prepare a working solution (100x final concentration). The concentrations of the working solutions were: 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005 mM, respectively. At the same time, a mixed working solution of each positive inhibitor of P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and its specific substrate was prepared. Human liver microsomes frozen in a -80°C refrigerator were thawed on ice. After the human liver microsomes were completely thawed, they were diluted with PBS (phosphate buffer solution) to prepare a working solution of a given concentration (0.253 mg / ml). 20 μL of the mixed substrate solution was added to the reaction plate (20 μL of PB was added to the blank well), and 158 μL of the human liver microsomes were added to the reaction plate (20 μL of PB was added to the blank well). Add the chromosome working solution to the reaction plate, and place the reaction plate on ice until use. At this time, add 2μL of each concentration of test compound (N=1) and positive inhibitor (N=2) to the corresponding wells, add the organic solvent corresponding to the group without inhibitor (test compound or positive inhibitor) to serve as the control group sample, and after incubation in a 37℃ water bath for 10 minutes, add 20μL of NADPH solution to the reaction plate, and incubate in a 37℃ water bath for 10 minutes, add 400μL of cold acetonitrile solution to stop the reaction, place the reaction plate on a shaker, shake for 10 minutes, and centrifuge at 4,000rpm for 20 minutes, take 200μL of the supernatant and add it to 100μL of water to dilute the sample, and finally seal the plate, shake evenly, and perform LC / MS / MS detection.

[0140] C. Experimental Results and Conclusions The compounds of the present invention are free of the associated risks of CYP inhibition. [Technical effect] The compounds of the present invention have strong agonist activity against GLP-1R / GIPR, the compounds of the present invention have excellent pharmacokinetic properties, the compounds of the present invention have excellent plasma stability, the compounds of the present invention have very high plasma protein binding, and the compounds of the present invention have excellent in vivo efficacy.

[0141] [Definitions and Explanations] Unless otherwise stated, the following terms and phrases used herein have the following meanings: A particular term or phrase, unless otherwise defined, should be understood to have its ordinary definition, not to be indefinite or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0142] As used herein, "pharmaceutically acceptable salts" refer to those compounds, materials, compositions and / or dosage forms which are within the scope of sound medical judgment, suitable for contact with human and animal tissues, without significant toxicity, irritation, allergic response or other problem or complication, and consistent with a reasonable benefit / risk ratio.

[0143] The term "pharmaceutical acceptable salt" refers to a salt of a compound of the present invention, which is prepared with a relatively non-toxic acid or base with a compound having certain substituents discovered in this invention. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting these compounds with a sufficient amount of base in a separate solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting these compounds with a sufficient amount of acid in a separate solution or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include inorganic and organic acid salts, as well as salts of amino acids (such as arginine) and organic acids such as glucuronic acid, such as inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like, and organic acids including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Certain compounds of the present invention contain both basic and acidic functional groups and can therefore be converted into any suitable base or acid addition salt.

[0144] The pharma- ceutically acceptable salts of the present invention can be synthesized in a conventional manner from the parent compound which contains an acidic or basic group. Typically, such salts are prepared by reacting the compound in its free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both.

[0145] As used herein, A or Ala represents alanine and the structure is

[0146] [ka] where R or Arg represents arginine and the structure is

[0147] [ka] wherein N or Asn represents asparagine and the structure is

[0148] [ka] where D or Asp represents aspartic acid and the structure is

[0149] [ka] where C or Cys represents cysteine ​​and the structure is

[0150] [ka] where Q or Gln represents glutamine and the structure is

[0151] [ka] where E or Glu represents glutamic acid and the structure is

[0152] [ka] where G or Gly represents glycine and the structure is

[0153] [ka] where H or His represents histidine and the structure is

[0154] [ka] where I or Ile represents isoleucine and the structure is

[0155] [ka] where L or Leu represents leucine and the structure is

[0156] [ka] where K or Lys represents lysine and the structure is

[0157] [ka] where M or Met represents methionine and the structure is

[0158] [ka] where F or Phe represents phenylalanine and the structure is

[0159] [ka] where P or Pro represents proline and the structure is

[0160] [ka] where S or Ser represents serine and the structure is

[0161] [ka] where T or Thr represents threonine and the structure is

[0162] [ka] where W or Trp represents tryptophan and the structure is

[0163] [ka] and Y or Tyr represents tyrosine and the structure is

[0164] [ka] where V or Val represents valine and the structure is

[0165] [ka] It is.

