CN114075275A

CN114075275A - Long-acting insulin analogue

Info

Publication number: CN114075275A
Application number: CN202010826813.1A
Authority: CN
Inventors: 周述靓; 王鹏; 邓岚
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2022-02-22
Also published as: WO2022037470A2; WO2022037470A3

Abstract

The invention relates to the field of medicine synthesis, and discloses a long-acting insulin analogue. The long-acting insulin analogue is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for treating various diseases, including type I diabetes, type II diabetes and gestational diabetes.

Description

Long-acting insulin analogue

Technical Field

The invention relates to a long-acting insulin analogue and application thereof.

Background

Insulin is a protein hormone secreted by pancreatic islet beta cells in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc., and is the only hormone in the body for reducing blood sugar and promoting the synthesis of glycogen, fat and protein, and exogenous insulin is mainly used for treating diabetes.

Unlike other recombinant protein drugs, insulin is used as a hormone for regulating blood sugar, the therapeutic window is very narrow, the blood concentration must be strictly controlled, hypoglycemia and even shock and death are easily caused when the blood concentration is too high, and hyperglycemia is easily caused when the blood concentration is too low. Therefore, insulin cannot be injected intravenously and is only slowly released into the blood by subcutaneous injection. In addition, the sugar intake of three meals a day needs the insulin regulation during meals, and the basal blood sugar is regulated by the basal insulin, so the physiological insulin curve is very complex, which is the design significance of quick-acting insulin and long-acting insulin.

Insulin drugs are generally formulated in 100IU/ml, and at this concentration insulin is usually present as a dimer and thus hexamer. After subcutaneous injection of insulin, the hexamer gradually releases the dimer-monomer, which then enters the blood for onset. Because the action time of the insulin is 4-6 hours, the insulin has longer action time compared with the insulin at meal, is easy to hypoglycemia after 2 hours after meal, and has shorter action time compared with the basal insulin. Protamine and insulin are combined to form precipitates, so that the insulin is slowly dissolved and released after subcutaneous injection, the action time is prolonged to more than ten hours, and the protamine and the insulin are generally injected twice a day. Protamine insulin partially solves the problems, but the insulin secretion curves in the physiological state are still quite different, and the precipitation property of the protamine insulin determines that the protamine insulin is a suspension, so that the dosage control is difficult to ensure accurately.

The sinophilin insulin glargine is similar to protamine in slow releasing effect, and has isoelectric point changed from 5.4 to 6.7 via adding 2 basic amino acids arginine to the end of B chain, forms precipitate near the isoelectric point after subcutaneous injection, and is slowly dissolved to introduce into blood. However, the change of isoelectric point determines that the insulin glargine must be prepared into an acidic preparation to be soluble, asparagine which is easy to deamidate A21 is mutated into glycine, which is also the source of the name of the insulin glargine, the half-life period of the insulin glargine is 12 hours, the action time is 20-24 hours, and the injection is carried out once a day.

Novonide develops a new generation of long-acting insulin degummed insulin, and successfully gets on the market in 2013, the degummed insulin removes threonine at B30, and a 16-carbon fatty acid chain is connected to lysine at B29 through a glutamic acid linker, and can be combined with albumin in plasma to prolong half-life, and the mechanism is the same as that of insulin detemir. Besides, by optimizing the proportion of the linker to the fatty acid chains and the zinc ions, degummed insulin is in a double hexamer form in the preparation, after subcutaneous injection, phenol diffuses, and the degummed insulin undergoes conformational change and is rapidly assembled into linear polyhexamers from the double hexamers, generally more than thousands of molecules, and the polyhexamers slowly release hexamer-dimer-monomers to enter blood for effect. By these two mechanisms, deglutated insulin has a half-life of up to 24 hours and a duration of action of up to 42 hours.

The long-acting insulin achieves the purpose of once-a-day administration, but cannot achieve a longer administration time and cannot achieve once-a-week administration. It is an object of the present invention to provide a long acting insulin analogue with a longer half-life.

Disclosure of Invention

The invention provides a long-acting insulin analogue and application thereof.

To achieve the above object, the present invention provides, in a first aspect, a compound of structure I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or any mixture thereof.

