CN111848778A - Teriparatide analogues - Google Patents

Abstract

The invention belongs to the field of medicines, and particularly relates to a teriparatide analogue. The invention obtains a new teriparatide analogue by modifying and/or substituting amino acid at 13, 26 and/or 27 positions of the N end of teriparatide, has enhanced cell membrane penetrating capacity, and can be used for oral administration.

Description

Teriparatide analogues

Technical Field

The invention belongs to the field of medicines, and particularly relates to a teriparatide analogue.

Background

At present, the incidence of osteoporosis in the elderly population is second to diabetes and senile dementia and leaps the third place of senile diseases, so the development of the medicine for treating osteoporosis is also a hot point of research. The drugs for treating osteoporosis can be mainly classified into the following three types: 1. bone mineralization promoting agents such as vitamin D and calcium preparations; 2. drugs that inhibit bone resorption, such as bisphosphonates, dinolizumab, estrogens, calcitonin, and the like; 3. the medicines for promoting bone formation mainly include parathyroid hormone (PTH) and its analogs, calcium ion receptor antagonists, anti-sclerostin monoclonal antibodies, etc. Among them, parathyroid hormone has become the first choice drug for the treatment of osteoporosis in postmenopausal women.

Parathyroid hormone is a polypeptide consisting of 84 amino acids synthesized, stored and secreted by parathyroid epithelial cells, and has the main physiological functions of promoting bone formation, mobilizing blood calcium into bone, and promoting reabsorption of calcium by renal tubules and gastrointestinal tract, thereby achieving the purpose of treating osteoporosis. When parathyroid hormone PTH (1-84) enters the human body, it is rapidly metabolized into N-terminal PTH (1-34) fragment, which has full physiological activity and can bind to the corresponding specific receptor of the tissue to exert biological effects. PTH (1-34) has been also developed as a drug for the treatment of osteoporosis because it has the same physiological activity as parathyroid hormone.

Teriparatide (trade name Forteo) is a recombinant PTH (1-34) developed by Eli Lilly corporation, usa, approved by the FDA for marketing in 2002, marketed in china in 2013 (futao), the amino acid sequence is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH, the product is approved for the treatment of postmenopausal women osteoporosis, primary or hypogonadal male osteoporosis patients, and persistent, systemic glucocorticoid-related osteoporosis patients.

The existing teriparatide on the market is an injection, a low-dose intermittent administration mode is adopted, subcutaneous injection is needed to be performed once a day, and great pain is brought to patients. If oral administration of teriparatide can be realized, clinical compliance of patients can be greatly improved.

However, for oral polypeptide drugs, their bioavailability in vivo is low, mainly due to: (1) the complex enzyme environment of the gastrointestinal tract makes the polypeptide easily degraded in the gastrointestinal tract; (2) the molecular weight is large and the adhesive film layer is not easy to pass; (3) lack of lipid solubility, poor permeability and difficulty in entering systemic circulation through small intestinal epithelial cells. The lipophilicity of the polypeptide can be increased by modifying the polypeptide through lipid acylation, so that the polypeptide can enter the systemic circulation through gastrointestinal tract cells. Meanwhile, receptors exist on the surface of gastrointestinal tract cells, and the polypeptide is chemically modified by using a ligand, and the ligand and the receptor realize specific binding so as to facilitate the polypeptide to penetrate the cells, such as cholic acid (sodium ion-dependent bile acid transporter ASBT) and biotin (sodium ion-dependent biotin receptor). In the US9993430B2 patent, the lipophilicity of the polypeptide is increased by carrying out lipid acylation modification on GLP-1, and the oral bioavailability of the GLP-1 polypeptide is improved by the combined action of the polypeptide and a penetration enhancer SNAC; y.w.cho et al/Lancet 2(2012) reports an oral insulin IN-105, wherein a section of PEG-fatty acid amphiphilic side chain is modified on a beta chain Lys29 to increase the lipophilicity of the insulin, thereby realizing the oral administration mode of the insulin; in US20110014247, 5-CNAC is added as a penetration enhancer to form a compound with polypeptide in a non-covalent bond manner, so that the lipophilicity of the polypeptide is increased, and the gastrointestinal permeation of the polypeptide is improved; in US8962015, biotin is used as a target to assist in penetrating the drug-loaded liposome into a single cell layer, so that the bioavailability of oral polypeptide is improved. Increasing cell permeation of polypeptides by increasing lipid solubility has now found widespread use in the field of oral polypeptides, however, chemical modifications may cause changes in the conformation of the polypeptide, resulting in loss of activity of the polypeptide. Therefore, when modifying a polypeptide, it is also critical to maintain the activity of the polypeptide.

Disclosure of Invention

The present patent application relates to teriparatide analogues which are modified and/or substituted with amino acids at position 13, 26 and/or 27 of the N-terminus of teriparatide.

In one aspect, the present application provides teriparatide analogs that are modified at one, two, or three lysines at position 13, 26, and/or 27 of the N-terminus of teriparatide.

In some embodiments, the modification is an acylation modification.

In some embodiments, the modification sites are located at one, two, or three of the three lysines at positions 13, 26, and 27 of the N-terminus of teriparatide.

In some embodiments, the modification site is located at one, two, or three of the three free-amino groups distributed over the three lysines at positions 13, 26, and 27 of the N-terminus of teriparatide.

In some embodiments, the general structural formula of the aliphatic chain for acylation modification of teriparatide is: HOOC- (AEEA)_m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) or default. m is one of 0,1,2,3,4 and 5. n is one of 0,1,2 and 3. R ₁Is aliphatic straight chain, branched chain C₆-C₂₀Deoxycholic acid, biotin or a deletion.

