HK1136837B - An erythropoietin mimetic peptide derivatives and its pharmaceutical salt, the preparation and uses thereof - Google Patents

Abstract

A erythropoietin mimetic peptide derivative with the general formula (I) and its pharmaceutically acceptable salt, as well as their preparation method, are provided, wherein R1, R2, R3, R4, R5, n1, n2 are as defined in the specification.A pharmaceutical composition containing a derivative of erythropoietin mimetic peptide of formula (I) and its pharmaceutically acceptable salt is also provided.The provided erythropoietin mimetic peptide derivatives and their pharmaceutically acceptable salts, or pharmaceutical compositions containing erythropoietin mimetic peptide derivatives and their pharmaceutically acceptable salts, can be widely used in the treatment of diseases characterized by a lack of erythropoietin or a deficiency or defect in the red blood cell population.R1-R2-(CH2)n1-R3-(CH2)n2-R4-R5 (I)

Description

Erythropoietin mimic peptide derivative and its medicinal salt, preparation method and use

Technical Field

The invention relates to an erythropoietin simulated peptide derivative which can be combined with an erythropoietin receptor and activate the erythropoietin receptor or can play the role of erythropoietin stimulation and a medicinal salt, in particular to an erythropoietin simulated peptide derivative modified by active methoxy polyethylene glycol and a preparation method thereof; also disclosed are methods of using the above peptidomimetic derivatives and pharmaceutically acceptable salts to treat diseases characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population.

Technical Field

Erythropoietin (EPO) is a glycoprotein hormone with a molecular weight of about 34 kD. Erythropoietin present in plasma consists of 165 amino acids, is glycosylated to a high degree, and the glycosyl component is predominantly sialic acid. Naturally occurring erythropoietin is classified into two types, alpha and beta, depending on the carbohydrate content, wherein the alpha form contains 34% carbohydrate and the beta form contains 26% carbohydrate. The two types are identical in biological characteristics, antigenicity and clinical application effect. The human erythropoietin gene is located in chromosome 7, region 22. In 1985, cDNA was successfully cloned, and large-scale production of recombinant human erythropoietin (rHuEPO) was started by gene recombination technology, and widely used in clinic. Erythropoietin (Egrie, JC, Strickland, TW, Lane, J, etc. (986) immunobiology (1 mmunobal) 72: 213-224), which is the product of a cloned human erythropoietin gene inserted into and expressed in ovarian tissue cells (CHO cells) of the Chinese hamster, has been biosynthesized using recombinant DNA techniques. Naturally occurring human erythropoietin is first translated into a polypeptide chain containing 166 amino acids with arginine at position 166. Arginine at position 166 is cleaved by hydroxypeptidase in post-translational modifications. The molecular weight of the polypeptide chain of human EPO without a sugar group is 18236da in the intact erythropoietin molecule, the sugar group represents approximately 40% of the total molecular weight (j.biol.chem.262: 12059).

Erythropoietin is the first cytokine to be used clinically and is the hemoglobin-increasing preparation which has the single action and is safe and reliable to date. Has certain curative effect on renal anemia, aplastic anemia, multiple myeloma, paroxysmal nocturnal hematuria and the like; in addition, the application of erythropoietin can reduce the blood transfusion amount in operation and can correct anemia caused by malignant tumor, chemotherapy and rheumatoid arthritis to a certain extent. Since erythropoietin is mainly produced by tubular endothelial cells, anemia caused by renal disorders is the first indication for erythropoietin; erythropoietin has almost 100% efficacy in correcting renal anemia, but does not improve renal function. The treatment of the erythropoietin is safe and effective, is suitable for long-term treatment and can also avoid the blood source tension. In the world biotech drug market in 2006, recombinant drugs of the erythropoietin class account for $ 119 billion, with enormous market capacity.

As early as 1989, the U.S. FDA approved recombinant human erythropoietin (EPOGEN) for the treatment of renal anemia, but it was not marketed in China until 1992. The annual incidence rate of chronic nephritis in China is about 0.25%, a considerable number of patients can be finally converted into renal failure, and about 50-60 ten thousand of patients with annual renal anemia are treated. According to the conservative drug consumption estimation, if the drug is taken by other patients with 30-40 yuan per patient according to the current price and cancer-related anemia and the like, the domestic market volume is about 12-16 million yuan or more (the average weight of the patients is calculated by 50 Kg). Since the later 90 s in the 20 th century, erythropoietin has entered the popular drug line in hospitals in key cities in China, and in 2003, the sample hospitals in key cities in China had a medication amount of 6213 ten thousand yuan, and the rank was 56. In 2004, the medicine purchase amount of sample hospitals in key cities in China is increased to 8049 ten thousand yuan, which is increased by 30% on year-on-year basis.

Erythropoietin plays an important role in regulating and controlling the oxygen supply condition of the organism as an endocrine hormone which acts on bone marrow hematopoietic cells and promotes the proliferation and differentiation of erythroid progenitor cells and the final maturation. Erythropoietin is produced by the liver in the early embryonic stage and then gradually migrates to the kidney and is secreted mainly by tubular interstitial cells after birth.

During the process of erythropoietin-induced erythroid differentiation, globins are induced, which enable the cells to take up more iron to synthesize functional hemoglobin, which can bind with the oxygen in mature red blood cells, and thus, red blood cells and hemoglobin play an extremely important role in providing body oxygen. This process is caused by the interaction between erythropoietin and surface receptors of the erythroid cells.

When a person is in a healthy state, the tissue can absorb enough oxygen from the existing red blood cells, where the body's concentration of erythropoietin is low, such a normal low erythropoietin concentration is completely stimulating to promote normal loss of red blood cells due to age problems.

When the level of oxygen transport by red blood cells in the circulatory system is reduced and hypoxia occurs, the amount of erythropoietin in the body will increase and the hypoxic state of the body can be caused by: excessive radiation, decreased oxygen intake due to high altitude or long-term coma, various types of anemia, and the like. In response to the tissue being subjected to hypoxic pressure, an increase in erythropoietin levels stimulates the differentiation of the red blood cells to the point of increasing erythropoiesis. When the number of red blood cells in the body is greater than that required by normal tissue, the levels of erythropoietin in the circulatory system are reduced. Because erythropoietin plays a crucial role in erythropoiesis, this class of hormones holds great promise for the treatment and diagnosis of hematological disorders characterized by poor and defective erythropoiesis. Recent studies have provided the basis for the speculation of the utility of erythropoietin therapy in a variety of diseases, disorders and hematological abnormalities, including: the use of Erythropoietin in the treatment of anemia in patients with Chronic Renal Failure (CRF) and Erythropoietin in the treatment of anemia in AIDS and cancer patients undergoing chemotherapy (Danna, RP, Rudnick, SA, Abels, RI, in: MB, Garnic eds., Erythropoietin in Clinical Applications-International patent application. Marcel Dekker; 1990: p 301-324).

Some of the biological effects of erythropoietin can be modulated by intrinsic interactions with receptors on the cell membrane surface. Initially, when immature red blood cells isolated from the spleen of a mouse were used to study cell surface-bound erythropoietin protein, it was found that this protein is composed of two polypeptides with a molecular weight of approximately 85000-100000 KD (Sawyer, et al (1987) Proc. Natl. Acad. Sci. USA 84: 3690-. The number of binding sites for erythropoietin was also calculated, approximately 800-1000 sites per cell membrane. Approximately 300 of these binding sites had a Kd level of 90pM, while the remaining binding sites were less binding, and approximately 570pM. showed that approximately 400 binding sites were found from spleen erythrocytes of mice infected with the anemia strain of the friend virus, with a high Kd level of 100pM and a low Kd level of 800pM, in response to EPO.