Structure I

When X1 is S in structure I, X2 is S, or CH 2;

when X1 in structure I is CH2, X2 is S, or CH 2;

x1 is NH and X2 is CO in structure I;

when X1 in structure I is CO, X2 is NH;

AA1 in structure I is any encodable amino acid other than Cys, or any non-encodable amino acid that does not contain an SH group;

AA2 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA3 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA4 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA5 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA6 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA7 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA8 in structure I is Lys, or Dah, or Orn, or Dab, or Dap;

AA9 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA10 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA11 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA12 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain an SH group, or is absent;

AA13 in structure I is Lys, or Dah, or Orn, or Dab, or Dap, or is absent;

AA14 in structure I is NH2, or OH;

r1 and R2 in the structure I are succinic acid cholesterol monoester or 2-cholesterol acetic acid, or 2-cholesteryl propionic acid, or 3-cholesteryl propionic acid, or 2-cholesteryl butyric acid, or 2-cholesterol isobutyric acid, or 3-cholesterol butyric acid, or 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, or 2-cholesteryl valeric acid, or 2-cholesteryl isovaleric acid, or 3-cholesteryl valeric acid, or 5-cholesteryl pentanoic acid, or 2-cholesteryl hexanoic acid, or 6-cholesteryl hexanoic acid, or 2-cholesterol heptanoic acid, or 7-cholesterol heptanoic acid, or 2-cholesterol octanoic acid, or 8-cholesterol octanoic acid, or HO2C (CH2) n1CO- (gamma Glu) n2- (PEGn3 (CH).₂)n4CO)n5-；

Wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5;

r1 and R2 in structure I cannot be present at the same time;

when AA14 is OH and X1 is S, X2 is CH2 in structure I, R1 and R2 may be absent at the same time;

when AA14 is OH, X1 is CH2, and X2 is S in structure I, R1 and R2 may be absent at the same time;

when AA14 is OH, X1 is CH2, and X2 is CH2 in structure I, R1 and R2 can be absent at the same time;

when AA14 in structure I is OH, X1 is NH, and X2 is CO, R1 and R2 may be absent at the same time;

when AA14 is OH, X1 is CO, and X2 is NH in structure I, R1 and R2 may be absent at the same time;

when AA14 is NH2 and X1 is S, X2 is S in structure I, R1 and R2 may be absent at the same time;

when AA14 is NH2 and X1 is S, X2 is CH2 in structure I, R1 and R2 may be absent at the same time;

when AA14 in structure I is NH2, X1 is CH2, and X2 is S, R1 and R2 can be absent at the same time;

when AA14 is NH2, X1 is CH2, and X2 is CH2 in structure I, R1 and R2 may be absent at the same time;

when AA14 is NH2, X1 is NH, and X2 is CO in structure I, R1 and R2 may be absent at the same time;

when AA14 is NH2, X1 is CO, and X2 is NH in structure I, R1 and R2 may be absent simultaneously.

The invention also provides compositions comprising a compound according to the invention and Zn²⁺The complex formed.

The invention also provides compounds comprising a compound according to the invention and Zn²⁺The pharmaceutical compositions of the complexes formed, and the use of the pharmaceutical compositions of the compounds of the invention for the preparation of pharmaceutical compositions for the treatment of diseases.

The use of the pharmaceutical composition in the treatment of various diseases, including type I diabetes, type II diabetes, gestational diabetes.

Further details of the invention are set forth below, or some may be appreciated in embodiments of the invention. Unless otherwise indicated, the amounts of the various ingredients, reaction conditions, and the like used herein are to be construed in any case to mean "about". Accordingly, unless expressly stated otherwise, all numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the standard deviation found in the respective experimental conditions.

Herein, when a chemical structural formula and a chemical name of a compound are ambiguous or ambiguous, the compound is exactly defined by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description, whether partial or complete, including similar structures as described above, includes all possible enantiomers and diastereomers of the compound, including any stereoisomer alone (e.g., pure geometric isomers, pure enantiomers, or pure diastereomers), as well as any mixture of such stereoisomers. Mixtures of these racemates and stereoisomers may also be further resolved into the enantiomers or stereoisomers of their constituent members by those skilled in the art using non-stop separation techniques or methods of chiral molecular synthesis.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above case, a single enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or racemate resolution. Resolution of the racemates can be accomplished by various methods, such as conventional recrystallization from resolution-assisting reagents, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, the compounds of formula I and all of their pharmaceutically acceptable different forms. The pharmaceutically acceptable different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above and any mixtures of these forms.