In some embodiments, m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2; n is the number 0,1,2,3, preferably 0 or 1, more preferably 1. When n is 1, (Xaa)_nIs D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-ammonia(ii) one of isobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), γ -glutamic acid (γ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably gamma-glutamic acid (gamma-Glu), proline (Pro). R₁Selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin; preferably lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin; more preferably, octadecanoyl group and 17-carboxyheptadecanoyl group.

The invention provides teriparatide lipidated derivatives, which have the following structures (polypeptide sequences are in the sequence from N end to C end):

when two or three lysine modifications are present at positions 13, 26 or 27, the T in the modifying group is the same structure, wherein the general chemical structure T can be represented as:

wherein m is a number of 0,1,2,3,4,5, n is a number of 0,1,2,3, p is an integer of 6 to 20, R₂is-H or-COOH; xaa is one or more or the default of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met).

More specifically, the side chain structure can be represented by the following structural formula:

wherein m is selected from an integer of 0 to 5, preferably an integer of 0 to 2, more preferably 2; xaa ₁Selected from the group consisting of D-alanine (D-Ala), β -alanine (β -Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), γ -glutamic acid (γ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably gamma-glutamic acid (gamma-Glu), proline (Pro).

In another aspect, the present application provides a class of teriparatide analogs having a substitution of a lysine at position 13, 26 or 27 of the N-terminus of teriparatide, the analogs having the general formula:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X₁-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X₂-X₃-Leu-Gln-Asp-Val-His-Asn-Phe-OH

wherein, X₁，X₂，X₃Not simultaneously Lys, and X₁，X₂，X₃At least one of which is Lys; in some embodiments, X is ₁，X₂，X₃Any one or two of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), preferably D-alanine (D-Ala), beta-alanine (beta-Ala), 2-aminoisobutyric acid (Aib), arginine (Arg), cysteine (Cys), glycine (Gly), more preferably arginine (Arg).

In another aspect, the present application provides acylated modifications of teriparatide analogues having a substitution of lysine at position 13, 26 and/or 27 of the N-terminus of teriparatide, said analogues having the general formula:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X₁-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X₂-X₃-Leu-Gln-Asp-Val-His-Asn-Phe-OH

wherein, X₁，X₂，X₃Not simultaneously Lys, and X₁，X₂，X₃At least one of which is Lys; in some embodiments, X is₁，X₂，X₃Either or both of which areOne or two of D-alanine (D-Ala), β -alanine (β -Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), γ -glutamic acid (γ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably D-alanine (D-Ala), beta-alanine (beta-Ala), 2-aminoisobutyric acid (Aib), arginine (Arg), cysteine (Cys), glycine (Gly), more preferably arginine (Arg);

Wherein the acylation modification site is positioned at X₁，X₂Or X₃Lys of (3), further, the acylation site is located at X₁，X₂Or X₃Lys at (a) is free-amino.

In some embodiments, the fatty chain structure for acylation modification is of the general formula: HOOC- (AEEA)_m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) or default. m is one of 0,1,2,3,4 and 5. n is one of 0,1,2 and 3. R₁Is aliphatic straight chain, branched chain C₆-C₂₀Deoxycholic acid, biotin or a deletion.

In some embodiments, m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2; n is the number 0,1,2,3, preferably 0 or 1, more preferably 1. When n is 1, (Xaa) _nIs D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA),2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), γ -glutamic acid (γ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably gamma-glutamic acid (gamma-Glu), proline (Pro). R₁Selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin. Preferably lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin. More preferably, octadecanoyl group and 17-carboxyheptadecanoyl group.

In another aspect, the present application provides an acylated modification of a teriparatide analogue, said modification being represented by the general formula (polypeptide sequence in order from N-terminus to C-terminus):

X₂ ²⁶,X₃ ²⁷,Lys¹³-PTH(1-34)

or

X₁ ¹³,X₃ ²⁷,Lys²⁶-PTH(1-34)

Or

X₁ ¹³,X₂ ²⁶,Lys²⁷-PTH(1-34)

Wherein, X₁Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x₂Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x₃Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg;

wherein, the chemical structural general formula T can be represented as:

wherein m is a number of 0,1,2,3,4,5, n is a number of 0,1,2,3, p is an integer of 6 to 20, R₂is-H or-COOH; xaa is one or more or the default of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met).

More specifically, the side chain structure T can be represented by the following structural formula:

wherein m is selected from 0-5, preferably 0-2, more preferably 2; xaa₁Selected from the group consisting of D-alanine (D-Ala), β -alanine (β -Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), γ -glutamic acid (γ -Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib) D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys) gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably gamma-glutamic acid (gamma-Glu), proline (Pro).

