CN116970042A - Antiviral polypeptide analogues - Google Patents
Antiviral polypeptide analogues Download PDFInfo
- Publication number
- CN116970042A CN116970042A CN202310486422.3A CN202310486422A CN116970042A CN 116970042 A CN116970042 A CN 116970042A CN 202310486422 A CN202310486422 A CN 202310486422A CN 116970042 A CN116970042 A CN 116970042A
- Authority
- CN
- China
- Prior art keywords
- glu
- ala
- aeea
- arg
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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Abstract
The invention provides an antiviral polypeptide analogue, and also provides a pharmaceutical composition containing the antiviral polypeptide analogue, a preparation method and application thereof. The antiviral polypeptide analogue provided by the invention has good half-life and good solubility while maintaining good virus infection inhibition activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to the field of long-acting biological drug development, in particular to an antiviral polypeptide analogue.
Background
Chronic hepatitis b virus infection is a global important public health problem, and about 2.4 hundred million people worldwide are infected with viruses. Cirrhosis and liver cancer caused by hepatitis b virus (Hepatitis B Virus, HBV) also have high morbidity and mortality. Antiviral drugs approved for use in the treatment of hepatitis b include pegylated interferon alpha and 6 nucleoside analogues. The former acts by inducing the production of antiviral proteins by neighboring cells, and the latter blocks viral replication by inhibiting the activity of HBV-DNA polymerase. However, pegylated interferon alpha has side effects such as influenza-like symptoms and abnormal blood circulation, and nucleoside analogues have disadvantages such as drug resistance and inapplicable population. Therefore, there is a great need for anti-hepatitis B virus therapeutic drugs with new mechanism of action in clinic.
Myrcludex B was licensed by MYR GmbH from Vision7 and developed by Heidelberg university and Hepatera cooperation. Myrcludex B is derived from HBV-L protein at positions 2-48, consists of 47 natural amino acid residues and N-terminal myristoyl, and is a viral invasion inhibitor targeting sodium-taurocholate co-transporting polypeptide (NTCP).
In 9 months 2020, myrcludex B was marketed in Europe as a drug for treating hepatitis D. The low-dose intermittent administration mode is adopted, and the administration is carried out by injecting 1 time a day, so that great pain is brought to patients. The clinical compliance of patients can be greatly improved if the half-life of Myrcludex B can be extended.
Disclosure of Invention
The invention provides a polypeptide analogue, and also provides a pharmaceutical composition containing the polypeptide analogue, a preparation method and application thereof.
Polypeptide analogues
The invention provides a polypeptide analogue, which has a structural general formula of C 14 -P 1 -U 1 ；
Wherein P is 1 Comprising the amino acid sequence:
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX 1 DHWPEANX 2 VG (SEQ ID NO: 1), wherein X 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, glu, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val or Met; x is X 2 For Lys, the epsilon-amino group of the Lys has an acylated modified group; c (C) 14 To at P 1 Is a myristoyl group at the N-terminus of (2); u (U) 1 Is equal to P 1 A C-terminal linked PAS sequence of (2) consisting of Pro, ala and/or Ser, having from 0 to 300 amino acid residues.
In some embodiments, the polypeptide analog has the following structural formula:
wherein C is 14 Is myristoyl, X 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, glu, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val or Met, T 1 For the acylating modified modifying group, U 1 Is equal to P 1 A C-terminal linked PAS sequence of (2) consisting of Pro, ala and/or Ser, having from 0 to 300 amino acid residues.
In some embodiments, the X 1 Selected from beta-Ala, GABA, aib, abu, arg or Cys, preferably Arg.
In some embodiments, the polypeptide analog has the following structural formula:
wherein C is 14 Is myristoyl, T 1 For the acylating modified modifying group, U 1 Is equal to P 1 A C-terminal linked PAS sequence of (2) consisting of Pro, ala and/or Ser, having from 0 to 300 amino acid residues.
In some embodiments, the acylated modified group comprises a fatty acid. The fatty acid can be non-covalently bound to albumin, extending the half-life of the polypeptide analog.
In some embodiments, the X 2 The modified group of (C) is of the general formula- (AEEA) m -(Xaa) n -CO-(CH 2 ) p -R 1 The structural formula is as follows:
in some embodiments, p is 6-20, 10-20, 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.
In some embodiments, the m is 0, 1, 2, 3, 4, or 5. In some embodiments, the m is 0, 1, 2, or 3. In some embodiments, the m is 0. In other embodiments, m is 2.
In some embodiments, the n is 0, 1, 2, or 3. In some embodiments, n is 0. In other embodiments, n is 1.
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, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe. In some embodiments, xaa is gamma-Glu. In other embodiments, xaa is Pro.
In some embodiments, the R 1 is-CH 3 or-COOH.
In some embodiments, the X 2 The general formula of the modifying group is: -CO- (CH) 2 ) p -R 1 The structural formula is as follows:
in some embodiments, p is 6-20, 10-20, 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.
In some embodiments, the R 1 is-CH 3 or-COOH.
In some specific embodiments, the X 2 The modifying group of (C) is selected from-CO- (CH) 2 ) 6 -CH 3 、-CO-(CH 2 ) 8 -CH 3 、-CO-(CH 2 ) 10 -CH 3 、-CO-(CH 2 ) 12 -CH 3 、-CO-(CH 2 ) 14 -CH 3 、-CO-(CH 2 ) 16 -CH 3 or-CO- (CH) 2 ) 18 -CH 3 The method comprises the steps of carrying out a first treatment on the surface of the Specifically, the structural formulas are as follows:
in other embodiments, the X 2 The general formula of the modified group in (a) is:
in some embodiments, p is 6-20, 10-20, 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.
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, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe. In some embodiments, xaa is gamma-Glu. In other embodiments, xaa is Pro.
In some embodiments, the R 1 is-CH 3 or-COOH.
In some specific embodiments, the X 2 Is selected from the group consisting of-AEEA-AEEA-gamma-Glu-CO- (CH) 2 ) 6 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH、-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH; specifically, the structural formulas are as follows:
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.
In some embodiments, pro comprises 4% or more, 5% or more, 6% or more, or 8% or more, preferably 10% or more, or 15% or more, more preferably 20% or more of the total number of amino acid residues in the PAS sequence. In some embodiments, pro comprises 40% or less, or 35% or less, preferably 30% or less of the total number of amino acid residues in the PAS sequence. In some embodiments, pro comprises 4% -40%,10% -40%,15% -40%,15% -35%,20% -35% or 20% -30% of the total number of amino acid residues in the PAS sequence. In some embodiments, the PAS sequence comprises 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.
In some embodiments, the PAS sequence comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the PAS sequence comprises the amino acid sequence shown in SEQ ID NO. 2, 3, 4, 5, 6, 7 or 8, orAn amino acid sequence obtained by repeating and/or arranging all or part of one or more of these sequences. In some embodiments, the PAS sequence has the amino acid sequence shown in SEQ ID NO. 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the PAS sequence has the amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the PAS sequence has the amino acid sequence shown in SEQ ID NO. 3. In some embodiments, the PAS sequence has the amino acid sequence shown in SEQ ID NO. 5. In some embodiments, the PAS sequence C terminal is carboxyl form (i.e., C terminal with-COOH group) or free carboxyl after amidation formed in the form of an amide (i.e., C terminal with-CONH) 2 A group), preferably in the form of a carboxyl group.
The amino acid sequences of exemplary PAS sequences of the present invention are provided in Table S1 below.
