CN109232743B - Long-acting hypoglycemic weight-loss peptide, preparation method thereof and application thereof as medicine - Google Patents

Abstract

The invention relates to a long-acting glucose-reducing weight-losing Oxyntomodulin (OXM) hybrid peptide, application thereof and a synthetic method thereof. By changing the peptide sequence of the OXM and hybridizing with the peptide sequence of Exenatide, the OXM hybrid peptide with longer pharmacological action time and better weight-reducing effect is obtained. The synthesis of the target polypeptide is realized rapidly by an orthogonal protection strategy solid-phase synthesis method, and a crude product is purified and freeze-dried to obtain a target compound.

Description

Long-acting hypoglycemic weight-loss peptide, preparation method thereof and application thereof as medicine

Technical Field

The invention relates to the field of medicinal chemistry, in particular to long-acting hypoglycemic weight-loss peptides, a preparation method thereof and application thereof as medicaments.

Background

The cause of the metabolic syndrome is metabolic abnormality of various substances such as protein, fat, carbohydrate, and the like. Excess nutrition, reduced physical activity, etc. can lead to obesity and obesity related diseases, such as diabetes, etc. In recent years, the incidence of type 2 diabetes and dyslipidemia has been increasing.

Gastrin regulin (Oxyntomodulin, OXM) is a 37 amino acid polypeptide secreted by L cells of the small intestine, comprising the entire 29 amino acid sequence of glucagon and an 8 amino acid portion extending C-terminally, with 50% homology to glucagon-like peptide-1 (GLP-1), the peptide sequence being: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA are provided. The OXM can simultaneously activate a glucagon-like peptide-1 receptor (GLP-1R) and a glucagon receptor (GCGR), and has certain effects of reducing weight gain and reducing blood sugar. After the GCGR is activated by the OXM, the effects of hepatic glycogenolysis and gluconeogenesis can be promoted, and lipolysis and fatty acid oxidation are promoted; accelerating the amino acid to enter liver cells, playing a role in heat production and having better weight loss and appetite suppression. Compared with a pure GLP-1R agonist, the OXM has better effects of interfering weight, regulating lipid metabolism and improving glucose tolerance, but has weaker hypoglycemic activity and shorter half-life.

GLP-1 is a glucose-dependent incretin hormone. It can activate GLP-1R and reduce blood sugar. The most obvious function is to promote the regeneration and repair of beta cells, increase the number of islet beta cells, and avoid the hypoglycemia risk frequently occurring in the diabetes treatment, and the application prospect in the diabetes treatment field is wide. Exenatide is a typical short-acting GLP-1 receptor agonist for reducing DPP-IV enzyme metabolism, and the partial peptide sequence of the Exenatide is introduced into OXM, so that the receptor agonistic activity of the compound on GLP-1R can be improved.

According to the invention, by virtue of a partial peptide sequence structure of hybrid OXM and Exenatide (Exenatide), the affinity of a peptide chain to GLP-1R is enhanced, the agonistic activity to GLP-1R is improved, the moderate GCGR agonistic activity is maintained, and an OXM analogue modified by a peptide sequence is synthesized. The fatty acid and the dicoumarin micromolecules have high serum albumin binding rate, the micromolecules are conjugated with the OXM analogue, the maintenance time of the hypoglycemic effect is greatly prolonged, and the hypoglycemic effect exceeds that of the existing marketed medicaments of liraglutide and exenatide, so that a series of long-acting polypeptide medicaments with good hypoglycemic activity and weight reducing effect are obtained.

Disclosure of Invention

In thatFirst aspectThe invention relates to a hypoglycemic polypeptide or pharmaceutically acceptable salt thereof, wherein the polypeptide amino acid sequence is as follows:

His-Gly-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂；

wherein

Xaa is taken from

Wherein the content of the first and second substances,

n is selected from natural number 1-20;

m is taken from natural numbers 1-20.

In a preferred embodiment of the present invention, the present invention is characterized in that,

xaa is taken from

Wherein n is taken from natural numbers 11, 15;

m is taken from natural number 11.

The hypoglycemic polypeptide or the pharmaceutically acceptable salt thereof related to the invention can also be expressed as follows:

in thatSecond aspect of the inventionThe present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the above compounds and pharmaceutically acceptable salts thereof, or a pharmaceutically acceptable carrier or diluent. Meanwhile, the invention further provides application of the compound and pharmaceutically acceptable salts thereof, or pharmaceutically acceptable carriers or diluents in preparing medicines for treating and preventing diabetes.

In thatThird aspect of the inventionThe invention also provides a preparation method of the compound, and the target compound is efficiently and quickly synthesized by adopting a solid-phase synthesis strategy.

