WO2014194835A1

WO2014194835A1 - Erythropoietin mimic peptide chemical dimmer and application thereof

Info

Publication number: WO2014194835A1
Application number: PCT/CN2014/079221
Authority: WO
Inventors: 刘克良; 梁远军; 许笑宇; 颜玲娣; 董华进; 宫泽辉; 冯思良; 郄建坤; 贾启燕
Original assignee: 中国人民解放军军事医学科学院毒物药物研究所
Priority date: 2013-06-07
Filing date: 2014-06-05
Publication date: 2014-12-11
Also published as: CN104231067A; CN104231067B

Abstract

Disclosed are erythropoietin mimic peptide chemical dimmer and an application thereof, and especially, disclosed are erythropoietin mimic peptide chemical dimmer shown in formula (I) or medicinal salt thereof, and a composition containing the erythropoietin mimic peptide chemical dimmer or the medicinal salt thereof. Also disclosed is an application of the erythropoietin mimic peptide chemical dimmer or the medicinal salt thereof. The erythropoietin mimic peptide chemical dimmer or the medicinal salt thereof of the present invention has desirable erythropoiesis activity.

Description

Erythropoietin mimetic peptide chemical dimer and use thereof

The present invention relates to an erythropoietin mimetic peptide chemical dimer, a process for preparing the same, a pharmaceutical composition containing the same, and the erythropoietin mimetic peptide chemical dimer in prevention or

Background technique

Erythropoietin (EPO) is a hematopoietic growth factor (HGF) that is required for erythropoiesis. It contains 165 amino acids and has a molecular weight of about 30.4 kD. 40% of the molecular weight is the result of glycosylation modification, and the side chain hydroxyl group is 0-glycosylated. In humans, EPO is mainly produced in the kidneys of the kidneys and fetuses and is synthesized and released by the perivascular capillary endothelial cells of the renal tubules. EPO acts as a cytokine in bone marrow hematopoietic tissue to promote erythropoiesis, thereby increasing the concentration of hemoglobin to ensure adequate oxygen transport from the lungs to aerobic tissue. Under normal circumstances, the production of EPO is inversely related to the oxygen concentration in the tissue. When tissue is hypoxic, the production of EPO is increased to ensure that all tissues and organs receive sufficient oxygen supply. When the plasma EPO concentration is in the range of 10-25 mU/mL, the normal level of hemoglobin (12-17 g/dL) can be maintained with a half-life of about 5 h. In addition to promoting red blood cell production in hematopoietic tissue, EPO also plays an important role in non-hematopoietic tissues and organs, such as promoting the growth and development of some tissues and organs, participating in cardiovascular formation, repair and repair of myocardial membrane damage, and EPO is also involved in brain-pairing. The response process of neuronal damage and the process of wound healing.

In the process of erythropoiesis, hematopoietic progenitor cells are first produced from bone marrow, and granulocytes (Gerulocyte, G)-Erythroid (E)-monocyte (Monocyte, M)-megakaryocytes are formed by various cytokines. The colony forming units of ( Megakaryocyte , M ) are further differentiated into erythroid colony forming units (CFUe) by Erythroid burst-forming units (BFUe). EPO The EPO receptor (EPOR), which binds to the surface of CFUe, forms young red blood cells, which further differentiate into reticulocytes and finally form red blood cells. This process requires not only nutrients such as iron, folic acid, and vitamin B12, but also many cytokines, and EPO is the most important regulator of erythropoiesis. Therefore, lack of EPO is likely to cause anemia. A variety of secondary anemias are caused by insufficient production of cytokine EPO. Studies have shown that EPO can be used to treat anemia caused by multiple causes (Dai Xiaoying, Chen Xiaonong, Research progress in the treatment of anemia caused by recombinant human erythropoietin, Zhejiang Clinical Medicine, Vol. 6, No. 9, September 827-828).

In the 1980s, research on the treatment of secondary anemia was gradually paid more attention. Through the study of its pathophysiology, some Erythropoietic-stimulating Agents (ESAs) drugs, mainly EPO receptors, were developed. Agonist. Since then, EPO drugs have been a hot spot in drug research, sales of EPO products continue to be the fourth highest-selling drug, and the EPO market continues to grow. ESAs drugs have dominated the market for treatment of secondary anemia.

