CN106674209B - Programmed death receptor 1 gene inhibitor and preparation method and application thereof - Google Patents

Abstract

The invention provides a programmed death receptor 1 gene inhibitor and a preparation method and application thereof. In particular to a compound for inhibiting the expression of a programmed death receptor 1 and pharmaceutically acceptable salt thereof, which has a structure shown in a formula I

Wherein A is selected from C or N; r₁Selected from H, CH₃、(CH₂)₃‑N(CH₃)₂、(CH₂)₂‑N(CH₃)₂、CH₂‑N(CH₃)₂、(CH₂)₃‑N(CH₃)CH₂CH₂NHR₃；R₂Selected from H, CH₃、

R₃Selected from H, CH₃、

The polyamide molecule disclosed by the invention belongs to polypeptide micromolecules, can realize chemical synthesis, is favorable for large-scale production, and provides more methods for inhibiting the function of PD 1.

Description

Programmed death receptor 1 gene inhibitor and preparation method and application thereof

technical field

The invention relates to the field of medicine, in particular to an expression inhibitor of programmed death receptor 1.

Background technique

PD-1 (programmed death receptor 1) is an immune checkpoint protein, a type I transmembrane protein, composed of 268 amino acids encoded by the PDCD1 gene, and an important member of the CD28/CTLA4 family. The main function of PD-1 is to suppress the activity of T cells during an immune response. Its protein structure is divided into three parts: extracellular immunoglobulin variable region, transmembrane domain and intracellular domain; ITSM (immunorecptor tyrosine-based switch motif) and ITIM (immunorecptor tyrosine-based inhibitory) in the intracellular domain motif) two motifs, which contain phosphorylation sites, in which the ITSM motif is related to the inhibitory function of PD-1. When the PD-1 molecule on the surface of the T cell binds to the receptor PDL-1/PDL-2 on the surface of the antigen-presenting cell, ITSM is phosphorylated, and then the phosphatase SHP-1 and SHP-2 bind to ITSM, which directly leads to Dephosphorylation of signaling molecules downstream of the TCR complex inhibits T cell activation. In the tumor microenvironment, a large number of PDL-1/PDL-2 molecules are expressed on the surface of tumor cells. When T cells infiltrate into the tumor tissue, the PD-1 molecules on the surface of T cells can recognize PDL-1/PDL-2. T cells cannot be activated by tumor antigens and cannot function to kill tumor cells.

The basic research on the transcriptional regulatory elements of the PDCD1 gene promoter region provides the target basis for the design of this patent. In 1997, Finger et al. studied the structure of the human PDCD1 gene and predicted many cis-acting elements in the promoter region, such as Sp1, PU.1box, AP-2, E-box MYC site and so on. In 2008, Oestreich et al. found two tissue-specific regulatory sites in the PDCD1 gene promoter region in mouse EL4 cell line and mouse primary CD8 ⁺ T cell model: CR-B (Conserved Region-B) and CR- C (Conserved Region-C); while the N1NFAT cis-element site located in the CR-C region is a regulatory element necessary for PDCD1 transcription. In the same year, Cho et al. found that the IFN-α response element ISRE (IFN-Sensitive Response Element) was involved in the expression of PDCD1 in response to IFN-α in the mouse macrophage cell line RAW264.7; then in 2011, Terawaki et al. Similar results were obtained, and treatment with IFN-α combined with PD-1 blockade to treat tumor-bearing mice effectively enhanced the anti-tumor activity of IFN-α. c-Fos is a subunit of AP1 protein, and abnormal expression of this protein in T cells can promote tumor growth. Xiao et al. found that c-Fos can bind to the AP1 binding site in the CR-B region of the PDCD1 promoter region, thereby activating the expression of PDCD1 gene in T cells. T cells that abnormally express PD-1 bind to PDL1/PDL2 receptors on the surface of tumor cells, greatly reducing the ability to recognize and kill tumor cells, thereby promoting tumor growth. Xiao et al. elucidated the mechanism by knockout mice of the AP1 binding site. The expression of PDCD1 gene is not only regulated by a series of cis-acting elements in the promoter region, but also some elements in the range of about 10-50kb upstream and distal end of the promoter are regulated by transcription factors such as STAT3, STAT4, NFATc1 and CTCF.

PD1 monoclonal antibody blocks the binding of PD1 protein to its receptors PDL1/PDL2 through the principle of antibody-antigen binding, thereby blocking the inhibition of T cell immune activation. Many monoclonal antibody drugs have been successfully launched.

The production process of antibody drugs is complex and the yield is low, which is not conducive to large-scale production; antibody drugs are poor in protein stability and have high storage conditions, which are not conducive to transportation and storage.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a variety of compounds, which block the expression of PDCD1 gene from the gene transcription level through pyrrolimidazole polyamide molecules, so as to achieve the inhibitory effect of PD1.

The small polypeptide molecules used in this patent are pyrrole-imidazole polyamide polypeptides (PIPs), which have strong DNA sequence recognition specificity. It is composed of N-methylpyrrole (N-methylpyrrole, Im) and N-methylimidazole (N-methylimidazole, Py). Im/Py pairing can recognize G:C base pairs; Py/Py pairing can recognize A:T and T:A base pairs; Hp(hydroxypyrrole)/Py pairing can recognize T:A base pairs; N-terminal Ct The (3-chlorothiophene)/Py pairing is more likely to bind T:A base pairs. According to this binding law, PIPs molecules can recognize specific DNA sequences and bind in the minor groove of the double helix structure. This patent designs and synthesizes a pyrrole-imidazole polyamide polypeptide molecule based on the DNA sequence of the cis-acting element in the PDCD1 gene promoter region. Polypeptide molecules compete with transcription factors to bind these cis-acting elements, block the transcriptional activation of PDCD1 gene, and inhibit the expression of PD1 from the gene transcription level.