The invention has the beneficial effects that:

1. the compound provided by the invention has obvious effects of reducing blood sugar and weight, has stable chemical properties, and has activity obviously superior to that of the prototype peptide OXM.

2. The hypoglycemic effect of part of the compounds provided by the invention can be maintained for more than 40h, and is remarkably improved compared with endogenous GLP-1 (half-life period of 2-3 min) or marketed drug exenatide (half-life period of 2.4 h).

3. The purity of the crude product of the peptide chain obtained by solid-phase synthesis of the OXM hybrid peptide by adopting an orthogonal protection strategy is more than 85 percent, and compared with the conventional synthesis method, the method is greatly improved, and the subsequent purification work is convenient.

4. The method adopts a solid phase method to synthesize the OXM heterozygous peptide, and has low cost. Because the coupling efficiency is higher, the amino acid required to be protected only needs 2 times excess on average, while the amino acid needs 4 to 5 times excess in the conventional synthetic method, thereby greatly saving the cost.

5. The method for synthesizing the OXM hybrid peptide by adopting the Fmoc/tBu orthogonal protection solid-phase synthesis strategy is easy to realize automation and large-scale production, so that the method is more suitable for industrial production.

Therefore, the OXM hybrid peptide prepared by the solid phase synthesis technology has good activity of reducing blood sugar and weight, long drug effect time, high yield, short synthesis period, easy purification of crude products, low production cost and easy industrial automatic production. The prepared OXM hybrid peptide is suitable to be used as an active ingredient of a medicament for treating diabetes and obesity.

The following are related pharmacological experimental methods and results of OXM hybrid peptides involved in the present invention:

1. abdominal glucose tolerance test of OXM hybrid peptide

Normal kunming mice, randomly grouped, 8 mice per group, were housed in standardized animal houses. Fasted for 12 hours prior to the experiment, only drinking water was given. In each group of mice, prior to administration of the OXM hybrid peptide, an initial blood glucose level was measured and set to-30 min, followed by intraperitoneal injection of 50nmol/kg of the OXM hybrid peptide. After 30min, 18mmol/kg glucose solution was intraperitoneally injected for 0min, and the control group was injected with the same volume of physiological saline or 50nmol/kg exenatide. Measuring blood glucose level with a glucometer at 0, 15, 30, 45, 60, 120min, and testing the hypoglycemic activity of the OXM hybrid peptide.

TABLE 1 results of intraperitoneal glucose tolerance experiments for OXM hybrid peptides

Results are expressed as mean±SD，*P＜0.05，**P＜0.01，***P＜0.001 vs saline.

As shown in Table 1, the results of blood sugar reduction experiments show that when the administration concentration of the OXM hybrid peptide disclosed by the invention is 50nmol/kg, the blood sugar reduction effect is equivalent to that of exenatide.

2. Stable blood glucose assay of OXM hybrid peptides

Blood glucose was measured in STZ-induced diabetic model mice, and mice with values higher than 20mmol/L were selected for random grouping of six mice per group, with free feeding during the experiment. The positive control group is injected with exenatide or liraglutide in the abdominal cavity, the dosage is 50nmol/kg, the negative control group is injected with normal saline in the abdominal cavity, and the administration group is respectively injected with 50nmol/kg of OXM hybrid peptide. Compound was administered at 0h and blood glucose levels were determined using a glucometer at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48 and 60h, respectively. The evaluation index is the time when the blood sugar value of the mice is lower than 8.35mmol/L after the compound is injected into the abdominal cavity.

As can be seen from the figures 1 and 2, the blood sugar stabilizing time of the exenatide is only 4.7 hours, the blood sugar stabilizing time of the liraglutide is 12.3 hours, and the blood sugar stabilizing time of the long-acting blood sugar reducing polypeptide can reach more than 40 hours. The blood sugar stabilizing experiment shows that the OXM hybrid peptide has good long-acting blood sugar reducing effect, can achieve better long-acting blood sugar reducing effect, and has the potential of being developed into a blood sugar reducing medicament which is administrated once every two days.

3. Experiment of reducing body weight gain with OXM hybrid peptide

Male C57bl/6 mice were fed on high-fat diet for 4 weeks, and mice weighing more than 30g were selected for the experiment. Mice were randomly grouped into groups of 8 mice, 7 groups in total, and OXM hybrid peptide (50nmol/kg, 10mL/kg) was administered daily for 56 consecutive days, with the negative control group being administered daily with physiological saline, and the positive control group being administered OXM. Fasting body weights were tested for each group of mice on day 1 and day 56, and the average body weight change for each group of mice was examined.

TABLE 2 weight gain reduction experiment of OXM hybrid peptides

Results are expressed as mean±SD.