The subsequent work is to transcribe the two erythropoietin receptors from a single gene, which has been cloned. For example, the DNA sequences of the mouse and human erythropoietin receptors and the sequences encoding the peptides have been described in WO 90/08822. Current models indicate that binding of erythropoietin to the erythropoietin receptor results in activation and dimerization of the two erythropoietin receptors, which further results in the initiation of signaling.

The use of the erythropoietin cloning gene further aids in the search for agonists and antagonists of these important receptors. Peptides that are capable of acting to some extent on the erythropoietin receptor have been identified and described. In particular, a group of peptides containing a major peptide segment is identified which bind to the erythropoietin receptor and stimulate differentiation and proliferation of erythropoietin cells. However, peptides capable of stimulating the proliferation and differentiation of erythrocytes have a very low EC50, between 20nM and 250 nM. Therefore, the peptides are greatly limited in clinical application, and in order to overcome the defects of the prior art, the invention provides erythropoietin mimic peptide derivatives with better biological activity and higher bioavailability, the pharmaceutically acceptable salts thereof and the preparation methods thereof

Disclosure of Invention

The invention aims to provide an erythropoietin mimic peptide derivative with better biological activity and higher bioavailability, a pharmaceutically acceptable salt thereof and a preparation method thereof.

The invention also aims to provide a pharmaceutical composition containing the erythropoietin mimetic peptide derivative and the pharmaceutically acceptable salts thereof, which is used for treating diseases characterized by the deficiency of erythropoietin or the deficiency or defect of erythrocyte groups.

The invention discloses an erythropoietin mimic peptide derivative with in-vivo biological activity and a pharmaceutical salt thereof,

R₁-R₂-(CH₂)_n1-R₃-(CH₂)_n2-R₄-R₅

(I)

wherein R is₁、R₅Selected from the group consisting of monomeric peptides of erythropoietin mimetic peptides and analogs thereof having biological activity in vivo; n is₁、n₂The number of (a) is independently an integer selected from 0 to 10; r₂、R₄Selected from-CO, -CH₂；R₃Selected from O, S, CH₂、N(CH₂)_n3NHR₆、NCO(CH₂)_n4NHR₆、CHOCONH(CH₂)_n5NHR₆、CHSCON(CH₂)_n5NHR₆Or CHNHCON (CH)₂)_n5NHR₆(ii) a Wherein n is₃The number of (a) is selected from an integer of 1 to 10, n₄The number of (a) is selected from an integer of 2 to 10, n₅The number of (a) is selected from an integer of 2 to 10, R₆Selected from H or methoxy polyethylene glycol derivatives.

In this embodiment, a preferred embodiment is: r₁、R₅Each independently selected from the group consisting of formula Y₁X₁X₂X₃GX₄X₅TWX₆X₇Y₂Y₃The erythropoietin mimetic peptide monomer peptide and the analog thereof having in vivo biological activity of (1), wherein each amino acid is represented by a standard one-letter code, R₁、R₅The amino acid sequences of (A) may be identical or different, and R is more preferably₁、R₅Is identical, and R₁、R₅The N-terminus of (A) is acetylated; x₂、X₃、X₄、X₅、X₆、Y₃Each independently selected from any one of 20 genetically encoded L-amino acids or unnatural amino acids; y is₁、Y₂Each independently selected from any one of 20 genetically encoded L-amino acids or unnatural amino acids or peptide fragments consisting of the amino acids; x₁、X₇Selected from C, K, D, E, Orn or Hoc.

Further optimized for the preferred embodiments described above, R₁、R₅Is a cyclic peptide cyclized by a disulfide bond or an amide bond; wherein R is₁、R₅If cyclic peptide cyclized by disulfide bond, X₁、X₇Each independently is selected from C or Hoc; likewise, R₁、R₅If cyclic peptides cyclized via an amide bond, X₁、X₇Each independently selected from K, D, E or Orn.

In this embodiment, including the preferred embodiments described above, Y₃Preferably K, H or R, more preferably Y₃Is K.

In this scheme, including the preferred embodiments described above, R₁、R₅Is preferably from 13 to 40 amino acids, more preferably 22 amino acids, more preferably but not limited to cyclic peptides having the following structure, most preferably but not limited to NO: 1 to NO: 8 Cyclic peptides, i.e.

Ac-GGLYAQPLRGGK-NH2(SEQ ID NO：1)

Ac-GGLYArn-QPLRGGK-NH2(SEQ ID NO：2)

Ac-GGLYAQPLRGGK-NH2(SEQ ID NO：3)

Ac-GGLYAQPLRGGK-NH2(SEQ ID NO：4)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：5)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：6)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：7)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：8)

Ac-GGLYAQPLRGGK-NH2(SEQ ID NO：9)

Ac-GGLYArn-QPLRGGK-NH2(SEQ ID NO：10)

Ac-GGLYAQPLRGGK-NH2(SEQ ID NO：11)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：12)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：13)

Ac-GGTYS-RPQRG-βAla-K-NH2(SEQ ID NO：14)

Ac-GGTYS-RPQRG-βAla-K-NH2(SEQ IDNO：15)

Ac-GGTYS-RPQRG-βAla-K-NH2(SEQ ID NO：16)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：17)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：18)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：19)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：20)

Ac-GGTYRLPMAGGK-NH2(SEQ ID NO：21)

Ac-GGTYRLPMAGGK-NH2(SEQ ID NO：22)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：23)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：24)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：25)

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：26)

Ac-GGMYSGPSRGGK-NH2(SEQ ID NO：27)

Ac-GGMYSGPSRGGK-NH2(SEQ ID NO：28)

Ac-GGTYS-RPQRG-beta Ala-K-NH2(SEQ ID NO: 29) or

Ac-GGTYSRPQRG-βAla-K-NH2(SEQ ID NO：30)。

In this embodiment, there are four preferred embodiments:

【1】n₁，n₂is 2, R₂、R₄is-CO, R₃Selected from CHOCONH (CH)₂)_n5NHR₆，n₅Is 2, R₆Is H or methoxy polyethylene glycol derivative.

【2】n₁，n₂Is 1, R₂、R₄is-CO, R₃Selected from NCO (CH)₂)_n4NHR₆，n₄Is 2, R₆Is H or methoxy polyethylene glycol derivative.

【3】n₁，n₂Is 2, R₂、R₄is-CH₂，R₃Selected from CHOCONH (CH)₂)_n5NHR₆，n₅Is 2, R₆Is H or methoxy polyethylene glycol derivative.

【4】n₁，n₂Is 1, R₂、R₄is-CH₂，R₃Selected from NCO (CH)₂)_n4NHR₆，n₄Is 2, R₆Is H or methoxy polyethylene glycol derivative.

The four preferred embodiments described above are in a side-by-side relationship, with no inclusion or progression. For the above four preferred embodiments, we can further optimize, R₆Is a methoxypolyethylene glycol derivative, most preferably R₆The compound is a methoxy polyethylene glycol derivative, wherein the molecular weight of the methoxy polyethylene glycol derivative is selected from 5,000-100,000 daltons, and the structure of the methoxy polyethylene glycol derivative is selected from a branched type or a linear type.

In this scheme, we can obtain the following four preferred embodiments by further comprehensive optimization:

【1】n₁，n₂is 2, R₁、R₅Selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, R₂、R₄Selected from-CO, -CH₂，R₃Selected from CHOCONH (CH)₂)_n5NHR₆Wherein n is₅Is selected from 2-10, preferably 2; r₆Is a methoxy polyethylene glycol derivative with a linear structure and a molecular weight of 20,000 daltons.

【2】n₁，n₂Is 1, R₁、R₅Selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, R₂、R₄Selected from-CO, -CH₂，R₃Selected from NCO (CH)₂)_n4NHR₆Wherein n is₄Is selected from 2-10, preferably 2; r₆Is a methoxy polyethylene glycol derivative with a linear structure and a molecular weight of 20,000 daltons.