Detailed Description

The invention discloses a long-acting insulin analogue and application thereof, and a person skilled in the art can appropriately improve related parameters to realize the long-acting insulin analogue by taking the contents as reference. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the compounds and processes described herein, as well as other changes and combinations of the foregoing, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The Chinese names corresponding to the English abbreviations related in the invention are shown in the following table:

english abbreviation	Name of Chinese	English abbreviation	Name of Chinese
				Fmoc	9-fluorenylmethoxycarbonyl group	OtBu	Tert-butoxy radical
tBu	Tert-butyl radical	Boc	Boc-acyl
				Trt	Trityl radical	Pbf	(2, 3-dihydro-2, 2,4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl group
Ala	Alanine	Leu	Leucine
				Arg	Arginine	Lys	Lysine
Asn	Asparagine	Met	Methionine
				Asp	Aspartic acid	Phe	Phenylalanine
Cys	Cysteine	Pro	Proline
				Gln	Glutamine	Ser	Serine
Glu	Glutamic acid	Thr	Threonine
				Gly	Glycine	Trp	Tryptophan
His	Histidine	Tyr	Tyrosine
				Ile	Isoleucine	Val	Valine
Dap	2, 3-diaminopropionic acid	Dab	2, 4-diaminobutyric acid
				Orn	Ornithine	Dah	2, 7-Diaminoheptanoic acid
Acm	S-acetyl group	DAS	(S, S) -2, 7-diaminooctanedioic acid
				Mtt	4-Methyltriphenylmethyl group

EXAMPLE 1 preparation of Compound 1

The preparation method comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, carrying out acidolysis on the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to sequentially insert corresponding protective amino acids or fragments in the following sequences on a carrier resin by the solid-phase coupling synthesis method to prepare the peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid is 1.2-6 times of the total mole number of charged resin; preferably 2.5 to 3.5 times.

In the preparation method, the substitution value of the carrier resin is 0.2-1.0 mmol/g resin, and the preferable substitution value is 0.3-0.5 mmol/g resin.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The deprotection time for removing Fmoc protection is 10-60 minutes, and preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.

The coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from one of DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate, 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3, 3-tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar consumption of the condensation reagent is 1.2-6 times of the total molar number of amino groups in the amino resin, and preferably 2.5-3.5 times.

The coupling reaction needs to add an activating reagent, wherein the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, and 1-hydroxybenzotriazole is preferred. The amount of the activating agent is 1.2 to 6 times, preferably 2.5 to 3.5 times of the total mole number of the amino groups in the amino resin.

As a preferable scheme of the invention, the reagent for removing Fmoc protection is PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution, and the piperidine content in the mixed solution is 10-30% (V). The dosage of the Fmoc protection removing reagent is 5-15 mL per gram of amino resin, and preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acidolysis while removing the resin and side chain protecting groups to obtain a crude product:

more preferably, the acidolysis agent used in the acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL per gram of the peptide resin; preferably, 7-10 mL of acidolysis agent is required per gram of peptide resin.

The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.

Further, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

1. Synthesis of peptide resins

Rink Amide BHHA resin is used as carrier resin, and is coupled with protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare peptide resin. The protected amino acids used in this example correspond to the protected amino acids shown below:

(1) 1 st protected amino acid inserted into main chain

Dissolving 0.03mol of the 1 st protected amino acid and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value about 0.3mmol/g) is taken, deprotected by 20% PIP/DMF solution for 25 minutes, washed and filtered to obtain Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.

(2) The 2 nd to 29 th protected amino acids of the main chain are grafted into

And sequentially inoculating the corresponding 2 nd to 29 th protected amino acids by the same method for inoculating the 1 st protected amino acid of the main chain to obtain the resin containing 29 amino acids of the main chain.

(3) Side chain insertion of the 1 st protected amino acid

Deprotection of the side chain using 50% HFIP/DCM solution was repeated 5 times for 35 min each time, and the filter washing yielded the Mtt deprotected resin for use.

Dissolving 0.3mol of 2,2' -dithiodipyridine with a proper amount of DMF, adding the solution into the resin with Mtt protection removed, stirring for reaction for 3 hours, filtering and washing to obtain SH activated resin for later use.