[0166] The term "treating" includes inhibiting, slowing, halting or reversing the progression or severity of an existing symptom or condition. Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, enantiomers and tautomers.

[0167] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures enriched in enantiomers or non-enantiomers, and all such mixtures are within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers and mixtures thereof are both within the scope of the present invention.

[0168] Unless otherwise stated, the terms "enantiomers" or "optical isomers" are stereoisomers that are mirror images of each other. Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" refer to the inability to freely rotate about double bonds or single bonds of ring carbon atoms.

[0169] Unless otherwise explained, the term "diastereomer" is a stereoisomer in which the molecules have two or more centers of chirality and the molecules are not mirror-images of each other. Unless otherwise specified, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

[0170] Unless otherwise stated, solid wedge connections (

[0171] [ka] ) and dotted wedge bond (

[0172] [ka] ) to indicate the absolute configuration of one stereocenter, and the solid rod bond (

[0173] [ka] ) and dotted bar bonds (

[0174] [ka] ) to indicate the relative configuration of stereocenters, and the wavy line (

[0175] [ka] ) with solid wedge connection (

[0176] [ka] ) or dotted wedge bond (

[0177] [ka] ), or a wavy line (

[0178] [ka] ) with solid bar connection (

[0179] [ka] ) or dotted bar bond (

[0180] [ka] )

[0181] Unless otherwise explained, the terms "enriched in one isomer," "isomer-enriched," "enriched in one enantiomer," or "enantiomer-enriched" mean that the amount of one isomer or enantiomer is less than 100% and that the amount of such isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0182] Unless otherwise stated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, then the isomeric or enantiomeric excess (ee value) is 80%.

[0183] Optically active (R)- and (S)-isomers, as well as D- and L-isomers, can be prepared using asymmetric synthesis or chiral reagents or other conventional techniques. A single enantiomer of a compound of the invention can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary is decomposed to provide the desired isolated enantiomer. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxyl) functional group, the diastereomeric salt can be formed with an appropriate optically active acid or base, and the diastereomers can be resolved by conventional methods known in the art, followed by recovery to provide the isolated enantiomers. Separation of enantiomers and diastereomers is also typically accomplished by chromatographic techniques using chiral stationary phases and any chemical derivatization techniques (e.g., formation of carbamates from amines).

[0184] The compounds of the present invention may contain unnatural atomic isotopes at one or more atoms constituting the compounds. For example, tritium ( 3 H), iodine-125( 125 I) or C-14( 14 Compounds can be labeled with radioisotopes such as radioisotopes C). Also, for example, deuterium can be replaced with hydrogen to form deuterated drugs, where the bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon, and compared to non-deuterated drugs, deuterated drugs have the advantages of reduced toxic side effects, increased drug stability, improved therapeutic efficacy, and extended biological half-life of the drug. Conversion of the isotopic composition of the compounds of the invention, whether radioactive or not, is within the scope of the invention.

[0185] Unless otherwise defined, the term "C 1-3 "Alkyl" refers to a saturated hydrocarbon group consisting of 1 to 3 carbon atoms in a straight or branched chain. 1-3 Alkyl has C 1-2 and C. 2-3 Alkyl, which may be monovalent (e.g., methyl), divalent (e.g., methylene) and polyvalent (e.g., methine). 1-3 Examples of alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl).

[0186] Unless otherwise defined, "S-NH2" in the compounds of the invention is used to indicate replacement of the carboxyl group on serine with an amide. The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art, and when the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means of those skilled in the art. For example, single crystal X-ray diffraction (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, after collecting the relevant data, the absolute configuration can be confirmed by direct method (Shelxs97) crystal structure analysis.

