CN103483428B

CN103483428B - Small molecule-polypeptide conjugate capable of inhibiting HIV infection

Info

Publication number: CN103483428B
Application number: CN201210189957.6A
Authority: CN
Inventors: 刘克良; 王潮; 史卫国; 蔡利锋; 王昆; 冯思良; 徐亮
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2012-06-11
Filing date: 2012-06-11
Publication date: 2017-05-17
Anticipated expiration: 2032-06-11
Also published as: CN103483428A; WO2013185575A1

Abstract

The invention belongs to the field of biological medicine, relates to an anti-HIV infection polypeptide, and specifically relates to a polypeptide represented by the formula I: Ism-L1-XNNYTSLIHSLIEESQNQQEKNEQELL-L2-Chol (formula I), derivatives, stereoisomer, or physiological-toxicity-free salt thereof. The invention also relates to a drug composition containing the formula I polypeptide, derivatives, stereoisomer or physiological-toxicity-free salt thereof, and applications of the formula I polypeptide, derivatives, stereoisomer or physiological-toxicity-free salt thereof on curing or preventing HIV infection related diseases especially the acquired immunodeficiency syndrome (AIDS).

Description

Small molecule-polypeptide conjugate for inhibiting HIV infection

Technical Field

The invention belongs to the field of biomedicine, relates to a small molecule and polypeptide conjugate for inhibiting HIV infection, and particularly relates to a conjugate shown in formula I, a derivative, a stereoisomer or a physiologically-nontoxic salt thereof. The invention also relates to a pharmaceutical composition containing the conjugate shown in the formula I, the derivative, the stereoisomer or the physiologically nontoxic salt thereof, and application of the conjugate shown in the formula I, the derivative, the stereoisomer or the physiologically nontoxic salt thereof in treating or preventing related diseases caused by HIV infection, in particular acquired immunodeficiency syndrome (AIDS).

Sm-L₁-XNNYTSLIHSLIEESQNQQEKNEQELL-L₂-Chol formula I.

Background

AIDS is a fatal infectious disease mainly caused by infection of human immunodeficiency virus type I (HIV-1), and is prevalent worldwide. The anti-HIV-1 drugs clinically applied at present can prolong the survival time of HIV infected persons and improve the life quality of the HIV infected persons to a certain extent by being assisted by high-efficiency antiretroviral therapy. However, due to the slow progress of HIV vaccine research and the increasing apparent problem of drug resistance, the development of new anti-HIV drugs is still an urgent task. HIV fusion inhibitors (HIV fusinihibitors) are novel anti-HIV drugs that interfere with viral entry into target cells, cut off viral transmission at the initial stage of infection, are of particular significance for the prevention and control of HIV-1 infection, and thus are the focus of research on new-mechanism anti-HIV drugs.

Gp41 is a specific protein mediating the fusion of HIV-1 with target cell membrane, and is the target of fusion inhibitor. The extracellular region of Gp41 has two functional regions of helical structure closely related to membrane fusion, namely an N-terminal repeat (HR1) and a C-terminal repeat (HR 2). During membrane fusion, HR2 interacts with HR1 to form a hexaspirochete core structure (6-HB). T20 is a fusion inhibitor polypeptide of 36 amino acid residues derived from the gp41 HR2 region, which was approved by the U.S. FDA in 2003 to market and is currently the only HIV-1 fusion inhibitor on the market. T20 competes with the helix trimer formed by HR1, occupies the action site of HR2, and further inhibits the formation of 6-HB, so that the membrane fusion process cannot be completed.

The marketing of T20 opens up a new field for the control of HIV-1 by polypeptide drugs. However, T20 has several drawbacks and deficiencies in its own right. The first is the problem of drug resistance: since T20 is derived completely from the native HR2 sequence, resistance to the target mutation is low and resistance easily develops. Residues 36-45 (GIVQQQNNLL) of HR1 are the major sites for T20 binding, with mutations at single residues resulting in a 5-10 fold decrease in T20 sensitivity, and mutations at two residues resulting in a 100-fold decrease in sensitivity. Secondly, T20 has poor in vivo stability, is easily degraded by protease and has low bioavailability. Again, T20 has a higher synthesis cost. Therefore, on the premise of ensuring the biological activity, how to solve the drug resistance, improve the enzymatic stability and reduce the synthesis cost of the polypeptide HIV-1 fusion inhibitor is the main direction of the research of the novel HIV-1 fusion inhibitor.