In 1996, phage display technology was used to scan EPO receptors with a combinatorial library consisting of random peptide sequences, and many short peptides with no sequence correlation with endogenous EPO were obtained. These short peptides can bind to EPO receptors and trigger A series of signal transductions promote the proliferation of cell lines and have the same mechanism of action as EPO. However, the half effective concentration (EC ₅₀ ) of these short peptides is at least 200 nM, which is much higher than the EC ₅ of EPO measured at the same time. (20pM), in addition, their in vivo stability is also poor, and therefore, the drug-making conditions cannot be satisfied. However, the discovery of these peptide sequences has served as a guide for subsequent research.

In Chinese Patent 201210170307.7, wherein the formula (la) has a good effect of promoting the proliferation of red blood cells, hemoglobin and reticulocytes.

Ac-Gly-Gly-Leu-Tyr-Ala-Cys-His-Met-Gly-Pro-lle-Thr-1 -Nal-Val-Cys-Gln-Pro- Leu-Cit-Sar-Lys-NH ₂ La )

Since the EPO receptor is activated by the dimerization of the EPO receptor by the ligand i, in 1999, Oded Livnah confirmed from the crystal structure that the dimerization conformation of the ligand (the erythropoietin peptide herein) is also The key to stimulating the biological effects of EPO receptors (Science 1999, 283: 987-990). Therefore, in order to further increase the activity of the sequence (la), the present invention obtains a chemical dimer based on the sequence (la) by chemical means on the δ3⁄4 of the Chinese patent 201210170307.7, thereby providing a red blood cell with higher biological activity. The peptone mimetic peptide and its pharmaceutically acceptable salt. Summary of the invention

The present inventors have surprisingly found that the erythropoietin peptidomimetic peptide obtained by chemical dimerization of the single-chain erythropoiesis mimetic peptide has a more active EPO receptor agonistic activity. The present invention has been completed based on the above findings. Summary of invention

The first aspect of the present invention provides an erythropoietin mimetic peptide chemical dimer having the following formula (I), or a pharmaceutically acceptable salt thereof

Formula (I)

, wherein each symbol is as described herein.

A second aspect of the invention provides a process for the preparation of the erythropoietin mimetic peptide dimer of the first aspect of the invention, or a pharmaceutically acceptable salt thereof.

A third aspect of the invention provides the use of the erythropoietin mimetic peptide dimer of any one of the first aspects of the invention, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament.

A fourth aspect of the invention provides a pharmaceutical composition comprising the erythropoietin mimetic peptide dimer of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

A fifth aspect of the invention provides a method of activating an EPO receptor in a subject in need thereof and a method of treating a disease or condition associated with low activity of EPO and its receptor. A sixth aspect of the present invention provides the erythropoietin mimetic peptide dimer of the first aspect of the present invention, which is a preventive and/or therapeutic agent for an EPO receptor agonist or a disease or symptom associated with low activity of EPO and its receptor, Or a pharmaceutically acceptable salt thereof.

A seventh aspect of the invention provides a composition for activating an EPO receptor in a subject in need thereof, or for preventing and/or treating a combination of diseases and/or symptoms associated with low activity of EPO and its receptor And the erythropoietin mimetic peptide dimer of the first aspect of the invention, or a pharmaceutically acceptable salt thereof. Detailed description of the invention

The first aspect of the present invention provides an erythropoietin mimetic peptide represented by the following formula (I), or a pharmaceutically acceptable salt thereof,

Formula (I)

among them,

P is a erythropoiesis-producing peptide:

Ac-Gly-Gly-Leu-Tyr-Ala-Cys-His-Met-Gly-Pro-lle-Thr-1 -Nal-Val-Cys-Gln-Pro- Leu-Cit-Sar-Lys-NH ₂

Wherein the C-terminal Lys side chain amino group forms an amide bond with the tether;

n, m is any integer between 1 and 5 (for example, 1, 2, 3, 4, 5);

R is H, or R is a natural amino acid of β-alanine, γ-aminobutyric acid, or L-form or D-form or a derivative thereof, and its carboxyl group forms an amide structure with hydrazine.

An erythropoietin peptidomimetic, or a pharmaceutically acceptable salt thereof, according to any one of the first aspects of the invention, which is selected from the group consisting of: SEQ-01:

SEQ - 02

In the compounds of the above formulas, the meanings of the various amino acids or unnatural amino acids are, if not specified, the L-forms, the amino acid symbols are expressed according to the general provisions, and the non-natural acid symbols are carried out in the specification. The explanation.

In the present invention, the connecting arm means

, where R, n, m are defined by the same formula (I).