[0187] The compounds of the present invention can be prepared by various synthetic methods familiar to those skilled in the art, including the specific embodiments given below, embodiments in combination with other chemical synthetic methods, and equivalent alternative methods familiar to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0188] All solvents used in this invention can be obtained commercially. The present invention uses the following abbreviations: aq stands for water, eq stands for equivalent, DCM stands for dichloromethane, PE stands for petroleum ether, DMSO stands for dimethylsulfoxide, MeOH stands for methanol, BOC stands for tert-butoxycarbonyl, an amine protecting group, rt stands for room temperature, O / N stands for overnight, THF stands for tetrahydrofuran, Boc2O stands for di-tert-butyl dicarbonate, TFA stands for trifluoroacetic acid, DIPEA stands for diisopropylethylamine, DMF stands for N,N-dimethylformamide, amide, HBTU stands for O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HOBT stands for 1-hydroxybenzotriazole, HOAT stands for 1-hydroxy-7-azabenzotriazole, DIC stands for N,N'-diisopropylcarbodiimide, DBU stands for 1,8-diazabicyclo[5.4.0]undec-7-ene, PhSiH3 stands for phenylsilane, and Pd(PPh3)4 stands for tetrakis(triphenylphosphine)palladium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0189] The present invention will be specifically described below by way of examples, but is not intended to be an adverse restriction of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have been disclosed. It is obvious to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0190] Intermediate B-1

[0191] [ka]

[0192] Step 1: Weigh out 1.1 g of 4-(2',4'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamide-methylbenzhydrylamine resin (degree of substitution Sub=0.28 mmol / g) and add it to the reaction column, add DMF (50 mL) to the reaction column and blow in nitrogen gas for 2 hours, drain the waste liquid until no liquid flows out, add DMF (50 mL), wash 5 times for 1 minute each, and drain the waste liquid until no liquid flows out.

[0193] Step 2: Add 20% piperidine / DMF (50 mL) to the reaction column, flush with nitrogen gas for 20 minutes, and drain until no liquid flows out. Wash with DMF (50 mL) 5 times for 1 minute each, and drain until no liquid flows out. Perform ninhydrin detection, the resin was blue.

[0194] Step 3: Coupling of amino acids 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin, add DIEA (6.00 eq), add 10 mL of DMF to the reaction column, blow in nitrogen gas, and after the amino acid is dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas to blow in the resin evenly.

[0195] After reacting at 2.25°C for 0.5 hours, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was extracted and removed, and washed with DMF 5 times (50 mL each time), 1 min each time, and the waste liquid was drained until no liquid flowed out.

[0196] 3.2 Coupling of Fmoc-Pro-OH 1.20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid flowed out. DMF (50 mL) was added and washed 5 times, 1 min each time, and the waste liquid was drained until no liquid flowed out. Ninhydrin detection was performed, and the resin was blue.

[0197] 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin, add DIEA (6.00 eq), add 10 mL of DMF to the reaction column, blow in nitrogen gas, and after the amino acid is dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas to blow in the resin evenly. After reacting for 0.5 hours at 3.25°C, ninhydrin detection was performed and the resin was colorless and transparent.

[0198] 4. The reaction solution was removed, and the column was washed 5 times with DMF (50 mL each time), 1 min each time, and the waste liquid was drained until no liquid flowed out. Step 3.2 was repeated to complete the coupling of the following amino acids:

[0199] [Table 1] TIFF0007597934000095.tif245169TIFF0007597934000096.tif194169

[0200] 3.40 De-Alloc and OAll 1. PhSiH3 (20.0 eq) and DCM (10 mL) were added to a reaction column and sparged with nitrogen gas, then Pd(PPh3)4 (0.2 eq) was added and sparged with nitrogen gas for 20 minutes, reacted twice, and the waste liquid was discharged until no liquid flowed out.

[0201] 2. Wash with DMF 5 times (50 mL each time), 1 min each time, draining the waste liquid until no liquid came out. 3.41 Amide ring closure 1. DIEA (3.00 eq) was added, 10 mL of DMF was added to the reaction column, and nitrogen gas was blown in to dissolve the resin. HATU (1.5 eq) was then added. The nitrogen gas was adjusted to blow in the resin evenly.