In some embodiments, the present application provides acylated modifications of a class of teriparatide analogs selected from compounds having the following structure (polypeptide sequences in order from N-terminus to C-terminus):

1.N-²⁷-PTH(1-34)-AEEA-AEEA-Pro-C₁₇-COOH：

2.N-¹³-PTH(1-34)-AEEA-AEEA-α-Glu-C₁₆：

3.N-²⁶-PTH-γ-Glu-C₁₂：

4.N-²⁷-PTH (1-34) -AEEA-deoxycholic acid:

5.N-¹³-(Arg²⁶，Gly²⁷)PTH(1-34)-γ-Glu-C₁₆：

6.N-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-Gly-AEEA-Gly-γ-Glu-C₁₇-COOH：

7.N-²⁶-(Cys¹³，Ala²⁷)PTH(1-34)-Phe-AEEA-AEEA-AEEA-C₁₂：

8.N-²⁶-(Arg¹³，Arg²⁷)PTH(1-34)-AEEA-AEEA-C₁₁-biotin:

9.N-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-C₁₇-COOH：

10.N-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-Glu-C₁₇-COOH：

11.N-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-Ala-C₁₇-COOH：

12.N-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-C₁₆：

compared with teriparatide, the in vitro bioactivity of the teriparatide analogue in the application is not changed greatly, the bioactivity of the polypeptide cannot be influenced by modifying the side chain, and the polarity is obviously reduced, in reverse chromatography, the elution ratio of the acetonitrile phase of the analogue is increased from 40% to 55% -60% of PTH, which shows that the polarity is reduced after the lipid acylation, thereby being beneficial to realizing transmembrane transport of the polypeptide.

The teriparatide analogue has the advantages that the cell permeability is remarkably improved, the PTH derivative has higher capability of penetrating a monolayer cell layer than a PTH reference substance in an in-vitro Caco-2 cell transmembrane transport experiment, and the apparent permeability coefficient P of the PTH derivative_appThe value is 1.89-4.03 times of that of a PTH standard product, and the modification of lipoylation can effectively enhance the cell membrane penetrating capacity.

In the application, only one lysine is left in the sequence of teriparatide by replacing the amino acid of teriparatide, so that only one modification site for lipoylation is reserved, the purification process is simple, and the purity of the product is improved.

The present application provides pharmaceutically acceptable salts of the aforementioned teriparatide analogs or uses thereof.

The polypeptide related to the application can be used for treating osteoporosis.

The teriparatide analogue can be used as an effective medicinal ingredient of an oral preparation; can also be used as an effective pharmaceutical ingredient for injection, such as intravenous injection, subcutaneous injection, intramuscular injection and the like; can also be used as effective component of topical medicine

The teriparatide analogue can be prepared into a pharmaceutically effective dosage unit by the existing pharmaceutical technology, and the form of the effective dosage unit can be oral administration, tablets, capsules or liquid and other dosage forms.

The teriparatide analogue components of the present application can be formulated in an aqueous formulation wherein the water content is not less than 50%.

In some embodiments, the oral preparation can be in the form of tablets, troches, pills, capsules (e.g., hard capsules, soft capsules, enteric capsules, microcapsules), elixirs, granules, syrups, granules, emulsions, suspensions, solutions, dispersions, and sustained-release preparations for oral or non-oral administration, wherein tablets containing various excipients (e.g., calcium carbonate, calcium phosphate, etc.) can also be formulated as disintegrating preparations.

In some embodiments, the pharmaceutical composition can be released in a controlled manner, including sustained or rapid release, and the controlled release dosage of the relevant pharmaceutical composition can be achieved by known pharmaceutical techniques.

The present application provides a pharmaceutical composition comprising a polypeptide of the present application, or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions of the present application also include a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical compositions described herein comprise an amount of a teriparatide analog of the present application of at least about 0.1mg, or at least about 0.2mg, or at least about 0.3mg, or at least about 0.4mg, or at least about 0.6mg, or at least about 0.8mg, or at least about 1mg, or at least about 1.5mg, or at least about 2mg, or at least about 2.5mg, or at least about 3mg, or at least about 5mg, or at least about 7mg, or at least about 10mg, or at least about 12mg, or at least about 15mg, or at least about 20mg, or at least about 30mg, or at least about 50mg, or at least about 70mg, or at least about 100 mg.

In some embodiments, the content of the teriparatide analogs of the present application in the pharmaceutical compositions described herein is in the range of 2.5 to 99.4% by weight. In some embodiments, the pharmaceutical composition described herein comprises a teriparatide analog in an amount in the range of 2.5 to 10 weight percent, or in the range of 8 to 15 weight percent, or in the range of 10 to 20 weight percent, or in the range of 15 to 30 weight percent, or in the range of 20 to 40 weight percent, or in the range of 30 to 50 weight percent, or in the range of 40 to 60 weight percent, or in the range of 50 to 70 weight percent.

When used in a treatment for treating osteoporosis, the teriparatide analogs of the present application, in some embodiments, are administered orally at least 100 μ g; in some embodiments, at least 200 μ g is administered orally; in some embodiments, at least 500 μ g is administered orally; in some embodiments, at least 100 μ g is administered orally; in some embodiments, at least 100 μ g is administered orally. When used in therapy for the treatment of osteoporosis, the teriparatide analogs of the present application, in some embodiments, are administered orally in an amount of 20mg or less; in some embodiments, 10mg or less is administered orally; in some embodiments, 5mg or less is administered orally; in some embodiments, 3mg or less is administered orally; in some embodiments, an amount of 2000 μ g or less is administered orally; in some embodiments, an amount of 1000 μ g or less is administered orally. The teriparatide analogs of the present application, when used in therapy for treating osteoporosis, in some embodiments, are administered orally from 200 μ g to 20 mg; in some embodiments, 200 μ g to 10mg is administered orally; in some embodiments, 200 μ g to 5mg is administered orally; in some embodiments, 200 μ g to 3000 μ g is administered orally; in some embodiments, 200 μ g to 2000 μ g is administered orally; in some embodiments, 500 μ g to 1000 μ g is administered orally; in some embodiments, 750 μ g is administered orally.