Table S1 amino acid sequence of PAS sequence
In some embodiments, the PAS sequence has 0 amino acid residues, i.e., the PAS sequence is deleted. In these embodiments, the P 1 In the form of a carboxyl group at the C-terminus (i.e., having a-COOH group at the C-terminus) or an amide form formed by amidation of the free carboxyl group (i.e., having a-CONH at the C-terminus) 2 A group).
In some embodiments, the polypeptide analog is selected from the following structural formulae:
wherein C is 14 Is myristoyl, p is 6-20, 10-20, 14-18 or 16-18, m is 0, 1, 2, 3, 4 or 5, n is 0, 1, 2 or 3, R 1 is-CH 3 or-COOH, xaa is selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met.
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.
In some embodiments, the m is 0, 1, 2, or 3. In some embodiments, the m is 0. In other embodiments, m is 2.
In some embodiments, the n is 0, 1, 2, or 3. In some embodiments, n is 0. In other embodiments, n is 1.
In some embodiments, the Xaa is selected from GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe. In some embodiments, xaa is gamma-Glu. In other embodiments, xaa is Pro.
In some specific embodiments, the polypeptide analog is selected from the following structural formulas:
wherein C is 14 Is myristoyl, p is 6-20, 10-20, 14-18 or 16-18, R 1 is-CH 3 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 embodimentsAnd p is 16. In some embodiments, p is 18. In some embodiments, p is 20.
In some specific embodiments, the polypeptide analog is selected from the following structural formulas:
wherein C is 14 Is myristoyl, p is 6-20, 10-20, 14-18 or 16-18, R 1 is-CH 3 or-COOH, xaa is selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met.
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.
In some embodiments, the Xaa is selected from GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe. In some embodiments, xaa is gamma-Glu. In other embodiments, xaa is Pro.
Exemplary polypeptide analogs provided herein have the following structural formula:
1.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 6 -CH 3 )-PAS100
2.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 8 -CH 3 )-PAS100
3.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 10 -CH 3 )-PAS100
4.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 12 -CH 3 )-PAS100
5.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 14 -CH 3 )-PAS100
6.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 16 -CH 3 )-PAS100
7.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 18 -CH 3 )-PAS100
8.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 6 -COOH)-PAS100
9.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH)-PAS100
10.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH)-PAS100
11.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 -COOH)-PAS100
12.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH)-PAS100
13.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-PAS100
14.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH)-PAS100
15.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 6 -CH 3 )-PAS50
16.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 8 -CH 3 )-PAS50
17.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 10 -CH 3 )-PAS50
18.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 12 -CH 3 )-PAS50
19.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 14 -CH 3 )-PAS50
20.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 16 -CH 3 )-PAS50
21.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 18 -CH 3 )-PAS50
22.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 6 -COOH)-PAS50
23.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH)-PAS50
24.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH)-PAS50
25.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 -COOH)-PAS50
26.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH)-PAS50
27.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-PAS50
28.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH)-PAS50
29.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 6 -CH 3 )-PAS30
30.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 8 -CH 3 )-PAS30
31.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 10 -CH 3 )-PAS30
32.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 12 -CH 3 )-PAS30
33.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 14 -CH 3 )-PAS30
34.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 16 -CH 3 )-PAS30
35.Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 18 -CH 3 )-PAS30
36.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 6 -COOH)-PAS30
37.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH)-PAS30
38.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH)-PAS30
39.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 -COOH)-PAS30
40.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH)-PAS30
41.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-PAS30
42.Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH)-PAS30
the polypeptide analogues of the invention exhibit a good half-life while maintaining good viral infection inhibitory activity.
The polypeptide analogues of the invention have good solubility. In some embodiments, the polypeptide analogs of the invention, the P 1 Presence of the enzyme except Lys45 (X 2 ) In addition to the epsilon aminoacylation modification, the polypeptide analog may be inactive.
Pharmaceutical composition
The present invention provides pharmaceutical compositions comprising the polypeptide analogs or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.
In some embodiments, the pharmaceutical composition contains at least about 0.1mg, at least about 0.2mg, or at least about 0.3mg, or at least about 0.4mg, or at least about 0.6mg, or at least about 0.8mg, or at least about 1mg, or at least about 1.5mg, or at least about 2mg, or at least about 2.5mg, or at least about 3mg, or at least about 5mg, or at least about 7mg, or at least about 10mg, or at least about 12mg, or at least about 15mg, or at least about 20mg, or at least about 30mg, or at least about 50mg, or at least about 70mg, or at least about 100mg of the polypeptide analog, or pharmaceutically acceptable salt thereof.
Preparation method
In some embodiments, the peptide chain portion consisting of amino acids in the polypeptide analogs of the invention is obtained by recombinant expression methods. Specifically, escherichia coli is constructed by adopting a mode of fusion expression of a tag and a target polypeptide, fusion protein is obtained by microbial culture, induction and amplification, and the target polypeptide is obtained by purifying means after the tag is specifically excised by using proteolytic enzymes such as enterokinase (enterokinase), ubiquitin-like specific proteinase 1 (Ulp-1) and the like.
In some embodiments, the peptide chain moiety consisting of amino acids in the polypeptide analogs of the invention is obtained by chemical synthesis.
In some embodiments, the peptide chain portion consisting of amino acids in the polypeptide analogs of the invention is obtained by a combination of recombinant expression and chemical synthesis.
In some embodiments, the method of making a polypeptide analog of the invention comprises the steps of: (a) Preparation of the P 1 A peptide chain portion consisting of amino acids and a modifying group for acylation modification; (b) Carrying out myristoylation reaction at the N-terminal of the peptide chain part; (c) Amino acid residue X of the peptide chain portion with the modifying group 2 Performing acylation modification on epsilon-amino groups to prepare the polypeptide analogue; and (d) isolating and purifying the polypeptide analog.
Use of the same
The present invention provides a method of inhibiting Hepatitis B Virus (HBV) and/or Hepatitis Delta Virus (HDV) infection of a cell comprising administering to the cell an effective amount of a polypeptide analog or pharmaceutical composition of the invention.
The present invention provides the use of a polypeptide analogue or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a patient infected with HBV and/or HDV.
The present invention provides the use of a polypeptide analogue or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a patient suffering from hepatitis b, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and/or nonalcoholic fatty liver disease.
The present invention provides a method of 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 invention.
The present invention provides a method of treating a patient suffering from hepatitis b, hepatitis d, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and/or nonalcoholic fatty liver disease comprising administering to the patient a therapeutically effective amount of a polypeptide analog or pharmaceutical composition of the present invention.
The present disclosure also provides some specific embodiments below, but the scope of protection of the present disclosure is not limited thereto:
embodiment 1A polypeptide analog having the general structural formula C 14 -P 1 -U 1 ；
Wherein,,
P 1 comprising the amino acid sequence:
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX 1 DHWPEANX 2 VG (SEQ ID NO: 1), wherein X 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, glu, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val or Met, preferably beta-Ala, GABA, aib, abu, arg or Cys, more preferably Arg; x is X 2 For Lys, the epsilon-amino group of the Lys has an acylated modified group;
C 14 to at P 1 Is a myristoyl group at the N-terminus of (2);
U 1 is equal to P 1 A C-terminal linked PAS sequence of (2) consisting of Pro, ala and/or Ser, having from 30 to 300 amino acid residues.
Embodiment 2. The polypeptide analog according to embodiment 1, wherein the X 2 Binding of modifying groups of (2)The structure is as follows:
wherein,,
p is 6-20, 10-20, 14-18 or 16-18,
m is 0, 1, 2, 3, 4 or 5,
n is 0, 1, 2 or 3,
xaa is selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met,
R 1 is-CH 3 or-COOH.