【3】n₁，n₂Is 2, R₁、R₅Selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, R₂、R₄Selected from-CO, -CH₂，R₃Selected from CHOCONH (CH)₂)_n5NHR₆Wherein n is₅Is selected from 2-10, preferably 2; r₆Is methoxy polyethylene glycol derivative with branched structure and molecular weight of 40,000 Dalton.

【4】n₁，n₂Is 1, R₁、R₅Selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, R₂、R₄Selected from-CO, -CH₂，R₃Selected from NCO (CH)₂)_n4NHR₆Wherein n is₄Is selected from 2-10, preferably 2; r₆Is methoxy polyethylene glycol derivative with branched structure and molecular weight of 40,000 Dalton.

Wherein, the structure of the erythropoietin mimetic peptide derivative and the pharmaceutical salt thereof is selected from the following:

the erythropoietin mimetic peptide derivative provided by the invention belongs to an amphoteric compound, and can be reacted with an acidic or basic compound to form a salt by the known technology, and the commonly adopted acid for forming an acid addition salt is as follows:

hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid; salts include sulfate, pyrosulfate, trifluoroacetate, sulfite, bisulfite, phosphate, hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, bromide, iodide, acetate, propionate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, bromide, iodide, propionate, octanoate, propanesulfonate, and mixtures thereof, Naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like, preferably trifluoroacetate.

Basic substances, which may also form salts with erythropoietin mimetic peptide derivatives, include ammonium, alkali or alkaline earth metal hydroxides, and carbonates, bicarbonates, typically sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, and the like.

The invention also discloses a preparation scheme of the erythropoietin mimetic peptide derivative and the medicinal salt thereof, which comprises the following steps:

(1) preparation of R by genetic engineering or chemical synthesis₁、R₅，R₁、R₅Selected from peptide mimetics and analogues thereof having the biological function of erythropoietin,

(2) preparing functional small molecules with the general formula (II)

R₇-CO-(CH₂)_n1-Z₂-(CH₂)_n2-CO-R₈

(II)

Wherein n is₁、n₂The number of (a) is independently an integer selected from 0 to 10;

R₇、R₈selected from OH or H;

Z₂selected from O, S, CH₂、N(CH₂)_n6NHR₉、NCO(CH₂)_n7NHR₉、CHOCONH(CH₂)_n8NHR₉、CHSCON(CH₂)_n8NHR₉Or CHNHCON (CH)₂)_n8NHR₉Wherein n is₆The number of (a) is selected from an integer of 1 to 10, n₇The number of (a) is selected from an integer of 2 to 10, n₈The number of (a) is selected from an integer of 2 to 10, R₉Is selected from the group consisting of Boc or Cbz,

(3) r is to be₁、R₅Derivatizing with functional micromolecules with a general formula (II) to prepare a compound with a general formula (III);

R₁-R₂-(CH₂)_n1-Z₂-(CH₂)_n2-R₄-R₅

(III)，

wherein R is₂、R₄Each independently selected from-CO or-CH₂，

(4) After removing Boc or Cbz, the active methoxy polyethylene glycol is connected with the active methoxy polyethylene glycol through a covalent bond.

The invention also relates to a pharmaceutical composition comprising:

(1) a therapeutic amount of an erythropoietin mimetic peptide derivative of the general formula (I) and pharmaceutically acceptable salts thereof;

(2) a pharmaceutically acceptable pharmaceutical carrier.

The invention also discloses a medical application scheme, namely the erythropoietin mimic peptide derivative containing any therapeutic quantity and the medicinal salt thereof are used for treating diseases characterized by the deficiency of erythropoietin or the deficiency or defect of erythrocyte groups. In particular for the treatment of the following diseases: end-stage renal failure or dialysis; AIDS-related anemia, autoimmune diseases, or malignancies; cystic fibrosis; early stage prematurity anemia; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss; aging and neoplastic diseases accompanied by abnormal red blood cell production.

The erythropoietin mimic peptide derivative and the medicinal salt thereof provided by the invention can obviously stimulate the increase of the count of mouse peripheral blood reticulocytes, so that the erythropoietin mimic peptide derivative and the medicinal salt thereof can stimulate the generation of red blood cells and can also greatly prolong the half-life period of a conjugate in vivo. The erythropoietin mimetic peptide derivative and the erythropoietin protein have no obvious influence on mature red blood cells, hematocrit and hemoglobin content, and have no obvious influence on peripheral blood leukocyte counting liquid.

The synthesis of the erythropoietin mimetic peptide monomer peptide adopts a solid phase synthesis technology, and the basic principle is as follows: the hydroxyl group of the hydroxyl terminal amino acid of the peptide chain to be synthesized is firstly connected with insoluble polymer resin by a covalent bond structure, and then the amino acid combined on the solid phase carrier is taken as an amino component to be subjected to amino protecting group removal and reaction with excessive activated carboxyl component, so as to lengthen the peptide chain. Repeating (condensation → washing → deprotection → washing → the next round of condensation) operation to reach the length of the peptide chain to be synthesized, finally cracking the peptide chain from the resin, and carrying out purification and other treatments to obtain the polypeptide. The intermediate control of the condensation and deprotection steps is the ninhydrin test, which shows blue color when free amino groups are present on the resin peptide chain, and no color when free amino groups are absent (the ninhydrin reagent itself is yellow). Therefore, after the condensation reaction is finished, if the color is yellow (the color of the ninhydrin reagent per se), the ninhydrin detection shows that the deprotection operation before the coupling of the next amino acid can be carried out after the coupling of the step is finished, and if the color is blue, the peptide chain is proved to have some free amino groups, and further repeated coupling or the existing condensing agent is changed until the resin peptide is yellow through the ninhydrin detection.

Methods for cyclisation of monomeric peptides are well known to those skilled in the art, and cyclisation of disulfide bonds is effected primarily by oxidizing the amino groups in monomeric peptides with an oxidizing agentOxidizing the acid side chain sulfhydryl group into disulfide bond, wherein the specific method comprises placing monomer peptide in DMSO solution, or placing in 5% ammonium bicarbonate solution for autooxidation, or adding I₂Oxidation in acetic acid solution, preferably with addition of I₂Oxidizing in acetic acid solution. The cyclization of the amide bond is mainly carried out by forming the amide bond through the carboxyl group and the amino group of the side chain of the amino acid in the monomer peptide in the presence of a condensing agent, and the condensing agent to be added is well known to those skilled in the art, and generally, DIC, EDC, HATU, Pybop and the like are available.

The dimeric peptide is synthesized by forming-NH-CH through amino group on side chain of amino acid residue of erythropoietin mimetic peptide monomer peptide and functional small molecule₂-linkage or-NH-CO-linkage, and one skilled in the art can readily synthesize a functional small molecule and link it to a monomeric peptide cyclic peptide by known techniques.

The reaction system can be selected to be carried out in an organic solvent or an available buffer system, and when the pegylation reaction of the dimeric peptide is carried out in the organic solvent, the following appropriate amount of base can be added, such as triethylamine, diisopropylethylamine, pyridine, and 2, 4, 6-trimethylpyridine. When the pegylation derivatization reaction is performed in a buffer system, the buffer system may be selected from various known buffers available, and a phosphate buffer solution having a pH of 7.7 is preferred.

The biological activity of erythropoietin or the erythropoietin mimetic peptide derivatives provided by the present invention and pharmaceutically acceptable salts thereof can be determined by a variety of assays known in the art. In vivo activity test by subcutaneous injection of erythropoietin and the erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof provided by the present invention into mice for three consecutive days, then the mice are sacrificed, whole blood is taken for peripheral blood cell and reticulocyte counting, and blood cell counting is performed by a full-automatic blood cell counter. The pharmacodynamics study is carried out on the intravenous injection of macaques, the single administration dose is 1.35mg/kg, the administration dose of erythropoietin protein used as a contrast medicament is 240 mu/kg, the medicine is taken three times per week for six weeks continuously, and blood samples are collected for carrying out the analysis of related hematology indexes.