Based on the above problems, the current main solution strategy is to avoid the target binding site of T20 and introduce a new functional sequence different from T20 to overcome drug resistance; meanwhile, a helix forming and stabilizing factor is added, so that the helicity and stability of the sequence are improved, and the enzymolysis stability and the inhibitory activity are improved. For example, a second-generation polypeptide fusion inhibitor T-1249 is added with an N-trimer hydrophobic pocket binding sequence (WQEWEQKI) at the N end, so that the activity of the second-generation polypeptide fusion inhibitor is improved by one order of magnitude compared with T20; as another example, the third generation fusion inhibitor T-1144, and T20 have completely different target sites of action, mainly the hydrophobic pocket region (WEAWERAI) of HR 1. Phase I clinical research results show that T-1144 can obviously inhibit a T20 drug-resistant strain, and simultaneously shows higher activity and better pharmacokinetic property than T20. In addition, the 5HR series of polypeptides opens up a new idea of using computer-aided design of completely non-native alpha helical peptides based on the three-dimensional crystal structure of the target gp41 HR1 helical trimer. 5HR is taken as a lead structure, a pocket binding region (WMEWWRRE) is introduced at the N end of the lead structure, and a lipid membrane binding region (WASLWNWF) is introduced at the C end of the lead structure, so that the fusion inhibition activity is obviously improved.

The hydrophobic pocket of the Gp 41N-trimer surface is simultaneously small molecule fusion inhibitionThe target of action of the agent. Such as NB-2, its EC₅₀The value reached 1.04. mu.M; a. the₁₂Also shown to inhibit HIV replication activity at micromolar levels, EC₅₀The value was 0.69. mu.M; natural small molecule compound Hydroxytyrosol (HT) extracted from folium Canarii albi has HIV replication inhibiting activity of 50 μ M. However, small molecule fusion inhibitors are far less active than peptide fusion inhibitors. The reason for this is that: (1) the single small molecule only partially occupies a hydrophobic pocket, has low binding force with a target and is not enough to competitively inhibit the formation of 6-HB; (2) the recognition ability of single small molecules is not strong, and the local concentration near the target point is not high.

Peptides and non-peptides, which have pharmacodynamic activities respectively, are combined into the same molecule, and the effect of 1+1 > 2 is often obtained. The earliest applications demonstrated permissive effects derived from hormones. In the so-called permissive response, the biological activity of peptide hormones is significantly increased by the presence of steroid hormones. Based on the concept, Pengzhi and the like design new compounds with immunosuppressive effect, such as ureoxin tripeptide His-Gly-Glu and the like conjugated with hydrocortisone. The activity experiment proves that the conjugated hormone peptide can prolong the survival time of different mouse ectopic transplantation cardiac muscle. The activity is stronger than that of a control group of a mixture of tripeptide and hydrocortisone; in addition, the combination of ligand and receptor can be obviously enhanced by covalently bonding the leucineide, the enkephalin and the analgesic oxymorphone with analgesic activity; the kyotol is H-Tyr-Arg-OH dipeptide and has analgesic activity. The double-valence conjugated peptide generated by bonding the succinyl-containing conjugate with the 21-site amino group of hydrocortisone shows good analgesic activity.

In conclusion, the inventor conjugates peptide pharmacophores derived from gp41 HR2 with non-peptide pharmacophores to ensure that the peptide pharmacophores and the non-peptide pharmacophores play a synergistic effect, designs an HIV fusion inhibitor with a brand-new structure, and explores a new idea of inhibiting drug resistance, improving the stability of peptide drugs and reducing the synthesis cost of peptides. Thus, the present invention has been completed.

Disclosure of Invention

One aspect of the invention relates to polypeptide-small molecule conjugates of formula I, derivatives thereof, stereoisomers thereof, or physiologically non-toxic salts thereof,

Sm-L₁-XNNYTSLIHSLIEESQNQQEKNEQELL-L₂-Chol formula I

Wherein,

sm is a small molecular compound capable of being specifically combined with an HIV-1gp 41N-trimer surface hydrophobic pocket region, or Sm is deleted;

L₁linking arms between the linking peptide and the small molecule, or L, for binding to the target to enable the small molecule to retain spatial flexibility₁Deletion;

x is L-isoleucine (L-Ile), or X is deleted;

NNYTSLIHSLIEESQNQQEKNEQELL is derived from the native sequence C34;

L₂is a linking arm for linking the polypeptide and the cholesterol molecule;

chol is cholesterol.

In one embodiment of the invention, Sm is selected from the following small molecule compounds:

wherein, NB2-L and M-A12 are linkers which are used for connecting polypeptide and introduce a carboxyl group at the 3-position of the pyrrole ring of NB-2 and A12 molecules respectively; M-NB2 and A12-L respectively introduce carboxymethyl as Linker on the phenolic hydroxyl of NB-2 and A12 molecules, so that the carboxymethyl is connected with the polypeptide.

L₁Can be as follows: natural or unnatural amino acids; a diacid; a diamine;a diol; polyethylene glycol with one end being amino and the other end being carboxyl; specifically, L₁Can be as follows:

glycine (Gly), alanine (Ala), leucine (Leu), isoleucine (Ile), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), valine (Val), lysine (Lys), serine (Ser), threonine (Thr), arginine (Arg), histidine (His), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), cysteine (Cys), methionine (Met) in L-or D-form.