Embodiment 3. The polypeptide analogue according to embodiment 2, wherein m is 0, 1, 2 or 3, preferably 0 or 2.
Embodiment 4. The polypeptide analogue of embodiment 2 or 3, wherein n is 0 or 1.
Embodiment 5. The polypeptide analog of embodiment 2, wherein the modifying group has a structural formula shown in formula 1 or formula 2:
embodiment 6. The polypeptide analogue according to embodiment 5, wherein Xaa is selected from GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe, preferably gamma-Glu or Pro.
Embodiment 7. The polypeptide analogue according to embodiment 5 or 6, wherein p is 6, 8, 10, 12, 14, 16, 18 or 20, preferably 16 or 18.
Embodiment 8. The polypeptide analog of embodiment 1, wherein the X 2 The modifying group of (2) is selected from the following structural formula:
embodiment 9. The polypeptide analogue according to any one of embodiments 1-8, wherein the PAS sequence has 30-100 amino acid residues, preferably 30, 50 or 100 amino acid residues.
Embodiment 10. The polypeptide analog of embodiment 9, wherein Pro comprises 10% -40% of the total number of amino acid residues in the PAS sequence.
Embodiment 11. The polypeptide analog of embodiment 9 or 10, wherein the PAS sequence comprises the amino acid sequence shown in SEQ ID NO. 2, 3, 4, 5, 6, 7 or 8.
Embodiment 12. The polypeptide analog of embodiment 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.
Embodiment 13. The polypeptide analog according to embodiment 1, wherein the polypeptide analog is selected from the group consisting of the following structural formulas:
wherein C is 14 Is myristoyl; p is 6-20, 10-20, 14-18 or 16-18, preferably 6, 8, 10, 12, 14, 16, 18 or 20; xaa is selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met, preferably GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe, more preferably gamma-Glu or Pro; r is R 1 is-CH 3 or-COOH.
Embodiment 14. The polypeptide analog according to embodiment 1, wherein the polypeptide analog is selected from the group consisting of the following structural formulas:
wherein C is 14 Is myristoyl.
Embodiment 15. A method of making the polypeptide analog of any of embodiments 1-14, comprising the steps of:
a) Preparation of P according to any of embodiments 1-14 1 A peptide chain portion consisting of amino acids and a modifying group for acylation modification;
b) Carrying out myristoylation reaction at the N-terminal of the peptide chain part;
c) Amino acid residue X of the peptide chain portion with the modifying group 2 Performing acylation modification on epsilon-amino groups to prepare the polypeptide analogue; and
d) Isolating and purifying the polypeptide analogue.
Embodiment 16. A pharmaceutical composition comprising the polypeptide analog of any of embodiments 1-14, and a pharmaceutically acceptable carrier.
Embodiment 17. A method of inhibiting HBV and/or HDV infection in a cell comprising administering an effective amount of a polypeptide analog of any of embodiments 1-14 or a pharmaceutical composition of embodiment 16 to said cell.
Embodiment 18. Use of the polypeptide analogue of any one of embodiments 1-14 or the pharmaceutical composition of embodiment 16 in the manufacture of a medicament for treating a patient infected with HBV and/or HDV.
Embodiment 19. Use of the polypeptide analog of any of embodiments 1-14 or the pharmaceutical composition of embodiment 16 in the manufacture of a medicament for treating a patient with hepatitis b, hepatitis d, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia, and/or nonalcoholic fatty liver disease.
Embodiment 20. A method of treating a patient infected with HBV and/or HDV comprising administering to the patient a therapeutically effective amount of a polypeptide analog of any of embodiments 1-14 or a pharmaceutical composition of embodiment 16.
Embodiment 21. A method of treating a patient with hepatitis b, hepatitis d, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia, and/or nonalcoholic fatty liver disease comprising administering to the patient a therapeutically effective amount of a polypeptide analog of any one of embodiments 1-14 or a pharmaceutical composition of embodiment 16.
Drawings
FIG. 1 shows Myrcludex B and Myristonyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH) -PAS100 blood concentration-time profile in beagle dogs.
Detailed Description
Definition and description
The following terms used in the present application have the following meanings unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art.
Unless otherwise indicated, with solid wedge bondsAnd wedge-shaped dotted bond->Representing the absolute configuration of a stereogenic center.
When a group has a bondable site, the bond of the site to other groups may be by wavy lines, unless otherwise specifiedAnd (3) representing.
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 part of the present disclosure. The asymmetric carbon atom containing compounds of the present disclosure may be isolated in optically active pure or racemic forms. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.
When referring to the description of amino acid positions, this means that the starting amino acids according to the corresponding sequences are numbered sequentially as the first position. For example, "Lys at position 45", "Lys45" or "K45" in SEQ ID NO. 1 refers to Lys at position 45 in the sequence numbered sequentially starting with the first amino acid in SEQ ID NO. 1 as position 1; as another example, "37Arg" or "37R" in SEQ ID NO. 1 means that the sequence is numbered starting with position 1 as the first amino acid in SEQ ID NO. 1, and Arg is at position 37 in the sequence.
As used herein "(a group) n "means that there are n of such groups in the moiety. For example, as used herein (AEEA) n Or (Xaa) n Meaning that the moiety has n attached AEEA groups or Xaa groups.
The term "IC 50 "refers to the concentration of the test compound that inhibits the reaction to a level of 50% of the maximum inhibition reaction (i.e., intermediate between the maximum inhibition reaction and the untreated reaction) in an in vitro or in vivo assay. IC (integrated circuit) 50 "can be measured by ELISA or FACS analysis or any other method known in the art.
The term "AEEA" means 2- [2- (2-aminoethoxy) ethoxy ] acetic acid.
"C" as used herein 14 "means Myristoyl (Myristonyl), also known as Myristoyl, of the structure-CO- (CH) 2 ) 12 -CH 3 。
As used herein, a "PAS sequence" is a polypeptide fragment consisting of three amino acids, proline, alanine and serine, which is highly soluble, uncharged, and exhibits an irregular coil structure under physiological conditions. When the PAS sequence has 0 amino acid residues, it means that the PAS sequence is deleted.
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 compound described herein. Such substances are administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which they are contained.
The term "pharmaceutically acceptable salt" includes salts of a base ion with a free acid or salts of a acid ion with a free base, including 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, potassium, ammonium, amino acid salts and the like. In some embodiments, the pharmaceutically acceptable salt of the polypeptide analog of the invention is an acetate salt of the polypeptide analog.
The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as is well known to those skilled in the art (Remington' sPharmaceutical Sciences,18th Ed.Mack Printing Company,1990,pp.1289-1329). In addition to carriers that are incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
The term "treatment" refers to attempting to alter the natural course of a disease in a treated individual, and may be for the purpose of preventing or clinical intervention performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
The term "therapeutically effective amount" refers to the amount of a polypeptide analog, pharmaceutical composition, or other administration necessary to provide a therapeutic and/or prophylactic benefit to a subject.
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, and the like. Preferably, the subject according to the application is a human. Unless indicated, the terms "patient" or "subject" are used interchangeably.
The application also includes isotopically-labeled compounds of the application which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as, respectively 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, and the like.
Certain isotopically-labeled compounds of the application (e.g., with 3 H is H 14 C-labeled) can be used in compound and/or substrate tissue distribution analysis. Tritiation (i.e 3 H) And carbon-14 (i.e 14 C) Isotopes are particularly preferred for their ease of preparation and detectability. In addition, the use of heavier isotopes (such as deuterium (i.e. 2 H) Substitution may provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances. Positron emitting isotopes, such as 15 O、 13 N、 11 C and C 18 F can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Can be generally passed throughIsotopically-labeled compounds of the present application are prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.