In the present invention, the natural amino acid includes alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Th), cysteine (Cys), tyrosine (Tyr), aspartame Amide (Asn), glutamine (Gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu). According to the method of any of the second aspects of the present invention, the erythropoietin peptidomimetic chemical dimer is prepared according to the following steps: (1) using Rink-amide resin as a solid phase carrier, and HBTU-HOBt As a condensing agent, according to the amino acid sequence of the erythropoietin mimetic peptide to be obtained, the corresponding amino acid protected by Fmoc is used as a raw material, and the peptide resin is synthesized according to a standard Fmoc solid phase polypeptide synthesis method; (2) then trifluoroacetic acid: benzene Methyl sulfide: m-cresol: ethanedithiol: a mixture of water (for example, the volume ratio of these five is 8.25:0.5:0.5:0.25:0.5) as a lysate, at 0-40. The peptide is deprotected by C and cleaved from the resin (for example, at about 0 C for about 30 minutes, then at room temperature for 90 minutes); (3) Dissociation of cysteine in the resulting linear peptide molecule The thiol is oxidized to form a cyclic peptide; (4) an optional purification step to obtain a single-stranded erythropoiesis mimetic peptide la; (5) using a suitable tether, reacting with two peptide chains to form a dimer, optionally obtaining a purification step Dimer.

The third aspect of the invention provides the use of the erythropoietin mimetic peptide chemical dimer of any one of the first aspects of the invention, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament as an EPO receptor agonist.

The present invention also provides the erythropoietin mimetic peptide chemical dimer of any one of the first aspects of the present invention, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment for lack of erythropoietin or red blood cell deficiency or Use in a drug characterized by a defect, or a drug associated with a disease and/or symptom associated with an EPO or EPO receptor activity.

In the present invention, the disease characterized by lack of erythropoietin or red blood cell deficiency or deficiency, or diseases and/or symptoms associated with low activity of EPO or EPO receptors means anemia caused by various causes.

In an embodiment of the invention, the various causes of anemia include, but are not limited to, red blood cell defects, low red blood cell count, low hemoglobin content, myelodysplastic syndrome, human immunodeficiency virus infection, autologous blood collection, bone marrow transplantation, Anemia caused by hemoglobinopathy, renal anemia, tumor or cancer-related anemia, anemia in premature infants, anemia after surgery, maternal anemia, aplastic anemia, or other anemia.

In the present invention, the anemia caused by the hemoglobinopathy is, for example, thalassemia, sickle cell anemia.

In the present invention, the renal anemia mainly refers to anemia caused by chronic renal failure. In the present invention, the tumor or cancer-related anemia can be caused by various factors, and mainly includes tumor factors (such as blood loss, hemolysis, bone marrow involvement) or factors for tumor treatment (such as bone marrow of chemotherapy). Inhibition, tumor radiation therapy, etc.) two aspects.

In the present invention, the other anemia is, for example, anemia caused by chronic inflammation or infection. A fourth aspect of the invention provides a pharmaceutical composition comprising the erythropoietin peptidomimetic chemical dimer of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

A fifth aspect of the invention provides a method of activating an EPO receptor in a subject in need thereof, and a method of treating a disease or condition associated with low activity of EPO and its receptor, the method comprising preventing or treating a subject in need thereof A therapeutically effective amount of the erythropoietin mimetic peptide chemical dimer, or a pharmaceutically acceptable salt thereof, of the first aspect of the invention.

A sixth aspect of the present invention provides the erythropoietin mimetic peptide chemical dimer of the first aspect of the present invention, which is a preventive and/or therapeutic agent for an EPO receptor agonist or a disease or symptom associated with EPO and its receptor activity Or a pharmaceutically acceptable salt thereof.

A seventh aspect of the invention provides a composition for activating an EPO receptor in a subject in need thereof, or for preventing and/or treating a combination of diseases and/or symptoms associated with low activity of EPO and its receptor And the erythropoietin mimetic peptide chemical dimer of the first aspect of the invention, or a pharmaceutically acceptable salt thereof. In one embodiment, the disease and/or condition associated with low activity of the EPO or EPO receptor is renal anemia.

Any aspect of the invention or any one of the aspects of the invention is equally applicable to any of the other aspects or any of the other aspects, as long as they do not contradict each other, of course, when applied to each other If necessary, the corresponding features can be appropriately modified. In the present invention, for example, when referring to "any of the first aspects of the present invention, the "any" refers to any of the sub-aspects of the first aspect of the invention; when otherwise mentioned in a similar manner, It also has the same meaning.

Various aspects and features of the present invention are further described below.