The code number and structure correspondence table used in the invention:

drawings

FIG. 1: effect of erythropoietin mimetic peptide derivatives (HH-EPO-018) on macaque hematocrit.

FIG. 2: effect of erythropoietin mimetic peptide derivatives (HH-EPO-018) on cynomolgus hemoglobin content.

Detailed Description

In order to illustrate the present invention in more detail, the following examples are given. The scope of the invention is not limited thereto.

The first embodiment is as follows: synthesis of erythropoietin mimetic peptide derivative monomer peptide

Erythropoietin mimetic peptide derivative monomeric peptides are synthesized by solid phase peptide synthesis, which is reported in many documents, see stewart, j.m., and Young, j.d., solid peptide synthesis 2d edition, novabiochem peptide synthesis nos. the erythropoietin mimetic peptide derivative monomeric peptides provided by the present invention are synthesized by hand, resin is rink amino, alpha amino group of the amino acid derivative is protected by Fmoc (fluorenylcarbonyl), cysteine side chain mercapto group, glutamine side chain amino group, histidine side chain imidazolyl group is protected by Trt (trityl), arginine side chain guanidino group is protected by Pbf (2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl), tryptophan side chain indolyl group, lysine amino group is protected by Boc (t-butyloxycarbonyl group), threonine side chain hydroxyl, tyrosine side chain phenol group and serine side chain hydroxyl are protected by tBu (tert-butyl). The carboxyl of C-terminal amino acid of the peptide chain of the erythropoietin mimic peptide derivative monomer to be synthesized is connected with high-molecular insoluble resin (rink amino resin) by a covalent bond structure, then the amino acid bonded on the solid phase carrier is taken as an amino component, the amino protecting group is removed by 20 percent piperidine/DMF solution, and then the peptide chain is connected by reaction with excessive amino acid derivative. Repeating (condensation → washing → deprotection → washing → the next round of condensation) operation to reach the length of the peptide chain to be synthesized, finally cracking the peptide chain from the resin by using a mixed solution of trifluoroacetic acid, water, ethanedithiol and triisopropylsilane 92.5: 2.5, and settling by using ethyl ether to obtain a crude product of the monomeric peptide of the derivative of the erythropoietin mimic peptide, wherein the crude product of the monomeric peptide is separated and purified by using a C18 reversed-phase preparation column to obtain the monomeric peptide of the derivative of the erythropoietin mimic peptide. The intermediate control of the condensation and deprotection steps is the ninhydrin test, which shows blue color when free amino groups are present on the resin peptide chain, and no color when free amino groups are absent (the ninhydrin reagent itself is yellow). Therefore, after the condensation reaction is finished, if the color is yellow (the color of the ninhydrin reagent per se), the ninhydrin detection shows that the deprotection operation before the coupling of the next amino acid can be carried out after the coupling of the step is finished, and if the color is blue, the peptide chain is proved to have some free amino groups, and further repeated coupling or the existing condensing agent is changed until the resin peptide is yellow through the ninhydrin detection.

Example two: preparation of functional small molecule (LG-1)

The first step is as follows: preparation of LG-1-A

Diethyl iminodiacetate (10.0g, 52.8mmol), Boc-beta-alanine (10.0g, 52.8mmol) were dissolved in 100mL of dichloromethane, DIC (8.0mL, 52.8mmol) was added, the mixture was stirred at room temperature overnight, the reaction was filtered, and the filtrate was successively diluted with 100mL of saturated NaHCO₃50ml of a 0.5N HCl solution, and 100ml of a saturated brine were washed, and the organic layer was separated and washed with anhydrous MgSO₄And (5) drying. Filtering the organic layer, and concentrating to obtain colorless oily liquid LG-1-A: 17 g.

The second step is that: preparation of LG-1-B

17g of LG-1-A are dissolved in a mixture of 100mL of LEOH: THF (1: 1), and 25mL of water and 5g of NaOH (125mmol) are added. After stirring at room temperature for 2h, the pH was adjusted to 1 with 6N HCl solution. The reaction solution was extracted 4 times with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give a white semi-solid. The product was dissolved in 50mL of dichloromethane, 300mL of n-hexane was added, and the solution was a white slurry. Concentrating under reduced pressure to obtain white solid LG-1-B: 14g (yield about 90%).

The third step: preparation of LG-1-C

7gLG-1-B (23mmol) was dissolved in 80ml of tetrahydrofuran, and 4.6g of 4.6g N, N-methoxymethylamine hydrochloride (46mmol) and 5.1g of triethylamine (51mmol) were added with stirring, and 4.4g of DIC (32mmol) and 4.7g of HOBT (32mmol) were further added, and the reaction was stirred at room temperature overnight. The next day, pouring the reaction solution into water, extracting with 350ml ethyl acetate, sequentially washing the organic layer with 200ml of 2N HCl aqueous solution, 200ml of saturated sodium bicarbonate solution and 100ml of saturated saline solution, separating out the organic layer, drying the organic layer with anhydrous magnesium sulfate for 2 hours, filtering, concentrating the filtrate under reduced pressure to obtain oily substance, performing column chromatography, and collecting the target product LG-1-C: 4.2g, yield: 70 percent.

The fourth step: preparation of LG-1

Dissolving 4.0gLG-1-C (10.2mmol) in 60ml tetrahydrofuran, cooling to zero degree in ice salt bath, adding LiAlH4(340mg, 8.9mmol), reacting at zero degree for 30 min, sequentially adding 4ml water and 4ml 15% NaOH aqueous solution, filtering the reaction solution, washing the filter cake with tetrahydrofuran, concentrating to dryness, and performing silica gel column chromatography to obtain LG-I: 1.63g (6mmol, yield: 58.8%)

Example three: preparation of functional small molecule (LG-2)

4g of LG-1-B (13mmol) was dissolved in 100mL of N, N-dimethylformamide and hydroxysuccinimide (3.1g, 21mmol), DIC (4mL, 26mmol) and DMAP (4-dimethylaminopyridine) (12mg, 0.08mmol) were added. After stirring overnight, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 80ml of ethyl acetate, and insoluble matter was filtered off. The organic phase was washed once with 40ml of a saturated sodium bicarbonate solution, 40ml of a saturated saline solution, 40ml of a 0.5N HCl solution, and 40ml of a saturated saline solution in this order, and the organic layer was separated and dried over anhydrous magnesium sulfate. Filtering the organic layer, and concentrating the filtrate under reduced pressure to obtain white solid LG-2: 4.4g (yield about 68%).

Example four: preparation of functional small molecule (LG-3)

The first step is as follows: preparation of LG-3-A

7.0g pentanone pimelic acid (0.04mol) is dissolved in 100ml methanol, 5% CsCO3 methanol solution is added under stirring, the adding amount is controlled to enable the pH of the reaction solution to be about 8.5 (measured by a precision pH test paper), stirring is carried out for 30 minutes after the adding is finished, then the reaction solution is filtered, the filtrate is concentrated in vacuum to obtain oily matter, the oily matter is heated to 60 ℃ by using about 100ml DMSO, 14g (0.08mol) bromobenzyl is added, the reaction solution is filtered after 8 hours of reaction, the solid is washed by a small amount of diethyl ether, 400ml diethyl ether is added into mother liquor, the mother liquor is washed by using 200ml saturated saline three times, an organic layer is separated and is dried by anhydrous magnesium sulfate for 2 hours and then filtered, and when the filtrate is concentrated to 1/5 of the original volume under reduced pressure, the mother liquor is placed in a. The next day, the solid was filtered off and dried to give white solid LG-3-A: 10.5 g. (yield 74%)

The second step is that: preparation of LG-3-B

2g of LG-3-A (0.0056mol) was dissolved in 20ml of tetrahydrofuran, the internal temperature of the solution was kept at less than-10 ℃ and 626mgNaBH was added with stirring₄(0.0168mol), after 1h of reaction, 200ml of frozen diethyl ether is added, then 150ml of saturated aqueous sodium bicarbonate solution is added to stop the reaction, the mixture is kept still for layering, and the organic layer is washed once with saturated brine and then with anhydrous Na₂Drying SO4 for 2 hours, filtering, and concentrating the filtrate under reduced pressure to obtain LG-3-B: 1.9g (yield: 94.6%).