As can be seen from table 2, after long-term administration, all compounds showed better weight control effect, which was significantly better than OXM.

4. Lipid lowering experiment of OXM hybrid peptide

Male C57bl/6 mice were fed on high-fat diet for 4 weeks, and mice weighing more than 30g were selected for the experiment. Mice were randomly grouped into groups of 8 mice, 7 groups in total, and OXM hybrid peptide (50nmol/kg, 10mL/kg) was administered daily for 56 consecutive days, with the negative control group being administered daily with physiological saline, and the positive control group being administered OXM. After the administration, serum of the mice is taken and the content of Total Cholesterol (TC) and Triglyceride (TG) is detected.

From fig. 3-4, it can be seen that the lipid parameter content of the mice in the saline group was increased, while the lipid parameter content of the mice in the administered group was decreased, indicating that the OXM analogues had therapeutic effect on hyperlipidemia.

5. Experiment on treatment of nonalcoholic fatty liver disease with OXM hybrid peptide

Male C57bl/6 mice were fed with high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly grouped into groups of 8 mice, 7 groups in total, and OXM hybrid peptide (50nmol/kg, 10mL/kg) was administered daily for 56 consecutive days, with the negative control group being administered daily with physiological saline, and the positive control group being administered OXM. After the administration, the serum of the mouse is taken and the glutamic-pyruvic transaminase content is detected. As can be seen from fig. 5, the level of glutamic pyruvic transaminase (ALT) was increased in the mice of the saline group, which was consistent with the pathological characteristics of non-alcoholic fatty liver disease, while the level of glutamic pyruvic transaminase was decreased in the mice of the administered group, indicating that OXM analogue had therapeutic effect on non-alcoholic fatty liver disease.

Drawings

Fig. 1 is OXM hybrid peptide seq.id NO: 1-3 results of experiments on stabilizing blood sugar.

Fig. 2 is OXM hybrid peptide seq.id NO: 4-5 of results of experiments on stabilizing blood sugar.

Fig. 3 is OXM hybrid peptide seq.id NO: 1-5 TC detection results.

Fig. 4 is OXM hybrid peptide seq.id NO: 1-5 TG detection results.

Fig. 5 is OXM hybrid peptide seq.id NO: 1-5 ALT detection results.

Detailed Description

The following abbreviations are used throughout the specification:

english abbreviation	Chinese character
		DCM	Methylene dichloride
NMP	N-methyl pyrrolidone
		HBTU	benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
HOBt	1-hydroxy-benzotriazole
		DIEA/DIPEA	N, N' -diisopropylethylamine
Fmoc	Fmoc-9-carboxylic acid
		ESI-MS	Electrospray mass spectrometry
EDT	Ethanedithiol
		HPLC	High performance liquid chromatography
TFA	Trifluoroacetic acid
		tBu	Tert-butyl radical
Boc	Tert-butyloxycarbonyl radical
		EDC·HCl	1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
DMAP	4-dimethylaminopyridine
		DIC	N, N-diisopropylcarbodiimide
Na2SO4	Sodium sulfate
		DMSO	Dimethyl sulfoxide
K2CO3	Potassium carbonate
		HCl	Hydrogen chloride

The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.

Example 1

Solid phase synthesis of

1. Synthesis of peptide chains

1.1 swelling of the resin

Weighing 50mg of Fmoc-Rink amide-MBHA Resin (the substitution degree is 0.4mmol/g), swelling with 7mL of DCM for 30min, filtering off DCM by suction, swelling with 10mL of NMP for 30min, and washing with 7mL of NMP and DCM respectively.

1.2 removal of Fmoc protecting group

And (3) putting the swelled resin into a reactor, adding a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt into the resin to remove Fmoc, and washing the resin with NMP after the reaction is finished. The resin was obtained with the Fmoc protecting group initially attached removed.

1.3 Synthesis of Fmoc-Ser (tBu) -Rink amide-MBHA Resin

Fmoc-Ser (tBu) -OH (15.4mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9. mu.L, 0.08mmol) were dissolved in NMP 10mL, and this solution was added to the resin obtained in the previous step, reacted for 2 hours, after which the reaction solution was filtered off and the resin was washed 3 times with 7mL each of DCM and NMP.

1.4 elongation of peptide chain

Repeating the steps of deprotection and coupling according to the sequence of the peptide chain, sequentially connecting corresponding amino acids, and sequentially connecting corresponding amino acids until the peptide chain is synthesized, so as to obtain the peptide with the sequence shown in SEQ ID NO: 1 backbone peptide sequence.