All documents cited in the present invention are hereby incorporated by reference in their entirety, and if the meanings expressed by these documents are inconsistent with the present invention, the expression of the present invention shall prevail. Moreover, the various terms and phrases used herein have the generic meaning well-known to those skilled in the art. It is to be understood that the present invention is intended to be more fully described and explained herein, and the terms and phrases referred to herein are intended to be inconsistent with the meaning of the invention.

The term "about" as used herein, for example, when preparing the yield of a product, generally refers to a range of tolerances permitted in the art, such as ± 10%, such as ± 5%, such as ± 2%.

As used herein, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.

As used herein, the term "pharmaceutical composition" refers to a substance that can be used to effect treatment, prevention, alleviation and/or alleviation of the diseases, conditions, symptoms of the invention in a subject.

As used herein, the term "subject," or "patient," may refer to an animal that receives the compositions and extracts of the invention to treat, prevent, ameliorate, and/or alleviate the diseases, disorders, symptoms of the invention. , especially mammals, such as humans, dogs, monkeys, cows, horses, etc.

As used herein, the term "disease or condition" refers to a physical condition of the subject that is associated with the disease or condition described herein.

As used herein, "%", unless otherwise specified, is generally a percentage by weight/weight for the total material solids and generally a weight/volume percentage for the total material liquid. Of course, for a total material that is liquid and the solute is a liquid, the percentage that characterizes the liquid solute is generally a percentage of volume/volume.

As described herein, the term "erythropoietin mimetic peptide" refers to a sequence of

Ac-Gly-Gly-Leu-Tyr-Ala-Cys-His-Met-Gly-Pro-lle-Thr-1 -Nal-Val-Cys-Gln-Pro- Leu-Cit-Sar-Lys-NH ₂ Ten - "cyclic peptide. The term "erythropoietin mimetic peptide chemical dimer" refers to a dimer formed by chemically linked "erythropoietin mimetic peptide".

Unless otherwise specified, the acid of the present invention is an L-type acid, and the D-type acid is specifically indicated.

As described herein, the term "a disease characterized by a deficiency or deficiency in erythropoietin or red blood cells, or a disease and/or symptom associated with an EPO or EPO receptor" means achieving prevention and prevention by agonizing EPO receptors. / or related diseases for treatment purposes, mainly refers to various causes Anemia caused by diseases such as red blood cell defects, low red blood cell count, low hemoglobin content, tumor release, and chemotherapy leading to anemia. Red blood cell defects, low red blood cell count, low hemoglobin content, myelodysplastic syndrome, human immunodeficiency virus infection, autologous blood collection, bone marrow transplantation, anemia caused by hemoglobinopathy, renal anemia, tumor or cancer-related anemia, anemia in premature infants , postoperative anemia, maternal anemia, aplastic anemia, or other anemia. It is an object of the present invention to provide a class of erythropoietin mimetic peptide chemical dimers which are useful in the development of therapeutic drugs and preparations for renal anemia. The inventor's patent in China

Based on 201210170307.7, a chemical (di)-based chemical dimer is obtained by chemical means, which provides a more biologically active erythropoietin mimetic peptide and its usable salt and a preparation method thereof. The present inventors have found that by the formation of a chemical dimer, a higher EPO receptor agonistic activity is obtained, which provides a possibility of obtaining good pharmaceutical activity.

One of the objects of the present invention is to provide an erythropoietin mimetic peptide chemical dimer having a structure in which a dimer is formed by a chemical tether in addition to a erythropoiesis-producing peptidomimetic. The erythropoietin mimetic peptide chemical dimer of the present invention is described in detail in the structure of formula (I) and its definitions described herein.

The invention further relates to a pharmaceutical composition comprising at least one of the above erythropoietin peptidomimetic chemical dimers, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient.

The invention further relates to the use of a compound of the invention in the manufacture of a medicament for the treatment and prevention of anemia. The anemia is, for example, anemia caused by renal anemia, tumor release, and chemotherapy.

The invention further relates to the use of a compound of the invention for the treatment and prevention of anemia and associated red blood cell defects or symptoms.

In one aspect of the invention, there is provided a erythropoietin mimetic peptide chemical dimer having the structure of formula (I):

Formula (I)

Or a pharmaceutically acceptable salt thereof, wherein

P is a erythropoiesis-producing peptide:

n, m is any integer between 1 and 5;

R is H, or R is a natural amino acid of β-alanine, γ-aminobutyric acid or L-form or D-form or a derivative thereof, and its carboxyl group forms an amide structure with hydrazine.