The teriparatide analogues or pharmaceutical compositions thereof, when used in the treatment of osteoporosis, are administered orally from 1 to 3 times per day according to any of the various embodiments described herein; in some embodiments, the oral administration according to any of the various embodiments described herein is performed 1 or 2 times per day; in some embodiments, the oral administration according to any of the various embodiments described herein is performed 1 time per day.

The scheme of this application still includes:

1. a teriparatide analogue characterized in that said analogue is modified and/or substituted with an amino acid at position 13, 26 and/or 27 of the N-terminus of teriparatide.

2. The teriparatide analogue according to claim 1, which is acylated at position 13, 26 and/or 27 of the N-terminus of teriparatide.

3. The teriparatide analogue of any one of the preceding claims, wherein the acylation modification sites are located at one, two or three of the lysines at position 13, 26 or 27 of the N-terminus of teriparatide.

4. The teriparatide analogue as described in any one of the preceding claims, wherein the fatty chain used for acylation modification has the general structural formula: HOOC- (AEEA)_m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or is absent; m is a number of 0,1,2,3,4, 5; n is one of 0,1,2 and 3; r ₁Selected from aliphatic straight chain and branched chain C₆-C₂₀Acyl group of deoxycholic acid, biotin or a deletion.

5. Teriparatide analogues according to any of the preceding claims, wherein m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2.

6. Teriparatide analogues according to any of the preceding claims, wherein n is the number 0,1,2,3, preferably 0 or 1, more preferably 1.

7. The teriparatide analogue of any one of the preceding claims, wherein n is 1, said (Xaa)_nSelected from 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, prolineAlanine, phenylalanine, preferably gamma-glutamic acid or proline.

8. Teriparatide analogues according to any of the preceding claims, wherein R is₁Selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin, preferably lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin, more preferably octadecanoyl, 17-carboxyheptadecanoyl.

9. A teriparatide analogue according to any one of the preceding claims, characterised in that the analogue has a structure according to one of the following:

wherein, the chemical structural general formula T can be represented as:

when two or three lysine modifications are present at positions 13, 26 or 27, the T in the modifying group are of the same structure, wherein m is a number from 0,1,2,3,4,5, n is a number from 0,1,2,3, p is an integer from 6 to 20, R is an amino acid residue₂is-H or-COOH.

10. The teriparatide analogue according to any one of the preceding claims, characterized in that T may represent the following structure:

wherein m is selected from an integer of 0 to 5, preferably an integer of 0 to 2, more preferably 2; xaa₁Selected from the group consisting of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine, methionine; preferably 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine; more preferably gamma-glutamic acid or proline.

11. A teriparatide analogue according to any one of the preceding claims, wherein the analogue has a substitution of a lysine at position 13, 26 or 27 of the N-terminus of teriparatide, the analogue having the general formula:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X₁-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X₂-X₃-Leu-Gln-Asp-Val-His-Asn-Phe-OH, wherein X₁，X₂，X₃Not simultaneously Lys, and X₁，X₂，X₃At least one of which is Lys.

12. Teriparatide analogues according to any of the preceding claims, characterized in that said X₁，X₂，X₃Any one or two of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionineOne or two of (a); preferably D-alanine, beta-alanine, 2-aminoisobutyric acid, arginine, cysteine, glycine; more preferably arginine.

13. The teriparatide analogue according to any one of the preceding claims, characterized in that the teriparatide analogue is provided with an acylation modification at the site of X₁，X₂Or X₃Lys of (a).

14. The teriparatide analogue according to any one of the preceding claims, wherein the acylation modification is to link an aliphatic chain structure of the general formula: HOOC- (AEEA) _m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or is absent; m is a number of 0,1,2,3,4, 5; n is one of 0,1,2 and 3; r₁Selected from aliphatic straight chain and branched chain C₆-C₂₀Acyl group of deoxycholic acid, biotin or a deletion.

15. Teriparatide analogues according to any of the preceding claims, wherein m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2.

16. Teriparatide analogues according to any of the preceding claims, wherein n is the number 0,1,2,3, preferably 0 or 1, more preferably 1.

17. Teriparatide analogues according to any of the preceding claims, wherein said n is 1, Xaa_nSelected from 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine, preferably gamma-glutamic acid or proline.

18. Teriparatide analogues according to any of the preceding claims, wherein R is₁Selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoylExamples of the substituent include a methyl group, a methylnonanoyl group, a decanoyl group, a methyldecanoyl group, a lauroyl group, a myristoyl group, a palmitoyl group, an octadecanoyl group, a 17-carboxyheptadecanoyl group, a 15-carboxypentadecanoyl group, a 13-carboxytridecanoyl group, an 11-carboxyundecanoyl group, a deoxycholic acid group and a biotin group, and the preferable examples are a lauroyl group, a myristoyl group, a palmitoyl group, an octadecanoyl group, a 17-carboxyheptadecanoyl group, a deoxycholic acid group and a biotin group, and the more.

19. A teriparatide analogue according to any one of the preceding claims, characterised in that the analogue has the structure as described below:

wherein, X₁Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x₂Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x ₃Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg;

wherein, the chemical structural general formula T can be represented as:

wherein m is a number of 0,1,2,3,4,5, n is a number of 0,1,2,3, p is a number of 6-20An integer of (A), R₂is-H or-COOH.

20. The teriparatide analogue of any one of the preceding claims, wherein T may be of the structure:

wherein m is selected from an integer of 0 to 5, preferably an integer of 0 to 2, more preferably 2; xaa₁Selected from the group consisting of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine, methionine, preferably 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine, more preferably gamma-glutamic acid or proline.

