CN108299553B - Oxyntomodulin modifier - Google Patents

Abstract

The invention discloses a modification similar to oxyntomodulin and having double excitation effects on a GLP-1 receptor and a glucagon receptor, and a pharmaceutical composition comprising the modification as an active ingredient. The modifier is shown as a formula (I): (OXM ') -L-Y (I) wherein OXM ' is a natural oxyntomodulin or an analogue of an oxyntomodulin, Y is a fatty acid, and L is a linking group of OXM ' to Y.

Description

Oxyntomodulin modifier

Technical Field

The invention relates to a novel oxyntomodulin analogue and a modifier thereof, in particular to an oxyntomodulin analogue with double excitation effects on a GLP-1 receptor and a glucagon receptor and a modifier thereof, and a pharmaceutical composition taking the analogue and the modifier as active ingredients.

Background

Obesity is a common group of metabolic disorders. When the eating calorie of the human body is more than the consumed calorie, the excessive calorie is stored in the body in the form of fat, the amount of which exceeds the normal physiological requirement, and when the excessive calorie reaches a certain value, obesity develops. The weight of the adipose tissues of normal male adults accounts for 15-18 percent of the body weight, and the weight of the adipose tissues of the female adults accounts for 20-25 percent. With age, the proportion of body fat increases correspondingly. The obesity assessment method includes various methods such as anthropometry, dual-energy X-ray absorption method, ultrasound, CT, and infrared induction method. If the disease has no obvious cause, the disease is called simple obesity, and the disease with definite cause is called secondary obesity.

The World Health Organization (WHO) estimates that more than 10 billion people in the world are overweight, and at least 3 billion of them are clinically obese. Specifically, 250,000 people annually in europe and over 250 million people annually in the world die due to being overweight.

The probability of coronary heart disease and hypertension of a patient suffering from obesity is obviously higher than that of a patient suffering from non-obesity, the incidence rate of the patient is generally 5-10 times that of the patient suffering from non-obesity, and particularly, the patient suffering from central obesity with a thick waist (90 cm for male and 85cm for female) is suitable for patients suffering from obesity. The decreased lung capacity and decreased lung compliance of obese patients can lead to a variety of pulmonary dysfunction, such as obese hypopnea syndrome, clinically characterized by lethargy, obesity, alveolar hypopnea, and often associated obstructive sleep dyspnea. Severe cases may cause pulmonary heart syndrome. The excessive calories consumed promote triglyceride synthesis and catabolism, and the lipid metabolism of obesity appears to be more active and the metabolic changes involved in the development of insulin resistance are suppressed relative to carbohydrate metabolism. Obesity is accompanied by active lipid metabolism and metabolic disorders, such as hypertriglyceridemia, hypercholesterolemia, and hypo-HDL cholesterolemia. Fat cells of obese people secrete a plurality of fat factors and inflammatory factors, and can aggravate the occurrence of osteoporosis and bone fracture of obese people. Obesity can also cause undesirable changes in the endocrine system.

Obesity is difficult to treat because obesity is a complex disorder involving appetite regulation and energy metabolism. In the case of obesity treatment, the efforts of obese patients should be combined to treat abnormal manifestations related to appetite regulation and energy metabolism. Drugs that have been used or are being used in clinical practice for the treatment of obesity include Rimonabant (Rimonabant, Sanofi-Aventis), sibutramine (sibutrin, Abbott), continve (takeda), Orlistat (Orlistat, Roche), etc., but have the disadvantage of serious side effects or a very weak anti-obesity effect. For example, rimonabant (Sanofi-Aventis) is reported to show side effects of central nervous disorders, sibutramine (Abbott) and contextual (takeda) to show cardiovascular side effects, and orlistat (Roche) to show weight loss of only 4kg for 1 year. At present, no safe and effective obesity treatment medicine exists.

The regulation of appetite and energy metabolism in human body is mainly regulated by hypothalamus, brain stem solitary nucleus, insulin, adiponectin, insulin and other islets, fat metabolism signal molecules, gastrointestinal hormones such as gastric growth hormone, glucagon-like peptide and gastrin regulating hormone, vagus nerve and the like. Hormones and neurotransmitters secreted by hypothalamus and the nucleus of the fasciculatus of the Yangtze brain and fat metabolism signal molecules need to play a role through a nervous system and a blood circulation system, and weight-reducing medicines aiming at the regulating pathways need to play a role through a central nerve, so that the influence is large, and the side effect is serious. Only the gastrointestinal hormones act directly on the gastrointestinal tract, regulating appetite and energy metabolism. Therefore, the weight-reducing medicine target aiming at the gastrointestinal hormone is limited, and the side effect is small.

Oxyntomodulin (oxntomodulin, OXM) is a short peptide hormone secreted by L-cells of the intestinal epithelium. There is a massive distribution of OXM at the end of the human intestinal tract, with concentrations of OXM increasing progressively in the rodent gastrointestinal tract from the duodenum to the ileum and decreasing after the cecum. The secretion of oxyntomodulin by the intestinal epithelium is regulated by nutrient and energy metabolism, and OXM levels begin to rise 5-10 minutes after feeding and reach a peak 30 minutes later. The secretion level is low in the early morning and high in the evening. OXM is a peptide hormone consisting of 37 amino acids, which is a product of post-transcriptional processing of glucagon gene, and includes the entire sequence of glucagon in the 33-69 th amino acid region of its precursor, and extends an 8-peptide (Lys-Arg-Asn-Lys-Asn-Ile-Ala) at the C-terminus, thus being called "glucagon-37", and its typical amino acid sequence is as follows: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA are provided.

Previous studies also showed that OXM is a potent slimming drug because it inhibits food intake, promotes satiety, like GLP-1, and has lipolytic activity like glucagon. Long-term central and peripheral administration of OXM reduced the weight gain of the rats. Intravenous administration of OXM above physiological levels can reduce food intake in a subject. Dhilfocs et al found in four week study period that overweight or obese subjects self-administered OXM had significantly less intake than saline control group and also had significantly faster weight loss than control group, and this effect was sustained over the course of the four week study period. OXM slows the emptying of nutrients in the stomach. Has the functions of reducing food absorption, suppressing appetite, mobilizing fat, reducing energy intake, increasing energy consumption, and making energy metabolism in negative balance state, thereby reducing weight.

However, OXM has a short half-life and is rapidly inactivated by cell surface dipeptidyl peptidase IV (DP-IV), whereas DP-IV inhibitors have intermediate body weight effects in the clinic, suggesting that supra-physiological levels of OXM may be required to achieve weight loss in humans. Therefore, oxyntomodulin shows potential as a therapeutic means for metabolic diseases such as diabetes and obesity, but because OXM has poor in vivo stability, it is required to develop a modification that can replace OXM.

WO2012169798 discloses a series of novel peptides showing superior activity to the glucagon-like peptide-1 receptor and glucagon receptor than the natural oxyntomodulin. The disclosed polypeptides of SEQ ID NO.24 and 25 have excellent dual stimulation effects of GLP-1 and glucagon receptors.

WO2012173422 discloses a conjugate of a plurality of oxyntomodulin peptides, an immunoglobulin Fc region and a non-peptidyl polymer, wherein the conjugate is obtained by covalently linking the oxyntomodulin peptides to the immunoglobulin Fc region by the non-peptidyl polymer.