An erythropoietin mimetic peptide chemical dimer according to any one of the invention, which is selected from the group consisting of:

SEQ - 01:

Ac-Gly-Gly-Leu-Tyr-Ala-c s-His-Met-Gly-Pro-lle-Thr-l-Nal-Val-C^s-Gln-Pro-Leu-Cit-Sar-N^- --""

SEQ - 02

Unless otherwise specified, the amino acids are all L-type amino acids. The preparation of the compound of the present invention adopts a conventional polypeptide synthesis method, including a solid phase polypeptide synthesis method, a liquid phase polypeptide synthesis method, and a solid phase-liquid phase polypeptide synthesis method, and the amino acid adopts a Fmoc-/tBu- or Boc-/Bzl-protection strategy. The connection method adopts a method of sequentially connecting from the N-terminus to the C-terminus, or synthesizing the fragment first, and then connecting the fragments, and solid-phase synthesis uses various resins capable of forming an amide end as a carrier (such as MBHA, PAL, Rink amide resin, etc.) The condensation reaction (such as DCC/HOBT, BOP/DIE A, HBTU/HOBt, TBTU, etc.) is carried out by various common condensing agents, and after the reaction, the peptide is cleaved from the resin with trifluoroacetic acid or without HF. The crude peptide forms a cyclic peptide by oxidizing two thiol groups in the molecule, and is isolated and purified to obtain a single-chain peptide, which is then chemically reacted with a suitable tether to form a dimer product, and the final product is determined by MALDI-TOF-MS.

The compounds of the invention have an agonistic effect on EPO receptors at the cellular level.

The compounds of the present invention have an effect of increasing the number of red blood cells and the hemoglobin content in normal mice.

The invention further relates to a pharmaceutical composition comprising as an active ingredient an effective amount of at least one erythropoietin mimetic peptide chemical dimer or a non-physiologically toxic salt thereof, together with a conventional pharmaceutical excipient or adjuvant. Here, "conventional pharmaceutical excipients or adjuvants" include any or all solvents, dispersion shields, coatings, antibacterial or antifungal agents, isotonic and sustained release agents, and similar physiologically compatible preparations, suitable for intravenous Injection, intramuscular, subcutaneous, or other non-digestive administration is preferred. Depending on the mode of administration, the active compound may be coated to protect the compound from the action of acid or other natural conditions.

The term "pharmaceutically acceptable salt", "pharmaceutically acceptable salt" or "physiologically acceptable salt" as used in the present invention means that the expected physiological activity of the parent compound can be retained without any expectation. Salts with side effects, or compositions containing them, for example: hydrochloride, hydrobromide, sulfate, phosphate, nitrate, and acetate, oxalate, tartrate, succinate, apple Acid salts, benzoates, pamoate, alginate, sulfonate, naphthalene sulfonate, and the like.

The erythropoietin peptidomimetic chemical dimer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same may be administered in any manner known in the present invention, such as oral, intramuscular, subcutaneous, nasal administration, etc., administration agent Types such as tablets, capsules, buccal tablets, chewable tablets, tinctures, suspensions, transdermal agents, microencapsulated agents, implants, syrups and the like. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems. In order to formulate a unit dosage form into tablets, various biodegradable or biocompatible carriers well known in the art can be used. Examples of the carrier are, for example, a saline base and various buffered aqueous solutions, ethanol or other polyols, liposomes, polylactic acid, vinyl acetate, polyhepatic, polyglycolic acid, collagen, polyorthoesters and the like.

The dose of the erythropoietin mimetic peptide chemical dimer or a pharmaceutically acceptable salt thereof in the present invention depends on many factors such as the sexual shield and severity of the disease to be prevented or treated, the sex, age, and weight of the patient or animal. , sensitivity and individual response, the particular compound used, the route of administration, the number of doses, and the desired therapeutic effect. The above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.

Some of the abbreviations used in the present invention have the following meanings:

Ala represents alanine

Cys representation

Gin means glutamine

Gly means glycine

His indicates histidine

He represents isoleucine

Leu means leucine

Lys stands for lysine

Met represents methionine

Pro stands for proline

Sar stands for N-mercapto glycine

Thr represents threonine Tyr represents tyrosine

Val for proline

Cit means citrulline

1-Nal represents 3-(1-naphthyl)-alanine

Fmoc means 芴methoxy

DMF represents dimercaptoacetamide

DCC represents dicyclohexylcarbodiimide

HOBt stands for 1-hydroxybenzotriazole

DMAP stands for 4-N,N-Dimethylaminopyridine

TFA stands for trifluoroacetic acid

EDT stands for mercaptoethanol

HF stands for hydrogen fluoride

HBTU stands for 2-(1Η-1-hydroxybenzotriazole)-1,1,3,3-tetradecylurea

RP-HPLC RP-HPLC

Other unlabeled abbreviations have the meanings well known in the art.