The natural amino acid residues in a protein or polypeptide are abbreviated as follows: phenylalanine is Phe or F, leucine is Leu or L, isoleucine is Ile or I, methionine is Met or M, valine is Val or V, serine is Ser or S, proline is Pro or P, threonine is Thr or T, alanine is Ala or A, tyrosine is Tyr or Y, histidine is His or H, glutamine is Gln or Q, asparagine is Asn or N, lysine is Lys or K, aspartic acid is Asp or D, glutamic acid is Glu or E, cysteine is Cys or C, tryptophan is Trp or W, arginine is Arg or R, glycine is Gly or G; unnatural amino acid residues are abbreviated as follows: d-alanine is D-Ala, D-glutamic acid is D-Glu, beta-alanine (i.e., 3-aminopropionic acid) is beta-Ala, 4-aminobutyric acid is GABA, 2-aminoisobutyric acid is Aib, and 2-aminobutyric acid is Abu.
The gamma-carboxyl group is involved in peptide bond formation and the alpha-carboxyl group is a free carboxyl group, which is gamma-Glu (gamma-glutamic acid) having the following structure:
the words "comprise", "comprising" or "includes" and variations thereof such as include or comprise are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to".
In this document, singular terms encompass plural referents and vice versa, unless the context clearly dictates otherwise.
As used herein, "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation per the practice of the art. Alternatively, "about" may mean a range of up to + -5%, e.g., fluctuating within + -2%, within + -1%, or within + -0.5% of a given specific numerical range. When a particular value is given in the disclosure or claims, unless otherwise indicated, the meaning of "about" is to be considered within the acceptable error range for that particular value. In this context, the values of the dose, time, step parameters or conditions of all drugs are by default modified by "about" unless otherwise indicated.
The chemical reactions of the embodiments of the present application are accomplished in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.
The application will be further described with reference to specific examples, which are, however, only intended to illustrate and not limit the scope of the application. Also, the application is not limited to any particular preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications of the technical features of the present application are included in the scope of the present application. The reagents used in the examples below are commercially available products, and the solutions may be formulated using techniques conventional in the art, unless otherwise specified.
Unless otherwise specified, the practice of the present application will employ, or are in accordance with the product specification, conventional techniques of organic synthesis, biochemistry, protein purification and the like, which are within the skill of the art, well-defined in the literature. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for.
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- (fluorenylmethoxycarbonyl amino) ethoxy ] acetic acid;
Fmoc-Gly-OH: fluorenylmethoxycarbonyl-glycine;
Fmoc-Arg (Pbf) -OH: n-fluorenylmethoxycarbonyl-2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine;
Fmoc-Ser (tBu) -OH: N-fluorenylmethoxycarbonyl-O-tert-butyl-L-serine;
Fmoc-Pro-OH: fluorenylmethoxycarbonyl-L-proline;
Fmoc-Ala-OH: N-fluorenylmethoxycarbonyl-L-alanine;
Fmoc-Ile-OH: fluorenylmethoxycarbonyl-L-isoleucine;
Fmoc-Leu-OH: N-fluorenylmethoxycarbonyl-L-leucine;
Fmoc-Trp (Boc) -OH: fluorenylmethoxycarbonyl-tryptophan (t-butoxycarbonyl);
Fmoc-Gln (Trt) -OH: fluorenylmethoxycarbonyl-glutamic acid (t-butyl ester);
Fmoc-Val-OH: fluorenylmethoxycarbonyl-L-valine;
Fmoc-Phe-OH: fluorenylmethoxycarbonyl-L-phenylalanine;
Fmoc-Glu (OtBu) -OH: fluorenylmethoxycarbonyl-glutamic acid (t-butyl ester);
Fmoc-Thr (tBu) -OH: n- (9-fluorenylmethoxycarbonyl) -tert-butyl-L-threonine;
Fmoc-Tyr (tBu) -OH: fluorenylmethoxycarbonyl-oxy-tert-butyl-tyrosine;
Fmoc-Asp (OtBu) -OH: fluorenylmethoxycarbonyl-aspartic acid (tert-butyl ester);
Fmoc-Lys (Boc) -OH: N-fluorenylmethoxycarbonyl-N' -tert-butoxycarbonyl-L-lysine;
Fmoc-His (Trt) -OH: fluorenylmethoxycarbonyl-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-fluorenylmethoxycarbonyl;
TFA: trifluoroacetic acid;
TFE: trifluoroethanol;
tis: triisopropylsilane;
HOSU: n-hydroxysuccinimide;
m/z: mass to charge ratio.
PBS (pH 7.4): containing potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.27g/L, disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 ·12H 2 O) 2.85g/L, sodium chloride (NaCl) 8.5g/L and potassium chloride (KCl) 0.2g/L.
1 2 16 Example 1: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH) -PAS100 has the following structure:
(1) Materials and reagents
2-CTC resin (Sean blue Xiao technology New Material Co., ltd.) with a substitution value (SD) of 1.15mmol/g.
Materials: fmoc-Glu-OMe (CAS number: 145038-49-9), fmoc-AEEA-OH (CAS number: 166108-71-0), mono-tert-butyl octadecanedioate (CAS number: 843666-40-0), succinimidyl myristate (Han Xiang Bio, CAS number: 69888-86-4).
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM, PIP, DIEA.
Activating reagent: DCC, HOSU.
P 1 (37 Arg) -PAS100 was obtained by expression of Escherichia coli, m/z=1212.0 [ M+11H ]] 11+ ，P 1 The sequence of (37 Arg) -PAS100 is as follows:
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPAPSAPAAASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS(SEQ ID NO:9)。
(2) Instrument for measuring and controlling the intensity of light
CS-BIO type polypeptide synthesizer, waters 600 semi-preparative high performance liquid chromatograph, beckman centrifuge, BUCHI vacuum distillation instrument.
(3) Operating procedure
a.P 1 (37 Arg) -PAS 100N-terminal myristoylation reaction
50mg of P 1 (37 Arg) -PAS100 was dissolved in 20mmol/L PBS (pH 7.4) to a final concentration of 5mg/mL, and an equal volume of acetonitrile was added thereto, and 10 volumes of an acetonitrile solution (2 mg/mL) containing succinimidyl myristate was slowly added dropwise thereto, and the reaction was stirred at room temperature for 2 hours, and was terminated by adjusting pH to 4.0 with 0.1mol/L hydrochloric acid to give a crude product Myristonyl-P 1 (37Arg)-PAS100。
The crude product was purified by Waters 600 semi-preparative hplc using gradient elution parameters as shown in table 1: the chromatographic column is Kromasil 300-10-C 4 10 x 250mm, flow rate of 5mL/min, detection wavelength of 215nm, 280nm, mobile phase A of 0.5% HAC (v/v) aqueous solution, mobile phase B of 0.5% HAC (v/v) acetonitrile solution.
TABLE 1 gradient elution parameters
The collected purified product was analyzed using gradient elution parameters as shown in table 2: the chromatographic column is Kromasil 100-3.5-C 4 4.6X105 mm, a flow rate of 1mL/min, a detection wavelength of 215nm, mobile phase A of 0.05% TFA (v/v) in water, and mobile phase B of 0.05% TFA (v/v) in acetonitrile.