The invention discloses a pyrrole-imidazole polyamide polypeptide molecule for inhibiting the transcription of programmed death receptor 1 gene. The sequence of the molecule is: 1. PyPyPyIm-γ-PyPyImIm; 2. PyPyImIm-γ-PyPyImIm. Im is methylimidazole, Py is methylpyrrole, and γ is γ-aminobutyric acid. 1A, 1B, 1C, 2A, 2B, 2C and 2D are different modifications on 1 and 2, respectively.

One aspect of the present invention provides a compound that inhibits the expression of programmed death receptor 1 and a pharmaceutically acceptable salt thereof, which has the structure shown in formula I

wherein, A is selected from C or N;

R ₁ is selected from H, CH ₃ , (CH ₂ ) ₃ -N(CH ₃ ) ₂ , (CH ₂ ) ₂ -N(CH ₃ ) ₂ , CH ₂ -N(CH ₃ ) ₂ , (CH ₂ ) ₃ -N( _{CH3 ) CH2CH2NHR3 ;}

R ₂ is selected from H, CH ₃ ,

R ₃ is selected from H, CH ₃ ,

In a technical scheme of the present invention, there is provided a compound that inhibits the expression of programmed death receptor 1 and a pharmaceutically acceptable salt thereof, which has the structure shown in formula I

Among them, A is C;

或者R ₁ is selected from H, CH ₃ , (CH ₂ ) ₃ -N(CH ₃ ) ₂ , (CH ₂ ) ₂ -N(CH ₃ ) ₂ , CH ₂ -N(CH ₃ ) ₂ ; and R ₂ is selected from

or

R ₁ is selected from (CH ₂ ) ₃ -N(CH ₃ )CH ₂ CH ₂ NHR ₃ ; and R ₂ is selected from H, CH ₃ ; R ₃ is selected from H, CH ₃ ,

In the technical scheme of the present invention, there is provided a compound that inhibits the expression of programmed death receptor 1 and a pharmaceutically acceptable salt thereof, which has the structure shown in formula I

Among them, A is N;

或者R ₁ is selected from H, CH ₃ , (CH ₂ ) ₃ -N(CH ₃ ) ₂ , (CH ₂ ) ₂ -N(CH ₃ ) ₂ , CH ₂ -N(CH ₃ ) ₂ ; and R ₂ is selected from

or

R ₁ is selected from (CH ₂ ) ₃ -N(CH ₃ )CH ₂ CH ₂ NHR ₃ ; and R ₂ is selected from H, CH ₃ ; R ₃ is selected from H, CH ₃ ,

In a specific embodiment, the structure of formula I is selected from one of 1A, 1B, 1C, 2A, 2B, 2C or 2D.

Another aspect of the present invention provides the use of the above compounds and pharmaceutically acceptable salts thereof in the preparation of a medicament for inhibiting the expression of PD-1 on the cell surface.

Another aspect of the present invention provides a method for regulating the expression of PD1 gene in cells in vitro, the method comprising the step of applying the compound of the present invention or a pharmaceutically acceptable salt thereof to the cells to be treated.

Another aspect of the present invention provides the use in the preparation of a medicine for treating a disease with abnormal expression or regulation of PD-1 gene, preferably, the disease with abnormal expression or regulation of PD-1 gene is selected from cancer, viral infection or self immune disease.

Cancer is selected from diseases in which immortal cell proliferation exists in an organ or body tissue. Preferred are ovarian cancer, leukemia, lung cancer, colorectal cancer/colon cancer, CNS cancer, melanoma, renal cell carcinoma, plasmacytoma/myeloma, prostate cancer, breast cancer, and the like.

Autoimmune diseases are selected from diseases in which a subject develops a damaging immune response to its own tissues. Preferred are Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple Sclerosis, myasthenia gravis and diabetes.

Yet another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a preparation method of the aforementioned compound, comprising the steps of:

1) Coupling 2 pieces of 4-amino-1-methyl-2-carboxy-pyrrole and 2 pieces of 4-amino-1-methyl-2-carboxy-imidazole in sequence on a solid-phase resin; or

1) Coupling 3 pieces of 4-amino-1-methyl-2-carboxy-pyrrole and 1 piece of 4-amino-1-methyl-2-carboxy-imidazole sequentially on the solid phase resin;

2) coupling the carboxyl group of 2,4-diaminobutyric acid on the 4-amino group of the imidazolyl group obtained in step 1);

3) Coupling 2 pieces of 4-amino-1-methyl-2-carboxy-pyrrole and 1 piece of 4-amino-1 on the 4-amino group of 2,4-diaminobutyric acid of the intermediate obtained in step 2) in turn - methyl-2-carboxy-imidazole and one 1-methyl-2-carboxy-imidazole;

4) cracking the resin, and modifying isophthalic acid, Hoechst acid derivative Ht-1, Hoechst acid derivative Ht-2 or folic acid FA on the intermediate obtained in step 3) to obtain a final product; or

4) Modifying isophthalic acid, Hoechst acid derivative Ht-1, Hoechst acid derivative Ht-2 or folic acid FA on the intermediate obtained in step 3), and cleaving the resin to obtain the final product.

