Antiviral polypeptide analogue

Abstract

An antiviral polypeptide analogue, a pharmaceutical composition containing the antiviral polypeptide analogue, a preparation method, and use thereof. The antiviral polypeptide analogue exhibits a good half-life and good solubility while maintaining good inhibitory activity against viral infections.

Description

Antiviral peptide analogs Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and in particular to the field of long-acting biological drug development, and specifically to an antiviral peptide analog. Background Art

[0002] Chronic hepatitis B virus infection is a major global public health issue, with approximately 240 million people infected worldwide. Liver cirrhosis and liver cancer caused by hepatitis B virus (HBV) also have high morbidity and mortality rates. Approved antiviral drugs for the treatment of hepatitis B include pegylated interferon α and six nucleoside analogs. The former works by inducing adjacent cells to produce antiviral proteins, while the latter blocks viral replication by inhibiting the activity of HBV-DNA polymerase. However, pegylated interferon α has side effects such as flu-like symptoms and abnormal blood routine tests, and nucleoside analogs have disadvantages such as drug resistance and a large number of people who are not suitable for the disease. Therefore, there is an urgent need for anti-hepatitis B virus therapeutic drugs with new mechanisms of action in clinical practice.

[0003] Myrcludex B, licensed from Vision7 by MYR GmbH, is being developed in collaboration between the University of Heidelberg and Hepatera. Derived from positions 2-48 of the HBV-L protein, Myrcludex B consists of 47 naturally occurring amino acid residues and an N-terminal myristoyl group. It is a viral entry inhibitor that targets the sodium-taurocholate co-transporting polypeptide (NTCP).

[0004] In September 2020, Myrcludex B was launched in Europe as a treatment for hepatitis D. Its low-dose, intermittent, once-daily injection regimen is quite painful for patients. Extending the half-life of Myrcludex B could significantly improve patient compliance.

[0005] Summary of the Invention

[0006] The present disclosure provides polypeptide analogs, and also provides pharmaceutical compositions comprising the polypeptide analogs, preparation methods, and uses.

[0007] Peptide analogs

[0008] The present disclosure provides a polypeptide analogue, the general structural formula of which is C 14 -P1-U1;

[0009] Wherein, P1 comprises the following amino acid sequence:

[0010] GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX1DHWPEANX2VG (SEQ ID NO: 1), wherein X1 is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, Glu, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met; X2 is Lys, and its ε-amino group has an acylated modification group; C 14 is a myristoyl group at the N-terminus of P1; U1 is a PAS sequence connected to the C-terminus of P1, wherein the PAS sequence consists of Pro, Ala and / or Ser and has 0-300 amino acid residues.

[0011] In some embodiments, the polypeptide analog has the following structural formula:

[0012] Among them, C 14 is a myristoyl group, X1 is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, Glu, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, T1 is the modifying group of the acylation modification, and U1 is a PAS sequence connected to the C-terminus of P1, wherein the PAS sequence consists of Pro, Ala and / or Ser and has 0-300 amino acid residues.

[0013] In some embodiments, the X1 is selected from β-Ala, GABA, Aib, Abu, Arg or Cys, preferably Arg.

[0014] In some embodiments, the polypeptide analog has the following structural formula:

[0015] Among them, C 14 is a myristoyl group, T1 is the acylation modification group, U1 is a PAS sequence connected to the C-terminus of P1, and the PAS sequence consists of Pro, Ala and / or Ser and has 0-300 amino acid residues.

[0016] In some embodiments, the acylation-modified modifying group comprises a fatty acid, which can non-covalently bind to albumin to extend the half-life of the polypeptide analog.

[0017] In some embodiments, the general formula of the modification group of X2 is -(AEEA) m-(Xaa) n -CO-(CH2) p -R1, whose structural formula is:

[0018] Among them, the wavy line represents the connection position between the modifying group and the ε-amino group of X2.

[0019] In some embodiments, p is 6-20, 10-20, 14-20, 14-18, or 16-18. In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0020] In some embodiments, m is 0, 1, 2, 3, 4, or 5. In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 0. In other embodiments, m is 2.

[0021] In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0. In other embodiments, n is 1.

[0022] In some embodiments, the Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val, or Met. In some embodiments, the Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro, or Phe. In some embodiments, the Xaa is γ-Glu. In other embodiments, the Xaa is Pro.

[0023] In some embodiments, said R1 is -CH3 or -COOH.

[0024] In some embodiments, the general formula of the modification group of X2 is: -CO-(CH2) p -R1, whose structural formula is:

[0025] Among them, the wavy line represents the connection position between the modifying group and the ε-amino group of X2.

[0026] In some embodiments, p is 6-20, 10-20, 14-20, 14-18, or 16-18. In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0027] In some embodiments, said R1 is -CH3 or -COOH.

[0028] In some specific embodiments, the modification group of X2 is selected from -CO-(CH2)6-CH3, -CO-(CH2)8-CH3, -CO-(CH2) 10 -CH3, -CO-(CH2) 12 -CH3, -CO-(CH2) 14 -CH3, -CO-(CH2) 16 -CH3 or -CO-(CH2) 18 -CH3; specifically, their structural formulas are:

[0029] Among them, the wavy line represents the connection position between the modifying group and the ε-amino group of X2.

[0030] In other embodiments, the modifying group of X2 is of the general formula:

[0031] Among them, the wavy line represents the connection position between the modifying group and the ε-amino group of X2.

[0032] In some embodiments, p is 6-20, 10-20, 14-20, 14-18, or 16-18. In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0033] In some embodiments, the Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val, or Met. In some embodiments, the Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro, or Phe. In some embodiments, the Xaa is γ-Glu. In other embodiments, the Xaa is Pro.

[0034] In some embodiments, said R1 is -CH3 or -COOH.

[0035] In some specific embodiments, the modification group of X2 is selected from -AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH, -AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH, -AEEA-AEEA-γ-Glu-CO-(CH2) 10 -COOH, -AEEA-AEEA-γ-Glu-CO-(CH2) 12 -COOH, -AEEA-AEEA-γ-Glu-CO-(CH2) 14 -COOH, -AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH, -AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH; specifically, their structural formulas are:

[0036] Among them, the wavy line represents the connection position between the modifying group and the ε-amino group of X2.

[0037] In some embodiments, the PAS sequence has 10-300, 30-300, 50-300 or 30-100 amino acid residues. In some embodiments, the PAS sequence has 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 amino acid residues. In some embodiments, the PAS sequence has 30, 50, 70, 100, 150, 200 or 300 amino acid residues. In some embodiments, the PAS sequence has 30 amino acid residues. In some embodiments, the PAS sequence has 50 amino acid residues. In some embodiments, the PAS sequence has 100 amino acid residues.

[0038] In some embodiments, in the PAS sequence, Pro accounts for 4% and above, 5% and above, 6% and above or 8% and above of the total number of amino acid residues, preferably 10% and above or 15% and above, more preferably 20% and above. In some embodiments, in the PAS sequence, Pro accounts for 40% and below or 35% and below of the total number of amino acid residues, preferably 30% and below. In some embodiments, in the PAS sequence, Pro accounts for 4%-40%, 10%-40%, 15%-40%, 15%-35%, 20%-35% or 20%-30% of the total number of amino acid residues. In some embodiments, in the PAS sequence, Pro accounts for about 4%, about 10%, about 15%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 35% or about 40% of the total number of amino acid residues.

[0039] In some embodiments, the PAS sequence comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the PAS sequence comprises an amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8, or an amino acid sequence obtained by repeating and / or permuting one or more of these sequences in whole or in part. In some embodiments, the amino acid sequence of the PAS sequence is as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the amino acid sequence of the PAS sequence is as shown in SEQ ID NO: 2. In some embodiments, the amino acid sequence of the PAS sequence is as shown in SEQ ID NO: 3. In some embodiments, the amino acid sequence of the PAS sequence is as shown in SEQ ID NO: 5. In some embodiments, the C-terminus of the PAS sequence is in the carboxyl form (ie, the C-terminus has a -COOH group) or in the amide form formed by amidation of a free carboxyl group (ie, the C-terminus has a -CONH2 group), preferably in the carboxyl form.

[0040] The amino acid sequences of exemplary PAS sequences of the present disclosure are provided in Table S1 below.