1.6 cleavage of Polypeptides on resins

And (3) connecting the obtained polypeptide with SEQ ID NO: the resin with the main chain peptide sequence of 1 is put into a reaction bottle, 10mL of a cleavage agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5: 5: 2.5, V/V) is added respectively, the mixture is firstly shaken for 30min at 0 ℃, and then reacted for 3h at normal temperature. After the reaction was completed, the reaction mixture was filtered with suction, washed three times with a small amount of TFA and DCM, and the filtrates were combined. Adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, freezing and centrifuging to obtain a crude product of the target polypeptide. The crude product was finally obtained in 77.1mg, yield 90.2%. The reaction was monitored using HPLC with chromatographic conditions: a C18 column (150 mm. times.4.6 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 35-85% of mobile phase B for 20 min; the flow rate is 1 mL/min; the column temperature is 40 ℃; the detection wavelength was 214 nm. After the reaction is finished, purifying by adopting a preparative liquid chromatography, wherein the chromatographic conditions are as follows: a C18 column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 40-90% of mobile phase B for 20 min; the flow rate was 6mL/min and the detection wavelength was 214 nm. The collected solution was lyophilized to give pure 28.5 mg. The theoretical relative molecular mass is 4256.6. ESI-MS m/z: calcd [ M +3H ]]³⁺1419.9，[M+4H]⁴⁺1065.2；Found[M+3H]³⁺1420.1，[M+4H]⁴⁺1065.5。

2. Synthesis of 12- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) dodecanoic acid

Dissolving 12-aminododecanoic acid (0.86g, 4mmol) and maleic anhydride (0.47g, 4.8mmol) in glacial acetic acid, ultrasonic dissolving, reflux reacting at 120 deg.C for 6 hr, detecting reaction on thin-layer plate, cooling the reaction liquid to room temperature, extracting with ethyl acetate three times (3 × 20mL), mixing the upper layer extractive solutions, washing with saturated saline solution for 3 times, and adding anhydrous Na₂SO₄Dry overnight. Vacuum rotary drying the extract to obtain crude product, and separating by column chromatography (ethyl acetate/petroleum ether) to obtain yellowish pure product 0.89g, yield 80%, mp 91-92 deg.C.

¹H-NMR(DMSO-d₆，300MHz)：δppm：12.45(s，1H，-COOH)，7.50(s，2H，-COCH＝CHCO-)，3.88(t，2H，J＝7.0Hz，-NC ₂H-)，2.68(t，J＝7.3Hz，2H，-C ₂HCOOH)，2.00-1.96(m，4H，-NCH₂C ₂H(CH₂)₇C ₂H)，1.73(s，14H，-NCH₂CH₂(C _{2 7}H)CH₂).ESI-MS m/z：294.1[M+H]⁺。

3. Synthesis and purification of chemically modified OXM conjugates

Dissolving the 12- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) dodecanoic acid obtained in the above step in DMSO to prepare a solution of about 10mg/mL, and reacting the obtained amino acid sequence of SEQ.ID NO: the 1-backbone peptide sequence was also dissolved in DMSO, and 20. mu.l DIEPA was added after ultrasonic mixing of the two, and the reaction was stirred at room temperature and monitored by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After completion of the reaction, the reaction mixture was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 μm microporous membrane, and purified by preparative liquid chromatography under the following conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate was 5ml/min and the detection wavelength was 214 nm. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain pure 8.1 mg. The theoretical relative molecular mass is 4551.7. ESI-MS m/z: calcd [ M +3H ]]³⁺1518.3，[M+4H]⁴⁺1138.9；Found[M+3H]³⁺1517.4，[M+4H]⁴⁺1138.8。

Example 2

1. Synthesis of 16- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) hexadecanoic acid

Will 1Dissolving 6-aminocaproic acid (1.09g, 4mmol) and maleic anhydride (0.47g, 4.8mmol) in glacial acetic acid, ultrasonic dissolving, reflux reacting at 120 deg.C for 6h, detecting reaction on thin layer plate, cooling reaction solution to room temperature, extracting with ethyl acetate for three times (3 × 20mL), mixing upper layer extractive solutions, washing with saturated salt water for 3 times, and collecting anhydrous Na₂SO₄Dry overnight. The extract is dried in vacuum to obtain a crude product, and the crude product is separated by column chromatography (ethyl acetate/petroleum ether) to obtain a light yellow pure product of 1.02g with the yield of 72 percent.