Wherein step 1) is:

Remove the protective group on the solid phase resin phenylhydrazine, couple 2 4-amino-1-methyl-2-carboxy-pyrrole, and 2 4-amino-1-methyl-2-carboxy-imidazole, or

Remove the protective group on the solid-phase resin phenylhydrazine, and couple 3 4-amino-1-methyl-2-carboxy-pyrrole and 1 4-amino-1-methyl-2-carboxy-imidazole;

Preferably, the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:

Activation of the carboxyl group on 4-tert-butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid, and coupling with the deprotected aniline solid-phase synthetic resin or the amino group on the intermediate; removal The amino protecting group of tert-butoxycarbonyl;

More preferably, the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:

4-tert-Butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid and triphosgene are dissolved together in an organic solvent, and trimethylpyridine is added dropwise. After the reaction is complete, an alkali agent is added and the protection is removed. The amino group on the base intermediate is coupled and mixed, and the reaction is completed in an inert atmosphere; TFA/phenol/H ₂ O (v:v:v=92:5:2.5) mixed solution removes the tert-butoxycarbonyl protecting group .

The step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) or 3) is:

Activation of the carboxyl group on 4-tert-butoxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid, and coupling with the deprotected aniline solid-phase synthetic resin or the amino group on the intermediate; removal The amino protecting group of tert-butoxycarbonyl;

More preferably, the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) or 3) is:

4-tert-Butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid and triphosgene are dissolved together in an organic solvent, and trimethylpyridine is added dropwise. After the reaction is complete, an alkali agent is added and the protection is removed. The aniline solid-phase synthetic resin based on the base or the amino group on the intermediate is coupled and mixed, and the reaction is completed in an inert atmosphere; the mixed solution of TFA/phenol/H ₂ O (v:v:v=92:5:2.5) is dehydrated. In addition to the tert-butoxycarbonyl protecting group.

In the technical scheme of the present invention, step 2) is to activate R-2-(9-fluorenemethoxycarbonylamino)-4-tert-butoxycarbonylaminobutyric acid, and to couple the amino group on the intermediate obtained in step 1). ; Removal of tert-butoxycarbonyl protecting group;

Preferably, step 2) is to jointly dissolve R-2-(9-fluorenylmethoxycarbonylamino)-4-tert-butoxycarbonylaminobutyric acid and triphosgene in an organic solvent, add collidine dropwise, and the reaction is complete Then, add alkali agent and condensing agent until the reaction is complete, and carry out coupling and mixing with the amino group on the deprotected intermediate, and under inert atmosphere until the reaction is complete; TFA/phenol/H ₂ O (v:v:v =92:5:2.5) mixed solution to remove the tert-butoxycarbonyl protecting group.

In the technical scheme of the present invention, the step of coupling 1-methyl-2-carboxy-imidazole in step 3) is:

The carboxyl group on 1-methyl-1H-imidazole-2-carboxylic acid is activated and coupled with the amino group on the intermediate obtained in the previous step of deprotection; then the Fmoc protecting group is removed.

Preferably, 1-methyl-1H-imidazole-2-carboxylic acid and a condensing agent are dissolved in an organic solvent, an alkali agent is added to react to completion, then added to the intermediate obtained in the previous step, and the condensation reaction is performed to completeness under an inert atmosphere; The Fmoc protecting group was removed with 20% piperidine/DMF.

The condensing agent used in the present invention is selected from one or more combinations of HATU, HOBt, HCTU, DCC, DIC, EDC, HBTU, HOAt, PyBOP, PyAOP, BOP-Cl, SOCl2, and oxalyl chloride.

The alkaline agent described in the present invention refers to a compound that can provide an alkaline environment, and is selected from one or more of DIEA, collidine, and triethylamine

In the technical scheme of the present invention, step 4) is to protect the 2-position amino group of butyric acid in the product of step 3) with a tert-butoxycarbonyl group; then add N,N-bis(3-aminopropyl)methylamine, and react until complete post-purification; compound A, a condensing agent and an alkali agent are added to the obtained product to react to complete and then purified to obtain a final product; the compound A is selected from isophthalic acid, folic acid, Hoechst acid derivative Ht-2 or Hoechst Acid derivative Ht-1.

In the technical scheme of the present invention, step 4) is to activate compound A with a condensing agent and an alkali agent, then react with the product obtained in step ₃ ) to completeness, and react with dimethylaminopropylamine and Cu(OAc) to completeness , purifying to obtain the final product; the compound A is selected from isophthalic acid, folic acid, Hoechst acid derivative Ht-2 or Hoechst acid derivative Ht-1.

beneficial effect

The polyamide molecule disclosed in the invention belongs to the small molecule of the polypeptide class, can realize chemical synthesis, is favorable for large-scale production, and provides more methods for inhibiting the function of PD1.

Description of drawings

Figure 1 is the HRMS spectrum of the compound of formula 1A.

Figure 2 is the HRMS spectrum of the compound of formula 1B

Figure 3 is the HRMS spectrum of the compound of formula 1C.

Figure 4 is the HRMS spectrum of the compound of formula 2A.

Figure 5 is the HRMS spectrum of the compound of formula 2B

Figure 6 is the HRMS spectrum of the compound of formula 2C.

Figure 7 is an HRMS spectrum of the compound of formula 2D.

Figure 8 is the HNMR spectrum of Ht-2.

Fig. 9 is a graph showing the results of the experiment of inhibiting the expression of the programmed death receptor 1 gene by the compounds of the present invention.