21. Teriparatide analogues according to any of the preceding claims, characterized in that said analogues are selected from compounds having one of the following structures:

1).

22. a pharmaceutical composition comprising any one of the teriparatide analogs or a pharmaceutically acceptable salt thereof.

23. The pharmaceutical composition of any of the preceding claims, further comprising a pharmaceutically acceptable carrier and/or excipient.

24. The pharmaceutical composition according to any one of the preceding claims, which is an injectable formulation or an oral formulation.

25. The pharmaceutical composition according to any one of the preceding claims, wherein the oral formulation is in the form of tablets, troches, pills, capsules, elixirs, granules, syrups, granules, emulsions, suspensions, solutions, dispersions and sustained release formulations for oral or non-oral administration.

26. Use of a pharmaceutical composition according to any of the preceding claims for the manufacture of a medicament for the treatment of osteoporosis.

Definition of

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F、CH₂CHF₂Etc.) or completely substituted (CF)₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

C as used herein_m-nMeaning that the moiety has m-n carbon atoms. For example, "carbon_3-10Cycloalkyl "means the ringThe alkyl group has 3 to 10 carbon atoms. "carbon_0-6Alkylene "means that the alkylene group has 0 to 6 carbon atoms, and when alkylene has 0 carbon atom, the group is a bond.

As used herein (AEEA)_mOr (Xaa)_nRefers to having m or n attached AEEA or Xaa groups in the moiety.

Numerical ranges herein refer to each integer in the given range. E.g. "C_1-6By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 2R, then there are separate options for each R.

The term "substituted" means that any one or more of the last hydrogen atoms on a particular atom is replaced with a substituent, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═ O), meaning that two hydrogen atoms are substituted, oxo does not occur on the aryl.

PTH in the present embodiment refers to PTH (1-34) or analogs having PTH (1-34) as the parent nucleus, unless otherwise specified.

AEEA in the scheme of the invention refers to 8-amino-3, 6 dioxyoctanoic acid.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned. Non-limiting examples of metal salts include, but are not limited to, salts of alkali metals, such as sodium, potassium, and the like; salts of alkaline earth metals such as calcium, magnesium, barium, and the like; aluminum salts, and the like. Non-limiting examples of salts with organic bases include, but are not limited to, salts with trimethylamine, triethylamine, pyridine, picoline, 2, 6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and the like. Non-limiting examples of salts with inorganic acids include, but are not limited to, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts with organic acids include, but are not limited to, salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Non-limiting examples of salts with basic amino acids include, but are not limited to, salts with arginine, lysine, ornithine, and the like. Non-limiting examples of salts with acidic amino acids include, but are not limited to, salts with aspartic acid, glutamic acid, and the like.

The term "pharmaceutical ingredient" refers to a formulation of one or more compounds of the present application or salts thereof with excipients, diluents, or carriers generally accepted in the art for delivering biologically active compounds to an organism (e.g., a human). The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "pharmaceutically acceptable excipient, diluent, or carrier" refers to those excipients, diluents, or carriers that do not significantly irritate the organism and do not impair the biological activity and performance of the active compound. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The present application also includes isotopically-labeled compounds of the present application, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include hydrogen, carbon, nitrogen, oxygen Isotopes of phosphorus, sulfur, fluorine, iodine and chlorine, such as respectively²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、¹²³I、¹²⁵I and³⁶cl, and the like.

Certain isotopically-labelled compounds of the present application (e.g. with³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Furthermore, substitution with heavier isotopes such as deuterium (i.e. 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g. increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances. Positron emitting isotopes, such as 15O, 13N, 11C, and 18F, can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

The following examples represent only one aspect of the present invention, and are not intended to limit the subject matter of the invention.

Description of the drawings:

FIG. 1: the teriparatide analogues with amino acid substitutions at positions 13,26 and 27 of PTH (1-34) without acylation modification are shown: (Arg)²⁶，Gly²⁷)Lys¹³-PTH，(Cys¹³，Ala²⁷)Lys²⁶-PTH and (Arg)¹³，Arg²⁶)Lys²⁷In vitro active EC50 value of PTH

The specific implementation scheme is as follows:

example 1N-²⁷-PTH(1-34)-AEEA-AEEA-Pro-C₁₇-COOH

(1) Materials and reagents

2-CTC resin, substitution value 1.15 mmol/g.

The amino acids are: Fmoc-Pro-OH, Fmoc-AEEA-OH, mono-tert-butyl octadecanedioate

Synthesizing a reagent: HOBt, DIC, DMF, DCM, piperidine, DIEA.

(2) Instrument for measuring the position of a moving object

CS-BIO type polypeptide synthesizer, Waters600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, and BUCHI rotary evaporator.