[0041] Table S1 Amino acid sequence of PAS sequence

[0042] In some embodiments, the PAS sequence has 0 amino acid residues, i.e., the PAS sequence is deleted. In some of these embodiments, the C-terminus of the P1 is in the form of a carboxyl group (i.e., the C-terminus has a -COOH group) or an amide form formed after amidation of a free carboxyl group (i.e., the C-terminus has a -CONH2 group).

[0043] In some embodiments, the polypeptide analog is selected from the following structural formula:

[0044] Among them, C 14is myristoyl, p is 6-20, 10-20, 14-20, 14-18 or 16-18, m is 0, 1, 2, 3, 4 or 5, n is 0, 1, 2 or 3, R1 is -CH3 or -COOH, and Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met.

[0045] In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0046] In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 0. In other embodiments, m is 2.

[0047] In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0. In other embodiments, n is 1.

[0048] In some embodiments, the Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro, or Phe. In some embodiments, the Xaa is γ-Glu. In other embodiments, the Xaa is Pro.

[0049] In some specific embodiments, the polypeptide analog is selected from the following structural formula:

[0050] Among them, C 14 In some embodiments, p is myristoyl, p is 6-20, 10-20, 14-20, 14-18, or 16-18, and R1 is -CH3 or -COOH. In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0051] In some specific embodiments, the polypeptide analog is selected from the following structural formula:

[0052] Among them, C 14is myristoyl, p is 6-20, 10-20, 14-20, 14-18 or 16-18, R1 is -CH3 or -COOH, and Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met.

[0053] In some embodiments, p is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 16. In some embodiments, p is 18. In some embodiments, p is 20.

[0054] In some embodiments, the Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro, or Phe.

[0055] In some embodiments, the Xaa is γ-Glu. In other embodiments, the Xaa is Pro.

[0056] Exemplary polypeptide analogs provided herein have the following structural formula:

[0057] 1.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)6-CH3)-PAS100

[0058] 2.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)8-CH3)-PAS100

[0059] 3.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 10 -CH3)-PAS100

[0060] 4.Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 12 -CH3)-PAS100

[0061] 5.Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 14 -CH3)-PAS100

[0062] 6.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 16 -CH3)-PAS100

[0063] 7.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 18 -CH3)-PAS100

[0064] 8.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS100

[0065] 9.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS100

[0066] 10.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 -COOH)-PAS100

[0067] 11.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 -COOH)-PAS100

[0068] 12.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 -COOH)-PAS100

[0069] 13.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH)-PAS100

[0070] 14.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100

[0071] 15.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)6-CH3)-PAS50

[0072] 16.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)8-CH3)-PAS50

[0073] 17.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 10 -CH3)-PAS50

[0074] 18.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 12 -CH3)-PAS50

[0075] 19.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 14 -CH3)-PAS50

[0076] 20.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 16 -CH3)-PAS50

[0077] 21.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 18 -CH3)-PAS50

[0078] 22.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS50

[0079] 23.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS50

[0080] 24.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10-COOH)-PAS50

[0081] 25.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 -COOH)-PAS50

[0082] 26.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 -COOH)-PAS50

[0083] 27.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH)-PAS50

[0084] 28.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS50

[0085] 29.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)6-CH3)-PAS30

[0086] 30.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2)8-CH3)-PAS30

[0087] 31.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 10 -CH3)-PAS3

[0088] 32.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 12 -CH3)-PAS3

[0089] 33.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 14 -CH3)-PAS3

[0090] 34.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 16 -CH3)-PAS3

[0091] 35.Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 18 -CH3)-PAS3

[0092] 36.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS30

[0093] 37.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS30

[0094] 38.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 -COOH)-PAS30

[0095] 39.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 -COOH)-PAS30

[0096] 40.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 -COOH)-PAS30

[0097] 41.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH)-PAS30

[0098] 42.Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS30

[0099] The polypeptide analogs disclosed herein have good NTCP binding ability. In some embodiments, the Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 (i.e., the polypeptide analogue shown in structural formula 14) has good NTCP binding ability.

[0100] The polypeptide analogs disclosed herein exhibit good half-life while maintaining good viral infection inhibitory activity. The polypeptide analogs disclosed herein have good in vivo antiviral activity. In some embodiments, the Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 has good in vivo antiviral activity. The polypeptide analogs disclosed herein have good half-life in mice. In some embodiments, the Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 has a good half-life in mice. The polypeptide analogs disclosed herein have a good half-life in dogs. In some embodiments, the Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 has a good half-life in dogs.

[0101] The polypeptide analogs disclosed herein have good solubility. In some embodiments, the Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 has good solubility in mice. In some embodiments, when the P1 in the polypeptide analog of the present disclosure has other acylation modifications except the ε-amino acid modification of Lys45 (X2), the polypeptide analog loses its activity.

[0102] Intermediate compounds

[0103] The present disclosure provides a polypeptide intermediate compound comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the polypeptide intermediate compound comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the polypeptide intermediate compound has an amino acid sequence as shown in SEQ ID NO: 9.

[0104] The present disclosure also provides a polypeptide intermediate compound comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the polypeptide intermediate compound comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the polypeptide intermediate compound has an amino acid sequence as shown in SEQ ID NO: 10.

[0105] The present disclosure also provides a myristoylated polypeptide intermediate compound, the structure of which is shown below:

[0106] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPA PSAPAAASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS,

[0107] Among them, C 14 Myristoyl.

[0108] The present disclosure also provides a myristoylated polypeptide intermediate compound, the structure of which is shown below:

[0109] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP,

[0110] Among them, C 14 Myristoyl.

[0111] Pharmaceutical composition

[0112] The present disclosure provides a pharmaceutical composition comprising the polypeptide analog or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

[0113] In some embodiments, the pharmaceutical composition contains at least about 0.1 mg, at least about 0.2 mg, or at least about 0.3 mg, or at least about 0.4 mg, or at least about 0.6 mg, or at least about 0.8 mg, or at least about 1 mg, or at least about 1.5 mg, or at least about 2 mg, or at least about 2.5 mg, or at least about 3 mg, or at least about 5 mg, or at least about 7 mg, or at least about 10 mg, or at least about 12 mg, or at least about 15 mg, or at least about 20 mg, or at least about 30 mg, or at least about 50 mg, or at least about 70 mg, or at least about 100 mg of a polypeptide analog or a pharmaceutically acceptable salt thereof.

[0114] In some embodiments, the pharmaceutical composition is a liquid preparation. In some embodiments, the pharmaceutical composition is an injection. In some embodiments, the pharmaceutical composition is a solid preparation. In some embodiments, the pharmaceutical composition is a lyophilized preparation.

[0115] Preparation method

[0116] In some embodiments, the amino acid peptide chain portion of the polypeptide analogs disclosed herein is obtained by recombinant expression. Specifically, Escherichia coli is constructed by fusion expression of a tag and a target polypeptide. The fusion protein is obtained through microbial culture, induction, and amplification. The tag is then specifically removed using a proteolytic enzyme such as enterokinase or ubiquitin-like specific protease 1 (Ulp-1), followed by purification to obtain the target polypeptide.

[0117] In some embodiments, the peptide chain portion composed of amino acids in the polypeptide analogs of the present disclosure is obtained by chemical synthesis.

[0118] In some embodiments, the peptide chain portion composed of amino acids in the polypeptide analogs of the present disclosure is obtained by a combination of recombinant expression and chemical synthesis.

[0119] In some embodiments, the preparation method of the polypeptide analog of the present disclosure includes the following steps: (a) preparing the peptide chain portion composed of amino acids and a modifying group for acylation modification in the P1; (b) performing a myristoylation reaction on the N-terminus of the peptide chain portion; (c) using the modifying group to acylate the ε-amino group of the amino acid residue X2 of the peptide chain portion to obtain the polypeptide analog; and (d) isolating and purifying the polypeptide analog.

[0120] use

[0121] The present disclosure provides a method for inhibiting hepatitis B virus (HBV) and / or hepatitis D virus (HDV) infection of cells, comprising administering an effective amount of the polypeptide analog or pharmaceutical composition of the present disclosure to the cells.

[0122] The present disclosure provides use of the polypeptide analog or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating patients infected with HBV and / or HDV.

[0123] The present disclosure provides use of the polypeptide analogs or pharmaceutical compositions of the present disclosure in the preparation of medicaments for treating patients with hepatitis B, hepatitis D, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease.