The third step: preparation of LG-3-C

3.2g of LG-3-B (0.009mol) were dissolved in 50ml of dichloromethane and 4.34g of triethylamine (0.043mol) were added with stirring below zero. 1.33g (0.0045mol) of triphosgene was dissolved in 25ml of methylene chloride and then added dropwise to the above solution, and after 1 hour, 2.8g of t-butoxycarbonylethylenediamine was added. After reacting for 3h, the reaction solution is adjusted to be neutral by glacial acetic acid, at this time, precipitate is generated, the precipitate is filtered, the filtrate is concentrated to be dry under reduced pressure, then ether is added for dissolving, 50ml of water is used for washing for three times, and 50ml of saturated saline solution is used for washing once. The organic layer was separated, dried over anhydrous magnesium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give an oil, which was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate: 10: 1). Combining and collecting target products, and concentrating to obtain white solid LG-3-C: 1.5g (yield 38.8%).

The fourth step: preparation of LG-3-D

13g of LG-3-C (0.031mol) are dissolved in 8ml of methanol, about 200mg of 10% palladium on carbon are added with stirring, and H is passed through at atmospheric pressure₂After reacting for 4h, filtering activated carbon, and concentrating the filtrate to obtain an oily substance LG-3-D: 8.28g (yield: 96.7%).

The fifth step: preparation of LG-3

5g of LG-3-D (0.018mol) was dissolved in 10ml of THF, 4.7g of p-nitrophenol (0.043mol) was added, and 4.2g (0.043) of DIC solution was added with stirring. The reaction was stirred overnight. The following day, the precipitate formed is filtered off, the filter cake is washed with a small amount of ethyl acetate, the filtrate is concentrated to dryness under reduced pressure, 100ml of ethyl acetate are added to the residue and dissolved, and then washed once with 50ml of saturated saline solution, the organic layer is separated and dried over anhydrous magnesium sulfate for 2 hours and then filtered, the filtrate is concentrated under reduced pressure to obtain an oil, the oil is purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate 20: 1 ═ 10: 1), the target product is collected in a combined manner, and the mixture is concentrated to dryness under reduced pressure to obtain a white solid LG-3: 3.5g (yield: 32%).

Example five: preparation of functional small molecule (LG-4)

The first step is as follows: preparation of LG-4-A

5g of LG-3-D (0.018mol) was dissolved in 60ml of THF, and 3.51g N, N-methoxymethylamine hydrochloride (0.036mol) and 4.0g of triethylamine (0.04mol) were added with stirring, and 3.4g of DIC (0.027mol) and 3.65g of HOBT (0.027mol) were further added, and the reaction was stirred at room temperature overnight. The next day, the reaction solution was washed with 200ml of water, and extracted twice with 200ml of ethyl acetate each time. The organic layers were combined and successively treated with 50ml of 2N HCl solution, 100ml of saturated NaHCO₃Washing the solution once with 100ml of saturated saline solution, separating an organic layer, drying the organic layer with anhydrous magnesium sulfate for 2 hours, filtering, and concentrating the filtrate under reduced pressure to obtain an oil, purifying the oil by column chromatography (eluent: n-hexane/ethyl acetate 10: 1), combining target components, and concentrating under reduced pressure to obtain a white solid LG-4-A: 6.24g (yield: 80%).

The second step is that: preparation of LG-4

4.0g of LG-4-A (9mmol) is dissolved in 50ml of tetrahydrofuran, 300mg of LiAlH4(7.9mmol) is added under cooling with an ice salt bath to zero, after a reaction is carried out for 30 minutes while maintaining the zero degree, 0.3ml of water, 0.9ml of a 15% NaOH solution and 0.3ml of water are added to the reaction solution in this order, a precipitate is produced, the precipitate is filtered off, the filter cake is washed once with 20ml of tetrahydrofuran, the filtrates are combined and concentrated to dryness under reduced pressure, and the residue is purified by column chromatography to give 1.65g of LG-4 (yield: 55.5%).

Example six: preparation of HH-EPO-005

The first step is as follows: SEQ ID NO: preparation of 5-Cyclic peptides

9g of erythropoietin mimetic peptide derivative monomer peptide SEQ ID NO: 5 (synthesized according to the method given in the example) was dissolved in 3000ml of 20% glacial acetic acid, and then 5% iodomethanol solution was slowly added dropwise until the yellow color did not disappear. The reaction solution is directly prepared and purified by reverse phase chromatography, and octadecylsilane chemically bonded silica is used as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying to obtain the product of SEQ ID NO: 5 Cyclic peptide 3.0g (yield: 15.6%)

The second step is that: preparation of HH-EPO-005

Converting SEQ ID NO: dissolving 3.0g (1.22mmol) of cyclic peptide 5 in 150ml of N, N-dimethylformamide, adding 147mg (1.46mmol) of triethylamine and 368mg (0.61mmol) of functional small molecule (LG-3), stirring at room temperature for 6 hours, concentrating a part of N, N-dimethylformamide under reduced pressure, adding 200ml of diethyl ether into the residue, standing in a refrigerator for 2 hours, centrifuging, drying under vacuum to obtain a white solid, dissolving the white solid in 50ml of 20% trifluoroacetic acid/dichloromethane solution, stirring at room temperature for 30 minutes, concentrating a part of solvent under reduced pressure to obtain a residue, and dissolving the residue in the solution of trifluoroacetic acid/dichloromethane, stirring at room temperature for 30 minutesAdding 200ml diethyl ether, standing in refrigerator for 2 hr, centrifuging, drying to obtain white solid, purifying by reverse phase chromatography using octadecylsilane chemically bonded silica as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-005: 1.0g (yield about 33%).

Example seven: preparation of HH-EPO-006

The first step is as follows: SEQ ID NO: preparation of 6-cyclic peptides

9g erythropoietin mimetic peptide derivative monomeric peptide of SEQ ID NO: 6 (synthesized according to the method given in the example) was dissolved in 3000ml of 20% glacial acetic acid, and then 5% iodomethanol solution was slowly added dropwise until the yellow color did not disappear. The reaction solution is directly prepared and purified by reverse phase chromatography, and octadecylsilane chemically bonded silica is used as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and then carrying out freeze drying to obtain the amino acid sequence shown in SEQ ID NO: 6 Cyclic peptide 3.0g (yield: 15.3%)

The second step is that: preparation of HH-EPO-006

Converting SEQ ID NO: 3.0g (1.23mmol) of 6-cyclic peptide is dissolved in 150ml of N, N-dimethylformamide, 147mg (1.46mmol) of triethylamine and 368mg (0.61mmol) of functional small molecule (LG-3) are added, the mixture is stirred at room temperature for 6 hours, partial DMF is concentrated under reduced pressure, 200ml of diethyl ether is added into the residue, the mixture is placed in a refrigerator for 2 hours, centrifuged and dried in vacuum to obtain white solidDissolving the white solid in 50ml of 20% trifluoroacetic acid/dichloromethane solution, stirring at room temperature for 30 min, concentrating under reduced pressure to obtain part of solvent, adding 200ml of diethyl ether into the residue, standing in a refrigerator for 2 hr, centrifuging, drying to obtain white solid, purifying by reverse phase chromatography, and using octadecylsilane bonded silica gel as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-006: 0.98g (yield about 32.7%).