(3) Procedure for the preparation of the

a. Solid phase chemical synthesis of polypeptides

Weighing 1.00g of 2-CTC resin, placing the 2-CTC resin in a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-AEEAc-OH and absorbing 4-6 times of DIEA, adding 10mLDCM, dissolving, putting the mixture into the reactor, reacting at room temperature for two hours, namely, coupling the first amino acid onto the resin, washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then adding 10ml of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DCM for 6 times, coupling a second amino acid, weighing three times of Fmoc-AEEAc-OH, HOBt and DIC, adding 10ml of DMF/DCM (1:1) mixed solvent for dissolving, reacting, wherein the reaction temperature is room temperature, the reaction progress is monitored by ninhydrin reaction, the reaction is completed when colorless is monitored, and washing the resin with DCM for 6 times. Then, the coupling reaction of proline and octadecanedioic acid can be continued according to the above coupling method, and the process is circulated until all amino acids are coupled.

b. Cracking and precipitation

Adding a cracking reagent according to the ratio of 5mL of the cracking reagent to 1g of resin, stirring and reacting for 1 hour at room temperature according to the ratio of TFE to DCM (1: 4) (V: V), filtering, carrying out rotary evaporation on the filtrate at 40 ℃ until the filtrate is dried, adding 10mLDCM into a rotary evaporation bottle, carrying out rotary evaporation again until the filtrate is dried, repeatedly carrying out the reaction for 2-3 times, finally adding 3mLDCM to dissolve polypeptide, adding 40mL of diethyl ether, placing in a refrigerator at-20 ℃ for 20min, centrifuging, carrying out vacuum drying, and weighing the crude peptide.

c. Preparation of teriparatide derivatives by liquid phase reaction

The dried crude peptide was weighed to about 0.1mmol, HOSU to about 0.095mmol, and DIC 15. mu.L, added to about 5mL tetrahydrofuran, reacted at room temperature for 1-2 hours, rotary evaporated, and THF removed to give a yellow oil. Adding TFA: H₂And (3) carrying out tert-butyl ester removal protection in a mixed solvent of O-90: 10, reacting at room temperature for 1 hour, then pouring into glacial ethyl ether for precipitation, centrifuging to obtain a solid, washing with ethyl ether for three times, drying under reduced pressure to obtain solid powder, weighing, and dissolving with DMF to obtain a side chain activated ester solution.

Dissolving 0.5mmol of teriparatide in 0.1mol/L triethylamine solution, slowly dropwise adding a side chain activated ester solution into the solution, wherein the feeding ratio is 1: 1.2, the reaction mixture was stirred at room temperature for 10 minutes, and the pH was adjusted to 8.0 with 0.1mol/L hydrochloric acid to terminate the reaction.

The crude product was purified by semi-preparative RP-HPLC.

1 purification of

A chromatographic column: nano Micro C18 preparation column (10 mm. times.250 mm,10 μm)

Flow rate: 5ml/min

Detection wavelength: 215nm

Mobile phase: phase A: 1% HAC/water solution

Phase B: 1% HAC/acetonitrile

Gradient elution procedure as in table 3:

TABLE 3 gradient elution Table

Analysis of the collected product by Agilent 1260HPLC

A chromatographic column: YMC-pack ODS-AQ C18 analytical column (4.6 mm. times.250 mm,5 μm)

Flow rate: 1ml/min

Detection wavelength: 215nm

Mobile phase: phase A: 0.1% TFA/water

Phase B: 0.1% TFA/acetonitrile

Gradient elution procedure as in table 4:

TABLE 4 gradient elution Table

Collecting target components with the purity of more than 90%, removing acetonitrile by rotary evaporation, and carrying out vacuum freeze drying. The molecular weight is confirmed by ESI-MS, and M/Z is 1201.62 [ M +4H ]⁺In line with the theoretical molecular weight.

Example 2N-¹³-PTH(1-34)-AEEA-AEEA-α-Glu-C₁₆

The procedure was as described in example 1, and the molecular weight was confirmed by ESI-MS, M/Z-1194.6 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 3: n-²⁶-PTH(1-34)-γ-Glu-C₁₂

The procedure was as described in example 1, and the molecular weight was confirmed by ESI-MS, M/Z-1108.2 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 4: n-²⁷-PTH (1-34) -AEEA-AEEA-deoxycholic acid

The procedure was as described in example 1, and the molecular weight was confirmed by ESI-MS, M/Z-1196.5 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 5: n-¹³-(Arg²⁶，Gly²⁷)PTH-γ-Glu-C₁₆

(1) Materials and reagents

2-CTC resin, substitution value 1.15 mmol/g.

Teriparatide analogues (Arg)²⁶，Gly²⁷) PTH is obtained by expression of engineering bacteria or chemical synthesis method, M/Z1019.2 (M +4H)⁺

The amino acids are: Fmoc-gamma-Glu-Otbu, palmitic acid

Synthesizing a reagent: HOBt, DIC, DMF, DCM, piperidine, DIEA.

(2) Instrument for measuring the position of a moving object

CS-BIO type polypeptide synthesizer, Waters600 semi-preparative high performance liquid chromatograph, Beckman centrifuge and bushi rotary evaporator.

(3) Procedure for the preparation of the

a. Solid phase chemical synthesis of polypeptides

Weighing 1.00g of 2-CTC resin, placing the 2-CTC resin in a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-gamma-Glu-OH and absorbing 4-6 times of DIEA, adding the mixture into 10mLDCM, dissolving, putting the mixture into the reactor, reacting for two hours at room temperature (preferably 25 ℃ or above, or prolonging the reaction time), namely, coupling the first amino acid onto the resin, then washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then adding 10ml of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DCM for 6 times, coupling a second amino acid, weighing three times of palmitic acid, HoBT and DIC, adding 10ml of DMF/DCM (1:1) mixed solvent together for dissolving, carrying out reaction, wherein the reaction temperature is room temperature, the reaction progress is monitored by ninhydrin reaction, and the reaction is completed when colorless is monitored, and washing the resin with DCM for 6 times.