Although a plurality of oxyntomodulin modifiers are disclosed in the prior art, the failure rate of research and development is high in the process of drug development, so that more lead drugs need to be researched to increase the success rate of drug development.

Disclosure of Invention

The inventor develops a safe and effective medicament for treating obesity, diabetes and hyperlipidemia related diseases with clinical prospect by researching and developing novel oxyntomodulin analogues and modifications thereof.

A typical natural oxyntomodulin is shown in SEQ ID NO: 1, and in the present invention, the polypeptide is referred to as P1. In one aspect, the invention provides a series of analogs of natural oxyntomodulin, which are polypeptides.

SEQ ID NO：1HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

The polypeptides of the invention are shown in table 1:

TABLE 1 oxyntomodulin analogs of the invention

In a second aspect, the present invention provides a modified product of the above analog. The modifier is shown as a formula (I):

(OXM‘)-L-Y(I)

wherein OXM 'is a natural oxyntomodulin or an analogue of oxyntomodulin, Y is a fatty acid, and L is a linking group of OXM' and Y.

Preferably, the modifier is represented by formula (II):

n is any integer between 6 and 20.

Formula (II) is preferably

In preferred modifications of formula (II), OXM' is:

a polypeptide of the amino acid sequence of SEQ ID No. 2;

a polypeptide of the amino acid sequence of SEQ ID No. 3;

a polypeptide of the amino acid sequence of SEQ ID No. 4;

a polypeptide of the amino acid sequence of SEQ ID No. 5;

a polypeptide of the amino acid sequence of SEQ ID No. 6;

a polypeptide of the amino acid sequence of SEQ ID No. 7;

a polypeptide of the amino acid sequence of SEQ ID No. 8;

a polypeptide of the amino acid sequence of SEQ ID No. 9;

a polypeptide of the amino acid sequence of SEQ ID No. 10;

a polypeptide of the amino acid sequence of SEQ ID No. 24;

a polypeptide of the amino acid sequence of SEQ ID No. 25.

Wherein the polypeptides of the amino acid sequences of SEQ ID No.24 and SEQ ID No.25 are the polypeptides disclosed in WO2012169798, wherein:

SEQ ID No.24 HAibQG TFTSD YSKYL DEKRAKEFVQ WLMNTC

SEQ ID No.25 HAibQG TFTSD YSKYL DEKRAKEFVQ WLMNTC (underlined indicates ring formation)

The polypeptides of the amino acid sequences of SEQ ID No.24 and SEQ ID No.25 are referred to herein as P2 and P3, respectively.

Specifically, the present invention provides the following modifications:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 2:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 3:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 4:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 5:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 6:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 7:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 8:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 9:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 10:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 24:

a modification of the polypeptide of the amino acid sequence of SEQ ID No. 25:

wherein m is any integer between 2 and 10; n is any integer between 6 and 20.

More specifically, the present invention provides the following modifications:

PP03b01：

PP03c01：

PP03d01：

PP03h01：

PP03h02：

PP03h03：

PP03j01：

PP03k01：

PP03m01：

PP03n01：

PP03s01：

PP03p01：

PP03q01：

in a third aspect, the invention provides the use of an oxyntomodulin analogue of the invention and a modification of an oxyntomodulin analogue of the invention in the manufacture of a medicament for the prevention and/or treatment of a disease and/or condition associated with low GLP-1 receptor activity; the use in the manufacture of a medicament for the treatment of obesity and related diseases; use in the manufacture of a medicament for weight loss; use in the manufacture of a medicament for a disease and/or condition associated with carbohydrate metabolism; the use in the preparation of medicaments for diabetes; the application in preparing the medicine for preventing or treating hyperlipemia, fatty liver disease or arteriosclerosis.

The fourth aspect of the present invention provides a pharmaceutical composition comprising the oxyntomodulin analog of the present invention or a modification of the oxyntomodulin analog of the present invention, and optionally a pharmaceutically acceptable carrier, which may be a pharmaceutically common tablet, capsule, syrup, injection, or the like.

In a fifth aspect, the present invention provides the use of a pharmaceutical composition comprising an oxyntomodulin analogue of the invention or a modification of an oxyntomodulin analogue of the invention in the manufacture of a medicament for the prevention and/or treatment of a disease and/or condition associated with low GLP-1 receptor activity; the use in the manufacture of a medicament for the treatment of obesity and related diseases; use in the manufacture of a medicament for weight loss; use in the manufacture of a medicament for a disease and/or condition associated with carbohydrate metabolism; the use in the preparation of medicaments for diabetes; the application in preparing the medicine for preventing or treating hyperlipemia, fatty liver disease or arteriosclerosis.

The oxyntomodulin has agonistic effect on GLP-1 and glucagon receptors. GLP-1 lowers blood glucose, reduces food intake, and inhibits gastric emptying, while glucagon increases blood glucose, promotes lipolysis, and reduces body weight by increasing energy metabolism. The different biological effects of the two peptides can lead to undesirable effects, increasing blood glucose if glucagon shows more dominant effects than GLP-1, or nausea and vomiting if GLP-1 shows more dominant effects than glucagon. Thus, the present invention of modifications of oxyntomodulin peptide analogues and analogues is not only intended to enhance these activities, but also has a balanced ratio of glucagon to GLP-1 activity, as illustrated by pharmacological tests:

the reagents, instruments, samples used in the following tests, unless otherwise indicated, are commercially available, wherein:

HEPES (high efficiency particulate air): 4-hydroxyethyl piperazine ethanesulfonic acid

HBSS: hank's balanced salt solution

BSA stabilizer: bovine serum albumin stabilizer

IBMX: 3-isobutyl-1-methylxanthines

EXPERIMENT I CAMP DETERMINATION TARGETING GLUCOPAGON AND GLP-1 RECEPTOR

Firstly, testing target:

cAMP assay of sample for glucagon and GLP-1 receptor activity

II, materials:

1. reagent

HEK293 cells overexpressing glucagon and GLP-1 receptors

cAMP detection kit, Cisbio (Cat #62AM4PEJ)

1M HEPES，Invitrogen(Cat#15630-106)

1X HBSS，Invitrogen(Cat#14025)

BSA stabilizer (7.5% solution), Perkinelmer (Cat # CR84-100)

IBMX, Sigma (catalog number I5879)

2. Consumable and instrument

OptiPlate-384 well plates, PerkinElmer (Cat #6007290)

384 well plate, Labcyte (Cat # P-05525)

EnVision Detector, PerkinElmer

Bravo liquid processor (liquid handler) (Agilent)

Vi-CELL cytometer, Beckman (Cat # Vi-CELL)^TMXR cell viability analyzer

3. Sample (I)

P1, P2, P3, PP03b, PP03c, PP03d, PP03h, PP03j, PP03k, PP03m, PP03n, PP03s, PP03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03P01, and PP03q01 are all synthesized by borrelid biomedical (suzhou) incorporated.

Glucagon was purchased from Sigma (Cat #91201) and exenatide was supplied by a co-operating clinical laboratory.

Third, test method

1. Configuration of Glucagon (GCGR) and GLP-1 cell suspensions:

1) assay buffer: 5mM HEPES; 0.1% BSA stabilizer; 0.5mM IBMX; HBSS.

2) One tube was thawed rapidly for each cell in a 37 ℃ water bath.