Detailed ways

The preparation method of embodiment 1 compound 1A

(a) Resin swelling: 400 mg of Fmoc-protected phenylhydrazine resin (0.66 mmol/g, 0.264 mmol) and 3 mL of CH ₂ Cl ₂ were added to a 10 mL solid-phase reactor, the resin was swelled for 30 min, and CH ₂ Cl ₂ was removed by suction ,spare;

(b) Removal of Fmoc protecting group: Add 3 mL of 20% piperidine/DMF solution to the swollen resin in step (a), bubbling and mixing with _N2 , after 10 min, remove the solvent, and then add 3 mL of 20% piperidine /DMF solution, _N2 bubbling and mixing, after 10min, the resin was washed with DMF (4 × 3mL), and then washed with 3mL of anhydrous DMF, for later use;

(c) Amino acid condensation: 4-tert-butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid (254 mg, 1.056 mmol) and triphosgene (BTC, 128 mg, 0.433 mmol) were dissolved in 2 mL of anhydrous THF, collidine (collidine, 488 μL, 3.696 mmol) was slowly added dropwise to the solution, and the reaction immediately produced a large amount of white precipitate. After the reaction was completed for 3 min, 2 mL of DIEA/DMF solution (5%, v/v) was added, The white precipitate disappeared completely, the reaction solution was transferred to the phenylhydrazine resin from which the protective group was removed in step (b), N ₂ was bubbled and mixed, and the condensation reaction was performed for 0.5-1 h. Wash the resin, spare;

(d) Removal of tert-butoxycarbonyl protecting group: wash with CH ₂ Cl ₂ (2×3 mL), extract CH ₂ Cl ₂ , add 3.0 mL of TFA/phenol/H ₂ O (v:v:v=92: 5:2.5) The tert-butoxycarbonyl protecting group on the condensation product obtained in step (b) was removed from the mixed solution, the solvent was removed after 2 min, and 3.0 mL of TFA/phenol/H ₂ O was added again (v:v:v=92: 5:2.5) The mixed solution was reacted for 20 min, the resin was washed with CH ₂ Cl ₂ (2×3 mL) and DMF (4×3 mL), and then the resin was washed with 3 mL of anhydrous DMF, for later use;

Repeat the above-mentioned condensation and deprotection steps (c) and (d) until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (1) is completed;

(e) Amino acid condensation: 4-tert-butoxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (254 mg, 1.056 mmol) and triphosgene (BTC, 128 mg, 0.433 mmol) were dissolved in 2 mL of anhydrous THF, collidine (collidine, 488 μL, 3.696 mmol) was slowly added dropwise to the solution, and the reaction immediately produced a large amount of white precipitate. After the reaction was completed for 3 min, 2 mL of DIEA/DMF solution (5%, v/v) was added, The white precipitate disappeared completely, the reaction solution was transferred to the phenylhydrazine resin from which the protective group was removed in step (b), N ₂ was bubbled and mixed, and the condensation reaction was performed for 0.5-1 h. The resin is washed to obtain the peptide supported on the phenylhydrazine resin represented by the formula (2);

(g) Condensation of γ-amino acids: R-2-(9-fluorenylmethoxycarbonylamino)-4-tert-butoxycarbonylaminobutyric acid (465 mg, 1.056 mmol) and triphosgene (128 mg, 0.433 mmol) were dissolved in 2mL of anhydrous THF, to this solution was slowly added dropwise collidine (488μL, 3.696mmol), the reaction immediately produced a large number of white precipitates, the reaction was completed for 1min, HOAt (144mg, 1.056mmol) was added, and then 2mL of DIEA/DMF was added The solution (5%, v/v) was reacted for 5 min, the white precipitate disappeared completely, and the reaction solution was transferred to the linear peptide (NH ₂ -Im-Im-Py) supported on the phenylhydrazine resin represented by formula (1). -Py-phenylhydrazine resin), N ₂ was bubbled and mixed, the condensation reaction was performed for 0.5-1 h, the reaction solution was removed, and the resin was washed with DMF (4×3 mL) to obtain the supported phenylhydrazine resin shown in formula (3). on the peptide;

(h) repeating the deprotection and condensation protection steps (d) and (c), wherein, until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (4) is completed;

(i) repeating deprotection and condensation protection steps (d) and (e), wherein, until the completion of formula (5)

Synthesis of the indicated peptide supported on a phenylhydrazine resin;

(j) Condensation of terminal amino acids: 1-Methyl-1H-imidazole-2-carboxylic acid (132 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) were dissolved in 3 mL of dry DMF, and DIEA (350 μL, 2.112 mmol) was added ), reacted for 5min, transferred the reaction solution to the peptide supported on the phenylhydrazine resin shown in the formula (4) obtained in step (h), bubbling and mixing with N ₂ , the condensation reaction was performed for 2h, and the reaction solution was removed, The resin was washed with DMF (4×3 mL), and the Fmoc protecting group in the peptide supported on the phenylhydrazine resin represented by the formula (5) was removed by the method of step (b) to obtain the supported peptide represented by the formula (6). Peptides on phenylhydrazine resin;

(k) Boc ₂ O (243 μL, 1.056 mmol) was dissolved in 3 mL of anhydrous DMF, DIEA (350 μL, 2.112 mmol) was added, and the reaction solution was transferred to the Fmoc-removed formula (6) supported on phenylhydrazine In the peptide on the resin, _N2 was bubbled and mixed, and the condensation reaction was carried out for 20min. The reaction solution was removed, and the resin was washed with DMF (4×3 mL) to obtain the Boc-protected peptide represented by the formula (7) supported on the phenylhydrazine resin;

The resin obtained above was taken out, 1 mL of DMF, 200 μL of N,N-bis(3-aminopropyl) methylamine were added, the reaction was shaken at 90° C. for 1 h, cooled to room temperature, the resin was filtered off, and the resin was washed with 20 mL of CH ₂ Cl ₂ The organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic elution over 5 min, 10% to 100% acetonitrile- _H2O (with 1% TFA) gradient elution for 25 min, retention time _TR = 16 min, the product was collected, freeze-dried to obtain a pale yellow solid, which was used for later use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add isophthalic acid IPA (0.99 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature for 2 h, and use Semi-preparative HPLC purification: 10% acetonitrile- _H2O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile- _H2O (containing 1% TFA) gradient elution 25 min, retention Time _TR = 18 min, collect the product, freeze-dry to obtain a solid; dissolve the solid in 1 mL of CH ₂ Cl ₂ , add 1 mL of TFA under ice bath, react at 0°C for 1 h, add 20 mL of cold ether, collect the precipitate by centrifugation, and use semi-prepared HPLC purification: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min, retention time T _R = 15min, the product was collected and lyophilized to obtain compound 1A as a pale yellow solid product,

Its HRMS is shown in Figure 1.