[0202] After reacting at 2.25°C for 0.5 hours, ninhydrin detection was performed and the resin was colorless and transparent. 3. Wash with DMF 5 times (50 mL each time), 1 min each time, draining the waste liquid until no liquid came out.

[0203] 3.42 De-Dde 1.3% hydrazine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled in for 15 minutes, and the waste liquid was drained until no liquid flowed out. DMF (50 mL) was added and washed 5 times, 1 min each time, and the waste liquid was drained until no liquid flowed out, to obtain intermediate B-1. Ninhydrin detection was performed, and the resin was blue.

[0204] Intermediate B-2

[0205] [ka]

[0206] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, amide ring closure and de-Dde protection were carried out to obtain B-2.

[0207] [Table 2] TIFF0007597934000099.tif245169TIFF0007597934000100.tif230169

[0208] Intermediate B-3

[0209] [ka]

[0210] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, amide ring closure and de-Dde protection were carried out to obtain B-3.

[0211] [Table 3] TIFF0007597934000103.tif244169TIFF0007597934000104.tif228169

[0212] Intermediate B-4

[0213] [ka]

[0214] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, cyclization of amide and de-Dde protection were carried out to obtain B-4.

[0215] [Table 4] TIFF0007597934000107.tif247169TIFF0007597934000108.tif230169

[0216] Intermediate B-5

[0217] [ka]

[0218] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, cyclization of amide and de-Dde protection were carried out to obtain B-4.

[0219] [Table 5] TIFF0007597934000111.tif244169TIFF0007597934000112.tif228169

[0220] Intermediate B-6

[0221] [ka]

[0222] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, amide ring closure and de-Dde protection were carried out to obtain B-6.

[0223] [Table 6] TIFF0007597934000115.tif246169TIFF0007597934000116.tif228169

[0224] Intermediate B-7

[0225] [ka]

[0226] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, amide ring closure and de-Dde protection were carried out to obtain B-7.

[0227] [Table 7] TIFF0007597934000119.tif245169TIFF0007597934000120.tif228169

[0228] Intermediate B-8

[0229] [ka]

[0230] Referring to the synthesis method of B-1, the coupling of the following amino acids was completed, and operations such as de-Alloc and OAll, amide ring closure and de-Dde protection were carried out to obtain B-8.

[0231] [Table 8] TIFF0007597934000123.tif245169TIFF0007597934000124.tif228169

[0232] Example 1

[0233] [ka]

[0234] 1. Coupling of long chain fatty acids: Synthesis of B-1. Refer to step 3.2 to complete the coupling of the following fragments:

[0235] [Table 9]

[0236] 2. Cleavage and Drying of Crude Peptide 2.1. A cutting fluid was prepared in the following volumes:

[0237] [Table 10]

[0238] The dried peptide resin was added to the prepared cutting fluid, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 volumes of ice-isopropyl ether, centrifuged, and washed three times with isopropyl ether. After drying in vacuum for 2 hours, the crude peptide was obtained and purified to obtain the polypeptide compound WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4811.4, and the detected value was 4812.0.

[0239] Example 2

[0240] [ka]

[0241] 1. Coupling of long chain fatty acids: Synthesis of B-1. Refer to step 3.2 to complete the coupling of the following fragments:

[0242] [Table 11]

[0243] 2. Cleavage and Drying of Crude Peptide 2.1. A cutting fluid was prepared in the following volumes:

[0244] [Table 12]

[0245] The dried peptide resin was added to the prepared cutting fluid, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 volumes of ice-isopropyl ether, centrifuged, and further washed with isopropyl ether three times. After drying in vacuum for 2 hours, the crude peptide was obtained and purified to obtain the polypeptide compound WX-002. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4940.5 and the detected value was 4940.6.

[0246] Example 3

[0247] [ka]

[0248] 1. Coupling of long chain fatty acids: Synthesis of B-1. Refer to step 3.2 to complete the coupling of the following fragments:

[0249] [Table 13]

[0250] 2. Cleavage and Drying of Crude Peptide 2.1. A cutting fluid was prepared in the following volumes:

[0251] [Table 14]

[0252] The dried peptide resin was added to the prepared cutting fluid, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 volumes of ice-isopropyl ether, centrifuged, and washed three times with isopropyl ether. After drying in vacuum for 2 hours, the crude peptide was obtained and purified to obtain the polypeptide compound WX-003. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4912.5 and the detected value was 4913.1.