3) Each tube of cell suspension was transferred to 10ml HBSS in a 15ml conical tube.

4) Centrifuging the cells at 1000rpm for 5 minutes at room temperature to obtain lower layer cells (pellet cells)

5) Gently aspirate supernatant, take care not to aspirate cells.

6) The lower layer cells were flicked loose and the cells were resuspended in 10ml of HBSS. And (4) blowing and beating up and down by using a sterile pipettor to uniformly mix the components.

7) The cell concentration was counted on Vi-cells and the cell activity was determined.

8) Resuspending the cells in the assayThe cell concentration in the assay buffer was 1.5X 10⁵/mL。

2. Sample preparation:

1) three-fold dilutions from the highest concentration of 0.2mM (sample dissolved in DMSO) were made using a Bravo liquid handler, configuring 10 concentrations of test samples.

2) Three-fold dilutions from the highest concentration of 0.002mM (control dissolved in DMSO) were made using Bravo liquid handler, configuring 10 concentrations of exenatide and glucagon control samples.

Assay for HTRF cAMP:

1) 50nL of exenatide, 50nL of glucagon, 50nL of sample, 50nLDMSO were transferred to OptiPlate-384 plates, respectively, using an acoustic pipetting system (Echo transfer).

2) Using an electronic multichannel pipettor (Electric Multiple Channel Pipette), 10 μ Ι _ of cell suspension was added to the designated area of the detection plate according to the plate map (plate map).

3) Rotate the plate at 1000rpm for 1 minute.

4) Cells were stimulated at room temperature for 60 min.

5) 8 concentrations of cAMP standard curve were configured, with a maximum concentration of 800nM, 4-fold dilution.

6) Draw cAMP standard curve according to detection plate.

7) Preparation of detection reagents and antibodies according to cAMP detection kit instructions.

8) Add 5 μ L of detection reagent to the detection plate by electronic multichannel pipettor (Electric Multiple Channel Pipette).

9) Cover 384 plates with TopSeal-a film and incubate for 60 min at room temperature.

10) Remove TopSeal-a and read on EnVision detector.

11) Data was analyzed by Prism.

% activity 100 × (calculated samples-Low controlled Average (Low control _ Average)/(High control _ Average) -Low controlled Average)

Fourthly, test results:

P1、P2、P3、PP03b、PP03c、PP03d、PP03h、PP03j、PP03k、PP03m、PP03n、PP03s、PP03b01、PP03c01、PP03d01、PP03h01、PP03h02、PP03h03、PP03j01、PP03k01、PP03m01、PP03n01、PP03s01、PP03p01、PP03q01

TABLE 2 cAMP assay for glucagon and GLP-1 receptor activity

The modified substances of the oxyntomodulin peptide analogue and the analogue have higher agonistic activity on GCGR receptor and GLP-1 receptor, and the agonistic activity is higher than that of natural oxyntomodulin P1. Wherein, the oxyntomodulin peptide analogues PP03b, PP03c, PP03d, PP03h, PP03j, PP03k, PP03m, PP03n and PP03s have higher activity on GCGR receptor and GLP-1 receptor than the polypeptides P2 and P3 with the amino acid sequences of SEQ ID No.24 and SEQ ID No.25 disclosed in WO2012169798, and have balanced activity ratio of glucagon and GLP-1.

It is known to those skilled in the art that in a biologically active molecule conjugated to a polymeric group, the biological activity of the conjugated biomolecule will decrease exponentially as the molecular weight of the conjugated group increases. The technical personnel of the invention unexpectedly discover that the modifiers PP03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03p01 and PP03q01 obtained by modifying the oxyntomodulin analogue of the invention retain most of agonist activity and have balanced activity ratio of glucagon to GLP-1.

In order to further verify the effects of the oxyntomodulin peptide analogues and modifications in weight loss, blood glucose reduction and lipolysis promotion, the inventors further performed the following pharmacological tests:

experiment II, lipid lowering Effect in hyperlipemia model hamster

Test animal group

Male hamsters (Golden Syrian hamsters, 120-. Hamsters are known to exhibit a similar blood lipid profile to humans but different from other rodents and are sensitive to high fat diets.

Animals were allowed to obtain either a sterilized high fat diet (Purina 5001 containing 11.5% corn oil, 11.5% coconut oil, 0.5% cholesterol and 0.25% deoxycholic acid; Dyets, Bethlehem, Pa.) or a standard rodent diet (low fat, 2018; Harlan Teklad, Madison, Wis.). The normal diet group was allowed to obtain filtered and UV sterilized tap water, and the high fat diet group was allowed to obtain water containing 10% fructose. Animals were housed in a housing meeting GLP standards under a 12 hour illumination/12 hour photophobic cycle (illumination: am 6 to pm6) and all experimental procedures were performed according to the animal experimental standards guidelines. Drug administration was started after 3 weeks of hyperlipidemia induction and animals were divided into 15 groups (n-5).

Specifically, group 1 (normal group) was fed with normal food and administered subcutaneously 5ml/kg of Dulbecco phosphate buffered saline (DPBS, Sigma) once or more weekly.

Group 2 (hyperlipidemia-induced group) was fed a high-fat diet to induce hyperlipidemia, followed by subcutaneous administration of 5ml/kg of Dulbecco phosphate buffered saline (DPBS, Sigma) once or more weekly.

Group 3 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03b01) was fed with a high fat diet to induce hyperlipidemia, and then administered with 5nmol/kg of PP03b01 once a week at an injection dose of 5 ml/kg.

Group 4 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03c01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03c01 once a week at an injection dose of 5 ml/kg.

Group 5 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03d01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03d01 once a week at an injection dose of 5 ml/kg.

Group 6 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03h01) was fed with a high fat diet to induce hyperlipidemia, followed by administration of 5nmol/kg of PP03h01 once a week at an injection dose of 5 ml/kg.

Group 7 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03h02) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03h02 once a week at an injection dose of 5 ml/kg.

Group 8 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03h03) was fed with a high fat diet to induce hyperlipidemia, followed by administration of 5nmol/kg of PP03h03 once a week at an injection dose of 5 ml/kg.

Group 9 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03j01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03j01 once a week at an injection dose of 5 ml/kg.

Group 10 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03k01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03k01 once a week at an injection dose of 5 ml/kg.

Group 11 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03m01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03m01 once a week at an injection dose of 5 ml/kg.

Group 12 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03n01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03n01 once a week at an injection dose of 5 ml/kg.

Group 13 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03s01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03s01 once a week at an injection dose of 5 ml/kg.

Group 14 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03p01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03p01 once a week at an injection dose of 5 ml/kg.

Group 15 (hyperlipidemia-inducing group + administration of 5.00nmol/kg of PP03q01) was fed with a high fat diet to induce hyperlipidemia, and then administered 5nmol/kg of PP03q01 once a week at an injection dose of 5 ml/kg.

The whole experimental period was 2 weeks and its effect of reducing lipid levels was analyzed. The test period was continued to be extended to 24 weeks and the anti-obesity effect was analyzed.

Second, experimental results

Lipid levels of blood were analyzed using HITACHI 7020, the results obtained were statistically processed, and mean values and standard deviations of the mean values were calculated. In the demonstration of inter-group significance, data were statistically processed using the Dunnett test (Dunnett's test) with one-way ANOVA, and values of p <0.05 were considered statistically significant differences, with the results as follows:

1. changes in blood triglyceride levels

See fig. 1, where groups 3-15 were significantly different from group 2 as compared to group 1.