Example eight: preparation of HH-EPO-007

The first step is as follows: SEQ ID NO: preparation of 7-cyclic peptides

9g of erythropoietin mimetic peptide derivative monomer peptide SEQ ID NO: 7 (synthesized according to the method given in example one) was dissolved in 3000ml of 20% glacial acetic acid, and then 5% iodomethanol solution was slowly added dropwise until the yellow color did not disappear. The reaction solution is directly prepared and purified by reverse phase chromatography, and octadecylsilane chemically bonded silica is used as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and then carrying out freeze drying to obtain the amino acid sequence shown in SEQ ID NO: 7 Cyclic peptide 3.15g (yield: 16.4%)

The second step is that: preparation of HH-EPO-007

Converting SEQ ID NO: 7 Cyclic peptide 3.0g (1.22mmol) was dissolved in 150ml N, N-dimethylformamide, and 147mg (1.46mmol) of triethylamine and 368mg (0.61 mm) of functional small molecule (LG-3) were addedol), stirring at room temperature for 6 hours, concentrating partial N, N-dimethylformamide under reduced pressure, adding 200ml of diethyl ether into the residue, standing in a refrigerator for 2 hours, centrifuging, drying under vacuum to obtain a white solid, dissolving the white solid in 50ml of 20% trifluoroacetic acid/dichloromethane solution, stirring at room temperature for 30 minutes, concentrating partial solvent under reduced pressure, adding 200ml of diethyl ether into the residue, standing in a refrigerator for 2 hours, centrifuging, drying to obtain a white solid, purifying the white solid by reverse phase chromatography, and using octadecylsilane bonded silica gel as a chromatographic column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; combining and collecting target components by taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-007: 1.0g (yield about 33%).

Example nine: preparation of HH-EPO-008

The first step is as follows: SEQ ID NO: preparation of 8-cyclic peptides

27g erythropoietin mimetic peptide derivative monomeric peptide of SEQ ID NO: 8 (synthesized according to the method given in example one) was dissolved in 3000ml of 20% glacial acetic acid, and then 5% iodomethanol solution was slowly added dropwise until the yellow color did not disappear. The reaction solution is directly prepared and purified by reverse phase chromatography, and octadecylsilane chemically bonded silica is used as column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and then carrying out freeze drying to obtain the amino acid sequence shown in SEQ ID NO: 8 Cyclic peptide 9.3g (yield: 15.7%)

The second step is that: preparation of HH-EPO-008

Converting SEQ ID NO: dissolving 8 cyclic peptide 3.0g (1.22mmol) in 150ml N, N-dimethylformamide, adding triethylamine 147mg (1.46mmol) and 368mg functional small molecule (LG-3) (0.61mmol), stirring at room temperature for 6 hr, concentrating under reduced pressure to obtain part of N, N-dimethylformamide, adding 200ml diethyl ether to the residue, standing in a refrigerator for 2 hr, centrifuging, vacuum drying to obtain white solid, dissolving the white solid in 50ml 20% trifluoroacetic acid/dichloromethane solution, stirring at room temperature for 30 min, concentrating under reduced pressure to obtain part of solvent, adding 200ml diethyl ether to the residue, standing in a refrigerator for 2 hr, centrifuging, drying to obtain white solid, purifying by reverse phase chromatography, and using octadecylsilane bonded silica gel as column filler (Waters SymmertyShield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-008: 1.12g (yield about 33%).

Example ten: preparation of HH-EP0-008A

Converting SEQ ID NO: 3.0g (1.22mmol) of 8-cyclic peptide is dissolved in 150ml of 20mmol acetic acid buffer solution (pH5.0), 201mg of functional small molecule (LG-4) (0.61mmol) and 10ml of acetonitrile are added, the reaction solution is stirred at room temperature for 30 minutes, and then the reaction solution is purified by reverse phase chromatography using octadecylsilane chemically bonded silica as a column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-008A: 0.75g (yield about 25%).

Example eleven: preparation of HH-EPO-008B

Converting SEQ ID NO: dissolving 8 cyclic peptide 3.0g (1.22mmol) in 150ml N, N-dimethylformamide, adding triethylamine 147mg (1.46mmol) and 322mg functional small molecule (LG-2) (0.61mmol), stirring at room temperature for 6 hr, concentrating under reduced pressure to obtain part of N, N-dimethylformamide, adding 200ml diethyl ether to the residue, standing in a refrigerator for 2 hr, centrifuging, vacuum drying to obtain white solid, dissolving the white solid in 50ml 20% trifluoroacetic acid/dichloromethane solution, stirring at room temperature for 30 min, concentrating under reduced pressure to obtain part of solvent, adding 200ml diethyl ether to the residue, standing in a refrigerator for 2 hr, centrifuging, drying to obtain white solid, purifying by reverse phase chromatography, and using octadecylsilane bonded silica gel as column filler (Waters SymmertyShield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases in different proportions, combining and collecting target components, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-008: 1.3g (yield about 43%).

Example twelve: preparation of HH-EPO-008C

Converting SEQ ID NO: 3.0g (1.22mmol) of 8-cyclic peptide is dissolved in 150ml of 20mmol acetic acid buffer solution (pH5.0), 165mg of functional small molecule (LG-1) (0.61mmol) and 10ml of acetonitrile are added, the reaction solution is stirred at room temperature for 30 minutes, and then the reaction solution is purified by reverse phase chromatography using octadecylsilane chemically bonded silica as a column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; with water (containing 0.05% trifluoroacetic acid) and acetonitrile (containing 0.05% trifluoroacetic acid)) The different proportions of (1) are mobile phases, the target components are combined and collected, most of acetonitrile is evaporated under reduced pressure, and then freeze-drying is carried out on the acetonitrile by adopting the method of HH-EPO-008C: 0.8g (yield about 27%).

Example thirteen: preparation of HH-EPO-018

0.5g of HH-EPO-008(0.98mmol) was dissolved in 100ml of N, N-dimethylformamide, and 39.6mg of triethylamine (0.196mmol), 3.8g of mPEG were added₂OSU (40K) (0.96mmol), stirred at room temperature for 6 hours. The reaction solution was directly eluted into 600ml of cold ether to precipitate a solid, which was left to stand in a refrigerator for 2 hours, centrifuged and dried to obtain a crude HH-EPO-018 product. Purifying HH-EPO-018 crude product by reverse phase chromatography using octadecylsilane chemically bonded silica as column packing material (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; combining and collecting target components by taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-018: 1.8g (yield about 47%).

Example fourteen: preparation of HH-EPO-018A

0.5g of HH-EPO-008(0.98mmol) was dissolved in 100ml of N, N-dimethylformamide, and 39.6mg of triethylamine (0.196mmol), 3.8g of mPEG were added₂OSU (40K) (0.96mmol), stirred at room temperature for 6 hours. The reaction solution was directly eluted into 600ml of cold ether to precipitate a solid, which was left to stand in a refrigerator for 2 hours, centrifuged and dried to obtain a crude HH-EPO-018 product. Purifying HH-EPO-018 crude product by reverse phase chromatography using octadecylsilane chemically bonded silica as column packing material (Waters symmetry Shield)^TM RP18 3.5μm，4.6 x 100mm), column temperature 60 ℃, detection wavelength 214 nm; combining and collecting target components by taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-018A: 1.5g (yield about 39%).