[0253] Example 4

[0254] [ka]

[0255] With reference to the synthesis of WX-001, WX-004 was obtained from intermediate B-2. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4797.4, and the detected value was 4797.3.

[0256] Example 5

[0257] [ka]

[0258] With reference to the synthesis of WX-001, WX-005 was obtained from intermediate B-3. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4869.5, and the detected value was 4869.3.

[0259] Example 6

[0260] [ka]

[0261] With reference to the synthesis of WX-001, WX-006 was obtained from intermediate B-4. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4825.5, and the detected value was 4825.5.

[0262] Example 7

[0263] [ka]

[0264] WX-007 was obtained from intermediate B-5 by referring to the synthesis of WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4811.4, and the detected value was 4811.1.

[0265] Example 8

[0266] [ka]

[0267] WX-008 was obtained from intermediate B-6 by referring to the synthesis of WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4811.4, and the detected value was 4811.4.

[0268] Example 9

[0269] [ka]

[0270] With reference to the synthesis of WX-001, WX-009 was obtained from intermediate B-7. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4854.5, and the detected value was 4854.9.

[0271] Example 10

[0272] [ka]

[0273] WX-010 was obtained from intermediate B-8 by referring to the synthesis of WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4839.4, and the detected value was 4839.6.

[0274] Biological Test Data Experimental Example 1: In vitro GLP-1R / GIPR agonist activity test A:Main materials: 1) Cell line The cell line was constructed by Shanghai WuXi PharmaTech. For details, please refer to the table below.

[0275] [Table 15]

[0276] 2) Reagents and consumables

[0277] [Table 16]

[0278] 3)Equipment

[0279] [Table 17]

[0280] B. Method 1) Experimental materials Experimental buffer

[0281] [Table 18]

[0282] Production of detection reagents

[0283] [Table 19]

[0284] 2) Experimental method a) Preparation of compound plates: Test compounds were diluted 4-fold in 10-point series with a starting concentration of 30 μM and dilutions were completed in a Bravo. b) Compound transfer: 1) 100 nL of compound was transferred to an OptiPlate-384 plate using an Echo. 2) The OptiPlate-384 plate was centrifuged at 1000 rpm for 5 seconds. c) Preparation of cell suspension 1) Place one GLP-1R / GIPR cell freezing tube in 37°C warm water and allow to thaw quickly. 2) The cell suspension was transferred to a 15 mL centrifuge tube and gently rinsed with 10 mL of HBSS. 3) The tube was centrifuged at 1000 rpm at room temperature for 1 minute. 4) The supernatant was discarded. 5) The cells at the bottom were gently dispersed, rinsed lightly with 10 mL of HBSS, centrifuged to sediment the cells, and finally resuspended in the test buffer. 6) Cell density and viability were measured using Vi-cell. 7) GLP-1R / GIPR cell concentration was 2.0 x 10 in test buffer. 5 / mL. 8) 100 nL of the diluted cell suspension was placed into an OptiPlate-384 plate. 9) Incubated at room temperature for 30 minutes. d) Addition of detection reagent: 1) 10 μL of 800 nM gradient diluted cAMP standard was added to empty wells of an OptiPlate-384 plate. 2) 10 μL of cAMP detection reagent was added. 3) The OptiPlate-384 plate was covered with TopSeal-A film and incubated at room temperature for 60 minutes. The TopSeal-A was removed and read on EnVision.

[0285] C. Experimental Results The experimental results are shown in Table 1.

[0286] [Table 20] Conclusion: The compounds of the present invention have strong agonistic activity against GLP-1R / GIPR.

[0287] Experimental Example 2: Pharmacokinetic evaluation of compounds in rats A. Purpose of the experiment The purpose is to test the pharmacokinetics of the compound in SD rats.