In the measurement results of blood triglyceride levels, triglyceride levels were significantly increased in the group of hamsters 2 fed with a high-fat diet, but when the modifications of the oxyntomodulin peptide analogues of the present invention, P03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03P01, PP03q01, were administered to hamsters, triglyceride levels were significantly decreased.

2. Changes in blood Total Cholesterol levels

See fig. 2, where groups 3-15 were significantly different from group 2 as compared to group 1.

In the measurement results of the total cholesterol level in blood, triglyceride level was significantly increased in the group of hamsters 2 fed with a high fat diet, but when the modifications of the oxyntomodulin peptide analogues of the present invention, P03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03P01, PP03q01, were administered to hamsters, total cholesterol level was significantly decreased.

3. Anti-obesity effect

See fig. 3, where groups 3-15 were significantly different from group 2 as compared to group 1.

In the measurement results of obesity, the body weight level was significantly increased in the group of hamsters 2 fed with a high-fat diet, but the body weight was significantly reduced when the modifications P03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03P01, and PP03q01 of the oxyntomodulin peptide analogues of the present invention were administered to hamsters.

From the above results, it can be seen that the modified oxyntomodulin peptide analog of the present invention can be used for the treatment of hyperlipidemia or associated fatty liver disease or arteriosclerosis, and can be used as an obesity therapeutic agent.

Experiment III, blood sugar reduction effect on spontaneous type 2 diabetes db/db mice

85C 57BL/6db/db mice (male) of 5-6 weeks old were purchased from Nanjing university model animal research institute, and the experimental animals were housed in SPF animal house. The animal room is well ventilated, an air conditioner is arranged, the temperature is kept at 20-25 ℃, the humidity is kept at 40% -70%, the ventilation frequency is 10-15 times/h, the lighting is carried out in a dark place for 12 hours respectively, the experimental animals eat and drink water freely, and each mouse is marked with an ear tag. Mice were used for one experiment per week, and mice were used for no more than three weeks. After one week of acclimation period, blood glucose in mouse tail tip capillaries was measured using a glucometer. 70 mice with blood sugar level more than 16.7mmol/L were selected and randomly divided into 14 groups according to blood sugar level, model control group was subcutaneously injected with 5mL/kg PBS (pH 7.4), positive control group 1 was subcutaneously injected with exenatide (10. mu.g/kg, 5mL/kg), administration group was subcutaneously injected with P03b01, PP03c01, PP03d01, PP03h01, PP03h02, PP03h03, PP03j01, PP03k01, PP03m01, PP03n01, PP03s01, PP03P01, PP03q01 (10. mu.g/kg, 5mL/kg), blood sugar after administration was measured with glucometer for 0, 1, 2, 4, 8, 12, 18, 24, 30, 36, 42, 48, 72h, and all data were inputted into Graphpad Prism to calculate average blood sugar. The maximum hypoglycemic effect (maximum rate of decrease compared to the model group), the maximum hypoglycemic time (last time point of significant decrease in blood glucose compared to the model group) were calculated.

TABLE 3 hypoglycemic effects on spontaneous type 2 diabetic db/db mice

Compared with exenatide, the modifier of the oxyntomodulin regulating peptide analogue has equivalent activity or only slightly reduced activity, so that the blood sugar level of a spontaneous type 2 diabetic db/db mouse is obviously reduced, the maximum blood sugar reduction time is prolonged from 4h to 30h-48h, the blood sugar reduction duration is long, and the better pharmacokinetic property is achieved. The modified oxyntomodulin peptide analogue can be effectively used for treating diabetes, and shows a remarkable effect of inhibiting weight gain, which shows that the modified oxyntomodulin peptide analogue can reduce cardiovascular complications of diabetes.

Drawings

FIG. 1: in experiment two of lipid lowering effect in hyperlipidemic model hamsters, changes in blood triglyceride levels in groups 1-15;

FIG. 2: in experiment two of lipid lowering effect in hyperlipidemic model hamsters, change in blood total cholesterol levels of groups 1-15;

FIG. 3: in experiment two of lipid lowering effect in hyperlipidemia model hamster, groups 1-15 had anti-obesity effect.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Polypeptide synthesis moieties

The amino acids and abbreviations and acronyms for English are shown in the following table:

EXAMPLE 1 preparation of PP03h

The PP03h was synthesized by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay to monitor the reaction, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Lys (alloc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Boc-OH, Fmoc-Ser (tBu) -OH, (Tyr-Tyr), (Tyr) (tBu) -OH, Asp-OtBu) OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, DMF washing, methanol washing, dichloromethane washing and drying, adding a cracking reagent (trifluoroacetic acid: benzyl sulfide: phenol: triisopropylsilane ═ 85: 5: 5), reacting for 2 hours, precipitating with ice-tert-butyl methyl ether, centrifuging to obtain crude PP03h, purifying by HPLC, and freeze-drying to obtain pure PP03 h.

ESI-MS M/z calculated 3623.31, found 1028.7[ M +3H ]]³⁺，906.8[M+4H]⁴⁺，725.8[M+5H]⁵⁺

EXAMPLE 2 preparation of PP03b

The procedure of example 1 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03b sequence amino acids onto the Resin as in example 1.

ESI-MS M/z calculated 3598.28, found 1200.6[ M +3H ]]³⁺，900.7[M+4H]⁴⁺

EXAMPLE 3 preparation of PP03f

The procedure of example 1 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03f sequence amino acids onto the Resin as in example 1.

ESI-MS M/z calculated 3495.14, found 1166.3[ M +3H ]]³⁺，875.0[M+4H]⁴⁺

Example 4 preparation of PP03k

The procedure of example 1 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03k sequence amino acids onto the Resin as in example 1.

ESI-MS M/z calculated 3570.99, found 1193.5[ M +3H ]]³⁺，893.9[M+4H]⁴⁺

Example 5 preparation of PP03n

The procedure of example 1 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03n sequence amino acids onto the Resin as in example 1.

ESI-MS M/z calculated 3596.29, found 1199.7[ M +3H ]]³⁺，900.1[M+4H]⁴⁺，720.4[M+5H]⁵⁺

EXAMPLE 6 preparation of PP03c

The PP03c was synthesized by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay to monitor the reaction, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OAll) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Boc-OH, Fmoc-Ser (tBu) -OH, (Tyr-Tyr), (Tyr) -OH, Asp-OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, followed by the addition of 3.0eq in chloroform: n-methylmorpholine: tetrakis (triphenylphosphine) palladium acetate (18:2:1), reacting for 2 hours to remove Lys (alloc), Glu (OAll)And (3) washing a protecting group with DMF, adding TBTU and DIEA to close a ring, washing the DMF after the ninhydrin detection reaction is almost completely finished, washing the protecting group with methanol, washing the protecting group with dichloromethane, drying the protecting group, adding a cracking reagent (trifluoroacetic acid: benzylthioether: phenol: triisopropylsilane: 85: 5: 5), reacting for 2 hours, precipitating the obtained product with ice-tert-butyl methyl ether, centrifuging the obtained product to obtain a crude product of PP03c, preparing and purifying the crude product by HPLC, and freeze-drying the crude product to obtain a pure product of PP03 c. ESI-MS M/z calculated 3580.28, found 1194.8[ M +3H ]]³⁺，896.5[M+4H]⁴⁺

Example 7 preparation of PP03d

The procedure of example 6 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03d sequence amino acids onto the Resin as in example 6.