HRMS(ESI) m/z: [M+H] ⁺ 1358.6041.

The preparation method of embodiment 2 compound 1B

The peptide supported on the phenylhydrazine resin represented by the formula (7) was prepared according to the same procedure as in Example 1,

The resin obtained above was taken out, 1 mL of DMF, 200 μL of N,N-bis(3-aminopropyl) methylamine were added, the reaction was shaken at 90° C. for 1 h, cooled to room temperature, the resin was filtered off, and the resin was washed with 20 mL of CH ₂ Cl ₂ The organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic elution over 5 min, 10% to 100% acetonitrile- _H2O (with 1% TFA) gradient elution for 25 min, retention time _TR = 16 min, the product was collected, freeze-dried to obtain a pale yellow solid, which was used for later use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add Hoechst acid derivative Ht-2 (2.7 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature The reaction was carried out for 2 h, and purified by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution for 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution Removed for 25 min, retention time _TR = 18 min, collected the product, freeze-dried to obtain a solid; the solid was dissolved in 1 mL of CH ₂ Cl ₂ , 1 mL of TFA was added under an ice bath, reacted at 0°C for 1 h, added with 20 mL of cold ether, and the precipitate was collected by centrifugation , purified by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min , retention time _TR = 17min, the product was collected and lyophilized to obtain compound 1B as a pale yellow solid product,

Its HRMS is shown in Figure 2.

HRMS(ESI)m/z:[M+H] ⁺ : 1644.7748

Wherein, the structure and synthetic route of Hoechst acid derivative Ht-2 are as follows:

Ht-2-B (11.4 g, 44.2 mmol) was combined with 4-(5-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)benzene-1,2 -Diamine (Ht-2-A, 10 g, 31.0 mmol, see Inorg. Chem. 1998, 37, 6018-6022 for the preparation method) was dissolved in acetic acid (100 mL), and the reaction was heated under reflux in an oil bath for 4 hours. The acetic acid was evaporated under reduced pressure, cooled to room temperature, and purified by column chromatography to obtain a grass-green solid. The obtained product was dissolved in methanol (100ml), magnetic stirring, nitrogen protection, ice-water bath cooling, slowly dropwise addition of 3.1g sodium hydroxide in 50ml aqueous solution, the dropping was completed, the reaction was carried out at room temperature for 8h, TLC detected that the reaction was complete, methanol was evaporated, diluted hydrochloric acid The pH was adjusted to be acidic, a solid was precipitated, filtered and dried to obtain a yellow-green solid Ht-2, whose HNMR were shown in Figure 8 respectively.

HNMR (DMSO-d ₆ , 400MHz): 2.44(s, 3H), 2.79(s, 2H), 3.23(s, 2H), 6.96(m, 1H), 7.05(br s, 1H), 7.47(m, 1H), 7.69-7.73(m, 2H), 8.08(m, 1H), 8.33-8.49(m, 2H), 8.83(s, 1H), 12.73(br s, 1H), 13.41(br s, 1H) .

The preparation method of embodiment 3 compound 1C

The peptide supported on the phenylhydrazine resin represented by the formula (7) was prepared according to the same procedure as in Example 1,

The resin obtained above was taken out, 1 mL of DMF, 200 μL of N,N-bis(3-aminopropyl) methylamine were added, the reaction was shaken at 90° C. for 1 h, cooled to room temperature, the resin was filtered off, and the resin was washed with 20 mL of CH ₂ Cl ₂ The organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic elution over 5 min, 10% to 100% acetonitrile- _H2O (with 1% TFA) gradient elution for 25 min, retention time _TR = 16 min, the product was collected, freeze-dried to obtain a pale yellow solid, which was used for later use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add folic acid FA (1.8 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature for 2 h, and use a semi-preparative HPLC purification: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min, retention time _TR =18min, collect the product, freeze-dry to obtain a solid; dissolve the solid in 1 mL of CH ₂ Cl ₂ , add 1 mL of TFA under an ice bath, react at 0°C for 1 h, add 20 mL of cold ether, collect the precipitate by centrifugation, and purify by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25min, retention time T _R =17min, The product was collected and lyophilized to obtain compound 1C as a pale yellow solid,

Its HRMS is shown in Figure 3.

HRMS(ESI) m/z: [M+H] ⁺ : 1633.7277.

Among them, the structure of folic acid FA:

The preparation method of embodiment 4 compound 2A

(a) Resin swelling: 400 mg of Fmoc-protected phenylhydrazine resin (0.66 mmol/g, 0.264 mmol) and 3 mL of CH ₂ Cl ₂ were added to a 10 mL solid-phase reactor, the resin was swelled for 30 min, and CH ₂ Cl ₂ was removed by suction ,spare;

(b) Removal of Fmoc protecting group: Add 3 mL of 20% piperidine/DMF solution to the swollen resin in step (a), bubbling and mixing with _N2 , after 10 min, remove the solvent, and then add 3 mL of 20% piperidine /DMF solution, _N2 bubbling and mixing, after 10min, the resin was washed with DMF (4 × 3mL), and then washed with 3mL of anhydrous DMF, for later use;