TABLE 2 gradient elution parameters
Collecting target component with purity higher than 90%, distilling under reduced pressure to remove acetonitrile, and vacuum freezingAnd (5) drying. Molecular weight confirmation by ESI-MS, m/z=1129.5 [ M+12H ]] 12+ Consistent with theoretical molecular weight.
b. Solid phase synthesis of acylated modified groups
1.00g of 2-CTC resin is weighed and placed in a CS-BIO type polypeptide synthesizer reactor, 10mL of DCM is added for soaking for 1h, fmoc-AEEA-OH and DIEA which are 2-3 times of the mole number of the resin and 4-6 times of DIEA are weighed and are added into the reactor after being dissolved, the reaction temperature is room temperature after the dissolution of 10mL of DCM, the coupling of the first amino acid is completed after the reaction is carried out for 2h, then the DCM is used for washing the resin for 6 times, then 10mL of 20% PIP (v/v) DMF solution is added, the Fmoc amino protecting group is removed after mixing for 30min, the DCM is used for washing the resin for 6 times, the second amino acid is coupled, and after the Fmoc-AEEA-OH, HOBt and DIC which are 3 times of the mole number of the resin are weighed, the mixed solvent which is added into 10mL of DMF/DCM (v: v=1:1) is dissolved, the reaction is put into the reactor for reaction at room temperature, the reaction progress is monitored, the progress of the ninhydrin reaction is monitored, and the colorless reaction is monitored to be completed after the reaction is washed for 6 times. Then, the coupling reaction of the gamma-glutamic acid and the octadecanedioic acid can be continued according to the coupling method, and the cycle is performed until all amino acid coupling is completed.
c. Cleavage and precipitation of acylated modified groups
Adding a cleavage reagent according to the proportion of 5mL of the cleavage reagent to 1g of resin, wherein the reagent proportion is TFE (TFE) and DCM=1:4 (v: v), stirring at room temperature for reaction for 1 hour, filtering, distilling the filtrate at 40 ℃ under reduced pressure to remove the solvent, adding 10mL of DCM into a reduced pressure distillation flask, distilling the solvent again under reduced pressure to remove the solvent, repeating for 2-3 times, finally adding 3mL of DCM to dissolve the polypeptide, adding 40mL of glacial ethyl ether, placing in a refrigerator at-20 ℃ for 20min, centrifuging, and drying in vacuum to obtain the acylated modified group.
d. Preparation of polypeptide analogue Myristonyl-P by liquid phase reaction 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-PAS100
Weighing about 0.1mmol of the dried acylated modified group, about 0.095mmol of HOSU, and sucking 15. Mu.L of DIC, adding about 5mL of Tetrahydrofuran (THF), and reacting at room temperature for 1-2 hoursTHF was removed by distillation under reduced pressure to give a yellow oil, which was then added with TFA: H 2 Removing tert-butyl ester protecting group by using the mixed solvent of O=90:10, reacting for 1 hour at room temperature, pouring in glacial ethyl ether for precipitation, centrifuging to obtain solid, washing with diethyl ether for three times, drying under reduced pressure to obtain solid powder, weighing, dissolving with DMF, and obtaining the acylation modified activated ester solution.
Will be 0.5mmol Myristonyl-P 1 (37 Arg) -PAS100 is dissolved in 1% (v/v) triethylamine solution, 1.2 times of the volume of the acylation modified activated ester solution is slowly dripped into the solution, the solution is stirred for 10 minutes at room temperature, the pH is regulated to 8.0 by 0.1mol/L hydrochloric acid, and the reaction is stopped, thus obtaining the crude product Myristonyl-P of the polypeptide analogue 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-PAS100。
Purification of the crude polypeptide analogs were performed using a Waters 600 semi-preparative high performance liquid chromatograph using gradient elution parameters as shown in table 1: the chromatographic column is Kromasil 300-10-C 4 10 x 250mm, flow rate of 5mL/min, detection wavelength of 215nm, 280nm, mobile phase A of 0.5% HAC (v/v) aqueous solution, mobile phase B of 0.5% HAC (v/v) acetonitrile solution.
The collected purified polypeptide analogs were analyzed using gradient elution parameters as shown in table 2: the chromatographic column is Kromasil 100-3.5-C 4 4.6X105 mm, a flow rate of 1mL/min, a detection wavelength of 215nm, mobile phase A of 0.05% TFA (v/v) in water, and mobile phase B of 0.05% TFA (v/v) in acetonitrile.
Collecting target component with purity higher than 90%, distilling under reduced pressure to remove acetonitrile, and vacuum freeze drying. As a result of molecular weight confirmation by ESI-MS, m/z=1296.17 [ M+11H ]] 11+ Consistent with theoretical molecular weight.
1 2 6 Example 2: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 6 -COOH) -PAS100 has the following structure:
the preparation and purification procedure was as described in reference to example 1, with ESI-MS molecular weight confirmation, m/z=1283.45 [ M+11H ]] 11+ Consistent with theoretical molecular weight.
1 2 8 Example 3: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH) -PAS100 has the following structure:
preparation and purification procedure reference was made to the procedure in example 1, as confirmed by ESI-MS molecular weight, m/z=1285.99 [ m+11h] 11+ Consistent with theoretical molecular weight.
1 2 10 Example 4: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH) -PAS100 has the following structure:
the preparation and purification procedure was as described in reference to example 1, as confirmed by ESI-MS molecular weight, m/z=1288.54 [ M+11H ]] 11+ Consistent with theoretical molecular weight.
1 2 12 Example 5: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 -COOH) -PAS100 has the following structure:
preparation and purification procedure reference the procedure in example 1, as confirmed by ESI-MS molecular weight, m/z=1291.08 [ M+11H ]] 11+ Consistent with theoretical molecular weight.
1 2 14 Example 6: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH) -PAS100 has the following structure:
preparation and purification procedure reference the procedure in example 1, as confirmed by ESI-MS molecular weight, m/z=1293.63 [ M+11H] 11+ Consistent with theoretical molecular weight.
1 2 18 Example 7: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS100 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH) -PAS100 has the following structure:
Preparation and purification procedure reference the procedure in example 1, as confirmed by ESI-MS molecular weight, m/z=1298.72 [ M+11H] 11+ Consistent with theoretical molecular weight.
1 2 6 3 Example 8: myristyl-Preparation of P (37 Arg, lys 45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 6 -CH 3 ) The structure of the PAS100 is as follows:
(1) Materials and reagents
Fatty acids used for the synthesis of the acylated modified groups are: n-octanoic acid.
The activating reagent is as follows: DCC, HOSU.
(2) Instrument for measuring and controlling the intensity of light
BUCHI vacuum distillation apparatus, waters 600 semi-preparative high performance liquid chromatograph.
(3) Operating procedure
In the procedure of example 1 (3), step b and step c were skipped and the obtaining of the acylation modified activated ester solution was modified as follows:
about 5mL of methylene chloride was added to 0.1mmol of n-octanoic acid, about 0.15mmol of HOSU and about 0.12mmol of DCC, and the mixture was reacted at room temperature for 1 to 2 hours, distilled under reduced pressure to remove methylene chloride, and dissolved in DMF to obtain an acylated activated ester solution.
The remaining preparation and purification steps were carried out by ESI-MS for molecular weight confirmation, m/z=1242.64 [ M+11H ] with reference to the procedure in example 1] 11+ Consistent with theoretical molecular weight.
1 2 8 3 Example 9: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 8 -CH 3 ) The structure of the PAS100 is as follows:
preparation and purification procedure reference is made to the procedure in example 8, molecular weight determination via ESI-MS Evidence, m/z=1245.19 [ m+11h] 11+ Consistent with theoretical molecular weight.
1 2 10 3 Example 10: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 10 -CH 3 ) The structure of the PAS100 is as follows:
preparation and purification procedure reference is made to the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1247.73 [ m+11h] 11+ Consistent with theoretical molecular weight.