¹H-NMR(DMSO-d₆，300MHz)：δppm：12.45(s，1H，-COOH)，7.50(s，2H，-COCH＝CHCO-)，3.88(t，2H，J＝7.0Hz，-NC ₂H-)，2.68(t，J＝7.3Hz，2H，-C ₂HCOOH)，2.00-1.96(m，4H，-NCH₂C ₂H(CH₂)₇C ₂H)，1.76(s，22H，-NCH₂CH₂(C _{2 11}H)CH₂).ESI-MS m/z：352.4[M+H]⁺。

2. Synthesis and purification of chemically modified OXM conjugates

Dissolving the 16- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) hexadecanoic acid obtained in the above step in DMSO to prepare a solution of about 10mg/mL, and reacting the obtained amino acid sequence of SEQ.ID NO: the 1-backbone peptide sequence was also dissolved in DMSO, and 20. mu.l DIEPA was added after ultrasonic mixing of the two, and the reaction was stirred at room temperature and monitored by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After completion of the reaction, the reaction mixture was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 μm microporous membrane, and purified by preparative liquid chromatography under the following conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/7 nitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min;the flow rate was 5ml/min and the detection wavelength was 214 nm. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain pure 8.7 mg. The theoretical relative molecular mass is 4607.8. ESI-MS m/z: calcd [ M +3H ]]³⁺1536.9，[M+4H]⁴⁺1152.9；Found[M+3H]³⁺1537.6，[M+4H]⁴⁺1153.5。

Example 3

1. Synthesis of chemically modified groups

Synthesis of 3, 3' - (4-carboxyphenylmethylene) -di-4-hydroxycoumarin

P-carboxybenzaldehyde (0.45g, 3mmol) was dissolved in 20ml of anhydrous ethanol, followed by addition of 4-hydroxycoumarin (0.98g, 6 mmol). Heating and refluxing for 12h, cooling the reaction solution to room temperature, filtering, washing the filter cake with 10ml of ethanol for 3 times to obtain 1.12g of a product, wherein the yield is 82.1%, ESI-MS m/z: 456.4[ M + H]⁺.

Synthesis of tert-butyl (12- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) dodecyl) carbamate

Dissolving N-Boc-dodecyl diamine (1.2g, 4mmol) and maleic anhydride (0.49g, 4.8mmol) in glacial acetic acid, heating at 120 deg.C for 6h, detecting reaction completion on thin layer plate, cooling reaction solution to room temperature, extracting with ethyl acetate (3 × 20mL), mixing upper layer extractive solutions, washing with saturated salt water for 3 times, and collecting anhydrous Na₂SO₄Dry overnight. Concentrating the extractive solution under reduced pressure, and purifying the obtained crude product by column chromatography to obtain light yellow pure product 1.10g, with yield of 72%, MS (ESI, m/z): 380.5[ M + H]⁺.

Synthesis of 4- (bis (4-hydroxy-2-oxo-2H-chromen-3-yl) methyl) -N- (12- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) dodecyl) benzamide

(12- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) dodecyl) carbamic acid tert-butyl ester (0.76g, 2mmol) is dissolved in HCl-saturated ethyl acetate, after stirring for 3H the solvent is distilled off under reduced pressure, DCM is redissolved and 3, 3' - (4-carboxyphenylmethylene) -bis (tert-butyl) is added-4-hydroxycoumarin (0.91g, 2mmol), DIC (0.30g, 2.4mmol) and HOBt (0.32g, 2.4mmol) and stirred at room temperature overnight. Pouring the reaction solution into water and extracting with ethyl acetate for three times after the thin-layer plate detection reaction is finished, combining the extracts, and respectively using saturated K₂CO₃The solution was washed three times with HCl1M and brine. Adding anhydrous Na into the extract₂SO₄Drying overnight, concentrating under reduced pressure to obtain crude product, and purifying by column chromatography to obtain pure product 0.93g with yield of 65%. ESI-MS m/z: 719.4[ M + H]⁺.

2. Synthesis and purification of chemically modified OXM conjugates

The 4- (bis (4-hydroxy-2-oxo-2H-chromen-3-yl) methyl) -N- (12- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) dodecyl) benzamide obtained in the above step was dissolved in DMSO to prepare a solution of about 10mg/mL, and seq.id NO: the 1-backbone peptide sequence was also dissolved in DMSO, and 20. mu.l DIEPA was added after ultrasonic mixing of the two, and the reaction was stirred at room temperature and monitored by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After completion of the reaction, the reaction mixture was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 μm microporous membrane, and purified by preparative liquid chromatography under the following conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate was 5ml/min and the detection wavelength was 214 nm. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 7.9mg of pure product. The theoretical relative molecular mass is 4974.9. ESI-MS m/z: calcd [ M +3H ]]³⁺1659.3，[M+4H]⁴⁺1244.7；Found[M+3H]³⁺1659.3，[M+4H]⁴⁺1245.8。

Example 4

1. Synthesis of chemically modified groups

Synthesis of 3, 3' - (4-nitrobenzylidene) -di-4-hydroxycoumarin

Weighing p-nitrobenzaldehyde (3.02g, 0.02mol), and dissolving with 35ml of absolute ethyl alcohol; 4-hydroxycoumarin (6.6g, 0.041mol) was added and 15ml of absolute ethanol was added to dissolve completely. Reacting for 4h at 80 ℃, filtering while hot, washing a filter cake for 3 times by using 10ml of hot ethanol to obtain 8.2g of a product, wherein the yield is 90.0 percent and the mp is 227 ℃.