[0124] The present disclosure provides a method for treating a patient infected with HBV and / or HDV, comprising administering to the patient a therapeutically effective amount of a polypeptide analog or pharmaceutical composition of the present disclosure.

[0125] The present disclosure provides a method for treating patients with hepatitis B, hepatitis D, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease, comprising administering to the patient a therapeutically effective amount of the polypeptide analog or pharmaceutical composition of the present disclosure.

[0126] Reagent test kit

[0127] The present disclosure provides a kit comprising a polypeptide analog or pharmaceutical composition of the present disclosure. In some embodiments, the kit further comprises instructions for use. The kit may also include other materials required from a commercial and user perspective, such as other buffers, diluents, needles, syringes, and the like.

[0128] The present disclosure also provides the following specific implementation schemes, but the protection scope of the present disclosure is not limited thereto:

[0129] Embodiment 1. A polypeptide analogue having the general structural formula C 14 -P1-U1;

[0130] in,

[0131] P1 contains the following amino acid sequence:

[0132] GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX1DHWPEANX2VG (SEQ ID NO: 1), wherein X1 is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, Glu, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, preferably β-Ala, GABA, Aib, Abu, Arg or Cys, more preferably Arg; X2 is Lys, and its ε-amino group has an acylated modification group;

[0133] C 14 is the myristoyl group at the N-terminus of P1;

[0134] U1 is a PAS sequence connected to the C-terminus of P1. The PAS sequence consists of Pro, Ala and / or Ser and has 30-300 amino acid residues.

[0135] Embodiment 2. The polypeptide analog according to embodiment 1, wherein the structural formula of the modification group of X2 is:

[0136] in,

[0137] p is 6-20, 10-20, 14-20, 14-18 or 16-18,

[0138] m is 0, 1, 2, 3, 4 or 5,

[0139] n is 0, 1, 2 or 3,

[0140] Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met,

[0141] R1 is -CH3 or -COOH,

[0142] Wavy lines represents the connection position between the modifying group and the ε-amino group of X2.

[0143] Embodiment 3. The polypeptide analog according to embodiment 2, wherein m is 0, 1, 2 or 3, preferably 0 or 2.

[0144] Embodiment 4. The polypeptide analog according to embodiment 2 or 3, wherein n is 0 or 1.

[0145] Embodiment 5. The polypeptide analog according to embodiment 2, wherein the modifying group has a structural formula shown in Formula 1 or Formula 2:

[0146] Embodiment 6. The polypeptide analog according to embodiment 5, wherein Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro or Phe, preferably γ-Glu or Pro.

[0147] Embodiment 7. The polypeptide analog according to embodiment 5 or 6, wherein p is 6, 8, 10, 12, 14, 16, 18 or 20, preferably 16 or 18.

[0148] Embodiment 8. The polypeptide analog according to embodiment 1, wherein the modification group of X2 is selected from the following structural formula:

[0149] Embodiment 9. The polypeptide analog according to any one of embodiments 1 to 8, wherein the PAS sequence has 30-100 amino acid residues, preferably 30, 50 or 100 amino acid residues.

[0150] Embodiment 10. The polypeptide analog according to any one of embodiments 1-9, wherein Pro accounts for 10%-40% of the total number of amino acid residues in the PAS sequence.

[0151] Embodiment 11. The polypeptide analog according to any one of embodiments 1-10, wherein the PAS sequence comprises the amino acid sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8.

[0152] Embodiment 12. The polypeptide analog according to any one of embodiments 1-11, wherein the amino acid sequence of the PAS sequence is as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8.

[0153] Embodiment 13. The polypeptide analog according to embodiment 1, wherein the polypeptide analog is selected from the following structural formula:

[0154] Among them, C 14is myristoyl; p is 6-20, 10-20, 14-20, 14-18 or 16-18, preferably 6, 8, 10, 12, 14, 16, 18 or 20; Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, preferably GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro or Phe, more preferably γ-Glu or Pro; R1 is -CH3 or -COOH.

[0155] Embodiment 14. The polypeptide analog according to embodiment 1, wherein the polypeptide analog is selected from the following structural formula:

[0156] Among them, C 14 Myristoyl.

[0157] Embodiment 15. A method for preparing the polypeptide analog of any one of embodiments 1-14, comprising the following steps:

[0158] a) preparing the peptide chain portion composed of amino acids and the modifying group for acylation modification in P1 according to any one of embodiments 1 to 14;

[0159] b) performing a myristoylation reaction on the N-terminus of the peptide chain portion;

[0160] c) acylating the ε-amino group of the amino acid residue X2 of the peptide chain portion with the modifying group to obtain the polypeptide analog; and

[0161] d) isolating and purifying the polypeptide analog.

[0162] Embodiment 16. A pharmaceutical composition comprising the polypeptide analog according to any one of embodiments 1-14, and a pharmaceutically acceptable carrier.

[0163] Embodiment 17. The pharmaceutical composition according to Embodiment 16, wherein the pharmaceutical composition is a liquid preparation.

[0164] Embodiment 18. The pharmaceutical composition according to embodiment 16 or 17, wherein the pharmaceutical composition is an injection solution.

[0165] Embodiment 19. The pharmaceutical composition according to Embodiment 16, wherein the pharmaceutical composition is a solid preparation.

[0166] Embodiment 20. The pharmaceutical composition according to embodiment 16 or 19, wherein the pharmaceutical composition is a lyophilized preparation.

[0167] Embodiment 21. A method for inhibiting HBV and / or HDV infection of cells, comprising administering to the cells an effective amount of the polypeptide analog of any one of embodiments 1-14 or the pharmaceutical composition of any one of embodiments 16-20.

[0168] Embodiment 22. Use of the polypeptide analogue of any one of Embodiments 1-14 or the pharmaceutical composition of any one of Embodiments 16-20 in the preparation of a medicament for treating patients infected with HBV and / or HDV.

[0169] Embodiment 23. Use of the polypeptide analog of any one of Embodiments 1-14 or the pharmaceutical composition of any one of Embodiments 16-20 in the preparation of a medicament for treating patients with hepatitis B, hepatitis D, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease.

[0170] Embodiment 24. A method of treating a patient infected with HBV and / or HDV, comprising administering to the patient a therapeutically effective amount of the polypeptide analog of any one of embodiments 1-14 or the pharmaceutical composition of any one of embodiments 16-20.

[0171] Embodiment 25. A method for treating a patient with hepatitis B, hepatitis D, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease, comprising administering to the patient a therapeutically effective amount of the polypeptide analog of any one of embodiments 1-14 or the pharmaceutical composition of any one of embodiments 16-20.

[0172] Embodiment 26. A polypeptide intermediate compound comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0173] Embodiment 27. A myristoylated polypeptide intermediate compound, the structure of which is shown below:

[0174] C 14-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPAPSAPAAASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS, or

[0175] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP,

[0176] Among them, C 14 Myristoyl. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Figure 1 shows the structure of Myrcludex B and Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 -COOH)-PAS100 plasma concentration-time curve in beagle dogs. DETAILED DESCRIPTION

[0178] Definition and Description

[0179] Unless otherwise specified, the following terms used in this disclosure have the following meanings: A particular term should not be considered ambiguous or unclear unless otherwise defined, but should be understood according to its ordinary meaning in the art.

[0180] Unless otherwise specified, use a solid wedge key. and dotted wedge key Indicates the absolute configuration of a stereocenter.

[0181] Unless otherwise specified, when a group has a linkable site, the bond connecting that site to another group is represented by a wavy line. express.

[0182] The compounds of the present disclosure (e.g., polypeptide analogs) may be asymmetric, e.g., have one or more stereoisomers. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present disclosure (including but not limited to diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures) are intended to be part of the present disclosure. Compounds of the present disclosure containing asymmetric carbon atoms may be isolated in optically pure or racemic forms. Optically pure forms may be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0183] When referring to amino acid positions, the reference to amino acid positions is based on the starting amino acid of the corresponding sequence being the first amino acid. For example, "Lys at position 45," "Lys45," or "K45" in SEQ ID NO:1 refers to the 45th Lys in the sequence, starting with the first amino acid in SEQ ID NO:1 as position 1. For another example, "37Arg" or "37R" in SEQ ID NO:1 refers to the 37th Arg in the sequence, starting with the first amino acid in SEQ ID NO:1 as position 1.