ESI-MS M/z calculated 3580.28, found 1194.6[ M +3H ]]³⁺，896.3[M+4H]⁴⁺

EXAMPLE 8 preparation of PP03j

The procedure of example 6 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03j sequence amino acids onto the Resin as in example 6.

ESI-MS M/z calculated 3605.31, found 1202.8[ M +3H ]]³⁺，902.4[M+4H]⁴⁺，722.3[M+5H]⁵⁺

Example 9 preparation of PP03m

The procedure of example 6 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03m sequence amino acids onto the Resin as in example 6.

ESI-MS M/z calculated 3552.98, found 1185.6[ M +3H ]]³⁺，889.6[M+4H]⁴⁺

EXAMPLE 10 preparation of PP03s

The procedure of example 6 was followed by solid phase synthesis using Fmoc method well known to those skilled in the art, Fmoc removal using 2-Cl-Trt Resin with 20% piperidine/DMF, coupling reagents HOBT/DIC with DMF as reaction solvent, and ninhydrin detection with sequential post-treatment of PP03s sequence amino acids onto the Resin as in example 6.

ESI-MS M/z calculated 3578.29, found 1193.8[ M +3H ]]³⁺，895.6[M+4H]⁴⁺，716.8[M+5H]⁵⁺

Example 11 preparation of PP03h01

Preparation of side chain Compound PEG 6-Glu-Octadecaneedioic Acid

Preparation of Compound 2

Under the protection of nitrogen, 200mL of pyridine and 50g of compound 1(1.0eq) are added into a 500mL three-necked flask, the mixture is stirred and cooled to 0 ℃, 35.5g of TsCl (1.0eq) is added in batches and stirred for 1h, then the temperature is slowly raised to room temperature, and the stirring is continued for 3-4 h. After the reaction, the reaction solution was poured into an ice diluted hydrochloric acid solution to produce a solid, ethyl acetate was added for extraction, the ethyl acetate layer was washed once with diluted hydrochloric acid, once with saturated sodium bicarbonate, with saturated saline, and with anhydrous Na₂SO₄Drying, vacuum evaporating to remove solvent, and performing silica gel column chromatography to obtain 38g of pure compound 2.

Preparation of Compound 3

To a 500mL three-necked flask, 38g of Compound 2(1.0eq) and 190mL of DMSO were added, stirred well, and then NaN was added₃11.5g (2.0eq), heated to 50 ℃ for reaction for 3 hours, cooled to room temperature, poured into water, extracted with ethyl acetate several times, combined organic phases, dried over anhydrous sodium sulfate, concentrated to obtain 340 g of colorless liquid compound.

Preparation of Compound 4

370 g of compound, 500mL of methanol and 8.0g of palladium on carbon are added into a 1L hydrogenation reaction kettle, the mixture is stirred and replaced by nitrogen, hydrogen is introduced for reaction for 3 to 4 hours, after the TLC monitoring reaction is finished, the reaction solution is filtered, and the filtrate is concentrated to obtain 52g of compound 4 oily matter.

Preparation of Compound 5

To a 250mL three-necked flask was added 410.0 g (1.0eq), (Boc)₂O15.5 g (2.0eq), methanol: and (2) stirring and heating the mixed solution 200ml of triethylamine (9: 1) to reflux, reacting for 1h, distilling off methanol and triethylamine after TLC monitoring reaction is finished, adding water to dissolve, extracting for 3 times by dichloromethane, combining organic layers, washing once by water, drying by anhydrous sodium sulfate, and concentrating to obtain 9.0g of an oily compound 5.

Preparation of Compound 6

Adding 57.0 g (1.0eq) of compound, 40mL each of toluene and tetrahydrofuran and 7.6g (3.0eq) of bromoacetic acid into a 250mL three-necked flask, stirring, heating to 45-50 ℃, adding 4.4g of sodium hydroxide, reacting overnight, monitoring by TLC after the reaction is finished, evaporating the reaction solution, adding water and ethyl acetate to extract impurities, adjusting the pH of an aqueous phase to 3, extracting the aqueous phase with dichloromethane, combining dichloromethane layers, drying with anhydrous sodium sulfate, and concentrating to obtain 4.2g of compound 6 oily matter.

Preparation of Compound 7

64.0 g of the compound was added to a 250mL single vial, 20mL of ethyl acetate was dissolved and then cooled to 0 ℃ and 20mL of ethyl acetate/HCl (7M/L) was added, and the reaction was monitored by TLC and then concentrated to give 74.2 g of the oily compound.

Preparation of Compound 9

To a 50mL three-necked flask, 1.0g of Compound 8(1.0eq), 10mL of dichloromethane, 10mL of t-butanol, 0.40g of DIC (1.0eq), and 0.39g of DMAP (1.0eq) were added, and the mixture was stirred at room temperature overnight, followed by TLC to monitor completion of the reaction, followed by dilution with diethyl ether, washing with water for 3 times, washing with saturated brine, drying over anhydrous sodium sulfate, and column chromatography to give 90.4 g of a foamy powdery compound.

Preparation of Compound 10

To a 100mL three-necked flask, 0.95g N-Hydroxysuccinimide (HOSU), 2.0g of compound 9 and 15mL of dichloromethane were added, 1.58g of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed with 50mmol/L potassium dihydrogen phosphate aqueous solution having pH of 6.0 for 2 times, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 2.6g of compound 10 as a white solid.

Preparation of Compound 11

Into a 250mL flask was added 3.0g of the compound H-Glu-OtBu (1.0eq), 70mL of water, 2.54g of NaHCO₃(2.05eq), stirring, dropwise adding a solution of 6.9g of compound 10 in 70ml of ethylene glycol dimethyl ether, adding 70ml of tetrahydrofuran, stirring overnight, monitoring by TLC, evaporating the organic solvent, adjusting the pH to 4 with dilute hydrochloric acid, extracting with dichloromethane, drying over anhydrous sodium sulfate, and concentrating to obtain an off-white solid, namely 7.7g of compound 11.

Preparation of Compound 12

To a 250mL three-necked flask, 2.4g N-Hydroxysuccinimide (HOSU), 7.7g of compound 11 and 77mL of dichloromethane were added, 4.1g of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed 2 times with 50mmol/L potassium dihydrogen phosphate aqueous solution having a pH of 6.0, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 7.9g of compound 12 as an off-white solid.

Preparation of Compound 13

Into a 250mL flask was added 4.4g of Compound 7(1.0eq), 80mL of water, 2.1g of NaHCO₃(2.05eq), stirring, dropwise adding a solution of 7.9g of compound 12 dissolved in 80ml of ethylene glycol dimethyl ether, adding 40ml of tetrahydrofuran, stirring overnight, monitoring by TLC after the reaction is finished, evaporating the organic solvent, adjusting the pH to 6 with acetic acid, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating to obtain an off-white solid, and performing column chromatography to obtain 6.5g of light yellow oily compound 13.