b. Cracking and precipitation

After the synthesis of the polypeptide is finished, the wet weight is weighed. Adding a cracking reagent according to the ratio of 5mL of the cracking reagent to 1g of resin, stirring and reacting for 1 hour at room temperature, filtering to a 25mL rotary evaporation bottle, carrying out rotary evaporation at 40 ℃ until no liquid exists, then adding 10mL of a mixed solution of a solvent and a solvent into the rotary evaporation bottle, carrying out rotary evaporation again until no liquid exists, repeating the reaction for 2-3 times, finally adding 3mL of the mixed solution of the solvent and the solvent, transferring the solution to a 50mL centrifugal tube, adding 40mL of diethyl ether, placing the solution in a refrigerator at-20 ℃ for 20min, centrifuging, drying in vacuum, and weighing the crude peptide.

c. Preparation of teriparatide derivatives by liquid phase reaction

Weighing about 0.1mmol of dried crude peptide, about 0.095mmol of HOSU and sucking DIC15 μ L, adding about 5mL of tetrahydrofuran to dissolve in a 5mLEP tube, reacting at room temperature for 1-2 hours, transferring the reaction liquid to a 10mL rotary evaporation bottle, removing THF through rotary evaporation, adding about 5mLDMF into the rotary evaporation bottle, and dissolving the reaction product in the rotary evaporation bottle to obtain the side chain activated ester.

Then 0.5mmol of teriparatide analogue is dissolved in 0.1mol/L triethylamine solution, and side chain activated ester solution is slowly dripped into the solution, wherein the feeding ratio is 1: 1.2, stirred at room temperature for 10 minutes, after which the reaction was terminated by adjusting the pH to 8.0 with 0.1N hydrochloric acid.

The crude product was purified by semi-preparative RP-HPLC.

1 purification of

A chromatographic column: nano Micro C18 preparation column (10 mm. times.250 mm,10 μm)

Flow rate: 5ml/min

Detection wavelength: 215nm

Mobile phase: phase A: 1% HAC/water solution

Phase B: 1% HAC/acetonitrile

Gradient elution procedure as in table 3:

TABLE 3 gradient elution Table

Analysis of the collected product by Agilent 1260HPLC

A chromatographic column: YMC-pack ODS-AQ C18 analytical column (4.6 mm. times.250 mm,5 μm)

Flow rate: 1ml/min

Detection wavelength: 215nm

Mobile phase: phase A: 0.1% TFA/water

Phase B: 0.1% TFA/acetonitrile

Gradient elution procedure as in table 4:

TABLE 4 gradient elution Table

Collecting the target component with purity of more than 90%, low pressure rotary steaming, and freeze drying. The molecular weight is confirmed by ESI-MS, and M/Z is 1111.1 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 6: n-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-Gly-AEEA-Gly-Glu-C₁₇-COOH

(Arg¹³，Arg²⁶)Lys²⁷the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method, wherein M/Z is 1044.3 [ M +4H ]⁺

Preparation of analogue derivatives molecular weight confirmation was carried out by ESI-MS as described in example 4, M/Z ═ 1251.8 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 7: n-²⁶-(Cys¹³，Ala²⁷)PTH(1-34)-Phe-AEEA-AEEA-AEEA-C12

(Cys¹³，Ala²⁷)Lys²⁶the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method, wherein M/Z is 1009.7 [ M +4H ] ⁺

The derivatives were prepared as described in example 4, and the molecular weight was confirmed by ESI-MS, M/Z-1200.7 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 8: n-²⁶-(Arg¹³，Arg²⁷)PTH(1-34)-AEEA-AEEA-C₁₁-biotin

(Arg¹³，Arg²⁷)Lys²⁶the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method, wherein M/Z is 1044.5 [ M +4H ]⁺

The derivatives were prepared as described in example 4, and the molecular weight was confirmed by ESI-MS, M/Z-1218.2 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 9: n-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-C₁₇-COOH

(Arg¹³，Arg²⁶)Lys²⁷the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method to obtain M/Z-1044.5 [ M +4H ]⁺

The derivatives were prepared as described in example 4, and the molecular weight was confirmed by ESI-MS, M/Z-1189.8 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 10: n-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-Glu-C₁₇-COOH

(Arg¹³，Arg²⁶)Lys²⁷Preparation of-PTH (1-34) analog using engineered bacteria expression or chemical synthesis to obtain M/Z ═ 1044.5 [ M +4H ] derivatives as described in example 4, molecular weight was confirmed by ESI-MS, M/Z ═ 1186.7 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 11: n-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-Ala-C₁₇-COOH

(Arg¹³，Arg²⁶)Lys²⁷the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method to obtain M/Z-1044.5 [ M +4H ]⁺The derivatives were prepared as described in example 4, and the molecular weight was confirmed by ESI-MS, M/Z-1208.5 [ M +4H ] ⁺Is consistent with the theoretical molecular weight.

Example 12: n-²⁷-(Arg¹³，Arg²⁶)PTH(1-34)-AEEA-AEEA-C₁₆

(Arg¹³，Arg²⁶)Lys²⁷the-PTH (1-34) analogue is obtained by adopting an engineering bacteria expression or chemical synthesis method to obtain M/Z-1044.5 [ M +4H ]⁺The derivatives were prepared as described in example 4, and the molecular weight was confirmed by ESI-MS, M/Z-1175.3 [ M +4H ]⁺Is consistent with the theoretical molecular weight.