[0184] As used herein, "(a group) n " means that there are n such groups in the moiety. For example, (AEEA) used herein n or (Xaa) n means that there are n linked AEEA groups or Xaa groups in the moiety.

[0185] The term "IC 50 "IC" refers to the concentration of the test compound that inhibits the reaction in an in vitro or in vivo assay to 50% of the maximal inhibitory response (i.e., a value halfway between the maximal inhibitory response and the untreated response). 50 " can be measured by ELISA or FACS analysis or any other method known in the art.

[0186] The term "AEEA" means 2-[2-(2-aminoethoxy)ethoxy]acetic acid.

[0187] As used herein, “C 14 "Myristoyl" means myristoyl, also known as myristoyl, whose structure is -CO-(CH2) 12 -CH3.

[0188] As used herein, a "PAS sequence" is a polypeptide fragment composed of three amino acids: proline, alanine, and serine, which is highly soluble under physiological conditions, has no charge, and exhibits a random coil structure. When a PAS sequence has 0 amino acid residues, it means that the PAS sequence is missing.

[0189] The term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds described herein. Such substances do not cause undesirable biological effects when administered to an individual or do not interact in a deleterious manner with any component of the composition in which they are contained.

[0190] The term "pharmaceutically acceptable salt" includes salts formed between an alkali ion and a free acid or between an acid ion and a free base, for example, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, trifluoroacetate, fumarate, oxalate, maleate, citrate, tartrate, succinate, methanesulfonate, benzoate, benzenesulfonate or p-toluenesulfonate, preferably hydrochloride, hydrobromide, sulfate, formate, acetate, trifluoroacetate, fumarate, maleate, methanesulfonate, p-toluenesulfonate, sodium salt, potassium salt, ammonium salt, amino acid salt, etc. In some specific embodiments, the pharmaceutically acceptable salt of the polypeptide analog of the present disclosure is the acetate salt of the polypeptide analog.

[0191] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are well known to those skilled in the art (Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for carriers that are incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0192] The term "treatment" refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis.

[0193] The term "therapeutically effective amount" refers to the amount of a polypeptide analog, pharmaceutical composition, or other administered substance necessary to provide a therapeutic and / or prophylactic benefit to a subject.

[0194] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject according to the present disclosure is a human. Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably.

[0195] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where 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 the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0196] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. In addition, heavier isotopes such as deuterium (i.e. 2 H)) substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and thus may be preferred in some circumstances. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0197] The abbreviations of the naturally occurring amino acid residues in proteins or polypeptides are as follows: Phe or F for phenylalanine, Leu or L for leucine, Ile or I for isoleucine, Met or M for methionine, Val or V for valine, Ser or S for serine, Pro or P for proline, Thr or T for threonine, Ala or A for alanine, Tyr or Y for tyrosine, His or H for histidine, Gln or Q for glutamine, Asn or N for asparagine, and L for lysine. The abbreviations for unnatural amino acid residues are as follows: D-alanine is D-Ala, D-glutamate is D-Glu, β-alanine (i.e., 3-aminopropionic acid) is β-Ala, 4-aminobutyric acid is GABA, 2-aminoisobutyric acid is Aib, and 2-aminobutyric acid is Abu.

[0198] The γ-carboxyl group participates in the formation of peptide bonds while the α-carboxyl group is a free carboxyl group, which has the following structure:

[0199] The words “comprise,” “comprise,” or “comprises,” and variations thereof such as comprises or comprising, should be construed in an open, non-exclusive sense, ie, “including but not limited to.”

[0200] Herein, singular terms encompass plural referents and vice versa unless the context clearly dictates otherwise.

[0201] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" can mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of the specific numerical range given. When a specific value is given in the present disclosure or claims, unless otherwise indicated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this document, unless otherwise indicated, all values ​​for drug doses, times, step parameters, or conditions are modified by "about" by default.

[0202] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0203] The present disclosure is further described below in conjunction with specific examples. However, these examples in the present disclosure are only used to illustrate and do not limit the scope of the present disclosure. Similarly, the present disclosure is not limited to any specific preferred embodiment described herein. Those skilled in the art should understand that equivalent replacements made to the technical features of the present disclosure, or corresponding improvements, still fall within the scope of protection of the present disclosure. Unless otherwise specified, the reagents used in the following examples are all commercially available products, and the preparation of the solutions can adopt conventional techniques in the art.

[0204] Unless otherwise specified, the operations disclosed herein will be performed using conventional techniques of organic synthesis, biochemistry, protein purification, etc. within the skill of the art, or according to product specifications, which are fully explained in the literature. In the following examples, efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account.

[0205] Abbreviations: 2-CTC: 2-chlorotrityl chloride; Fmoc-Glu-OMe: (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-methoxy-5-oxopentanoic acid; Fmoc-AEEA-OH: [2-[2-(Fmoc-Gly-OH): Fmoc-glycine; Fmoc-Arg(Pbf)-OH: N-Fmoc-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine; Fmoc-Ser(tBu)-OH: N-Fmoc-O-tert-butyl-L-serine; Fmoc-Pro-OH: Fmoc-L-proline; Fmoc-Ala-OH: N-Fmoc-L-alanine; Fmoc-Ile-OH: Fmoc-L-isoleucine; Fmoc-Leu-OH: N-Fmoc-L-leucine; Fmoc-Trp(Boc)-OH: Fmoc-Tryptophan (tert-butyloxycarbonyl); Fmoc-Gln(Trt)-OH: Fmoc-Glu (tert-butyl ester); Fmoc-Val-OH: Fmoc-L-valine; Fmoc-Phe-OH: Fmoc-L-phenylalanine; Fmoc-Glu(OtBu)-OH: Fmoc-Glu (tert-butyl ester); Fmoc-Thr(tBu)-OH: N-(9-Fmoc-T-butyl)-L-threonine; Fmoc-Tyr(tBu)-OH: Fmoc-tert-butyl-oxy-tyrosine; Fmoc-Asp(OtBu)-OH: Fmoc-tert-butyl-aspartate; Fmoc-Lys(Boc)-OH: N-Fmoc-N'-tert-butyloxycarbonyl-L-lysine; Fmoc-His(Trt)-OH: Fmoc-trityl-L-histidine; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DCC: dicyclohexylcarbodiimide; DIC: N,N'-diisopropylcarbodiimide; HOBt: 1-hydroxybenzotriazole; DMF: N,N-dimethylformamide; PIP: piperidine; HAC: acetic acid; Fmoc: 9-Fmoc- ... Tis: triisopropylsilane; HOSU: N-hydroxysuccinimide; m / z: mass-to-charge ratio.

[0206] PBS (pH 7.4): contains 0.27 g / L potassium dihydrogen phosphate (KH2PO4), 2.85 g / L disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 8.5 g / L sodium chloride (NaCl), and 0.2 g / L potassium chloride (KCl).

[0207] Example 1: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 Preparation of -COOH)-PAS100

[0208] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 The structure of -COOH)-PAS100 is as follows:

[0209] (1) Materials and reagents

[0210] 2-CTC resin (Xi’an Lanxiao Technology New Materials Co., Ltd.), with a substitution value (SD) of 1.15 mmol / g.

[0211] Materials: Fmoc-Glu-OMe (CAS No.: 145038-49-9), Fmoc-AEEA-OH (CAS No.: 166108-71-0), mono-tert-butyl octadecanoate (CAS No.: 843666-40-0), myristic acid succinimidyl ester (Hanxiang Biotechnology, CAS No.: 69888-86-4).

[0212] Synthesis reagents: HOBt, DIC, DMF, DCM, PIP, DIEA.

[0213] Activation reagents: DCC, HOSU.

[0214] P1(37Arg)-PAS100 was expressed in Escherichia coli, m / z = 1212.0 [M+11H] 11+ , the sequence of P1(37Arg)-PAS100 is as follows:

[0215] (2) Instruments

[0216] CS-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, BUCHI vacuum distillation apparatus.

[0217] (3) Operation steps

[0218] a. P1(37Arg)-PAS100 N-terminal myristoylation reaction

[0219] 50 mg of P1(37Arg)-PAS100 was dissolved in 20 mmol / L PBS (pH 7.4) to a final concentration of 5 mg / mL, and an equal volume of acetonitrile was added. 10 times the volume of acetonitrile solution containing myristoyl succinimidyl ester (2 mg / mL) was slowly added dropwise to the above solution. The reaction was stirred at room temperature for 2 hours. The reaction was terminated by adjusting the pH to 4.0 with 0.1 mol / L hydrochloric acid to obtain the crude product Myristoyl-P1(37Arg)-PAS100.