¹H-NMR(CDCl₃，300MHz)δppm：6.13(s，H，-CH-)，7.43(m，8H，Ar-H)，7.68(m，2H，Ar-H)，8.18(m，2H，Ar-H).ESI-MS m/z：456.0[M+H]⁺.

Synthesis of 3, 3' - (4-aminobenzylidene) -di-4-hydroxycoumarin

Weighing 3, 3' - (4-nitrobenzylidene) -di-4-hydroxycoumarin (1.14g, 0.0025mol), suspending with 30ml of acetic acid, adding 0.3g of 5% Pd/C, stirring, extracting for 3 times by a hydrogen tee joint, coating Vaseline on a bottle mouth, hydrogenating at normal temperature, reacting overnight, filtering, evaporating part of a solvent from a filtrate, recrystallizing with acetone to obtain 0.8g of a product, wherein the yield is 75.1%, and the mp is 220 ℃.

¹H-NMR(DMSO-d₆，300MHz)δppm：6.27(s，H，-CH-)，7.23(m，8H，Ar-H)，7.49(m，2H，Ar-H)，7.81(m，2H，Ar-H).ESI-MS m/z：426.0[M+H]⁺.

Synthesis of 3, 3' - (4- (12-maleimidododecanamido) benzylidene) -di-4-hydroxycoumarin

12-Maleamidododecanoic acid (294.1mg, 1mmol) was dissolved in tetrahydrofuran, DIC (17. mu.L, 1.1mmol) and HOBt (148.5mg, 1.1mmol) were added and stirred at room temperature for 30min, then a solution of 3, 3' - (4-aminobenzylidene) -bis-4-hydroxycoumarin and DIPEA (17.4. mu.L, 0.1mmol) in tetrahydrofuran was slowly added dropwise thereto and stirred at room temperature overnight. Pouring the reaction solution into water and extracting with ethyl acetate for three times after the thin-layer plate detection reaction is finished, combining the extracts, and respectively using K₂CO₃HCl1M, three times in saturated saline. Adding anhydrous Na into the extract₂SO₄Drying overnight, concentrating under reduced pressure to obtain crude product, purifying by column chromatography to obtain pure product with yield of 69%, mp 204-.

¹H-NMR(DMSO-d₆，300MHz)：δppm：10.17(s，1H，-CONH-)，8.31(d，J＝7.8Hz，2H，Ar-H)，8.00(t，J＝7.2Hz，2H，Ar-H)，7.84(d，J＝8.0Hz，2H，Ar-H)，7.76-7.72(m，6H，Ar-H)，7.49(s，2H，-COCH＝CHCO-)，6.70(s，1H，-CH-)，3.87(t，J＝7.0Hz，2H，-NCH₂-)，2.74(t，J＝7.2Hz，2H，-COCH₂-)，2.05-1.97(m，4H，-NCH₂CH₂(CH₂)₇CH₂-)，1.70(s，14H，-NCH₂CH₂(CH₂)₇CH₂-).ESI-MS m/z：703.1[M+H]⁺.

2. Synthesis and purification of chemically modified OXM conjugates

Dissolving the 3, 3' - (4- (12-maleimidododecanamido) benzylidene) -bis-4-hydroxycoumarin obtained in the above step in DMSO to prepare a solution of about 10mg/mL, and adding the amino acid sequence of seq.id NO: the 1 backbone peptide sequence was also dissolved in DMSO, 20ul DIEPA was added after ultrasonic mixing of the two, the reaction was stirred at room temperature and monitored by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After completion of the reaction, the reaction mixture was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 μm microporous membrane, and purified by preparative liquid chromatography under the following conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate was 5ml/min and the detection wavelength was 214 nm. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain pure 8.6 mg. The theoretical relative molecular mass is 4960.9. ESI-MS m/z: calcd [ M +3H ]]³⁺1654.6，[M+4H]⁴⁺1241.2；Found[M+3H]³⁺1654.2，[M+4H]⁴⁺1240.2。

Example 5

1. Synthesis of chemically modified groups

Synthesis of 3, 3' - (4-carboxyphenylmethylene) -di-4-hydroxycoumarin

P-carboxybenzaldehyde (0.45g, 3mmol) was dissolved in 20ml of anhydrous ethanol, followed by addition of 4-hydroxycoumarin (0.98g, 6 mmol). Heating and refluxing for 12h, cooling the reaction solution to room temperature, filtering, and washing the filter cake with 10ml of ethanol for 3 times to obtain 1.12g of the product with the yield of 82.1%.