1 2 12 3 Example 11: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 12 -CH 3 ) The structure of the PAS100 is as follows:
preparation and purification procedure reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1250.28 [ m+11h ]] 11+ Consistent with theoretical molecular weight.
1 2 14 3 Example 12: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 14 -CH 3 ) The structure of the PAS100 is as follows:
preparationAnd purification procedure reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1252.82 [ m+11h] 11+ Consistent with theoretical molecular weight.
1 2 16 3 Example 13: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 16 -CH 3 ) The structure of the PAS100 is as follows:
preparation and purification procedure reference was made to the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1255.37 [ m+11h] + Consistent with theoretical molecular weight.
1 2 18 3 Example 14: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS100
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 18 -CH 3 ) The structure of the PAS100 is as follows:
preparation and purification procedure reference is made to the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1257.91 [ m+11h] 11+ Consistent with theoretical molecular weight.
1 2 6 3 Example 15: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 6 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] + ，P 1 The sequence of (37 Arg) -PAS30 is as follows:
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP(SEQ ID NO:10)。
other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1591.22 [ m+5h] + Consistent with theoretical molecular weight.
1 2 8 3 Example 16: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 8 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1596.82 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 10 3 Example 17: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 10 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other systemsThe preparation and purification procedure was as described in example 8, with molecular weight confirmation via ESI-MS, m/z=1602 [ M+5H ] ] + Consistent with theoretical molecular weight.
1 2 12 3 Example 18: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 12 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1608.02 [ m+5h] + Consistent with theoretical molecular weight.
1 2 14 3 Example 19: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 14 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1613.62 [ m+5h] + Consistent with theoretical molecular weight.
1 2 16 3 Example 20: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 16 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1619.22 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 18 3 Example 21: preparation of Myristonyl-P (37 Arg, lys45-. Epsilon. -CO- (CH) -CH) -PAS30
Myristoyl-P 1 (37Arg，Lys45-ε-CO-(CH 2 ) 18 -CH 3 ) The structure of the PAS30 is as follows:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ] ] 5+ 。
Other preparation and purification procedures reference the procedure in example 8, molecular weight confirmation via ESI-MS, m/z=1624.82 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 6 Example 22: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 6 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1680.99 [ m+5h] + Consistent with theoretical molecular weight.
1 2 8 Example 23: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 8 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1686.59 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 10 Example 24: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 10 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1692.19 [ m+5h ] 5+ Consistent with theoretical molecular weight.
1 2 12 Example 25: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 12 The structure of-COOH) -PAS30 is as follows
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1697.79 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 14 Example 26: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 14 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1703.39 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 16 Example 27: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1708.99 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 2 18 Example 28: myristonyl-P (37 Arg, lys45- ε -AEEA-AEEA- γ -Glu-CO- (CH) -COOH) and method for preparing the same
PAS30 preparation
Myristoyl-P 1 (37Arg，Lys45-ε-AEEA-AEEA-γ-Glu-CO-(CH 2 ) 18 -COOH) -PAS30 has the following structure:
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89 [ M+5H ]] 5+ 。
Other preparation and purification procedures reference the procedure in example 1, molecular weight confirmation via ESI-MS, m/z=1714.59 [ m+5h] 5+ Consistent with theoretical molecular weight.
1 Example 29: preparation of Myristyl-P (37 Arg) -PAS100
Myristoyl-P 1 The structure of (37 Arg) -PAS100 is as follows:
C 14 -
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGSPASPAAPAPASPAAPAPSAPAA
ASAAAPAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPAS。
(1) Materials and reagents
Succinic acid succinimidyl esters (Variot, CAS number 69888-86-4);
P 1 (37 Arg) -PAS100 was obtained by expression of Escherichia coli, m/z=1212.0 [ M+11H ]] 11+ 。
(2) Instrument for measuring and controlling the intensity of light
Waters 600 semi-preparative high performance liquid chromatograph, BUCHI reduced pressure distillation apparatus.
(3) Operating procedure
Will be 0.5mmol P 1 (37 Arg) -PAS100 was dissolved in 20mmol/L PBS (pH 7.4) to a final concentration of 5mg/mL, and an equal volume of acetonitrile was added thereto, and 10 volumes of an acetonitrile solution (2 mg/mL) containing succinimidyl myristate was slowly added dropwise thereto, and the reaction was stirred at room temperature for 2 hours, followed by adjusting the pH to 4.0 with 0.1mol/L hydrochloric acid to terminate the reaction to give a crude product Myristyl-P 1 (37Arg)-PAS100。
The crude product was purified by semi-preparative high performance liquid chromatography using gradient elution parameters as shown in table 1: the chromatographic column is Kromasil 300-10-C 4 10 x 250mm, flow rate of 5mL/min, detection wavelength of 215nm, 280nm, mobile phase A of 0.5% HAC (v/v) aqueous solution, mobile phase B of 0.5% HAC (v/v) acetonitrile solution.
The collected purified product was analyzed using gradient elution parameters as shown in table 2: the chromatographic column is Kromasil 100-3.5-C 4 4.6X105 mm, a flow rate of 1mL/min, a detection wavelength of 215nm, mobile phase A of 0.05% TFA (v/v) in water, and mobile phase B of 0.05% TFA (v/v) in acetonitrile.
Collecting target component with purity higher than 90%, distilling under reduced pressure to remove acetonitrile, and vacuum freeze drying. Molecular weight confirmation by ESI-MS, m/z=1129.5 [ M+12H ]] + Consistent with theoretical molecular weight.
1 Example 30: preparation of Myristyl-P (37 Arg) -PAS30
Myristoyl-P 1 The structure of (37 Arg) -PAS30 is as follows:
C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVGASPAAPAPASPAAPAPSAPAAASAAAPAAP。
P 1 (37 Arg) -PAS30 was obtained by expression of Escherichia coli, m/z=1523.89[M+5H] 5+ 。
Preparation was carried out as described in example 29, molecular weight confirmation was carried out by ESI-MS, m/z=1565.96 [ M+5H ]] + Consistent with theoretical molecular weight.
1 Example 31: preparation of Myristonyl-P (37 Arg) -OH
Myristoyl-P 1 The structure of (37 Arg) -OH is as follows:
C 14 GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVG-OH, the C-terminus of which is the free carboxyl group.
(1) Materials and reagents
Fmoc-Gly-Wang Resin (Hischi Biotechnology Co., ltd.) with a substitution value of 0.29mmol/g;
Materials: fmoc-Gly-OH (CAS number: 29022-11-5), fmoc-Arg (Pbf) -OH (CAS number: 154445-77-9), fmoc-Ser (CAS number: 71989-8), fmoc-Pro-OH (CAS number: 71989-31-6), fmoc-Ala-OH (CAS number: 35661-39-3), fmoc-Ile-OH (CAS number: 71989-23-6), fmoc-Leu-OH (CAS number: 35661-60-0), fmoc-Trp (Boc) -OH (CAS number: 143824-78-6), fmoc-Gln (Trt) -OH (CAS number: 132327-80-1), fmoc-Val-OH (CAS number: 68858-20-8), fmoc-Phe-OH (CAS number: 35661-40-6), fmoc-OH (OtBu) -OH (CAS number: 3838-18-OH), fmoc-Trp (CAS number: 35-75-Glu-35 (CAS number: 35-35), fmoc-35-OH (CAS number: 35-37-75-6), fmoc-His (Trt) -OH (CAS number: 109425-51-6), succinimidyl myristate (Vast. RTM., CAS number: 69888-86-4).
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM, PIP, DIEA.