Preparation of PP03h01

The PP03h01 was synthesized by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay for reaction monitoring, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Lys (alloc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Boc-OH, Fmoc-Ser (tBu) -OH, (Tyr-Tyr), (Tyr) (tBu) -OH, Asp-OtBu) OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, 3.0eq dissolved in chloroform: n-methylmorpholine: reacting acetic acid (18:2:1) with tetrakis (triphenylphosphine) palladium for 2 hours, washing with dichloromethane, washing with DMF, condensing a connecting compound 13, after ninhydrin detection reaction, washing with DMF, washing with methanol, washing with dichloromethane, drying, adding a cracking reagent (T trifluoroacetic acid: benzylsulfide: phenol: triisopropylsilane: 85: 5: 5), reacting for 2 hours, precipitating with ice-tert-butyl methyl ether, centrifuging to obtain a crude product of PP03h01, purifying by HPLC, and freeze-drying to obtain a pure product of PP03h 01.

ESI-MS M/z calculated 4370.25, found 1458[ M +3H ]]³⁺，1094[M+4H]⁴⁺，875[M+5H]⁵⁺。

Example 12 preparation of PP03h02

Preparation of the side-chain Compounds

Preparation of Compound 21

Under the protection of nitrogen, 200mL of pyridine and 50.0g of compound 20(1.0eq) are added into a 500mL three-necked flask, stirred and cooled to 0 ℃, 89.9g of TsCl (1.0eq) is added in portions, stirred for 1h, then slowly heated to room temperature, and stirred for 3-4 h. After the reaction, the reaction solution was poured into an ice diluted hydrochloric acid solution to produce a solid, ethyl acetate was added for extraction, the ethyl acetate layer was washed once with diluted hydrochloric acid, once with saturated sodium bicarbonate, with saturated saline, and with anhydrous Na₂SO₄Drying, vacuum evaporating to remove solvent, and performing silica gel column chromatography to obtain 44.2g of pure compound 21.

Preparation of Compound 22

To a 500mL three-necked flask, 44.0g of Compound 21(1.0eq) and 200mL of DMSO were added, stirred well, and then NaN was added₃22.0g (2.0eq), heating to 50 ℃ for reaction for 3 hours, cooling to room temperature, pouring the reaction liquid into water, extracting with ethyl acetate for multiple times, combining organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain 45.1g of colorless liquid compound 22.

Preparation of Compound 23

2245 g of compound, 450mL of methanol and 6.0g of palladium carbon are added into a 1L hydrogenation reaction kettle, stirring and nitrogen replacement are carried out, hydrogen is introduced for reaction for 3-4h, after TLC monitoring reaction is finished, reaction liquid is filtered, and filtrate is concentrated to obtain 37.3g of compound 23 oily matter.

Preparation of Compound 24

To a 1000mL three-necked flask was added 2335.0 g (1.0eq), (Boc)₂O145.4 g (2.0eq), methanol: and (3) stirring and heating 700ml of mixed solution of triethylamine (9: 1) to reflux, reacting for 1h, distilling off methanol and triethylamine after TLC monitoring reaction is finished, adding water to dissolve, extracting for 3 times by dichloromethane, combining organic layers, washing once by water, drying by anhydrous sodium sulfate, and concentrating to obtain 33.6g of oily compound 24.

Preparation of Compound 25

2430.0 g (1.0eq) of compound, 150mL each of toluene and tetrahydrofuran and 61.0g (3.0eq) of bromoacetic acid were added to a 500mL three-necked flask, stirred, heated to 45 to 50 ℃, and added with 41.1g of sodium hydroxide to react overnight, after the completion of the reaction monitored by TLC, the reaction solution was evaporated, water and ethyl acetate were added to extract impurities, the pH of the aqueous phase was adjusted to 3, the aqueous phase was extracted with dichloromethane, the dichloromethane layers were combined, dried over anhydrous sodium sulfate and concentrated to obtain 2529.4g of compound.

Preparation of Compound 26

253.0 g of the compound was added to a 500mL single-neck flask, and after dissolving 100mL of ethyl acetate, the temperature was lowered to 0 ℃ and 100mL of ethyl acetate/HCl (7M/L) was added, and after completion of the TLC monitoring reaction, the mixture was concentrated, slurried with ethyl acetate, and filtered by suction to obtain 2.3g of off-white solid compound 26.

Preparation of Compound 28

To a 500mL three-necked flask, 15.0g of Compound 27(1.0eq), 150mL of dichloromethane, 150mL of t-butanol, 5.1g of DIC (1.0eq), 5.0g of DMAP (1.0eq) were added, the mixture was stirred overnight at room temperature, TLC was used to monitor the completion of the reaction, and the mixture was diluted with diethyl ether and then washed with water 3 times, saturated brine was used for washing, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain 285.1 g of a white-like solid compound.

Preparation of Compound 29

To a 100mL three-necked flask, 2.7g N-Hydroxysuccinimide (HOSU), 5.1g of compound 28 and 50mL of dichloromethane were added, 4.6g of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed with 50mmol/L potassium dihydrogen phosphate aqueous solution having pH of 6.0 for 2 times, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 5.2g of compound 29 as a white solid.

Preparation of Compound 30

Into a 250mL flask was added 2.0g of the compound H-Glu-OtBu (1.0eq), 50mL of water, 1.7g of NaHCO₃(2.05eq), stirring, dropwise adding 5.2g (1.0eq) of compound 29 in 50ml of DME (ethylene glycol dimethyl ether) solution, adding 50ml of tetrahydrofuran, stirring overnight, TLC monitoring the reaction, evaporating the organic solvent, adjusting the pH to 4 with dilute hydrochloric acid, extracting with dichloromethane, drying over anhydrous sodium sulfate, and concentrating to obtain 305.1 g of off-white solid compound

Preparation of Compound 31

To a 100mL three-necked flask, 1.44g (1.53eq) of N-hydroxysuccinimide (hou), 5.00g of compound 30 and 50mL of dichloromethane were added, 2.40g (1.53eq) of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed 2 times with 50mmol/L potassium dihydrogen phosphate aqueous solution having a pH of 6.0, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 5.82g of compound 31 as an off-white solid.

Preparation of Compound 32

To a 250mL flask was added 1.63g of Compound 26(1.0eq), 58mL of water, 1.41g of NaHCO₃(2.05eq), stirring, and 5.80g (1.0eq) of the compound is added dropwise31 in 58ml DME (ethylene glycol dimethyl ether), adding 58ml tetrahydrofuran, stirring overnight, TLC monitoring reaction, evaporating organic solvent, adjusting pH to 6 with acetic acid, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating to obtain off-white solid, and performing column chromatography to obtain 5.63g off-white solid compound 32.

Preparation of PP03h02

The PP03h02 was synthesized by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay for reaction monitoring, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Lys (alloc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Boc-OH, Fmoc-Ser (tBu) -OH, (Tyr-Tyr), (Tyr) (tBu) -OH, Asp-OtBu) OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, 3.0eq dissolved in chloroform: n-methylmorpholine: reacting acetic acid (18:2:1) with tetrakis (triphenylphosphine) palladium for 2 hours, washing with dichloromethane, washing with DMF, condensing a connecting compound 32, after ninhydrin detection reaction, washing with DMF, washing with methanol, washing with dichloromethane, drying, adding a cracking reagent (trifluoroacetic acid: benzylsulfide: phenol: triisopropylsilane: 85: 5: 5), reacting for 2 hours, precipitating with ice tert-butyl methyl ether, centrifuging to obtain a PP03h02 crude product, purifying by HPLC, and freeze-drying to obtain a PP03h02 pure product.