Example fifteen: preparation of HH-EPO-018B

0.5g of HH-EPO-008(0.98mmol) was dissolved in 100ml of N, N-dimethylformamide, and 39.6mg of triethylamine (0.196mmol), 3.8g of mPEG were added₂OSU (40K) (0.96mmol), stirred at room temperature for 6 hours. The reaction solution was directly eluted into 600ml of cold ether to precipitate a solid, which was left to stand in a refrigerator for 2 hours, centrifuged and dried to obtain a crude HH-EPO-018 product. Purifying HH-EPO-018 crude product by reverse phase chromatography using octadecylsilane chemically bonded silica as column packing material (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; combining and collecting target components by taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-018: 1.7g (yield about 45%).

Example sixteen: preparation of HH-EPO-018C

0.5gHH-EPO-008(0.98mmol) was dissolved in 100ml of N, N-dimethylformamide, and 39.6mg of triethylamine (0.196mmol), 3.8g of mPEG were added₂OSU (40K) (0.96mmol), stirred at room temperature for 6 hours. The reaction solution was directly eluted into 600ml of cold ether to precipitate a solid, which was left to stand in a refrigerator for 2 hours, centrifuged and dried to obtain a crude HH-EPO-018 product. Purification of the crude HH-EPO-018 fraction by reverse phase chromatographyThe product is prepared by using octadecylsilane bonded silica gel as chromatographic column filler (Waters symmetry Shield)^TMRP183.5 μm, 4.6 × 100mm), column temperature 60 ℃, detection wavelength 214 nm; combining and collecting target components by taking water (containing 0.05% of trifluoroacetic acid) and acetonitrile (containing 0.05% of trifluoroacetic acid) as mobile phases, evaporating most of acetonitrile under reduced pressure, and freeze-drying HH-EPO-018: 1.4g (yield about 37%).

Example seventeen: effect of erythropoietin mimetic peptide derivatives on mice

Purpose of the experiment:

the effect of erythropoietin mimetic peptide derivatives and erythropoietin protein on mouse erythropoiesis was evaluated and compared.

The material and the method are as follows:

the erythropoietin mimetic peptide derivatives HH-EPO-001, HH-EPO-002, HH-EPO-003, HH-EPO-004, HH-EPO-005, HH-EPO-006, HH-EPO-007, HH-EPO-008, HH-EPO-015, HH-EPO-016, HH-EPO-017 and HH-EPO-018 are provided by Jiangsu Haison pharmaceutical Co., Ltd; EPO: purchased from shenyang sansheng pharmaceutical llc; kunming mouse, purchased from Shanghai laboratory animal center of Chinese academy of sciences, with weight of 25-30 g, female parent and animal number of each group: 10 pieces of the Chinese herbal medicine.

The mice were injected subcutaneously with erythropoietin mimetic peptide derivatives and erythropoietin protein for three consecutive days, then the mice were sacrificed, whole blood was taken for peripheral blood cell and reticulocyte counting, and blood cell counting was performed with a full-automatic blood cell counter.

Results and discussion:

according to the current administration protocol, both the erythropoietin mimetic peptide derivatives and the erythropoietin protein significantly stimulate the increase in mouse peripheral blood reticulocyte count, indicating that they stimulate erythropoiesis (see Table I). The erythropoietin mimetic peptide derivatives and erythropoietin protein had no significant effect on mature red blood cells, hematocrit, hemoglobin content (see table two) and on peripheral blood leukocyte counts (see table three).

Epimedium, erythropoietin mimetic peptide derivatives influence on mouse reticulocyte production

EpineII, influence of erythropoietin mimic peptide derivative on mouse erythropoiesis, hematocrit and hemoglobin content

Epimeris, influence of erythropoietin mimic peptide derivatives on mouse thrombocyte and leucocyte generation

Example eighteen: effect of erythropoietin mimetic peptide derivatives on macaques

Purpose of the experiment:

the effect of erythropoietin mimetic peptide derivatives on macaque erythropoiesis was evaluated.

The material and the method are as follows:

erythropoietin mimetic peptide derivative HH-EPO-018, available from Jiangsu Haison pharmaceutical industries, Inc.;

erythropoietin: purchased from Shenyang Sansheng pharmaceuticals, Inc. Before use, the cells were diluted with 0.1% BSA in physiological saline.

The macaque is 5.5-8.5 kg in weight, is unlimited in male and female, and is purchased from the experimental animal center of the Zhongshan Zhongke Suzhou province. The macaques are grouped according to the basic hemoglobin, and each group comprises three macaques. HH-EPO-0181.35mg/kg, administered intravenously; EPO 240 mu/kg, three times per week, is continuously administrated for five weeks, and 1-2 hematology indexes are measured every week.

Results and discussion:

a single intravenous injection of HH-EPO-018 resulted in an increase in the peripheral blood hemoglobin content and an increase in the hematocrit of the macaques, indicating that HH-EPO-018 stimulated hemoglobin production, which peaked 35 days after administration and then slowly declined, with a stimulation of hemoglobin of approximately 33%. The positive control erythropoietin also increases the peripheral blood hemoglobin content of the macaque and increases the hematocrit, and the effect of the positive control erythropoietin is slowly weakened after stopping the medicine application. According to the current administration scheme, HH-EPO-018 and erythropoietin have equal stimulation effects on the generation of hemoglobin of macaques (see attached figures 1 and 2).

Example nineteenth: evaluation and comparison of the Effect of erythropoietin mimetic peptide derivatives HH-EPO-015, HH-EPO-018B and Positive control AF37702 on the production of mice

The material and the method are as follows: HH-EPO-015, HH-EPO-018, and HH-EPO-018B, AF37702 are available from Jiangsu Haison pharmaceutical Co. AF37702 is also an erythropoietin mimetic peptide derivative, and is a product of Affymax corporation (trade name: Hematide). Samples were prepared with 0.1% BSA in physiological salt prior to use. Kunming mouse, purchased from Shanghai laboratory animal center of Chinese academy of sciences, with a weight of 25 + -2 g and female parent, and the number of animals in each group: 10 pieces of the Chinese herbal medicine. After the animals were acclimated, HH-EPO-015, HH-EPO-018, and HH-EPO-018B, AF37702 were injected subcutaneously, the mice were sacrificed on day 6 after the 1 st administration, and whole blood was taken for peripheral blood cell and reticulocyte counts. Blood cell counts were performed using an ADVIA full-automatic hemocytometer.

Results and discussion: HH-EPO-015, HH-EPO-018 and HH-EPO-018B, AF37702 obviously increase the percentage and the count of peripheral blood reticulocytes of the mice through single subcutaneous injection; wherein HH-EPO-018B has relatively strong effect; HH-EPO-018, AF37702 have the next action; HH-EPO-015 is least effective; HH-EPO-018 and AF37702 were found to be essentially equivalent (see Table four). HH-EPO-015, HH-EPO-018 and HH-EPO-018B, AF37702 increased the mouse peripheral blood hematocrit and hemoglobin content, which were essentially equivalent, but had no significant effect on the mouse peripheral hemoglobin count (see Table five).

Table four: effect of HH-EPO-015, HH-EPO-018B, AF37702 on mouse peripheral blood reticulocyte production

**P＜0.01 vs control

Table five: effects of HH-EPO-015, HH-EPO-018B, AF37702 on peripheral red blood cell production, hematocrit, hemoglobin content in mice

*P＜0.05，**P＜0.01 vs control

Claims (25)

1. An erythropoietin mimetic peptide derivative with general formula (I) and its medicinal salt,

R₁-R₂-(CH₂)_n1-R₃-(CH₂)_n2-R₄-R₅

(I)

wherein R is₁、R₅Each independently selected from the group consisting of formula Y₁X₁X₂X₃GX₄X₅TWX₆X₇Y₂Y₃Erythropoiesis ofA peptidomimetic monomeric peptide, wherein each amino acid is represented by a standard single letter; x₂、X₃、X₄、X₅、X₆、Y₃Each independently selected from any one of 20 genetically encoded L-amino acids or unnatural amino acids; y is₁、Y₂Each independently selected from any one of 20 genetically encoded L-amino acids or unnatural amino acids or peptide fragments consisting of the amino acids; x₁、X₇Selected from C, K, D, E, Orn or Hoc; n is₁、n₂Each independently selected from integers of 0 to 10; r₂、R₄Each independently selected from-CO or-CH₂；R₃Selected from NCO (CH)₂)_n4NHR₆、CHOCONH(CH₂)_n5NHR₆Or CHSCON (CH)₂)_n5NHR₆Wherein n is₄An integer selected from 2 to 10, n₅An integer selected from 2 to 10, R₆Selected from H or methoxy polyethylene glycol derivatives.

2. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁、R₅Is identical or non-identical.

3. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁、R₅Is acetylated.

4. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁、R₅Is a cyclic peptide forming disulfide bond.

5. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein Y is₃Selected from K, H or R.

6. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein Y is₃Is K.

7. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁、R₅The length of the amino acid sequence of (a) is 13 to 40 amino acids.

8. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 7, wherein R is₁、R₅The length of the amino acid sequence of (a) is 22 amino acids.

9. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, wherein R is₁、R₅Selected from the group consisting of SEQ ID NOs having the sequence: 1-NO: 30 a cyclic peptide of structure:

10. the erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, wherein R is₁、R₅Each independently selected from the group consisting of SEQ ID NOs: 1-NO: 8 in the form of a cyclic peptide.

11. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, wherein the erythropoietin mimetic peptide derivatives are selected from the group consisting of peptides wherein

n₁，n₂Is 2, R₂、R₄is-CO, R₃Is CHOCONH (CH)₂)_n5NHR₅，n₅Is 2;

n₁，n₂is 1, R₂、R₄is-CO, R₃Is NCO (CH)₂)_n4NHR₆，n₄Is 2;

n₁，n₂is 2, R₂、R₄is-CH₂，R₃Is CHOCONH (CH)₂)_n5NHR₆，n₅Is 2; or

n₁，n₂Is 1, R₂、R₄is-CH₂，R₃Is NCO (CH)₂)_n4NHR₆，n₄Is 2.

12. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 11, wherein R is₆Is H.

13. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 11, wherein R is₆Is a methoxypolyethylene glycol derivative.

14. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 13, wherein the methoxypolyethylene glycol derivative has a molecular weight of 5,000 to 100,000 daltons.

15. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 13, wherein the methoxypolyethylene glycol derivatives have a branched or linear structure.

16. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to claim 15, wherein the methoxypolyethylene glycol derivative has a linear structure and a molecular weight of 20,000 daltons, or the methoxypolyethylene glycol derivative has a branched structure and a molecular weight of 40,000 daltons.

17. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, wherein the erythropoietin mimetic peptide derivatives are selected from the group consisting of peptides wherein

n₁，n₂Is 2, R₁、R₅Selected from the sequences SEQ ID NO: 5 to SEQ ID NO: 8, R₂、R₄Is selected from-CO or-CH₂，R₃Is CHOCONH (CH)₂)_n5NHR₆Wherein n is₅An integer selected from 2 to 10; r₆Is a methoxy polyethylene glycol derivative with a linear structure and a molecular weight of 20,000 daltons;

n₁，n₂is 1, R₁、R₅Selected from the sequences SEQ ID NO: 5 to SEQ ID NO: 8, R₂、R₄Is selected from-CO or-CH₂，R₃Is NCO (CH)₂)_n4NHR₆Wherein n is₄An integer selected from 2 to 10; r₆Is a methoxy polyethylene glycol derivative with a linear structure and a molecular weight of 20,000 daltons;

n₁，n₂is 2, R₁、R₅Selected from the sequences SEQ ID NO: 5 to SEQ ID NO: 8, R₂、R₄Is selected from-CO or-CH₂，R₃Is CHOCONH (CH)₂)_n5NHR₆Wherein n is₅An integer selected from 2 to 10; r₆Is methoxy polyethylene glycol derivative with branched structure and molecular weight of 40,000 daltons; or

n₁，n₂Is 1, R₁、R₅Selected from the sequences SEQ ID NO: 5 to SEQ ID NO: 8, R₂、R₄Is selected from-CO or-CH₂，R₃Is NCO (CH)₂)_n4NHR₆Wherein n is₄An integer selected from 2 to 10; r₆Is methoxy polyethylene glycol derivative with branched structure and molecular weight of 40,000 Dalton.

18. The erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, wherein the erythropoietin mimetic peptide derivatives and pharmaceutically acceptable salts thereof are

19. A process for the preparation of an erythropoietin mimetic peptide derivative according to any one of claims 1 to 18 and pharmaceutically acceptable salts thereof, comprising the steps of:

(1) preparation R₁H、R₅H, wherein R₁、R₅Selected from the group consisting of monomeric peptides of an erythropoietin mimetic peptide as defined in any one of claims 1 to 18,

(2) preparing functional small molecules with the general formula (II)

R₇-CO-(CH₂)_n1-Z₂-(CH₂)_n2-CO-R₈

(II)

Wherein n is₁、n₂Each independently selected from integers of 0 to 10;

R₇、R₈selected from OH or H;

Z₂selected from O, S, CH₂、N(CH₂)_n6NHR₉、NCO(CH₂)_n7NHR₉、CHOCONH(CH₂)_n8NHR₉、CHSCON(CH₂)_n8NHR₉Or CHNHCON (CH)₂)_n8NHR₉Wherein n is₆Is an integer selected from 1 to 10, n₇Is an integer selected from 2 to 10, n₈Is an integer selected from 2 to 10, R₉Is selected from the group consisting of Boc or Cbz,

(3) r is to be₁、R₅Carrying out amidation reaction or reductive amination reaction with functional micromolecules with a general formula (II) to prepare a compound with a general formula (III),

R₁-R₂-(CH₂)_n1-Z₂-(CH₂)_n2-R₄-R₅

(III)，

wherein R is₂、R₄Each independently selected from-CO or-CH₂，

(4) Removing Boc or Cbz, and carrying out amidation reaction with active methoxy polyethylene glycol derivative.

20. The process according to claim 19, wherein in the general formula (II),

n₁、n₂is 1 or 2, R₇、R₈Selected from OH, Z₂Selected from NCO (CH)₂)_n7NHR₉Or CHOCONH (CH)₂)_n8NHR₉，n₇An integer selected from 2 to 10, n₈An integer selected from 2 to 10, R₉Is Boc; or

n₁、n₂Is 1 or 2, R₇、R₈Selected from H, Z₂Selected from NCO (CH)₂)_n7NHR₉Or CHOCONH (CH)₂)_n8NHR₉，n₇An integer selected from 2 to 10, n₈An integer selected from 2 to 10, R₉Is Boc.

21. A pharmaceutical composition comprising:

(1) a therapeutic amount of an erythropoietin mimetic peptide derivative according to any one of claims 1 to 18 and pharmaceutically acceptable salts thereof, and

(2) a pharmaceutically acceptable pharmaceutical carrier.

22. Use of an erythropoietin mimetic peptide derivative according to any one of claims 1 to 18 and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of a disease characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population.

23. Use of a pharmaceutical composition according to claim 21 in the manufacture of a medicament for the treatment of a disorder characterized by a deficiency in erythropoietin or a deficiency or defect in the red blood cell population.

24. The use according to claim 23, characterized in that the disease characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population is end-stage renal failure or dialysis; AIDS-related anemia, autoimmune diseases, or malignancies; cystic fibrosis; early stage prematurity anemia; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss.

25. The use according to claim 23, characterized in that the diseases characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population are ageing and neoplastic diseases with abnormal red blood cell production.