Taking 0.03mol of the 1 st protected amino acid (Fmoc-Cys-Asn (Trt) -OtBu) of the side chain, dissolving with a proper amount of DMF, adding into the SH activated resin, adding 2ml of DIPEA, stirring for reaction for 3 hours, filtering and washing to finish the inoculation of the first amino acid of the side chain.

(4) Side chain insertion of the 2 nd protected amino acid

Dissolving 0.03mol of the 2 nd protected amino acid of the side chain and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.

And (3) adopting 20% PIP/DMF solution for deprotection for 25 minutes, washing and filtering, adding the activated side chain No. 2 protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the side chain No. 2 protected amino acid-containing resin.

(5) The 3 rd to 20 th protected amino acid of the side chain is inserted

And sequentially inoculating 3 rd to 20 th protected amino acids corresponding to side chains by the same method for inoculating the 1 st protected amino acid to the main chain to obtain the peptide resin.

2. Preparation of crude product

Adding a cleavage reagent (10 mL of cleavage reagent/g of resin) with a volume ratio of TFA: Tis: water of 95: 5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting a filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying to obtain the white-like powder.

Dissolving the obtained white-like powder in a 20% DMSO aqueous solution, adjusting the pH value to 7.5 with ammonia water, stirring for reaction for 10 hours, adding glacial acetic acid until the acetic acid content is 20%, dropwise adding an iodine/ethanol saturated solution while stirring until complete cyclization is achieved, and concentrating under reduced pressure at 35-40 ℃ to obtain a crude concentrated solution.

3. Preparation of the pure product

Filtering the crude concentrated solution with 0.45 μm mixed microporous membrane, and purifying;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a 30mm by 250mm chromatographic column is 20mL/min, eluting by a gradient system, circularly sampling for purification, sampling the crude product solution in the chromatographic column, starting the mobile phase for elution, collecting the main peak, and evaporating acetonitrile to obtain a purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use, and performing salt exchange by high performance liquid chromatography with 1% acetic acid/water solution-acetonitrile as mobile phase system, 10 μm reversed phase C18 as purification chromatographic filler, and 20mL/min of 30 mm/250 mm chromatographic column flow rate (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solutions of salt exchange, concentrating under reduced pressure to obtain pure acetic acid water solution, and freeze drying to obtain pure acetic acid 5.2g, purity 98.6% and total yield 8.0%. The molecular weight was 6522.6 (100% M + H).

EXAMPLE 2 preparation of Compound 2

The procedure is as in example 1, using the protected amino acids as in the following table:

3.9g of pure product is obtained, the purity is 97.8 percent, and the total yield is 6.2 percent. The molecular weight was 6275.3 (100% M + H).

EXAMPLE 3 preparation of Compound 3

(1) The preparation method was the same as in example 1 except that it was different from that of graft-side linker 1.

(2) Side chain insertion of the 1 st protected amino acid

Dissolving 0.03mol of the 1 st protected amino acid of the side chain and 0.03mol of HOBt in a proper amount of DMF; adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting under stirring at room temperature for 30min to obtain activated protected amino acid solution

And (3) removing side chain protection by adopting a 50% HFIP/DCM solution, repeating for 5 times, washing and filtering for 35 minutes each time, adding the activated side chain 1 st protected amino acid solution into the Mtt-protected resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the side chain 1 st protected amino acid-containing resin.

The protected amino acids used are as follows:

4.7g of pure product is obtained, the purity is 98.0 percent, and the total yield is 7.2 percent. The molecular weight was 6501.4 (100% M + H).

EXAMPLE 4 preparation of Compound 4

The procedure is as in example 3, using the protected amino acids as in the following table:

3.3g of pure product is obtained, the purity is 98.5 percent, and the total yield is 5.3 percent. The molecular weight was 6254.2 (100% M + H).

EXAMPLE 5 preparation of Compound 5

(2) Side chain insertion of the 1 st protected amino acid

Dissolving 0.03mol of the 1 st protected amino acid of the side chain in proper amount of DMF, adding the solution into the resin with the Mtt protection removed, stirring the solution evenly, adding 0.03mol of N-methylmorpholine and 0.03mol of lithium chloride, stirring the solution for reaction for 6 hours, filtering and washing the reaction product to obtain the resin with the 1 st protected amino acid of the side chain.