[0288] B. Experimental Procedure The pharmacokinetic properties of the compounds in rodents after subcutaneous injection were tested using standard protocols. In the experiments, the candidate compounds were prepared into clear solutions and administered to rats by a single subcutaneous injection (SC, 0.048 mpk). The injection solvent was citrate buffer (20 mM, pH=7). Whole blood was collected to prepare plasma, which was analyzed for drug concentration by LC-MS / MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0289] C. Experimental Results The experimental results are shown in Table 2.

[0290] [Table 21] Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in rats.

[0291] Experimental Example 3: Pharmacokinetic evaluation of compounds in mice A. Purpose of the experiment The purpose was to test the pharmacokinetics of the compound in C57BL / 6 mice.

[0292] B. Experimental Procedure The pharmacokinetic properties of the compounds in rodents after intravenous and subcutaneous injection were tested by standard protocols. In the experiments, the candidate compounds were prepared into clear solutions and administered to mice by single intravenous (IV, 0.048mpk) and subcutaneous (SC, 0.048mpk) injections. The solvent for intravenous injection was PBS buffer (pH=7), and the solvent for subcutaneous injection was citrate buffer (20mM, pH=7). Whole blood was collected to prepare plasma, and the drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software.

[0293] C. Experimental Results The experimental results are shown in Table 3.

[0294] [Table 22] Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in mice.

[0295] Experimental Example 4: Pharmacokinetic evaluation of compounds in cynomolgus monkeys A. Purpose of the experiment The purpose is to test the pharmacokinetics of the compound in the body of cynomolgus monkeys.

[0296] B. Experimental Procedure The mammalian pharmacokinetic properties of the compounds after intravenous and subcutaneous injection were tested by standard protocols. In the experiment, the candidate compounds were prepared into clear solutions and administered to cynomolgus monkeys by a single subcutaneous injection (SC, 0.02 mpk). The solvent for subcutaneous injection was citrate buffer (20 mM, pH=7). Whole blood was collected to prepare plasma, and the drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software.

[0297] C. Experimental Results The experimental results are shown in Table 4.

[0298] [Table 23] Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in monkeys.

[0299] Experimental Example 5: Plasma stability test (PLS) A. Purpose of the experiment To study the stability of the test compounds in normal mouse plasma.

[0300] B. Experimental Procedure 1. Before the experiment, the clotted frozen plasma was thawed in a water bath at 37° C. The plasma was centrifuged at 4000 rpm for 5 min, clots were removed if present, and the pH was adjusted to 7.4±0.1.

[0301] 2. Preparation of test compound solution: A 100 μM solution was prepared by diluting with DMSO. 2 μL of the test compound solution (100 μM) was added to 3.98 μL of blank control plasma so that the final concentration of the mixed solution of the two became 2 μM, and the mixture was incubated in a water bath at 37°C.

[0302] 4. At each time point (0, 10, 30, 60 and 120 min), 100 μL of H3PO4 solution and 800 μL of stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in 100% methanol) were added to precipitate proteins and mixed thoroughly.

[0303] 5. The samples were centrifuged at 4000 rpm for 20 min, and 100 μL of the supernatant was taken from each well for C-MS / MS analysis. C. Experimental Results The experimental results are shown in Table 5.

[0304] [Table 24] Conclusion: The compounds of the present invention have excellent plasma stability.

[0305] Experimental Example 6: Plasma Protein Binding (PPB) Test A. Purpose of the experiment The purpose is to study the binding rate of the test compound to human / mouse plasma albumin. B. Experimental Procedure 1. Preparation of the matrix: On the day of the experiment, the plasma was thawed in cold water and centrifuged at 3220 rpm for 5 minutes to remove any clots. The pH of the resulting plasma was measured and adjusted to 7.4±0.1 with 1% phosphoric acid or 1N sodium hydroxide, if necessary.

[0306] 2. Dilution step of test compound: Test compound was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with concentrations of 10 mM and 2 mM, respectively. 2 μL of stock solution (2 mM) was diluted with 98 μL of DMSO to prepare a working solution of 40 μM. 10 μL of stock solution was diluted with 240 μL of DMSO to prepare a working solution of 400 μM of control compound. Compound working solution (5 μL) and blank matrix (995 μL) were mixed uniformly in a ratio of 1:200 to prepare a loading matrix.