[0220] The crude product was purified using a Waters 600 semi-preparative high performance liquid chromatograph with the gradient elution parameters shown in Table 1: the chromatographic column was Kromasil 300-10-C4 10*250 mm, the flow rate was 5 mL / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC (v / v) in water, and the mobile phase B was 0.5% HAC (v / v) in acetonitrile.

[0221] Table 1 Gradient elution parameters

[0222] The collected purified product was analyzed using the gradient elution parameters shown in Table 2: the chromatographic column was Kromasil 100-3.5-C44.6*150 mm, the flow rate was 1 mL / min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v / v) in water, and the mobile phase B was 0.05% TFA (v / v) in acetonitrile.

[0223] Table 2 Gradient elution parameters

[0224] The target fraction with a purity greater than 90% was collected, acetonitrile was removed by vacuum distillation, and the product was freeze-dried. The molecular weight was confirmed by ESI-MS, m / z = 1129.5 [M+12H] 12+ It is consistent with the theoretical molecular weight.

[0225] b. Solid Phase Synthesis of Acylated Modifying Groups

[0226] 1.00 g of 2-CTC resin was weighed and placed in a CS-BIO peptide synthesizer reactor. 10 mL of DCM was added and soaked for 1 hour. Fmoc-AEEA-OH in an amount equivalent to 2-3 times the molar number of the resin and DIEA in an amount equivalent to 4-6 times the molar number of the resin were weighed and dissolved in 10 mL of DCM. The mixture was then placed in the reactor for reaction at room temperature for 2 hours to complete the coupling of the first amino acid. The resin was then washed with DCM 6 times. Subsequently, 10 mL of a 20% PIP (v / v) solution in DMF was added and mixed for 30 minutes to remove the Fmoc amino protecting group. The resin was washed with DCM 6 times to couple the second amino acid. Fmoc-AEEA-OH, HOBt and DIC in an amount equivalent to 3 times the molar number of the resin were weighed and dissolved in 10 mL of a DMF / DCM (v:v=1:1) mixed solvent. The mixture was placed in the reactor for reaction at room temperature. The reaction progress was monitored by ninhydrin reaction. The reaction was complete when colorless. The resin was washed with DCM 6 times. Then, the coupling reaction of γ-glutamic acid and octadecanedioic acid can be continued according to the above coupling method, and this cycle is repeated until all amino acids are coupled.

[0227] c. Cleavage and precipitation of acylated modifying groups

[0228] The cleavage reagent was added at a ratio of 5 mL of cleavage reagent / 1 g of resin, with a reagent ratio of TFE:DCM = 1:4 (v:v). The reaction was stirred at room temperature for 1 hour, filtered, and the filtrate was distilled under reduced pressure at 40°C to remove the solvent. 10 mL of DCM was added to the vacuum distillation flask, and the solvent was again distilled under reduced pressure. This was repeated 2-3 times. Finally, 3 mL of DCM was added to dissolve the polypeptide, and 40 mL of icy ether was added. The mixture was placed in a -20°C refrigerator for 20 minutes, centrifuged, and dried under vacuum to obtain the acylated modification group.

[0229] d. Liquid phase reaction to prepare peptide analogs Myristoyl-P1 (37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH)-PAS100

[0230] Weigh approximately 0.1 mmol of the dried acylated modifying group, approximately 0.095 mmol of HOSU, and 15 μL of DIC, add approximately 5 mL of tetrahydrofuran (THF), and react at room temperature for 1-2 hours. Distill under reduced pressure to remove THF to obtain a yellow oil. Add a mixed solvent of TFA:H2O = 90:10 to remove the tert-butyl ester protecting group. After reacting at room temperature for 1 hour, pour into icy ether for precipitation, centrifuge to obtain a solid, wash three times with ether, and dry under reduced pressure to obtain a solid powder, weigh it, and dissolve it in DMF to obtain an acylated activated ester solution.

[0231] 0.5 mmol Myristoyl-P1(37Arg)-PAS100 was dissolved in 1% (v / v) triethylamine solution, and 1.2 times the volume of acylated activated ester solution was slowly added dropwise to the solution. The mixture was stirred at room temperature for 10 minutes, and the pH was adjusted to 8.0 with 0.1 mol / L hydrochloric acid to terminate the reaction. The crude product of the peptide analogue Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 -COOH)-PAS100.

[0232] The crude product of the above-mentioned polypeptide analog was purified using a Waters 600 semi-preparative high performance liquid chromatograph with the gradient elution parameters shown in Table 1: the chromatographic column was Kromasil 300-10-C4 10*250 mm, the flow rate was 5 mL / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC (v / v) in water, and the mobile phase B was 0.5% HAC (v / v) in acetonitrile.

[0233] The collected purified peptide analogs were analyzed using the gradient elution parameters shown in Table 2: the chromatographic column was Kromasil 100-3.5-C44.6*150 mm, the flow rate was 1 mL / min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v / v) in water, and the mobile phase B was 0.05% TFA (v / v) in acetonitrile.

[0234] The target fraction with a purity greater than 90% was collected, acetonitrile was removed by distillation under reduced pressure, and the product was freeze-dried in a vacuum. The molecular weight was confirmed by ESI-MS, m / z = 1296.17 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0235] Example 2: Preparation of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS100

[0236] The structure of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS100 is as follows:

[0237] The preparation and purification process refers to the method in Example 1, and the molecular weight is confirmed by ESI-MS, m / z = 1283.45 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0238] Example 3: Preparation of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS100

[0239] The structure of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS100 is as follows:

[0240] The preparation and purification process was carried out according to the method in Example 1. The molecular weight was confirmed by ESI-MS, m / z = 1285.99 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0241] Example 4: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 Preparation of -COOH)-PAS100

[0242] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 The structure of -COOH)-PAS100 is as follows:

[0243] The preparation and purification process refers to the method in Example 1, and the molecular weight is confirmed by ESI-MS, m / z = 1288.54 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0244] Example 5: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 Preparation of -COOH)-PAS100

[0245] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 The structure of -COOH)-PAS100 is as follows:

[0246] The preparation and purification process refers to the method in Example 1, and the molecular weight is confirmed by ESI-MS, m / z = 1291.08 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0247] Example 6: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 Preparation of -COOH)-PAS100

[0248] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 The structure of -COOH)-PAS100 is as follows:

[0249] The preparation and purification process was carried out according to the method in Example 1. The molecular weight was confirmed by ESI-MS, m / z = 1293.63 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0250] Example 7: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 Preparation of -COOH)-PAS100

[0251] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 The structure of -COOH)-PAS100 is as follows:

[0252] The preparation and purification process was carried out according to the method in Example 1. The molecular weight was confirmed by ESI-MS, m / z = 1298.72 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0253] Example 8: Preparation of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)6-CH3)-PAS100

[0254] The structure of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)6-CH3)-PAS100 is as follows:

[0255] (1) Materials and reagents

[0256] The fatty acid used to synthesize the acylation-modified modifying group is: n-octanoic acid.

[0257] Activation reagents are: DCC, HOSU.

[0258] (2) Instruments

[0259] BUCHI vacuum distillation apparatus, Waters 600 semi-preparative high performance liquid chromatograph.

[0260] (3) Operation steps

[0261] In the operation steps of Example 1(3), steps b and c are skipped, and the preparation of the acylated activated ester solution is modified to the following steps:

[0262] Take 0.1 mmol of n-octanoic acid, about 0.15 mmol of HOSU, and about 0.12 mmol of DCC, add about 5 mL of dichloromethane, react at room temperature for 1-2 hours, distill under reduced pressure to remove dichloromethane, and dissolve with DMF to obtain an acylated activated ester solution.