(c) Amino acid condensation: 4-tert-butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid (254 mg, 1.056 mmol) and triphosgene (BTC, 128 mg, 0.433 mmol) were dissolved in 2 mL of anhydrous THF, collidine (collidine, 488 μL, 3.696 mmol) was slowly added dropwise to the solution, and the reaction immediately produced a large amount of white precipitate. After the reaction was completed for 3 min, 2 mL of DIEA/DMF solution (5%, v/v) was added, The white precipitate disappeared completely, the reaction solution was transferred to the phenylhydrazine resin from which the protective group was removed in step (b), N ₂ was bubbled and mixed, and the condensation reaction was performed for 0.5-1 h. Wash the resin, spare;

(d) Removal of tert-butoxycarbonyl protecting group: wash with CH ₂ Cl ₂ (2×3 mL), extract CH ₂ Cl ₂ , add 3.0 mL of TFA/phenol/H ₂ O (v:v:v=92: 5:2.5) The tert-butoxycarbonyl protecting group on the condensation product obtained in step (b) was removed from the mixed solution, the solvent was removed after 2 min, and 3.0 mL of TFA/phenol/H ₂ O was added again (v:v:v=92: 5:2.5) The mixed solution was reacted for 20 min, the resin was washed with CH ₂ Cl ₂ (2×3 mL) and DMF (4×3 mL), and then the resin was washed with 3 mL of anhydrous DMF, for later use;

Repeat the above-mentioned condensation and deprotection steps (c) and (d) until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (8) is completed;

(e) Amino acid condensation: 4-tert-butoxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (254 mg, 1.056 mmol) and triphosgene (BTC, 128 mg, 0.433 mmol) were dissolved in 2 mL of anhydrous THF, collidine (collidine, 488 μL, 3.696 mmol) was slowly added dropwise to the solution, and the reaction immediately produced a large amount of white precipitate. After the reaction was completed for 3 min, 2 mL of DIEA/DMF solution (5%, v/v) was added, The white precipitate disappeared completely, the reaction solution was transferred to the phenylhydrazine resin from which the protective group was removed in step (b), N ₂ was bubbled and mixed, and the condensation reaction was performed for 0.5-1 h. Wash the resin, spare;

Repeat the above-mentioned condensation and deprotection steps (e) and (d) until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (9) is completed;

(g) Condensation of γ-amino acids: R-2-(9-fluorenylmethoxycarbonylamino)-4-tert-butoxycarbonylaminobutyric acid (465 mg, 1.056 mmol) and triphosgene (128 mg, 0.433 mmol) were dissolved in 2mL of anhydrous THF, to this solution was slowly added dropwise collidine (488μL, 3.696mmol), the reaction immediately produced a large number of white precipitates, the reaction was completed for 1min, HOAt (144mg, 1.056mmol) was added, and then 2mL of DIEA/DMF was added The solution (5%, v/v) was reacted for 5 min, the white precipitate disappeared completely, and the reaction solution was transferred to the linear peptide (NH ₂ -Im-Im-Py) supported on the phenylhydrazine resin represented by formula (9). -Py-phenylhydrazine resin), N ₂ was bubbled and mixed, the condensation reaction was performed for 0.5-1 h, the reaction solution was removed, and the resin was washed with DMF (4×3 mL) to obtain the supported phenylhydrazine resin represented by formula (10). peptides on;

(h) repeating the deprotection and condensation protection steps (d) and (c), wherein, until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (11) is completed;

(i) repeating the deprotection and condensation protection steps (d) and (e), wherein, until the synthesis of the peptide supported on the phenylhydrazine resin represented by the formula (12) is completed;

(j) Condensation of terminal amino acids: 1-Methyl-1H-imidazole-2-carboxylic acid (132 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) were dissolved in 3 mL of dry DMF, and DIEA (350 μL, 2.112 mmol) was added ), reacted for 5min, transferred the reaction solution to the peptide loaded on the phenylhydrazine resin shown in the formula (12) obtained in step (h), bubbling and mixing N ₂ , the condensation reaction was performed for 2h, and the reaction solution was removed, The resin was washed with DMF (4×3 mL); and the Fmoc protecting group in the peptide supported on the phenylhydrazine resin represented by the formula (12) was removed by step (b) in Example 1 to obtain the formula (13). The indicated peptide supported on phenylhydrazine resin;

Boc ₂ O (243 μL, 1.056 mmol) was dissolved in 3 mL of anhydrous DMF, DIEA (350 μL, 2.112 mmol) was added, and the reaction solution was transferred to a phenylhydrazine resin supported by formula (13) to remove Fmoc. In the peptide, _N2 was bubbled and mixed, and the condensation reaction was carried out for 20min. The reaction solution was removed, and the resin was washed with DMF (4×3 mL) to obtain the Boc-protected peptide represented by formula (14) supported on phenylhydrazine resin;

The peptide supported on the phenylhydrazine resin represented by the above formula (14) was taken out, 1 mL of DMF and 200 μL of N,N-bis(3-aminopropyl) methylamine were added, and the reaction was shaken at 90 °C for 1 h, cooled to room temperature, The resin was filtered off and washed with 20 mL _{CH2Cl2 ;} the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic over 5 min, 10% Gradient elution to 100% acetonitrile-H ₂ O (containing 1% TFA) for 25 min, retention time _TR = 16 min, the product was collected and lyophilized to obtain a pale yellow solid for use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add isophthalic acid IPA (0.99 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature for 2 h, and use Semi-preparative HPLC purification: 10% acetonitrile- _H2O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile- _H2O (containing 1% TFA) gradient elution 25 min, retention Time _TR = 18 min, collect the product, freeze-dry to obtain a solid; dissolve the solid in 1 mL of CH ₂ Cl ₂ , add 1 mL of TFA under ice bath, react at 0°C for 1 h, add 20 mL of cold ether, collect the precipitate by centrifugation, and use semi-prepared HPLC purification: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min, retention time T _R =15min, the product was collected and lyophilized to obtain compound 2A as a pale yellow solid product,

Its HRMS is shown in Figure 4.