Example 13: detection of in vitro active EC50

CHO-K1 cells were used, teriparatide acetate was used as a control, and the assay was performed according to the United states Pharmacopeia biological activity. In vitro cell activity assays were performed on PTH (1-34) and derivatives of PTH (1-34). Teriparatide is capable of specific binding to the cell surface PTH receptor (PTH1R), resulting in activation of adenylate cyclase and thus production of cyclic adenosine monophosphate (cAMP), which stimulates cellular metabolic pathways. The quantitative analysis of cAMP formed by PTH1-34 concentration-dependently in these cells was performed using time-resolved fluorescence technique (TR-FRET) (detection Kit: LANCEUltracAMP Kit from Perkinelmer). And analyzing experimental data by Prism5 software, fitting by using a four-parameter equation with the sample concentration as an X axis and the corresponding fluorescence ratio as a Y axis, and drawing a dose response curve of an active reference substance and a test substance to obtain an EC50 value.

Example 14: study of transmembrane transport of derivatives

Caco-2 cells were plated at 5X10⁵Per cm²The density was measured by inoculating on the Apical side of a Millicell-CM insertion type cell culture dish (Transwell plate No. 3401, Corning Co.) with changing the solution every day, measuring the transmembrane resistance after the third week, and measuring the resistance when the resistance is more than 500. omega. CM²Can be used for transport experiments. Transport experiments on Caco-2 monolayers, teriparatide acetate (made by the laboratory) were used as a control. 0.3ml of PTH (1-34) or PTH (1-34) derivative solution (concentration 100. mu.g/ml) was added to the donor pool (AP-side), 0.7mg of Hank's buffer was added to the acceptor pool (BP-side), 0.1ml of the solution was taken out of the acceptor pool at various time points (0,20,45,60 and 90 minutes) and supplemented with an equal amount of blank Hank's solution, the concentration was measured using an ELISA kit and plotted against time using the apparent epithelial cell permeability coefficient P_appIs calculated by formula

A is the area of the permeable membrane, here the donor cell membrane area (cm)²)，C₀Is the initial sample concentration (ng/ml) of the donor cell and dQ/dt is the diffusion flux (ng/s).

Teriparatide has high polarity and poor cell permeability, and cannot realize trans-cell transport. Surprisingly, the P of the modified teriparatide derivatives (examples 1 and 3) and the derivatives of teriparatide analogues (examples 5 and 7) _appThe value is improved by 1.8-4 times, and according to the results of example 4 and example 8, the permeability of epithelial cell membranes is obviously improved after deoxycholic acid and biotin are modified, so that the transmembrane transport capacity of the polypeptide is enhanced, and a compound basis is effectively provided for polypeptide molecules to cross epithelial cell layers of gastrointestinal tracts.

While the compositions and methods of this invention have been described in terms of preferred embodiments in light of the present disclosure, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

The disclosures of all documents cited herein are incorporated by reference herein, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Claims (10)

1. A teriparatide analogue characterized in that said analogue is modified and/or substituted with an amino acid at position 13, 26 and/or 27 of the N-terminus of teriparatide.

2. The teriparatide analogue according to claim 1, characterized in that the modification is an acylation modification at one, two or three of the lysines at position 13, 26 or 27 of the N-terminus of teriparatide.

3. A teriparatide analogue according to claim 2, characterised in that the modification for acylation is to link an aliphatic chain structure of the general formula: HOOC-(AEEA)_m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or is absent; m is a number of 0,1,2,3,4, 5; n is one of 0,1,2 and 3; r₁Selected from aliphatic straight chain and branched chain C₆-C₂₀Acyl group of deoxycholic acid, biotin or a deletion.

4. A teriparatide analogue according to any one of claims 1-3, characterised in that the analogue has a structure according to one of the following:

when two or three lysine modifications are present at position 13, 26 or 27, the T in the modifying group are of the same structure,

wherein, the chemical structural general formula T can be represented as:

wherein m is a number of 0,1,2,3,4,5, n is a number of 0,1,2,3, p is an integer of 6 to 20, R ₂is-H or-COOH.

5. The teriparatide analogue of claim 1, wherein the analogue has a substitution of a lysine at position 13, 26 or 27 of the N-terminus of teriparatide, the analogue having the general formula:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X₁-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X₂-X₃-Leu-Gln-Asp-Val-His-Asn-Phe-OH, wherein X₁，X₂，X₃Not simultaneously Lys, and X₁，X₂，X₃At least one of which is Lys, said teriparatide analogue having an acylation modification at a site located at X₁，X₂Or X₃Lys of (a).

6. The teriparatide analogue according to claim 5, wherein the acylation modification is the attachment of an aliphatic chain structure of the general formula: HOOC- (AEEA)_m-(Xaa)_n-R₁Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or is absent; m is a number of 0,1,2,3,4, 5; n is one of 0,1,2 and 3; r₁Selected from aliphatic straight chain and branched chain C ₆-C₂₀Acyl group of deoxycholic acid, biotin or a deletion.

7. The teriparatide analogue according to claim 5, characterized in that the analogue has the structure as set forth in:

wherein, X₁Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x₂Selected from D-Ala. beta-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, gamma-Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably beta-Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg; x₃Selected from the group consisting of D-Ala, β -Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ -Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val, Met, preferably β -Ala, GABA, Aib, Abu, Arg, Cys, more preferably Arg;

wherein, the chemical structural general formula T can be represented as:

wherein m is a number of 0,1,2,3,4,5, n is a number of 0,1,2,3, p is an integer of 6 to 20, R₂is-H or-COOH.

8. Teriparatide analogue according to claim 1, characterized in that the analogue is selected from compounds having one of the following structures:

1).

2).

3).

4).

5).

6).

7).

8).

9).

10).

11).

12).

9. a pharmaceutical composition comprising a teriparatide analogue according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition according to claim 9, for use in the preparation of a medicament for the treatment of osteoporosis.

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