[0263] The remaining preparation and purification steps were carried out according to the method in Example 1. The molecular weight was confirmed by ESI-MS, m / z = 1242.64 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0264] Example 9: Preparation of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)8-CH3)-PAS100

[0265] The structure of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)8-CH3)-PAS100 is as follows:

[0266] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1245.19 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0267] Example 10: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 10 Preparation of -CH3)-PAS100

[0268] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 10 -CH3)-PAS100 has the following structure:

[0269] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1247.73 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0270] Example 11: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)12 Preparation of -CH3)-PAS100

[0271] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 12 -CH3)-PAS100 has the following structure:

[0272] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1250.28 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0273] Example 12: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 14 Preparation of -CH3)-PAS100

[0274] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 14 -CH3)-PAS100 has the following structure:

[0275] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1252.82 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0276] Example 13: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 16 Preparation of -CH3)-PAS100

[0277] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 16 -CH3)-PAS100 has the following structure:

[0278] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1255.37 [M+11H] + It is consistent with the theoretical molecular weight.

[0279] Example 14: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 18 Preparation of -CH3)-PAS100

[0280] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 18-CH3)-PAS100 has the following structure:

[0281] The preparation and purification process was carried out according to the method in Example 8. The molecular weight was confirmed by ESI-MS, m / z = 1257.91 [M+11H] 11+ It is consistent with the theoretical molecular weight.

[0282] Example 15: Preparation of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)6-CH3)-PAS30

[0283] The structure of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)6-CH3)-PAS30 is as follows:

[0284] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] + , the sequence of P1(37Arg)-PAS30 is as follows:

[0285] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1591.22 [M+5H] + It is consistent with the theoretical molecular weight.

[0286] Example 16: Preparation of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)8-CH3)-PAS30

[0287] The structure of Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2)8-CH3)-PAS30 is as follows:

[0288] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0289] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1596.82 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0290] Example 17: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 10 Preparation of -CH3)-PAS30

[0291] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 10 -CH3)-PAS30 has the following structure:

[0292] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0293] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1602.42 [M+5H] + It is consistent with the theoretical molecular weight.

[0294] Example 18: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 12 Preparation of -CH3)-PAS30

[0295] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 12 -CH3)-PAS30 has the following structure:

[0296] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0297] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1608.02 [M+5H] + It is consistent with the theoretical molecular weight.

[0298] Example 19: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 14 Preparation of -CH3)-PAS30

[0299] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 14 -CH3)-PAS30 has the following structure:

[0300] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0301] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1613.62 [M+5H] + It is consistent with the theoretical molecular weight.

[0302] Example 20: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 16 Preparation of -CH3)-PAS30

[0303] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 16 -CH3)-PAS30 has the following structure:

[0304] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0305] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1619.22 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0306] Example 21: Myristoyl-P1(37Arg, Lys45-ε-CO-(CH2) 18 Preparation of -CH3)-PAS30

[0307] Myristoyl-P1(37Arg,Lys45-ε-CO-(CH2) 18 -CH3)-PAS30 has the following structure:

[0308] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0309] Other preparation and purification processes refer to the method in Example 8. The molecular weight is confirmed by ESI-MS, m / z = 1624.82 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0310] Example 22: Preparation of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS30

[0311] The structure of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)6-COOH)-PAS30 is as follows:

[0312] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0313] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1680.99 [M+5H] + It is consistent with the theoretical molecular weight.

[0314] Example 23: Preparation of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS30

[0315] The structure of Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2)8-COOH)-PAS30 is as follows:

[0316] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0317] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1686.59 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0318] Example 24: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 Preparation of -COOH)-PAS30

[0319] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 10 The structure of -COOH)-PAS30 is as follows:

[0320] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0321] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1692.19 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0322] Example 25: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 Preparation of -COOH)-PAS30

[0323] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 12 -COOH)-PAS30 has the following structure:

[0324] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0325] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1697.79 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0326] Example 26: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 Preparation of -COOH)-PAS30

[0327] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 14 The structure of -COOH)-PAS30 is as follows:

[0328] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0329] Other preparation and purification processes refer to the method in Example 1, and the molecular weight is confirmed by ESI-MS, m / z = 1703.39 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0330] Example 27: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16Preparation of -COOH)-PAS30

[0331] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 16 The structure of -COOH)-PAS30 is as follows:

[0332] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0333] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1708.99 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0334] Example 28: Myristoyl-P1(37Arg, Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 Preparation of -COOH)-PAS30

[0335] Myristoyl-P1(37Arg,Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH2) 18 The structure of -COOH)-PAS30 is as follows:

[0336] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0337] Other preparation and purification processes refer to the method in Example 1. The molecular weight is confirmed by ESI-MS, m / z = 1714.59 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0338] Example 29: Preparation of Myristoyl-P1(37Arg)-PAS100

[0339] The structure of Myristoyl-P1(37Arg)-PAS100 is as follows:

[0340] C 14-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPAPSAPAAASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS.

[0341] (1) Materials and reagents

[0342] Myristate succinimidyl ester (Hanxiang Biotechnology, CAS No.: 69888-86-4);

[0343] P1(37Arg)-PAS100 was expressed in Escherichia coli, m / z = 1212.0 [M+11H] 11+ .

[0344] (2) Instruments

[0345] Waters 600 semi-preparative high performance liquid chromatograph and BUCHI vacuum distillation apparatus.

[0346] (3) Operation steps

[0347] 0.5 mmol P1(37Arg)-PAS100 was dissolved in 20 mmol / L PBS (pH 7.4) to a final concentration of 5 mg / mL, and an equal volume of acetonitrile was added. To the above solution, 10 times the volume of acetonitrile solution containing myristoyl succinimidyl ester (2 mg / mL) was slowly added dropwise. The reaction was stirred at room temperature for 2 hours, and then the pH was adjusted to 4.0 with 0.1 mol / L hydrochloric acid to terminate the reaction to obtain the crude product Myristoyl-P1(37Arg)-PAS100.

[0348] The crude product was purified using a semi-preparative high performance liquid chromatograph with the gradient elution parameters shown in Table 1: the chromatographic column was Kromasil 300-10-C4 10*250 mm, the flow rate was 5 mL / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC (v / v) in water, and the mobile phase B was 0.5% HAC (v / v) in acetonitrile.

[0349] The collected purified product was analyzed using the gradient elution parameters shown in Table 2: the chromatographic column was Kromasil 100-3.5-C44.6*150 mm, the flow rate was 1 mL / min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v / v) in water, and the mobile phase B was 0.05% TFA (v / v) in acetonitrile.

[0350] The target fraction with a purity greater than 90% was collected, acetonitrile was removed by vacuum distillation, and the product was freeze-dried. The molecular weight was confirmed by ESI-MS, m / z = 1129.5 [M+12H] + It is consistent with the theoretical molecular weight.

[0351] Example 30: Preparation of Myristoyl-P1(37Arg)-PAS30

[0352] The structure of Myristoyl-P1(37Arg)-PAS30 is as follows:

[0353] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP.

[0354] P1(37Arg)-PAS30 was expressed in Escherichia coli, m / z = 1523.89 [M+5H] 5+ .

[0355] The preparation process was as described in Example 29, and the molecular weight was confirmed by ESI-MS, m / z = 1565.96 [M+5H] + It is consistent with the theoretical molecular weight.

[0356] Example 31: Preparation of Myristoyl-P1(37Arg)-OH

[0357] The structure of Myristoyl-P1(37Arg)-OH is as follows:

[0358] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVG-OH, whose C-terminus is a free carboxyl group.

[0359] (1) Materials and reagents

[0360] Fmoc-Gly-Wang Resin (Xishi Biotechnology Co., Ltd.), substitution value 0.29 mmol / g;

[0361] Materials: Fmoc-Gly-OH (CAS No.: 29022-11-5), Fmoc-Arg(Pbf)-OH (CAS No.: 154445-77-9), Fmoc-Ser(tBu)-OH (CAS No.: 71989-33-8), Fmoc-Pro-OH (CAS No.: 71989-31-6), Fmoc-Ala-OH (CAS No.: 35661-39-3), Fmoc-Ile-OH (CAS No.: 71989-23-6), Fmoc-Leu-OH (CAS No.: 35661-60-0), Fmoc-Trp(Boc)-OH (CAS No.: 143824-78-6), Fmoc-Gln(Trt)-OH (CAS No.: 132327-80-1), Fmoc- Val-OH (CAS No.: 68858-20-8), Fmoc-Phe-OH (CAS No.: 35661-40-6), Fmoc-Glu(OtBu)-OH (CAS No.: 71989-18-9), Fmoc-Thr(tBu)-OH (CAS No.: 71989-35-0), Fmoc-Tyr(tBu)-OH (CAS No.: 71989-38-3), Fmoc-Asp(OtBu)-OH (CAS No.: 71989-14-5), Fmoc-Lys(Boc)-OH (CAS No.: 71989-26-9), Fmoc-His(Trt)-OH (CAS No.: 109425-51-6), myristate succinimidyl ester (Hanxiang Biotechnology, CAS No.: 69888-86-4).