HRMS (ESI) m/z: [M+H] ⁺ : 1359.5992.

The preparation method of embodiment 5 compound 2B

The peptide represented by the formula (14) supported on a phenylhydrazine resin was prepared in the same manner as in Example 4.

The peptide supported on the phenylhydrazine resin represented by the formula (14) obtained above was taken out, 1 mL of DMF, 200 μL of N,N-bis(3-aminopropyl) methylamine were added, and the reaction was shaken at 90 °C for 1 h, and cooled to room temperature. , the resin was filtered off, and the resin was washed with 20 mL of CH ₂ Cl ₂ ; the organic phase was concentrated, and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isogradient elution over 5 min, 10 % to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution for 25 min, retention time _TR = 16 min, the product was collected and lyophilized to obtain a pale yellow solid for use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add Hoechst acid derivative Ht-2 (2.7 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature The reaction was carried out for 2 h, and purified by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution for 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution Removed for 25 min, retention time _TR = 18 min, collected the product, freeze-dried to obtain a solid; the solid was dissolved in 1 mL of CH ₂ Cl ₂ , 1 mL of TFA was added under an ice bath, reacted at 0°C for 1 h, added with 20 mL of cold ether, and the precipitate was collected by centrifugation , purified by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min , retention time _TR = 17min, the product was collected and lyophilized to obtain compound 2B as a pale yellow solid product,

Its HRMS is shown in Figure 5.

HRMS(ESI)m/z:[M+H] ⁺ : 1645.7797.

The structure and synthetic route of Hoechst acid derivative Ht-2 are as follows:

Combine Ht-2 (11.4 g, 44.2 mmol) with 4-(5-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)benzene-1,2-di Amine (Ht-2-A, 10 g, 31.0 mmol, see Inorg. Chem. 1998, 37, 6018-6022 for the preparation method) was dissolved in acetic acid (100 mL), and the reaction was heated under reflux in an oil bath for 4 hours. The acetic acid was evaporated under reduced pressure, cooled to room temperature, and purified by column chromatography to obtain a grass-green solid. The obtained product was dissolved in methanol (100ml), magnetic stirring, nitrogen protection, ice-water bath cooling, slowly dropwise addition of 3.1g sodium hydroxide in 50ml aqueous solution, the dropping was completed, the reaction was carried out at room temperature for 8h, TLC detected that the reaction was complete, methanol was evaporated, diluted hydrochloric acid The pH was adjusted to be acidic, a solid was precipitated, filtered and dried to obtain a yellow-green solid Ht-2, whose HNMR were shown in Figure 8 respectively.

HNMR (DMSO-d ₆ , 400MHz): 2.44(s, 3H), 2.79(s, 2H), 3.23(s, 2H), 6.96(m, 1H), 7.05(br s, 1H), 7.47(m, 1H), 7.69-7.73(m, 2H), 8.08(m, 1H), 8.33-8.49(m, 2H), 8.83(s, 1H), 12.73(br s, 1H), 13.41(br s, 1H) .

The preparation method of embodiment 6 compound 2C

The peptide supported on the phenylhydrazine resin represented by the formula (14) was obtained by the same steps as in Example 4;

The peptide supported on the phenylhydrazine resin represented by the above formula (14) was taken out, 1 mL of DMF and 200 μL of N,N-bis(3-aminopropyl) methylamine were added, and the reaction was shaken at 90 °C for 1 h, cooled to room temperature, The resin was filtered off and washed with 20 mL _{CH2Cl2 ;} the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic over 5 min, 10% Gradient elution to 100% acetonitrile-H ₂ O (containing 1% TFA) for 25 min, retention time _TR = 16 min, the product was collected and lyophilized to obtain a pale yellow solid for use;

Dissolve 4 mg (3 μmol) of the above solid in 0.5 mL of anhydrous DMF, add folic acid FA (1.8 mg, 6 μmol), PyBOP (3.1 mg, 6 μmol) and DIEA (5 μL, 30 μmol), shake at room temperature for 2 h, and use a semi-preparative HPLC purification: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25 min, retention time _TR =18min, collect the product, freeze-dry to obtain a solid; dissolve the solid in 1 mL of CH ₂ Cl ₂ , add 1 mL of TFA under an ice bath, react at 0°C for 1 h, add 20 mL of cold ether, collect the precipitate by centrifugation, and purify by semi-preparative HPLC: 10% acetonitrile-H ₂ O (containing 1% TFA) isocratic elution 5min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution 25min, retention time T _R =17min, The product was collected and lyophilized to obtain compound 2C as a pale yellow solid,

Its HRMS is shown in Figure 6.

HRMS(ESI) m/z: [M+H] ⁺ : 1633.7277.

Structure of Folic Acid FA

The preparation method of embodiment 7 compound 2D

The peptide represented by the formula (13) supported on a phenylhydrazine resin was obtained by the same procedure as in Example 4.

(k) The Hoechst acid derivative Ht-1 (539 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) were dissolved in 3 mL of anhydrous DMF, DIEA (350 μL, 2.112 mmol) was added, and the reaction was carried out for 5 min. Transferred to the peptide supported on phenylhydrazine resin represented by formula (13), bubbling and mixing _N2 , condensation reaction for 1 h, and extracting the reaction solution to obtain the peptide supported on phenylhydrazine resin by formula (15).