ESI-MS M/z calculated 4250.15, found 1417.9[ M +3H ]]³⁺，1063.7[M+4H]⁴⁺。

Example 13 preparation of PP03h03

Preparation of the side-chain Compounds

Preparation of Compound 41

Under the protection of nitrogen, 200mL of pyridine and 50.0g of 40(1.0eq) are added into a 500mL three-necked flask, stirred and cooled to 0 ℃, 20.8g of TsCl (1.0eq) is added in batches, stirred for 1h, then slowly heated to room temperature, and stirred for 3-4 h. After the reaction, the reaction solution was poured into an ice diluted hydrochloric acid solution to produce a solid, ethyl acetate was added for extraction, the ethyl acetate layer was washed once with diluted hydrochloric acid, once with saturated sodium bicarbonate, with saturated saline, and with anhydrous Na₂SO₄Drying, evaporating the solvent under reduced pressure, and performing silica gel column chromatography to obtain 31g of pure compound 41.

Preparation of Compound 42

31g of Compound 41(1.0eq) and 155mL of DMSO were added to a 500mL three-necked flask, stirred well, and then NaN was added₃6.6g (2.0eq), heated to 50 ℃ for 3 hours, cooled to room temperature, poured into water, extracted several times with ethyl acetate, combined organic phases, dried over anhydrous sodium sulfate, concentrated to give 35.1g of compound 42 as a colorless liquid.

Preparation of Compound 43

Adding 35g of compound 42, 350mL of methanol and 4.5g of palladium carbon into a 1L hydrogenation reaction kettle, stirring, replacing with nitrogen, introducing hydrogen for reaction for 3-4h, monitoring by TLC, filtering the reaction solution after the reaction is finished, and concentrating the filtrate to obtain 4333.2 g of an oily compound.

Preparation of Compound 44

To a 500mL three-necked flask was added 4332.1 g (1.0eq), (Boc)₂O30.6 g (2.0eq), methanol: and (3) stirring and heating 300ml of mixed solution of triethylamine (9: 1) to reflux, reacting for 1h, distilling off methanol and triethylamine after TLC monitoring reaction is finished, adding water to dissolve, extracting for 3 times by dichloromethane, combining organic layers, washing once by water, drying by anhydrous sodium sulfate, and concentrating to obtain 4422.5 g of oily matter.

Preparation of Compound 45

22.5g (1.0eq) of compound 44, 230mL each of toluene and tetrahydrofuran, 16.8g (3.0eq) of bromoacetic acid were added to a 1000mL three-necked flask, stirred, heated to 45 to 50 ℃, and added with 11.3g of sodium hydroxide, reacted overnight, after the completion of TLC monitoring reaction, the reaction solution was distilled off, water and ethyl acetate were added to extract impurities, the pH of the aqueous phase was adjusted to 3, the aqueous phase was extracted with dichloromethane, the dichloromethane layers were combined, dried over anhydrous sodium sulfate, and concentrated to give 16.7g of compound 45 as an oily substance.

Preparation of Compound 46

455.2 g of compound was added to a 250mL single-neck flask, 20mL of ethyl acetate was dissolved and then cooled to 0 ℃ and 20mL of ethyl acetate/HCl (7M/L) was added, and after completion of the reaction monitored by TLC, the mixture was concentrated to give 465.5 g of an oily compound.

Preparation of Compound 48

To a 250mL three-necked flask, 10.0g of Compound 47(1.0eq), 100mL of dichloromethane, 100mL of t-butanol, 7.2g of DIC (1.0eq), 7.0g of DMAP (1.0eq) were added, the mixture was stirred overnight at room temperature, TLC was used to monitor the completion of the reaction, and the mixture was diluted with diethyl ether and then washed with water 3 times, saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain 484.2 g of an off-white solid.

Preparation of Compound 49

To a 100mL three-necked flask, 2.3g N-Hydroxysuccinimide (HOSU), 4.2g of compound 48 and 42mL of dichloromethane were added, 5.3g of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed with 50mmol/L potassium dihydrogen phosphate aqueous solution having pH of 6.0 for 2 times, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 5.9g of compound 49 as an off-white solid.

Preparation of Compound 50

A500 mL flask was charged with 3.7g of the compound H-Glu-OtBu (1.0eq), 60mL of water, 3.1g of NaHCO₃(2.05eq), stirred, 5.9g of compound 49 dissolved in 60ml of DME (ethylene glycol dimethyl ether) was added dropwise, 30ml of tetrahydrofuran was added, stirred overnight, TLC monitored the completion of the reaction, the organic solvent was evaporated, pH was adjusted to 4 with dilute hydrochloric acid, dichloromethane extracted, dried over anhydrous sodium sulfate, and concentrated to give 6.9g of compound 50 as a white solid.

Preparation of Compound 51

To a 250mL three-necked flask, 2.9g N-Hydroxysuccinimide (HOSU), 6.9g of compound 50 and 70mL of dichloromethane were added, 4.9g of EDC · HCl was added, the reaction was carried out at room temperature for 2 hours, TLC monitored for completion of the reaction, diluted with dichloromethane, washed with 50mmol/L potassium dihydrogen phosphate aqueous solution having pH of 6.0 for 2 times, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 8.6g of compound 51 as an off-white solid.

Preparation of Compound 52

To a 250mL flask was added 3.8g of Compound 46(1.0eq), 35mL of water, 1.2g of NaHCO₃(2.05eq), stirring, dropwise adding a solution of 3.5g of compound 51 in 35ml of DME (ethylene glycol dimethyl ether), adding 35ml of tetrahydrofuran, stirring overnight, monitoring by TLC after the reaction is finished, evaporating the organic solvent, adjusting the pH to 6 with acetic acid, extracting with ethyl acetate, drying over anhydrous sodium sulfate, concentrating to obtain an off-white solid, and performing column chromatography to obtain 4.3g of light yellow oily compound 52.

Preparation of PP03h03

The PP03h03 was synthesized by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay for reaction monitoring, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Lys (alloc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Boc-OH, Fmoc-Ser (tBu) -OH, (Tyr-Tyr), (Tyr) (tBu) -OH, Asp-OtBu) OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, 3.0eq dissolved in chloroform: n-methylmorpholine: reacting acetic acid (18:2:1) with tetrakis (triphenylphosphine) palladium for 2 hours, washing with dichloromethane, washing with DMF, condensing a connecting compound 51, after ninhydrin detection reaction, washing with DMF, washing with methanol, washing with dichloromethane, drying, adding a cracking reagent (trifluoroacetic acid: benzylsulfide: phenol: triisopropylsilane: 85: 5: 5), reacting for 2 hours, precipitating with ice-tert-butyl methyl ether, centrifuging to obtain a crude PP03h product, purifying by HPLC, and freeze-drying to obtain a pure PP03h03 product.