Where Sar, 1-

detailed description

The invention is further described by the following examples, however, the scope of the invention is not limited to the following examples. A person skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope of the invention. The present invention provides a general and/or specific description of the materials used in the test face and the test methods. While many of the materials and methods of operation used to accomplish the objectives of the present invention are well known in the art, the present invention is still described in detail herein. The solid phase synthesis carrier Rink-amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; HOBT, HBTU, DIEA and Fmoc-protected amino acids are provided by Shanghai Jill Biochemical Co., Ltd.

SEQ-01

a) Synthesis of single-strand erythropoiesis mimetic peptide Qa)

Ac-Gly-Gly-Leu-Tyr-Ala-Cys-His-Met-Gly-Pro-lle-Thr-1-Nal-Val-Cys-Gln-Pro- Leu-Cit-Sar-Lys-NH ₂ la

Using l.Og Rink-amide resin (0.25 mmol) as a solid phase carrier, Fmoc-Ala-OH, Fmoc-Cit-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-l-Nal-OH, Fmoc- Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Sar-OH, Fmoc-Val-OH as raw materials, HBTU-HOBt as condensing agent, according to 1&^^^ column, according to standard The Fmoc solid phase peptide synthesis method synthesizes a peptide resin. 20ml trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5: 0.5: 0.25: 0.5, volume ratio) as a lysate, 0 ° C reaction for 30 minutes, room temperature 90 reaction minutes The peptide is removed from the protecting group and cleaved from the resin. The crude peptide was dissolved in a 20% DMSO/H ₂ O solution to form a disulfide bond medium, and the crude peptide concentration was 0.1 mM, and the reaction was stirred at room temperature for 1 to 3 days. The solution was purified by RP-HPLC, RP-HPLC conditions, phase A: 0.05% TFA / water; phase B: 0.05% TFA 〃 0% ACN / water; column: C18 300A 4.6 x 250 mm; Gradient: 0-17 minutes B% 35-85, 17-21 minutes 8%85-35, 25 minutes End; flow rate: lmL / min; column temperature: 25. C. MALDI-Tof-MS: 2353.3 ₀ b ) Synthesis of N-tert-butoxy-rubber (lb)

8 g (0.2 mol) of NaOH was weighed into a 500 ml reaction flask, and dissolved in 200 ml of distilled water. 13.3 g (O.lmol) of iminodiacetic acid was weighed into the reaction solution, and dissolved completely. Add 200 ml of dioxane to the reaction flask and water bath. 25 ml (O.llmol) of Boc _{20 was} added dropwise to the reaction. The 2M NaOH7j solution controls the _P H value of the reaction solution to be stable at 9, and the reaction does not change until the pH value. Concentrated under reduced pressure to remove the organic solvent, 2, the aqueous layer extracted with ethyl acetate three times, the organic layers were combined, washed once with saturated NaCl solution adjusted with 10% HC1 pH, dried over anhydrous Na ₂ S0 _4. It was concentrated by filtration, and the residue was obtained from ethyl acetate / petroleum ether.

Take 0.23g (lmmol) of the above product in a 150ml eggplant-shaped bottle, add 15ml of dry acetonitrile to dissolve completely, weigh 0.29g (2.5mmol) of HOSu3⁄4V reaction, water bath, 0.4g (2mmol) DCC in the reaction solution, naturally return to temperature Stir the reaction. After one hour, 24 mg (0.2 mmol) of DMAP was added. Was stirred overnight, filtered, and concentrated to an oil which was dissolved in ethyl acetate was added, in a separatory funnel, the organic layer was washed with saturated NaHC0 _3, saturated NaCl, 10% HC1 solution, saturated NaCl, dried over anhydrous Na ₂ S0 _4. The mixture was concentrated under reduced pressure, ethyl acetate / petroleum ether / diethyl ether.

c Synthesis of SEO-01

250 mg (0.1 g) of la was accurately weighed into a 100 ml reaction flask, dissolved in 20 ml of dry DMF, and 21 mg (0.05 mmol) of lb was weighed into the reaction solution, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure, and the residue was evaporated, mjjjjjjjjjjjjjj The precipitate was purified by preparative high-performance liquid chromatography to obtain 225 mg of pure SEQ-01. MALDI-Tof-MS: 4805.0. Example 2: Synthesis of SEO-02

SEQ-02

a) N-tert-butoxy acyl-β-alanyl-iminodiacetic acid hydroxysuccinimide ester (2a)