[0362] Synthesis reagents: HOBt, DIC, DMF, DCM, PIP, DIEA.

[0363] (2) Instruments

[0364] CSIO-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, BUCHI vacuum distillation apparatus.

[0365] (3) Operation steps

[0366] a. Synthesis of Myristoyl-P1(37Arg)-OH

[0367] Weigh 0.5 g of Fmoc-Gly-Wang Resin resin, place it in a peptide synthesizer reactor, add 10 mL of DCM, soak for 1 hour, then add 10 mL of 20% PIP (v / v) DMF solution and mix for 30 minutes to remove the amino protecting group. Wash the resin with DCM 6 times, couple the first amino acid, weigh Fmoc-Val-OH, HOBt and DIC in an amount equivalent to 3 times the molar number of the resin, add 10 mL of DMF / DCM (v / v=1:1) mixed solvent to dissolve, and react. The reaction temperature is room temperature. The reaction progress is monitored by ninhydrin reaction. The reaction is complete when the monitoring is colorless. Wash the resin with DCM 6 times. Subsequently, 10 mL of a 20% (v / v) PIP solution in DMF was added and mixed for 30 minutes to remove the amino protecting group. The resin was washed six times with DCM. The second amino acid was coupled. Fmoc-Lys(BOC)-OH, HOBt, and DIC were weighed in an amount equivalent to three times the molar mass of the resin and dissolved in 10 mL of a DMF / DCM (v / v = 1:1) mixed solvent. The reaction was then carried out at room temperature. Reaction progress was monitored using ninhydrin. Completion was indicated by a colorless reaction. The resin was washed six times with DCM. The coupling reaction was then continued according to the above coupling method, repeating this cycle until all amino acids were coupled.

[0368] b. Lysis and precipitation

[0369] The cleavage reagent was added at a ratio of 5 mL of cleavage reagent / 1 g of resin, with a reagent ratio of TFA:H2O:Tis = 95:2.5:2.5 (v:v). The reaction was stirred at room temperature for 2 hours, filtered, and 130 mL of icy ether was added. The mixture was placed in a -20°C refrigerator for 20 minutes, centrifuged, and vacuum-dried. The crude product Myristoyl-P1(37Arg)-OH was weighed.

[0370] The crude product was purified using a semi-preparative high performance liquid chromatograph with the gradient elution parameters shown in Table 1: the chromatographic column was Kromasil 300-10-C4 10*250 mm, the flow rate was 5 mL / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC (v / v) in water, and the mobile phase B was 0.5% HAC (v / v) in acetonitrile.

[0371] The collected purified product was analyzed using the gradient elution parameters shown in Table 2: the chromatographic column was Kromasil 100-3.5-C44.6*150 mm, the flow rate was 1 mL / min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v / v) in water, and the mobile phase B was 0.05% TFA (v / v) in acetonitrile.

[0372] The target fraction with a purity greater than 90% was collected, acetonitrile was removed by vacuum distillation, and the product was freeze-dried. The molecular weight was confirmed by ESI-MS, m / z = 775.14 [M+7H] 7+ It is consistent with the theoretical molecular weight.

[0373] Example 32: Myristoyl-P1(37Arg)-NH2

[0374] The structure of Myristoyl-P1(37Arg)-NH2 is as follows:

[0375] C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVG-NH2, the C-terminus of which is amidated.

[0376] (1) Materials and reagents

[0377] MBHA resin (Xilanxiao Technology New Materials Co., Ltd.), substitution value is 0.58mmol / g

[0378] Materials: Fmoc-Gly-OH (CAS No.: 29022-11-5), Fmoc-Arg(Pbf)-OH (CAS No.: 154445-77-9), Fmoc-Ser(tBu)-OH (CAS No.: 71989-33-8), Fmoc-Pro-OH (CAS No.: 71989-31-6), Fmoc-Ala-OH (CAS No.: 35661-39-3), Fmoc-Ile-OH (CAS No.: 71989-23-6), Fmoc-Leu-OH (CAS No.: 35661-60-0), Fmoc-Trp(Boc)-OH (CAS No.: 143824-78-6), Fmoc-Gln(Trt)-OH (CAS No.: 132327-80-1), Fmoc- Val-OH (CAS No.: 68858-20-8), Fmoc-Phe-OH (CAS No.: 35661-40-6), Fmoc-Glu(OtBu)-OH (CAS No.: 71989-18-9), Fmoc-Thr(tBu)-OH (CAS No.: 71989-35-0), Fmoc-Tyr(tBu)-OH (CAS No.: 71989-38-3), Fmoc-Asp(OtBu)-OH (CAS No.: 71989-14-5), Fmoc-Lys(Boc)-OH (CAS No.: 71989-26-9), Fmoc-His(Trt)-OH (CAS No.: 109425-51-6), myristate succinimidyl ester (Hanxiang Biotechnology, CAS No.: 69888-86-4).

[0379] Synthesis reagents: HOBt, DIC, DMF, DCM, PIP, DIEA

[0380] (2) Instruments

[0381] CSIO-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, BUCHI vacuum distillation apparatus.

[0382] (3) Operation steps

[0383] a. Synthesis of Myristoyl-P1(37Arg)-NH2

[0384] Weigh 0.5 g of MBHA resin and place it in a peptide synthesizer reactor. Add 10 mL of DCM and soak for 1 hour. Then add 10 mL of 20% PIP (v / v) DMF solution and mix for 30 minutes to remove the amino protecting group. Wash the resin with DCM 6 times. Couple the first amino acid. Weigh Fmoc-Lys(BOC)-OH, HOBt and DIC in an amount equivalent to 3 times the molar number of the resin. Add 10 mL of DMF / DCM (v / v = 1:1) mixed solvent to dissolve them and react. The reaction temperature is room temperature. The reaction progress is monitored by ninhydrin reaction. The reaction is complete when the monitoring is colorless. Wash the resin with DCM 6 times. Subsequently, 10 mL of a 20% (v / v) PIP solution in DMF was added and mixed for 30 minutes to remove the amino protecting group. The resin was washed six times with DCM. The second amino acid was coupled. Fmoc-Lys(BOC)-OH, HOBt, and DIC were weighed in an amount equivalent to three times the molar mass of the resin and dissolved in 10 mL of a DMF / DCM (v / v = 1:1) mixture. The reaction was then carried out at room temperature. Reaction progress was monitored using ninhydrin, and completion was confirmed by a colorless reaction. The resin was then washed six times with DCM. The coupling reaction was then continued according to the above coupling method, repeating this cycle until all amino acids were coupled.

[0385] b. Lysis and precipitation

[0386] The cleavage reagent was added at a ratio of 5 mL of cleavage reagent per 1 g of resin, with a reagent ratio of TFA:H2O:Tis = 95:2.5:2.5 (v:v). The reaction was stirred at room temperature for 2 hours, filtered, and 130 mL of icy ether was added. The mixture was placed in a -20°C refrigerator for 20 minutes, centrifuged, and vacuum-dried. The crude product Myristoyl-P1(37Arg)-NH2 was weighed.

[0387] The crude product was purified using a semi-preparative high performance liquid chromatograph with the gradient elution parameters shown in Table 1: the chromatographic column was Kromasil 300-10-C4 10*250 mm, the flow rate was 5 mL / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC (v / v) in water, and the mobile phase B was 0.5% HAC (v / v) in acetonitrile.

[0388] The collected purified product was analyzed using the gradient elution parameters shown in Table 2: the chromatographic column was Kromasil 100-3.5-C44.6*150 mm, the flow rate was 1 mL / min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v / v) in water, and the mobile phase B was 0.05% TFA (v / v) in acetonitrile.

[0389] The target fraction with a purity greater than 90% was collected, acetonitrile was removed by vacuum distillation, and the product was freeze-dried. The molecular weight was confirmed by ESI-MS, m / z = 1085.40 [M+5H] 5+ It is consistent with the theoretical molecular weight.