¹H-NMR(DMSO-d₆，300MHz)：δppm：8.37(d，J＝7.8Hz，2H，Ar-H)，8.29(d，J＝8.0Hz，2H，Ar-H)，8.06(t，J＝7.2Hz，2H，Ar-H)，7.84-7.74(m，6H，Ar-H)，6.86(s，1H，-CH-).ESI-MS m/z：456.4[M+H]⁺.

Synthesis of tert-butyl 2- (2- (2-aminoethoxy) ethoxy) ethylcarbamate

1, 8-diamino-3, 6-dioxaoctane (10.7g, 72.3mmol) was dissolved in 70ml DCM, Boc anhydride (2.2g, 10.1mmol) was dissolved in 50ml DCM, and Boc anhydride was slowly added dropwise to the 1, 8-diamino-3, 6-dioxaoctane solution at 0 ℃. After the dropwise addition, the reaction solution was returned to room temperature, and the reaction was continued for 4 hours, after the reaction was completed, column chromatography was performed using basic alumina, and separation and purification were performed to obtain 1.8g of a colorless transparent oily substance with a yield of 72.0%.

¹H NMR(DMSO-d₆，300MHz)：δ4.96(s，1H，-NH-)，3.54(s，4H，-OC ₂H-)，3.42(dt，J＝5.1，5.1Hz，4H，-OC ₂HCH₂O-)，3.10(dt，J＝5.1，5.1Hz，2H，-C ₂HNH(Boc))，2.55(s，2H，-C ₂HNH₂)，1.45(s，2H，-N ₂H)，1.42(s，9H，-t-Bu).ESI-MS m/z：249.0[M+H]⁺.

Synthesis of tert-butyl (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) carbamate

2.2.1.2 portions of the 3- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) propionic acid (523.mg, 3.1mmoL) and tert-butyl 2- (2- (2-aminoethoxy) ethoxy) ethylcarbamate (843mg, 3.4mmoL) were weighed out and dissolved in 15ml of dichloromethane and, after cooling in an ice bath, EDC. HCl (680mg, 3.6mmoL) and DMAP (75mg, 0.6mmoL) were subsequently added. Slowly raising the temperature of the reaction solution from 0 ℃ to room temperature, reacting for 6h, and purifying by column chromatography to obtain a white paste pure product of 0.99g with the yield of 80.5%.

¹H NMR(DMSO-d₆，300MHz)：δ8.03(s，1H，-CH₂CONH-)，7.00(s，2H，-COCH＝CHCO-)，6.76(s，1H，-OCONH-)，5.75(t，J＝7.2Hz，2H，-NCH ₂CH₂-)，3.59(t，J＝4.4Hz，4H，-OC ₂HCH₂NH-)，3.48(s，4H，-CH₂O CH ₂CH ₂OCH₂-)，3.15(t，2H，J＝5.6Hz，-CH₂CONH CH ₂-)，3.06(t，2H，J＝5.8Hz，-OCONHCH ₂-)，2.33(t，J＝6.8Hz，2H，-CH ₂CONH-)，1.36(s，9H，-CH ₃).ESI-MS m/z：399.5[M+H]⁺.

Synthesis of 4- (bis (4-hydroxy-2-oxo-2H-benzopyran-3-yl) methyl) -N- (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) benzamide

Tert-butyl (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) carbamate (159.8mg, 0.4mmol) was dissolved in 3ml of acetonitrile, cooled to room temperature, 1ml of trifluoroacetic acid was added, after completion of the reaction, the solvent was distilled off under reduced pressure to give a pale yellow oil, which was redissolved in 3ml of tetrahydrofuran. 3, 3' - (4-carboxyphenylmethylene) -bis-4-hydroxycoumarin (182.6mg, 0.4mmol) was dissolved in 5ml of tetrahydrofuran, DIC (68. mu.L, 0.44mmol) and HOBt (59.4mg,0.44mmol), stirring at room temperature for 30min to activate the carboxyl group, slowly dropping the above solution into tetrahydrofuran solution of the above product obtained by removing Boc, and stirring at room temperature overnight to react. After the reaction, the reaction solution was poured into ice water and extracted three times with 20mL of dichloromethane, and the extracts were combined and separately saturated with K₂CO₃HCl1M, saturated brine washed three times. Adding anhydrous Na into the extract₂SO₄Drying overnight, concentrating under reduced pressure to obtain crude product, and separating by column chromatography to obtain pure white paste 132.8mg with yield of 45%.