(2) Instrument for measuring and controlling the intensity of light
A CSIO-BIO type polypeptide synthesizer, a Waters 600 semi-preparative high performance liquid chromatograph, a Beckman centrifuge and a BUCHI reduced pressure distiller.
(3) Operating procedure
a.Myristoyl-P 1 Synthesis of (37 Arg) -OH
0.5g of Fmoc-Gly-Wang Resin is weighed and placed in a reactor of a polypeptide synthesizer, 10mL of DCM is added for soaking for 1h, then 10mL of 20% PIP (v/v) DMF solution is added for mixing for 30min to remove amino protecting groups, the Resin is washed for 6 times by DCM, the first amino acid is coupled, 3 times of Fmoc-Val-OH, HOBt and DIC are weighed and the amount of Fmoc-Val-OH, HOBt and DIC is weighed, after 10mL of DMF/DCM (v/v=1:1) mixed solvent is added for dissolution, the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the colorless reaction is monitored, and the Resin is washed for 6 times by DCM. Then 10mL of 20% PIP (v/v) DMF solution is added, the mixture is mixed for 30min to remove amino protecting groups, the resin is washed by DCM for 6 times, the second amino acid is coupled, fmoc-Lys (BOC) -OH, HOBt and DIC which are 3 times of the mole number of the resin are weighed, 10mL of mixed solvent of MF/DCM (v/v=1:1) is added for dissolution, the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the colorless reaction is monitored, and the resin is washed by DCM for 6 times. And then, the coupling reaction can be continued according to the coupling method, and the cycle is performed until all amino acid coupling is completed.
b. Cleavage and precipitation
Adding the cracking reagent according to the proportion of 5mL of the cracking reagent/1 g of resin, wherein the reagent proportion is TFA to H 2 O: tis=95:2.5:2.5 (v: v), stirring at room temperature, reacting for 2 hours, filtering, adding 130mL of glacial ethyl ether, placing in a refrigerator at-20 ℃ for 20min, centrifuging, vacuum drying, and weighing crude product Myristonyl-P 1 (37Arg)-OH。
The crude product was purified by semi-preparative high performance liquid chromatography using gradient elution parameters as shown in table 1: the chromatographic column is Kromasil 300-10-C 4 10 x 250mm, flow rate of 5mL/min, detection wavelength of 215nm, 280nm, mobile phase A of 0.5% HAC (v/v) aqueous solution, mobile phase B of 0.5% HAC (v/v) acetonitrile solution.
The collected purified product was analyzed using gradient elution parameters as shown in table 2: the chromatographic column is Kromasil 100-3.5-C 4 4.6X105 mm, a flow rate of 1mL/min, a detection wavelength of 215nm, mobile phase A of 0.05% TFA (v/v) in water, and mobile phase B of 0.05% TFA (v/v) in acetonitrile.
Collecting target component with purity higher than 90%, distilling under reduced pressure to remove acetonitrile, and vacuum freeze drying. Molecular weight confirmation by ESI-MS, m/z=775.14[M+7H] 7+ Consistent with theoretical molecular weight.
1 2 Example 32: myristonyl-P (37 Arg) -NH
Myristoyl-P 1 (37Arg)-NH 2 The structure of (2) is as follows:
C 14 -GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNRDHWPEANKVG-NH 2 Its C-terminal amidation.
(1) Materials and reagents
MBHA resin (West Lan Xiao New Material Co., ltd.) with substitution value of 0.58mmol/g
Materials: fmoc-Gly-OH (CAS number: 29022-11-5), fmoc-Arg (Pbf) -OH (CAS number: 154445-77-9), fmoc-Ser (CAS number: 71989-8), fmoc-Pro-OH (CAS number: 71989-31-6), fmoc-Ala-OH (CAS number: 35661-39-3), fmoc-Ile-OH (CAS number: 71989-23-6), fmoc-Leu-OH (CAS number: 35661-60-0), fmoc-Trp (Boc) -OH (CAS number: 143824-78-6), fmoc-Gln (Trt) -OH (CAS number: 132327-80-1), fmoc-Val-OH (CAS number: 68858-20-8), fmoc-Phe-OH (CAS number: 35661-40-6), fmoc-OH (OtBu) -OH (CAS number: 3838-18-OH), fmoc-Trp (CAS number: 35-75-Glu-35 (CAS number: 35-35), fmoc-35-OH (CAS number: 35-37-75-6), fmoc-His (Trt) -OH (CAS number: 109425-51-6), succinimidyl myristate (Vast. RTM., CAS number: 69888-86-4).
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM, PIP, DIEA
(2) Instrument for measuring and controlling the intensity of light
A CSIO-BIO type polypeptide synthesizer, a Waters 600 semi-preparative high performance liquid chromatograph, a Beckman centrifuge and a BUCHI reduced pressure distiller.
(3) Operating procedure
a.Myristoyl-P 1 (37Arg)-NH 2 Is synthesized by (a)
0.5g of MBHA resin is weighed and placed in a reactor of a polypeptide synthesizer, 10mL of DCM is added and soaked for 1h, then 10mL of 20% PIP (v/v) DMF solution is added and mixed for 30min to remove amino protecting groups, the DCM is used for washing the resin for 6 times, the first amino acid is coupled, fmoc-Lys (BOC) -OH, HOBt and DIC which are 3 times of the mole number of the resin are weighed, 10mL of DMF/DCM (v/v=1:1) is added for dissolution, then the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the colorless reaction is monitored, and the resin is washed for 6 times by DCM. Then 10mL of 20% PIP (v/v) DMF solution is added, the mixture is mixed for 30min to remove amino protecting groups, the resin is washed with DCM for 6 times to couple with a second amino acid, fmoc-Lys (BOC) -OH, HOBt and DIC which are 3 times the mole number of the resin are weighed, 10mL of DMF/DCM (v/v=1:1) mixed solvent is added for dissolution, the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the monitoring of the colorless state is the completion of the reaction, and the resin is washed with DCM for 6 times. And then, the coupling reaction can be continued according to the coupling method, and the cycle is performed until all amino acid coupling is completed.
b. Cleavage and precipitation
Adding the cracking reagent according to the proportion of 5mL of the cracking reagent/1 g of resin, wherein the reagent proportion is TFA to H 2 O: tis=95:2.5:2.5 (v: v), stirring at room temperature, reacting for 2 hours, filtering, adding 130mL of glacial ethyl ether, placing in a refrigerator at-20 ℃ for 20min, centrifuging, vacuum drying, and weighing crude product Myristonyl-P 1 (37Arg)-NH 2 。
The crude product was purified by semi-preparative high performance liquid chromatography using gradient elution parameters as shown in table 1: the chromatographic column is Kromasil 300-10-C 4 10 x 250mm, flow rate of 5mL/min, detection wavelength of 215nm, 280nm, mobile phase A of 0.5% HAC (v/v) aqueous solution, mobile phase B of 0.5% HAC (v/v) acetonitrile solution.
The collected purified product was analyzed using gradient elution parameters as shown in table 2: the chromatographic column is Kromasil 100-3.5-C 4 4.6X105 mm, a flow rate of 1mL/min, a detection wavelength of 215nm, mobile phase A of 0.05% TFA (v/v) in water, and mobile phase B of 0.05% TFA (v/v) in acetonitrile.
Collecting target component with purity higher than 90%, distilling under reduced pressure to remove acetonitrile, and vacuum freeze drying. Molecular weight confirmation by ESI-MS, m/z=1085.40 [ M+5H ]] 5+ Consistent with theoretical molecular weight.