ESI-MS M/z calculated 4406.20, found 1470.2[ M +3H ]]³⁺，1102.9[M+4H]⁴⁺。

Example 14 Synthesis of PP03b01

Preparation of Compound 15

To a 100mL flask was added 0.73g of Compound 14(1.0eq) and 20mL of dichloromethane, and after stirring to dissolve, 0.8g of DIEA and 0.96g of Compound 10(1.0eq) were added, and after completion of the TLC reaction, the mixture was diluted with dichloromethane, washed with hydrochloric acid, sodium bicarbonate and brine, and then dried over anhydrous sodium sulfate and concentrated to give 1.34g of Compound 15.

Preparation of Compound 16

A100 mL flask was charged with 1.34g of Compound 15(1.0eq), 20mL of 30% trifluoroacetic acid in dichloromethane, stirred at 0 ℃ for 2h, after TLC reaction was complete, diluted with dichloromethane, washed with purified water, washed with brine, dried over anhydrous sodium sulfate and concentrated to give crude Compound 16. HPLC preparative purification gives 0.65g of pure compound 16.

Preparation of PP03b01

100mg of the compound PP03b was dissolved in 10ml of 20mM sodium phosphate buffer (pH 7.4), 20mg of compound 16(1.1eq) was added in 2ml of tetrahydrofuran, and the mixture was stirred at 20 ℃ for 4 hours, and after completion of the HPLC monitoring reaction, the compound was purified by HPLC, and lyophilized to obtain the coupling compound PP03b0151 mg.

ESI-MS M/z calculated 4254.28, found 1419.5[ M +3H ]]³⁺，1065.0[M+4H]⁴⁺，852.2[M+5H]⁵⁺

Example 15 preparation of PP03c01

The preparation process is referred to example 14.

ESI-MS M/z calculated 4237.13, found 1413.5[ M +3H ]]³⁺，1060.4[M+4H]⁴⁺，848.6[M+5H]⁵⁺

Example 16 preparation of PP03d01

The preparation process is referred to example 14.

ESI-MS M/z calculated 4237.13, found 1413.6[ M +3H ]]³⁺，1060.4[M+4H]⁴⁺，848.7[M+5H]⁵⁺

ESI-MS M/z calculation, found [ M +3H]³⁺，[M+4H]⁴⁺，[M+5H]⁵⁺

Example 17 preparation of PP03j01

The synthesis of PP03j01 was carried out by Fmoc solid phase synthesis, well known to those skilled in the art, using 2-Cl-Trt Resin, 20% piperidine/DMF to remove Fmoc, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay for monitoring, and the following protected amino acids were attached to the Resin in sequence: Fmoc-Lys (ivDde) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OAll) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Tyr-OH, Fmoc-Lys-OH, Fmoc (Boc) -OH, Fmoc-Ser (tBu) -OH, (Fmoc-Tyr) (Tyr) -OH, Fmoc-Asp-OtBu) OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH, 3.0eq dissolved in chloroform: n-methylmorpholine: reacting tetra (triphenylphosphine) palladium with acetic acid (18:2:1) for 2 hours to remove protecting groups of Lys (alloc) and Glu (OAll), washing DMF, adding TBTU and DIEA to close a ring, washing DMF after ninhydrin detection reaction is almost complete, removing protecting groups of Lys (ivDde) with hydrazine hydrate, detecting ninhydrin to be positive, condensing a connecting compound 13, washing DMF after ninhydrin detection reaction is finished, washing methanol, washing dichloromethane, drying, adding a cracking reagent (trifluoroacetic acid, benzyl sulfide, phenol, triisopropylsilane, 85: 5: 5: 5), precipitating with ice-tert-butyl methyl ether after reacting for 2 hours, centrifuging to obtain a crude PP03j 35 01, preparing and purifying by HPLC, and freeze-drying to obtain a pure PP03j 01.

ESI-MS M/z calculated 4352.25, found 1452.1[ M +3H ]]³⁺，1089.3[M+4H]⁴⁺。

Example 18 Synthesis of PP03k01

The preparation process is referred to example 17.

ESI-MS M/z calculated 4227.84, found 1410.3[ M +3H ]]³⁺，1058.0[M+4H]⁴⁺，846.5[M+5H]⁵⁺

EXAMPLE 19 Synthesis of PP03m01 (requiring supplemental ESI-MS)

The preparation process is referred to example 17.

ESI-MS M/z calculated 4209.83, found 1404.3[ M +3H]³⁺，1053.5[M+4H]⁴⁺，843.0[M+5H]⁵⁺

EXAMPLE 20 Synthesis of PP03n01 (requiring supplemental ESI-MS)

The preparation process is referred to example 17.

ESI-MS M/z calculated 4343.23, found 1448.9[ M +3H ]]³⁺，1087.0[M+4H]⁴⁺，869.7[M+5H]⁵⁺

EXAMPLE 20 Synthesis of PP03s01 (requiring supplemental ESI-MS)

The preparation process is referred to example 17.

ESI-MS M/z calculated 4325.23, found 1442.8[ M +3H ]]³⁺，1082.4[M+4H]⁴⁺

EXAMPLE 21 Synthesis of PP03p01 (requiring supplemental ESI-MS)

The preparation process is referred to example 14.

ESI-MS M/z calculated 4268.86, found 1424.0[ M +3H]³⁺，1068.3[M+4H]⁴⁺

EXAMPLE 22 Synthesis of PP03q01 (requiring supplemental ESI-MS)

The preparation process is referred to example 14.

ESI-MS M/z calculated 4250.86, found 1418.0[ M +3H ]]³⁺，1063.8[M+4H]⁴⁺，851.2[M+5H]⁵⁺

Claims (7)

1. A modification of an oxyntomodulin or an analogue thereof, which modification is as follows:

n is any integer between 6 and 20;

wherein OXM 'is a natural oxyntomodulin or an analogue of oxyntomodulin, and L is a linking group of OXM' and Y;

the OXM' is:

a polypeptide of the amino acid sequence of SEQ ID No. 2;

a polypeptide of the amino acid sequence of SEQ ID No. 3;

a polypeptide of the amino acid sequence of SEQ ID No. 4;

a polypeptide of the amino acid sequence of SEQ ID No. 5;

a polypeptide of the amino acid sequence of SEQ ID No. 6;

a polypeptide of the amino acid sequence of SEQ ID No. 7;

a polypeptide of the amino acid sequence of SEQ ID No. 8;

a polypeptide of the amino acid sequence of SEQ ID No. 9;

a polypeptide of the amino acid sequence of SEQ ID No. 10.

2. The finish of claim 1, which is:

wherein m is any integer between 2 and 10; n is any integer between 6 and 20.

3. A modification of an oxyntomodulin analogue which is:

wherein m is any integer between 2 and 10; n is any integer between 6 and 20; the underlined in the sequence indicates loop formation.

4. The finish of claim 3, which is:

5. the use of any of the oxyntomodulin analogues or modifications thereof as claimed in claims 1 to 4 which is: the use in the manufacture of a medicament for the treatment of obesity; the application in preparing medicines for treating diabetes; the application in preparing the medicine for preventing or treating hyperlipemia, fatty liver disease or arteriosclerosis.

6. A pharmaceutical composition comprising an oxyntomodulin analogue or a modification thereof as claimed in any of claims 1 to 4 and optionally a pharmaceutically acceptable carrier.

7. Use of the composition of claim 6 which is: the use in the manufacture of a medicament for the treatment of obesity; the application in preparing medicines for treating diabetes; the application in preparing the medicine for preventing or treating hyperlipemia, fatty liver disease or arteriosclerosis.

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