¹H-NMR(DMSO-d₆，300MHz)：δppm：8.94(s，1H，-NHCO-Ar)，8.38(s，1H，-NHCOCH₂-)，8.37(d，J＝7.8Hz，2H，Ar-H)，8.29(d，J＝8.25Hz，2H，Ar-H)，8.06(t，J＝7.2Hz，2H，Ar-H)，7.84-7.74(m，6H，Ar-H)，6.98(s，2H，-COCH＝CHCO-)，6.86(s，1H，-CH-).5.75(t，J＝7.2Hz，2H，-NCH ₂CH₂-)，3.59(t，J＝6.0Hz，4H，-OC ₂HCH₂NH-)，3.35(s，4H，-OC ₂HC ₂HO-)，3.15(t，4H，J＝6.0Hz，-CONH CH ₂-)，2.33(t，J＝7.2Hz，2H，-CH ₂CONH-).ESI-MS m/z：738.4[M+H]⁺.

2. Synthesis and purification of chemically modified OXM conjugates

The 4- (bis (4-hydroxy-2-oxo-2H-benzopyran-3-yl) methyl) -N- (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) benzamide obtained in the above step was dissolved in DMSO to prepare a solution of about 10mg/mL, and seq.id NO: the 1-backbone peptide sequence was also dissolved in DMSO, and 20. mu.l DIEPA was added after ultrasonic mixing of the two, and the reaction was stirred at room temperature and monitored by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After the reaction, the reaction mixture was diluted with acetonitrile containing 1% TFAAfter release, high-speed centrifugation and filtration using a 0.45 μm microfiltration membrane, purification was carried out using preparative liquid chromatography, the chromatographic conditions being: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate was 5ml/min and the detection wavelength was 214 nm. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain pure 8.3 mg. The theoretical relative molecular mass is 4993.8. ESI-MS m/z: calcd [ M +3H ]]³⁺1665.6，[M+4H]⁴⁺1249.5；Found[M+3H]³⁺1665.8，[M+4H]⁴⁺1249.1。

Sequence listing

<110> university of Chinese pharmacy

<120> long-acting hypoglycemic weight-reducing peptides, preparation method thereof and application thereof as medicine

<160>1

<210>1

<211>39

<212>PRT

<213> Artificial sequence

<220>

<221> synthetic construct

<222>(16)..(16)

<223> Xaa at position 16 is a small molecule engineered Cys

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp

1 5 10 15

Xaa Arg Arg Ala Gln Asp Phe Val Glu Trp Leu Lys Asn Gly Gly

16 20 25 30

Pro Ser Ser Gly Ala Pro Pro Pro Ser

31 35

Claims (7)

1. An OXM conjugate, characterized in that the OXM conjugate has the chemical structure:

2. a pharmaceutical composition comprising a therapeutically effective amount of an OXM conjugate according to any one of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: the salt is a combination of OXM conjugate with hydrochloric, hydrobromic, hydroiodic, sulfuric, pyrosulfuric, phosphoric, nitric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2- (4-hydroxybenzoyl) benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonic, itaconic, sulfamic, trifluoromethanesulfonic, dodecylsulfuric, 2-naphthalenesulfonic, naphthalenedisulfonic, camphorsulfonic, citric, tartaric, stearic, lactic, oxalic, malonic, tartaric, stearic, lactic, fumaric, succinic, tartaric, citric, tartaric, citric, tartaric, and mixtures thereof, Succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid, or thiocyanic acid.

3. The pharmaceutical formulation of any one of the OXM conjugates of claim 1, which is a pharmaceutically acceptable tablet, capsule, elixir, syrup, lozenge, inhalant, spray, injection, film, patch, powder, granule, block, emulsion or suppository.

4. A medicament prepared from the composition of claim 2, said medicament being a pharmaceutically acceptable tablet, capsule, elixir, lozenge, inhalant, spray, injection, film, patch, powder, granule, block or suppository.

5. Use of an OXM conjugate according to claim 1 for the preparation of a medicament for the treatment of diabetes, obesity, hyperlipidemia or non-alcoholic fatty liver disease.

6. Use of a pharmaceutically acceptable salt of an OXM conjugate according to claim 1 for the preparation of a medicament for the treatment of diabetes, obesity, hyperlipidemia or non-alcoholic fatty liver disease.

7. A process for the preparation of an OXM conjugate according to any one of claims 1, comprising a liquid phase synthesis or a solid phase synthesis preparation.

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