Example 33: HBV infection inhibitionExperiment
HepG2 NTCP cells at 2X 10 5 The culture was induced by plating at a density of 100. Mu.L/well, adding DME-maintaining medium containing 2.5% DMSO and 5% FBS the next day. HBV virus from HepAD38 cell supernatant was diluted to an infection amount of 100MOI, and the diluted medium was DMEM medium containing 2.5% DMSO and 4% PEG 8000. Compound dilution: the polypeptide analogue to be detected and the reference Myrcludex B are respectively diluted to 8 dilutions of 1000nmol/L, 200nmol/L, 40nmol/L, 8nmol/L, 1.6nmol/L, 0.32nmol/L, 0.064nmol/L and 0.0128nmol/L, and the dilution culture medium is DMEM culture medium of 2.5% DMSO and 4% PEG 8000. The infection-blocking groups were pre-diluted with different concentrations of compound, 100. Mu.L/well, and diluted virus, 100. Mu.L/well, and gently flicked 4-6 times. Centrifuge at 1000rpm for 30min at room temperature. Incubation for 16-24h, removing the infection solution, washing 3 times with PBS, and adding DMEM maintenance medium containing 2.5% DMSO and 5% FBS. Cell supernatants were collected on day 11 post-infection, centrifuged to discard cell pellet, and cell supernatant stock was assayed for HBeAg levels. Scanning the well plate with enzyme-labeled instrument to obtain corresponding light absorption value, and linearly fitting standard curve of concentration and signal value to calculate IC of different compounds for inhibiting HBV infection 50 The values are shown in table 3 below.
TABLE 3 inhibitory Activity of polypeptide analogs on HBV infection of HepG2 NTCP cells
Example 34: in vivo pharmacokinetic Studies in beagle dogs of HB-38
Beagle dogs (weight 9-11 kg) were randomized, 2 in each group, and Myrcludex B and HB-38 were subcutaneously injected at 1mg/kg dose, respectively.
The animals (beagle dogs) were fasted for 12h before dosing and were fed with food 4h after dosing, with free water before, after and during the experiment.
Blood was collected from the anterior extremity vein at 0h (pre-injection), and 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 24h, 30h, 48h, 99h, 120h, 144h, 168h, 196h, 216h, 240h and 264h post-subcutaneous injection, EDTA-K2 anticoagulated, and plasma was centrifuged at 4℃at 4000rpm for 10 min. All plasma was collected and stored at-80℃for testing.
100. Mu.L of the plasma sample to be tested and the standard yeast sample are sucked, 50. Mu.L of an internal standard solution is added, 150. Mu.L of a precipitant (solvent: acetonitrile: methanol (v: v=3:2) (containing 0.1% glacial acetic acid)) is added, vortex mixing is carried out, 18000g is centrifuged at 4 ℃ for 7min, 200. Mu.L of the supernatant is taken, 20. Mu.L of the supernatant is sucked for LC-MS/MS analysis, and a chromatogram is recorded.
Subcutaneous injection exposure of the compounds of the invention was assessed by in vivo pharmacokinetic experiments in beagle dogs and peak drug concentrations (C max ) Peak time of arrival (T) max ) Area under the curve of time of Administration (AUC) (0-264h) And AUC% 0-∞ ) Half-life (t) elimination 1/2 ) And average residence time (MRT #) 0-264h )). The above drug substitution parameters are shown in table 4. The plasma concentration versus time curve is shown in figure 1.
TABLE 4 pharmacokinetic parameters in HB-38 and Myrcludex B beagle dogs
Experimental results show that compared with the in vivo drug substitution behavior difference after the subcutaneous injection of 1mg/kg Myrcludex B and HB-38 in beagle, the in vivo drug substitution behavior difference is large, and the HB-38 group is C max Exposure AUC (0-t) Significantly higher than control Myrcludex B, HB-38 group half-life was significantly longer than control Myrcludex B.
Claims (10)
1. A polypeptide analog has a general structural formula of C 14 -P 1 -U 1 ；
Wherein,,
P 1 comprising the amino acid sequence:
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNX 1 DHWPEANX 2 VG (SEQ ID NO: 1), wherein X 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, glu, D-Glu, gamma-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; x is X 2 For Lys, the epsilon-amino group of the Lys has an acylated modified group;
C 14 to at P 1 Is a myristoyl group at the N-terminus of (2);
U 1 is equal to P 1 A C-terminal linked PAS sequence of (2) consisting of Pro, ala and/or Ser, having from 30 to 300 amino acid residues.
2. The polypeptide analog of claim 1 wherein the X is 2 The structural formula of the modifying group is as follows:
wherein,,
p is 6-20, 10-20, 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, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met,
R 1 is-CH 3 or-COOH;
preferably, the modifying group has a structural formula shown in formula 1 or formula 2:
or (b)
Wherein Xaa is selected from GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe, preferably gamma-Glu or Pro; the p is6. 8, 10, 12, 14, 16, 18 or 20, preferably 16 or 18.
3. The polypeptide analog of claim 1 wherein the X is 2 The modifying group of (2) is selected from the following structural formula:
4. a polypeptide analogue according to any one of claims 1-3, wherein the PAS sequence has 30-100 amino acid residues, preferably 30, 50 or 100 amino acid residues; preferably, pro in the PAS sequence is 10% -40% of the total number of amino acid residues; preferably, the PAS sequence comprises the amino acid sequence shown in SEQ ID NO. 2, 3, 4, 5, 6, 7 or 8.
5. The polypeptide analog of claim 1 wherein the polypeptide analog is selected from the following structural formulas:
wherein C is 14 Is myristoyl; p is 6-20, 10-20, 14-18 or 16-18, preferably 6, 8, 10, 12, 14, 16, 18 or 20; xaa is selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, lys, pro, phe, ser, tyr, thr, trp, val or Met, preferably GABA, aib, D-Ala, beta-Ala, asp, cys, gamma-Glu, gly, pro or Phe, more preferably gamma-Glu or Pro; r is R 1 is-CH 3 or-COOH;
preferably, the polypeptide analogue is selected from the following structural formulae:
wherein C is 14 Is myristoyl.
6. A method of preparing the polypeptide analog of any one of claims 1-5, comprising the steps of:
a) Preparation of P as defined in any one of claims 1 to 5 1 A peptide chain portion consisting of amino acids and a modifying group for acylation modification;
b) Carrying out myristoylation reaction at the N-terminal of the peptide chain part;
c) Amino acid residue X of the peptide chain portion with the modifying group 2 Performing acylation modification on epsilon-amino groups to prepare the polypeptide analogue; and
d) Isolating and purifying the polypeptide analogue.
7. A pharmaceutical composition comprising the polypeptide analog of any one of claims 1-5, and a pharmaceutically acceptable carrier.
8. A method of inhibiting HBV and/or HDV infection in a cell comprising administering an effective amount of a polypeptide analog of any of claims 1-5 or a pharmaceutical composition of claim 7 to the cell.
9. Use of the polypeptide analogue of any one of claims 1-5 or the pharmaceutical composition of claim 7 in the manufacture of a medicament for treating a patient infected with HBV and/or HDV.
10. Use of the polypeptide analogue of any one of claims 1-5 or the pharmaceutical composition of claim 7 for the manufacture of a medicament for the treatment of a patient with hepatitis b, hepatitis d, cirrhosis, portal hypertension, liver fibrosis, dyslipidemia and/or nonalcoholic fatty liver disease.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210475287 | 2022-04-29 | ||
| CN2022104752878 | 2022-04-29 |
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| CN116970042A true CN116970042A (en) | 2023-10-31 |
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| CN202310486422.3A Pending CN116970042A (en) | 2022-04-29 | 2023-04-28 | Antiviral polypeptide analogues |
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