The protected amino acids used are as follows:

7.9g of pure product is obtained, the purity is 98.4 percent, and the total yield is 12.1 percent. The molecular weight was 6504.5 (100% M + H).

EXAMPLE 6 preparation of Compound 6

The preparation method is the same as example 5. The protected amino acids used are as follows:

6.1g of pure product is obtained, the purity is 97.6 percent, and the total yield is 9.7 percent. The molecular weight was 6257.3 (100% M + H).

EXAMPLE 7 preparation of Compound 7

(2) Side chain insertion of the 1 st protected amino acid

And (2) removing side chain protection by adopting a 2% hydrazine hydrate/DMF solution, repeating for 3 times, washing and filtering for 10 minutes each time, adding the activated side chain 1 st protected amino acid solution into the Dde-removed resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the side chain 1 st protected amino acid-containing resin.

The protected amino acids used are as follows:

9.2g of pure product is obtained, the purity is 98.9 percent, and the total yield is 14.2 percent. The molecular weight was 6486.5 (100% M + H).

EXAMPLE 8 preparation of Compound 8

The preparation method is the same as example 7. The protected amino acids used are as follows:

8.7g of pure product is obtained, the purity is 98.1 percent, and the total yield is 13.9 percent. The molecular weight was 6239.3 (100% M + H).

EXAMPLE 9 determination of Primary pharmacokinetic Properties

Each compound was divided into two dosing groups: SD rats, 4 males per group, 8 in total.

Tail vein intravenous injection group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0h) and 30min, 1h, 2h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are centrifugally separated.

Subcutaneous administration group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0h) and 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are separated by centrifugation.

Plasma concentrations of the corresponding compounds in plasma samples of SD rats were measured by the liquid chromatography-mass spectrometry method, and the half-lives of the compounds after intravenous and subcutaneous administration in SD rats under Subcutaneous (SC) administration are shown in the following table:

compound (I)	t_1/2(h)
		Compound 1	10.2
Compound 2	13.4
		Compound 3	9.0
Compound 4	11.5
		Compound 5	10.4
Compound 6	12.7
		Compound 7	11.6
Compound 8	14.1

Claims

1. A long acting insulin analogue having the structural formula i:

when X1 is S in structure I, X2 is S, or CH 2;

when X1 in structure I is CH2, X2 is S, or CH 2;

x1 is NH and X2 is CO in structure I;

when X1 in structure I is CO, X2 is NH;

AA8 in structure I is Lys, or Dah, or Orn, or Dab, or Dap;

AA13 in structure I is Lys, or Dah, or Orn, or Dab, or Dap, or is absent;

AA14 in structure I is NH2, or OH;

r1 and R2 in the structure I are succinic acid cholesterol monoester or 2-cholesterol acetic acid, or 2-cholesteryl propionic acid, or 3-cholesteryl propionic acid, or 2-cholesteryl butyric acid, or 2-cholesterol isobutyric acid, or 3-cholesterol butyric acid, or 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, or 2-cholesteryl valeric acid, or 2-cholesteryl isovaleric acid, or 3-cholesteryl valeric acid, or 5-cholesteryl pentanoic acid, or 2-cholesteryl hexanoic acid, or 6-cholesteryl hexanoic acid, or 2-cholesterol heptanoic acid, or 7-cholesterol heptanoic acid, or 2-cholesterol octanoic acid, or 8-cholesterol octanoic acid, or HO2C (CH2) n1CO- (gamma Glu) n2- (PEGn3 (CH).₂) n4CO) n 5-; wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5;

r1 and R2 in structure I cannot be present at the same time;

2. A long acting insulin analogue according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the analogue, a prodrug based on the compound, or a mixture of any of the above forms.

3. A long acting insulin analogue with Zn according to claims 1 and 2²⁺The complex formed.

4. An insulin analogue or with Zn according to claims 1-3²⁺The formed complex is used for preparing medicines for treating diseasesThe pharmaceutical composition of (1).

5. The pharmaceutical composition according to claim 4, for use in the treatment of various diseases.

6. The various diseases according to claim 5 including type I diabetes, type II diabetes, gestational diabetes.