[0307] 3. Analysis step 3.1 An equal amount of 30 μL of loading matrix (n=2) was transferred to a sample collection plate to prepare test time 0 (T0) samples to be used for residue measurement. The samples were immediately matched with the corresponding blank buffer, and the final volume was 60 μL, with a 1:1 volume ratio of plasma to buffer in each well. Then, 60 μL of 4% H3PO4 in H2O and 480 μL of stop solution containing internal standard were added to each T0 sample of test compound. They were stored at 2-8 °C with other samples until further processing.

[0308] 3.2 The remaining plasma samples were pre-incubated for 30 minutes in a carbon dioxide incubator at 37±1°C. Protein-free samples (F samples) were prepared by transferring all of the loading matrix samples (230 μL) to polycarbonate tubes (n=2) and ultracentrifuging at 37°C and 155,000×g (35,000 rpm) for 4 hours.

[0309] 3.3 To prepare T samples (test samples), the samples containing additional matrix were transferred to another 96-well plate (sample incubation plate) and incubated at 37°C for 4 hours. 3.4 After centrifugation, 30 μL of protein-free sample and 30 μL of T sample were taken from the second layer of the supernatant (below the upper layer) and transferred to a new sample collection plate. Each sample was mixed with the corresponding blank buffer or matrix, with a final volume of 60 μL and a matrix:buffer volume ratio of 1:1. 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing internal standard) were added to all samples. The mixture was centrifuged at 4000 rpm for 20 min, and 100 μL of each sample supernatant was taken to perform LC-MS / MS analysis.

[0310] C. Experimental Results The experimental results are shown in Table 6. Note: NA indicates that plasma protein binding was too high to detect free drug at normal plasma protein concentrations.

[0311] [Table 25] Conclusion: The compounds of the present invention have extremely high plasma protein binding.

[0312] Experimental Example 7: Kidney Homogenate Stability Test (BHS) A. Purpose of the experiment To study the stability of test compounds in normal mouse kidney homogenates.

[0313] B. Experimental Procedure 1. Before the experiment, frozen kidney homogenates were thawed in a 37°C water bath. 2. Test compounds: Dilute 10 mM stock solution in DMSO to make a 1 mM intermediate solution; 3. Test compounds: Dilute 1 mM intermediate solution with DMSO to make 50 μM drug spiked solution; 4. At each time point (0, 10, 30, 60, 120, 180, 240 min), 100 μL of 4% H3PO4 and 800 μL of stop solution were added to precipitate proteins and mixed thoroughly.

[0314] 5. The samples were centrifuged at 4000 rpm for 10 minutes, and 100 μL of the supernatant from each well was collected onto the plate. The samples were shaken at 800 rpm for about 10 minutes, and then the LC-MS / MS test was performed.

[0315] C. Experimental Results The experimental results are shown in Table 7.

[0316] [Table 26]

[0317] Experimental Example 8: Intraperitoneal glucose tolerance (ipGTT) experiment of compounds in mice - in vivo efficacy evaluation A. Purpose of the experiment This is to study the improving effect of the test compound on glucose tolerance in normal mice.

[0318] B. Experimental Procedure 1. After dividing the mice into groups according to their body weight and blood glucose level, the animals in each group were injected with test compound (0.3 nmol / kg) and vehicle (20 mM Nguyen's buffer solution) respectively, fasted overnight, and then intraperitoneally injected with glucose solution (2 g / kg, 10 mL / kg) 18 hours later; 2. Blood glucose concentrations were measured using a blood glucose meter at -60, 0, 15, 30, 60 and 120 minutes after glucose administration.

[0319] C. Experimental Results The experimental results are shown in Table 8.

[0320] [Table 27] Conclusion: The compound of the present invention has an excellent effect of improving glucose tolerance.

[0321] Experimental Example 9: Pharmacodynamic studies in db / db mice - in vivo efficacy evaluation A. Purpose of the experiment To study the blood glucose control effect of the test compound on type II diabetic db / db mice.