2a

, ₀ A^ _{N c}

Dimethyl iminodiacetic acid 4.03 g (25 mmol) was placed in a 250 ml eggplant-shaped flask, 100 ml of dry dichloromethane was added, and BOC-P-Ala-OH 4.7 g (25 mmol) was added, 4.1 g (30 mmol) of HOBt was dissolved in 10 ml of DMF. The reaction flask was added, stirred under an ice bath, and finally 5.82 g (30 mmol) of EDC was added, and the mixture was naturally warmed and stirred overnight. The organic layer was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The mixture was washed with saturated NaHC0 ₃ and saturated NaCl, and dried over anhydrous Na ₂ SO ₄ . The oil was concentrated by filtration, and the oil was evaporated to dryness (yield: 7.07 g).

5 g (15 mmol) of the above solid was weighed into a 250 mL vial, and a mixed solution of tetrahydrofuran and methanol (1:1) 42 ml and 14 ml of water were added, and the mixture was stirred under a water bath. 2.4 g of potassium hydroxide was dissolved in 7 ml of pure water, slowly dropped into the reaction, naturally warmed up, and stirred overnight. The pH of the reaction solution was adjusted to neutrality with 10% HCl aqueous solution. The organic solvent was concentrated under reduced pressure. The residue was added to pure water. The aqueous layer was adjusted to pH 2 with 10% aqueous HCl solution, and the aqueous layer was extracted three times with ethyl acetate. Wash once and dry with anhydrous Na ₂ S0 ₄ . The mixture was concentrated by filtration, and ethyl acetate / petroleum ether was evaporated.

Lg ( 3.29 mmol) of the above diacid solid and 0.95 g ( 8.21 mmol) of HOSU in a 250 ml reaction flask were added, 50 ml of acetonitrile was added, and the mixture was stirred in an ice bath, then 1.36 g ( 6.59 mmol) of DCC was added, and the reaction was stirred with stirring overnight. The reaction mixture was filtered to remove insoluble matter, the organic solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate was added, in a separatory funnel successively with saturated NaHC0 _3, saturated NaCl, 10% HC1 aqueous solution, washed with saturated NaCl, the organic layer was dried over anhydrous Na ₂ S0 _{4 is} dry. Filtration and concentration gave 1.14 g of a blister 2a solid, yield 71%.

b Synthesis of SEO-02

217 mg (0.09 mmol) la and 26.7 mg (0.05 mmol) 2a were accurately weighed into a 150 ml reaction flask, dissolved in 20 ml of anhydrous DMF, and 140 μl (0.09 mmol) was added thereto with stirring, and the reaction was monitored by HPLC. After completion, the oil pump is concentrated under reduced pressure to remove DMF, and the residue is added with deprotection reagent 30 ml (trifluoroacetic acid: m-nonylphenol: dichloromethane 8:0.5:1.5). The reaction is completed, and the organic solvent is evaporated under reduced pressure, and anhydrous. Ethyl ether, solid precipitated, collected by solid diethyl ether, washed with 20% aqueous acetic acid, filtered and lyophilized to a crude peptide. High performance liquid phase purification obtained SEQ-02 pure product 200mgo MALDI-Tof-MS: 4874.8. Biological face example 1: EPO receptor-mediated cell proliferation activity evaluation

The method for detecting EPO activity in vitro is mainly based on the study of EPO-induced proliferation and/or differentiation of EPO-sensitive cells. TF-1 cells (purchased from GenScrip) are such a cell. The TF-1 cell line was first isolated from human erythroblastic leukemia cells, which expresses EPOR. Proliferation of TF-1 cells is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) Or interleukin 3 (IL-3). EPO also induces proliferation of TF-1 cells and has been widely accepted as a method for detecting EPO activity in vitro (T Kitamura, et al. Blood. 1989, 73: 375-380. S Chretien, et al. The EMBO J 1996, 15: 4174-4181). The pro-cell proliferative activity of the EPO receptor-mediated erythropoietin mimetic peptide was determined at the cellular level. The results are shown in Table 1. Table 1. Erythropoietin mimetic peptide cell proliferation activity

Compound structure sequence EC ₅₀ (nM) la Ac-Gly-Gly-Leu-Tyr-Ala-Cys-His-Met-Gly-Pro-lle-Thr-1-Nal-Val-Cys-Gln-Pro- 23.070

Leu-Cit-Sar-Lys-NH ₂

It can be seen that the erythropoietin mimetic peptide chemical dimers of the present invention both have the EPO receptor-mediated pro-cell proliferative activity, and the activity is more than 100 times higher than la. Biological face example 2: Evaluation of erythrocyte activity in normal mice

Animals: Mice (KM, male, 18-22g, Laboratory Animal Center, Academy of Military Medical Sciences); Drug preparation: The drug is now ready for use, with 0.1% BSA physiological saline as the vehicle. Automatic blood analyzer (YODER, France).