The structure of the Hoechst acid derivative Ht-1 is as follows (see J.AM.CHEM.SOC.2004, 126, 3736-3747 for the preparation method):

(1) The resin of formula (15) was taken out, 1 mL of DMF, 200 μL of dimethylaminopropylamine and 10 mg of Cu(OAc) ₂ were added, the reaction was shaken at room temperature for 12 h, the resin was filtered off, and the resin was washed with 20 mL of CH ₂ Cl ₂ ; Concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile- _H2O (with 1% TFA) isocratic over 5 min, 10% to 100% acetonitrile- _H2O (1% TFA) gradient Elution for 25min, retention time _TR = 15min, the product was collected and lyophilized to obtain compound 2D as a pale yellow solid,

Its HRMS is shown in Figure 7.

HRMS (ESI) m/z: Theoretical calculated for C ₈₂ H ₉₄ N ₂₉ O ₁₁ [M+H] ⁺ 1660.7682 found: 1660.7652.

Example 8 Compounds inhibit the expression of programmed death receptor 1 gene expression

1) Isolate peripheral blood mononuclear cells from fresh blood and culture with Takara+10% FBS+IL2;

2) The peripheral blood mononuclear cells were inoculated into a 96-well plate, and each well was inoculated with 500,000 cells, and the volume was 150ul; the concentration of the compound was 20uM, and the CD3 antibody was added for stimulation;

3) After culturing for 48 hours, the cells of the 72h group were equally divided into two groups, one of which was added with the same compound again for cultivation, one group was not added with the compound, and one group of the newly added compound was 20uM according to the drug concentration, and the total volume was 200ul;

4) Collect cell samples at 0, 24, 48, and 72 hours, stain the cells with PE-PD1 antibody and FITC-CD3 antibody, and then perform flow detection. The results are shown in Figure 9. From Figure 9, it can be found that the inhibition effect of 2A and 2D is better. The inhibition rate of 2A to PD1 reaches 40% in 48 hours of treatment, while the inhibition rate of 2D is close to 50%.

Claims (11)

1. A compound that inhibits the expression of programmed death receptor 1, the compound having the structure set forth in the following table:

2. use of a compound according to claim 1 and pharmaceutically acceptable salts thereof in the manufacture of a medicament for inhibiting the expression of PD-1 on the surface of a cell.

3. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of a disease in which PD-1 gene expression or regulation is abnormal; the disease with abnormal PD-1 gene expression or regulation is selected from cancer, virus infection or autoimmune disease;

cancer is selected from diseases in which there is unlimited proliferation of cells in an organ or body tissue;

the autoimmune disease is selected from a disease in which the subject exerts a destructive immune response to its own tissues.

4. The use according to claim 3, wherein the cancer is selected from ovarian cancer, leukemia, lung cancer, colorectal/colon cancer, CNS cancer, melanoma, renal cell carcinoma, plasmacytoma/myeloma, prostate cancer, breast cancer.

5. Use according to claim 3, wherein the autoimmune disease is selected from hashimoto's thyroiditis, systemic lupus erythematosus, sjogren's syndrome, Graves ' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and diabetes.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

7. A process for the preparation of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the steps of:

1) sequentially coupling 2 4-amino-1-methyl-2-carboxy-pyrroles and 2 4-amino-1-methyl-2-carboxy-imidazoles on a solid phase resin; or

Sequentially coupling 3 4-amino-1-methyl-2-carboxy-pyrroles and 1 4-amino-1-methyl-2-carboxy-imidazole on a solid phase resin;

2) coupling carboxyl of 2, 4-diaminobutyric acid on amino at 4-position of imidazolyl of the intermediate obtained in the step 1);

3) sequentially coupling 2 4-amino-1-methyl-2-carboxy-pyrroles, 1 4-amino-1-methyl-2-carboxy-imidazole and one 1-methyl-2-carboxy-imidazole to the 4-amino group of 2, 4-diaminobutyric acid of the intermediate obtained in step 2);

4) cracking the resin, and modifying isophthalic acid, a herstonic acid derivative Ht-1, a herstonic acid derivative Ht-2 or folic acid FA on the intermediate obtained in the step 3) to obtain a final product; or

Modifying isophthalic acid, a hoechst acid derivative Ht-1, a hoechst acid derivative Ht-2 or folic acid FA on the intermediate obtained in the step 3), and cracking the resin to obtain the final product.

8. The method of claim 7, wherein step 1) is:

removing the protective group from the solid-phase resin phenylhydrazine, coupling 2 4-amino-1-methyl-2-carboxy-pyrroles with 2 4-amino-1-methyl-2-carboxy-imidazoles, or

Removing the protective group on the solid-phase resin phenylhydrazine, and coupling 3 4-amino-1-methyl-2-carboxyl-pyrrole and 1 4-amino-1-methyl-2-carboxyl-imidazole.

9. The method according to claim 8, wherein the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:

activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid, and coupling with amino on aniline solid-phase synthetic resin or intermediate with a removed protecting group; removing the amino protecting group of the tert-butyloxycarbonyl group;

the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) or 3) is:

activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid, and coupling with amino on aniline solid-phase synthetic resin or intermediate with a removed protecting group; and removing the amino protecting group of the tert-butyloxycarbonyl group.

10. The preparation method according to claim 9, wherein step 2) is activating R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid, and coupling with an amino group on the intermediate obtained in step 1); and removing the amino protecting group of the tert-butyloxycarbonyl group.

11. The method of claim 10, wherein the step of coupling 1-methyl-2-carboxy-imidazole in step 3) is:

activating carboxyl on the 1-methyl-1H-imidazole-2-carboxylic acid, and coupling with amino on the intermediate obtained in the previous step of removing the protecting group.

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