[0322] B. Experimental Procedure 1. After the db / db mice arrived at the facility, they were kept in a strictly environmentally controlled animal room with a temperature of 20-24°C and humidity of 30-70%. The temperature and humidity in the room were monitored in real time with a thermohygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon). The light in the animal room was controlled by an electronic timing lighting system, with the lights on for 12 hours and off for 12 hours every day (lights on at 7:00 a.m. and off at 7:00 p.m.). During the experiment, the animals were kept in single cages, and toys were provided in each cage. During the experiment, the animals were given free access to food (rat and mouse growth / breeding chow) and drinking water. 2. The animals in each group were subcutaneously injected with the solvent and the test compound (15 nmol / kg), respectively. The administration time was 9:30 to 11:00 am, the administration frequency was once a day, and the administration was continued for 4 weeks.

[0323] C. Experimental Results The experimental results are shown in Table 9.

[0324] [Table 28] Conclusion: The compounds of the present invention show excellent hypoglycemic effects in db / db mice.

[0325] Experimental Example 10: Pharmacodynamic study in DIO mice - in vivo efficacy evaluation A. Purpose of the experiment To study the weight loss effect of the test compound in DIO mice.

[0326] B. Experimental Procedure 1. After the DIO mice arrived at the WuXi AppTec facility, they were housed in a strictly environmentally controlled animal room with a temperature of 20-24°C and humidity of 30-70%. The temperature and humidity in the animal room were monitored in real time with a thermohygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon). The light in the animal room was controlled by an electronic timing lighting system, with the lights on for 12 hours and off for 12 hours every day (lights on at 7:00 a.m. and off at 7:00 p.m.). During the experiment, the animals were housed in single cages, and toys were provided in each cage. During the experiment, the animals were provided with food (rat and mouse growth / breeding chow) and drinking water ad libitum. 2. The animals in each group were subcutaneously injected with the solvent and the test compound (10 nmol / kg), respectively, the administration time was 9:30 am, the administration frequency was once every 3 days, and the administration period was 22 days.

[0327] C. Experimental Results The experimental results are shown in Table 10.

[0328] [Table 29] Conclusion: The compounds of the present invention show excellent weight-reducing effects in DIO mice.

Claims

1. A compound of the formula: or a pharma- ceutically acceptable salt thereof. SEQ ID NO: 1: YAibEGT FTSDY SIAibLD KK １ A.Z. ０ K ０ Z １ FZ ２ E ０ W.L.Z. ３ AGG PSSGA PPPS ０ SEQ ID NO: 2: YAibEGT FTSDY SIAibLD KE ０ AQK ０ AFVK １ W LIAGG PSSGA PPPS ０ (however, The structure of Aib is 【Chemistry 1】 and S ０ teeth, 【Chemistry 2】 and Z ０ is selected from glutamine (Q) and asparagine (N); Z １ is selected from alanine (A) and glutamic acid (E); Z ２ is selected from valine (V) and isoleucine (I); Z ３ is selected from isoleucine (I) and leucine (L); E ０ and K. ０ represents that the carboxyl on the side chain of glutamic acid and the amino on the side chain of lysine are joined together to form a lactam, where K ０ Z １ FZ ２ E ０ The structure of 【Chemistry 3】 And E ０ AQK ０ The structure of 【Chemistry 4】 and K １ is the amino group on the side chain of lysine and -X-X １ -X ２ The structure is 【Chemistry 5】 and X is 【Chemistry 6】 and R １ and R ２ is H, X １ teeth, 【Chemistry 7】 is selected from X ２ teeth, 【Chemistry 8】 and m and n are 2; s is 2; q is selected from 15 and 17.

2. 2. The compound of claim 1 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 9】 (however, Aib, Z ０ , Z １ , Z ２ , Z ３ and K. １ is as defined in claim 1.

3. X １ teeth, 【Chemistry 10】 3. The compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, selected from:

4. X ２ teeth, 【Chemistry 11】 3. The compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, selected from:

5. -X-X １ -X ２ teeth, 【Chemistry 12】 3. The compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, selected from:

6. A compound represented by the following formula or a pharma- ceutically acceptable salt thereof: 【Chemistry 13】 【change】 【change】

7. 13. Use of a compound according to any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating diseases related to obesity and diabetes.

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