Test methods: Adult male mice were randomly divided into 5 groups, about 10 in each group. The animals were continuously administered for 7 days, once a day, subcutaneously (sc), and the blood was measured at the end of the test (trusted by 307 hospitals). RhEPO (recombinant EPO, Cyclone, 5000 IU/mL, Sihuan Biopharmaceutical Co., Ltd.) was used as a positive control.

Experimental results: See Table 2. Table 2 Effects of compounds on peripheral red blood cell (RBC), hemoglobin (Hb) and reticulocyte (RET) production

Drug dose RBC Hb RET

(mg/kg) (10 ¹² L) (g L) (10 ¹² /L)

Solvent - 8.74 ± 0.58 135.1 ± 9.5 0.455 ± 0.107 rhEPO 5xl0 ^{- 3} moving 1 ± 0.59** 159.0 ± 4.5** 0.985 ± 0.163 *** la 5 9.67 ± 0.64 * 158.1 ± 11.3** 0.893 ± 0.083 ***

SEQ-01 0.05 10.35±0.46** 164.4±7.4** 1.258±0.081***#

SEQ-02 0.05 9.89±0.57** 161.4±6.0** 1.152±0.095***# Note: The vehicle is 0.1% BSA in normal saline; animals are administered continuously for 7 days, once per day, sc; */;<0.05 ; ** /; <0.01; *** /; < 0.001 compared with the vehicle group; ^# /; < 0.05 compared with the solvent group compared with the rhEPO group; preliminary results show that la is below the 5mg / kg dose There is no significant reddening effect. The results showed that SEQ -01 and SEQ -02 were effective against the red blood cells of mice, and the dose was much lower than that of erythropoiesis producing peptidomimetic monomer la, indicating that the dimer titer was higher. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

Rights request

1. An erythropoietin mimetic peptided dimer of the formula (I), or a pharmaceutically acceptable salt thereof:

Formula (I)

among them,

P is a erythropoiesis-producing peptide:

n, m is any integer between 1 and 5;

The erythropoietin peptidomimetic chemical dimer of claim 1, or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of

SEQ - 01:

SEQ 02:

3. Use of the erythropoietin mimetic peptide chemical dimer of claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament as an EPO receptor agonist.

4. The erythropoietin mimetic peptide chemical dimer of claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the preparation of a disease for preventing and/or treating a deficiency or deficiency of erythropoietin or red blood cells, Or use in a medicament for diseases and/or symptoms associated with low activity of EPO or EPO receptors.

5. The use according to claim 4, wherein the disease characterized by lack of erythropoietin or red blood cell deficiency or deficiency, or diseases and/or symptoms associated with low activity of EPO or EPO receptors are caused by various causes. anemia.

6. The use according to claim 5, wherein the anemia caused by the various causes is, for example, red blood cell deficiency, low red blood cell count, low hemoglobin content, myelodysplastic syndrome, human immunodeficiency virus infection, autologous blood collection, bone marrow transplantation, hemoglobin Disease-induced anemia, renal anemia, tumor or cancer-related anemia, anemia in premature infants, postoperative anemia, maternal anemia, aplastic anemia, or other anemia.

A pharmaceutical composition comprising the erythropoietin mimetic peptide chemical dimer of claim 1 or 2, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

8. Prevention and / or treatment with a lack of erythropoietin or red blood cell deficiency or defects A method of afflicting a disease, or a disease and/or symptom associated with an activity of an EPO or EPO receptor, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the erythropoietin of claim 1 or 2. A step of mimicking a peptide chemical dimer, or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein said disease characterized by lack of erythropoietin or red blood cell deficiency or deficiency, or disease and/or symptom associated with low activity of EPO or EPO receptor is caused by various causes anemia.

10. The method of claim 9, wherein the various causes of anemia are, for example, red blood cell defects, low red blood cell count, low hemoglobin content, myelodysplastic syndrome, human immunodeficiency virus infection, autologous blood collection, bone marrow transplantation, hemoglobin Disease-induced anemia, renal anemia, tumor or cancer-related anemia, anemia in premature infants, postoperative anemia, maternal anemia, aplastic anemia, or other anemia.