[0390] Example 33: HBV infection inhibition experiment

[0391] HepG2NTCP cells were grown at 2×10 5 Cells were plated at a density of 100 μL / well. The next day, DMEM containing 2.5% DMSO and 5% FBS was added for 24 hours of induction. HBV virus derived from HepAD38 cell supernatant was diluted to an infection dose of 100 in DMEM containing 2.5% DMSO and 4% PEG8000. Compound dilutions: The peptide analogs to be tested and the control Myrcludex B were diluted to eight dilutions, namely 1000 nmol / L, 200 nmol / L, 40 nmol / L, 8 nmol / L, 1.6 nmol / L, 0.32 nmol / L, 0.064 nmol / L, and 0.0128 nmol / L, in DMEM containing 2.5% DMSO and 4% PEG8000. For the infection blocking group, different concentrations of diluted compounds were added 30 minutes in advance, 100 μL / well, and then diluted viruses were added, 100 μL / well, and gently pipetted 4-6 times. Centrifuged at 1000 rpm for 30 minutes at room temperature. Incubate for 16-24 hours, discard the infection solution, wash 3 times with PBS, and add DMEM maintenance culture medium containing 2.5% DMSO and 5% FBS. On the 11th day after infection, the cell supernatant was collected and the cell pellet was discarded by centrifugation. The HBeAg level was detected in the cell supernatant stock solution. The corresponding absorbance value was obtained by scanning the well plate with an enzyme reader, and the standard curve of concentration and signal value was linearly fitted to calculate the IC of different compounds for inhibition of HBV infection. 50 The values ​​are shown in Table 3 below.

[0392] Table 3 Inhibitory activity of peptide analogs on HBV-infected HepG2NTCP cells

[0393] Example 34: Pharmacokinetic Study of HB-38 in Beagle Dogs

[0394] Beagle dogs (weight 9-11 kg) were randomly divided into two groups, each group had two dogs, and were subcutaneously injected with Myrcludex B and HB-38 at a dose of 1 mg / kg.

[0395] The test animals (beagle dogs) were fasted for 12 h before administration and given food 4 h after administration. They had free access to water before, during and after the experiment.

[0396] Approximately 1 mL of blood was collected from a forearm vein at 0 h (before injection) and 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h, 48 h, 99 h, 120 h, 144 h, 168 h, 196 h, 216 h, 240 h, and 264 h after subcutaneous injection. The blood was anticoagulated with EDTA-K2 and centrifuged at 4000 rpm for 10 min at 4°C to separate plasma. All collected plasma was stored at -80°C until testing.

[0397] Pipette 100 μL of the plasma sample to be tested and the standard sample, add 50 μL of internal standard solution, add 150 μL of precipitant (solvent: acetonitrile:methanol (v:v=3:2) (containing 0.1% glacial acetic acid) solution), vortex mix, centrifuge at 18000g, 4°C for 7 min, take 200 μL of the supernatant, aspirate 20 μL for LC-MS / MS analysis, and record the chromatogram.

[0398] The pharmacokinetic study in beagle dogs was used to evaluate the exposure of the disclosed compounds after subcutaneous injection, and the peak drug concentration (C max ), peak time (T max ), area under the drug-time curve (AUC (0-264h) and AUC( 0-∞ )), elimination half-life (t 1 / 2 ) and mean residence time (MRT( 0-264 h )). The above pharmacokinetic parameters are shown in Table 4. The plasma concentration-time curve is shown in Figure 1.

[0399] Table 4 Pharmacokinetic parameters of HB-38 and Myrcludex B in beagle dogs

[0400] The experimental results showed that the pharmacokinetic behavior of beagle dogs after subcutaneous injection of 1mg / kg Myrcludex B and HB-38 was significantly different. max and exposure AUC (0-t) It was significantly higher than the Myrcludex B control group, and the half-life of the HB-38 group was significantly longer than that of the Myrcludex B control group.

Claims

1. A polypeptide analogue having the general structural formula C 14 -P1-U1; in, P1 contains the following amino acid sequence: GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX1DHWPEANX2VG (SEQ ID NO: 1), wherein X1 is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, Glu, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, preferably β-Ala, GABA, Aib, Abu, Arg or Cys, more preferably Arg; X2 is Lys, and its ε-amino group has an acylated modification group; C 14 is a myristoyl group at the N-terminus of P1; U1 is a PAS sequence connected to the C-terminus of P1, wherein the PAS sequence consists of Pro, Ala and / or Ser and has 30-300 amino acid residues.

2. The polypeptide analog according to claim 1, wherein the structural formula of the modification group of X2 is: in, p is 6-20, 10-20, 14-20, 14-18 or 16-18, m is 0, 1, 2, 3, 4 or 5, preferably 0 or 2, n is 0, 1, 2 or 3, preferably 0 or 1, Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, R1 is -CH3 or -COOH; Wavy Lines represents the connection position of the modifying group to the ε-amino group of X2.

3. The polypeptide analog according to claim 2, wherein the modifying group has a structural formula shown in Formula 1 or Formula 2: in, The Xaa is selected from GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro or Phe, preferably γ-Glu or Pro; the p is 6, 8, 10, 12, 14, 16, 18 or 20, preferably 16 or 18.

4. The polypeptide analog according to claim 1, wherein the modification group of X2 is selected from the following structural formula: Wavy Lines represents the connection position of the modifying group to the ε-amino group of X2.

5. The polypeptide analogue according to any one of claims 1 to 4, wherein the PAS sequence has 30-100 amino acid residues, preferably 30, 50 or 100 amino acid residues.

6. The polypeptide analog according to any one of claims 1 to 5, wherein Pro accounts for 10% to 40% of the total number of amino acid residues in the PAS sequence.

7. The polypeptide analog according to any one of claims 1-6, wherein the PAS sequence comprises the amino acid sequence shown in SEQ ID NO:2, 3, 4, 5, 6, 7 or 8; preferably, the amino acid sequence of the PAS sequence is as shown in SEQ ID NO:2, 3, 4, 5, 6, 7 or 8.

8. The polypeptide analog according to claim 1, wherein the polypeptide analog is selected from the following structural formula: in, C 14 is myristoyl; p is 6-20, 10-20, 14-20, 14-18 or 16-18, preferably 6, 8, 10, 12, 14, 16, 18 or 20; Xaa is selected from D-Ala, β-Ala, GABA, Aib, Abu, Arg, Asp, Asn, Cys, D-Glu, γ-Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Tyr, Thr, Trp, Val or Met, preferably GABA, Aib, D-Ala, β-Ala, Asp, Cys, γ-Glu, Gly, Pro or Phe, more preferably γ-Glu or Pro; R1 is -CH3 or -COOH.

9. The polypeptide analog according to claim 1, wherein the polypeptide analog is selected from the following structural formula: in, C 14 Myristoyl.

10. A method for preparing the polypeptide analogue according to any one of claims 1 to 9, comprising the following steps: a) preparing the peptide chain portion composed of amino acids and the modifying group for acylation modification in P1 according to any one of claims 1 to 9; b) performing a myristoylation reaction on the N-terminus of the peptide chain portion; c) acylation-modifying the ε-amino group of the amino acid residue X2 of the peptide chain portion with the modifying group to obtain the polypeptide analog; and d) separating and purifying the polypeptide analogs.

11. A pharmaceutical composition comprising the polypeptide analog according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is a liquid preparation or a solid preparation; preferably, the pharmaceutical composition is an injection or a lyophilized preparation.

13. A method for inhibiting HBV and / or HDV infection of cells, comprising administering an effective amount of the polypeptide analogue according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 11 or 12 to the cells.

14. Use of the polypeptide analogue according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for treating patients infected with HBV and / or HDV.

15. Use of the polypeptide analogue according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for treating patients with hepatitis B, hepatitis D, liver cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease.

16. A method for treating a patient infected with HBV and / or HDV, comprising administering to the patient a therapeutically effective amount of the polypeptide analogue of any one of claims 1 to 9 or the pharmaceutical composition of claim 11 or 12.

17. A method for treating patients with hepatitis B, hepatitis D, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and / or non-alcoholic fatty liver disease, comprising administering to the patient a therapeutically effective amount of the polypeptide analogue of any one of claims 1-9 or the pharmaceutical composition of claim 11 or 12.

18. A polypeptide intermediate compound comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

19. A myristoylated polypeptide intermediate compound, the structure of which is shown below: C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPAPSAPAAASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS, or C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP, in, C 14 Myristoyl.

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