Beta-alanine (beta-Ala);

gamma-aminobutyric acid (GABA)

6-aminocaproic acid (Aca);

oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid

Ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine

Ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol

NH₂-CH₂CH₂-O-CH₂CH₂-COOH(PEG₁)

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₂)

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₃)

L₂Can be as follows:

Chloroacetic acid

Bromoacetic acid

Beta-alanine (beta-Ala);

gamma-aminobutyric acid (GABA)

6-aminocaproic acid (Aca);

oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid

NH₂-CH₂CH₂-O-CH₂CH₂-COOH(PEG₁)

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₂)

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₃)

Chol is cholesterol.

In one embodiment of the invention, the polypeptide of formula I, a derivative thereof, a stereoisomer thereof, or a physiologically nontoxic salt thereof is selected from the following compounds:

1 HT-------NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：1)；

2 HT-βAla-NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：2)；

3 HT--Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：3)；

4 HT-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：4)；

5 HT--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：5)；

6 HT--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：6)；

7 HT--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：7)；

8 NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：8)；

9 HT-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：9)；

10 HT-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：10)；

11 HT--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：11)；

12 HT-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：12)；

13 HT--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：13)；

14 HT--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：14)；

15 HT--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：15)；

16 HT-------INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：16)；

17 HT-βAla-INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：17)；

18 HT--Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：18)；

19 HT-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：19)；

20 HT--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：20)；

21 HT--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：21)；

22 HT--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：22)；

23 INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：23)；

24 HT-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：24)；

25 HT-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：25)；

26 HT--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：26)；

27 HT-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：27)；

28 HT--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：28)；

29 HT--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：29)；

30 HT--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：30)；

31 MNB2-------NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：31)；

32 MNB2-βAla-NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：32)；

33 MNB2--Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：33)；

34 MNB2-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：34)；

35 MNB2--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：35)；

36 MNB2--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：36)；

37 MNB2--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：37)；

38 MNB2-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：38)；

39 MNB2-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：39)；

40 MNB2--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：40)；

41 MNB2-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：41)；

42 MNB2--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：42)；

43 MNB2--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：43)；

44 MNB2--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：44)；

45 MNB2-------INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：45)；

46 MNB2-βAla-INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：46)；

47 MNB2--Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：47)；

48 MNB2-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：48)；

49 MNB2--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ IDNO：49)；

50 MNB2--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：50)；

51 MNB2--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：51)；

52 MNB2-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：52)；

53 MNB2-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：53)；

54 MNB2--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：54)；

55 MNB2-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：55)；

56 MNB2--PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：56)；

57 MNB2--PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：57)；

58 MNB2--PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：59)；

59 A12L-------NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：59)；

60 A12L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：60)；

61 A12L--Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：61)；

62 A12L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：62)；

63 A12L--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：63)；

64 A12L--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：64)；

65 A12L--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：65)；

66 A12L-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：66)；

67 A12L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：67)；

68 A12L--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：68)；

69 A12L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：69)；

70 A12L-PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：70)；

71 A12L-PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：71)；

72 A12L-PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：72)；

73 A12L-------INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：73)；

74 A12L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：74)；

75 A12L--Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：75)；

76 A12L-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：76)；

77 A12L--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：77)；

78 A12L--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：78)；

79 A12L--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：79)；

80 A12L-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：80)；

81 A12L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：81)；

82 A12L--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：82)；

83 A12L-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：83)；

84 A12L-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：84)；

85 A12L--PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：85)；

86 A12L--PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：86)；

87 MA12-------NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：87)；

88 MA12-βAla-NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：88)；

89 MA12--Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：89)；

90 MA12-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：90)；

91 MA12--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：91)；

92 MA12--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：92)；

93 MA12--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：93)；

94 MA12-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：94)；

95 MA12-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：95)；

96 MA12--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：96)；

97 MA12-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：97)；

98 MA12--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：98)；

99 MA12--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：99)；

100 MA12--PEG₃-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：100)；

101 MA12-------INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：101)；

102 MA12-βAla-INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：102)；

103 MA12--Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：103)；

104 MA12-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：104)；

105 MA12--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：105)；

106 MA12--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：106)；

107 MA12--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：107)；

108 MA12-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：108)；

109 MA12-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：109)；

110 MA12-Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：110)；

111 MA12-2Aca-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：111)；

112 MA12-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：112)；

113 MA12-PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：113)；

114 MA12-PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：114)；

115 NB2L-------NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：115)；

116 NB2L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：116)；

117 NB2L--Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：117)；

118 NB2L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：118)；

119 NB2L--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：119)；

120 NB2L--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：120)；

121 NB2L--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：121)；

122 NB2L-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：122)；

123 NB2L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：123)；

124 NB2L-Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：124)；

125 NB2L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：125)；

126 NB2L-PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：126)；

127 NB2L-PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：127)；

128 NB2L-PEG₃-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQID NO：128)；

129 NB2L-------INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：129)；

130 NB2L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：130)；

131 NB2L--Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：131)；

132 NB2L-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：132)；

133 NB2L--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：133)；

134 NB2L--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：134)；

135 NB2L--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL(SEQ ID NO：135)；

136 NB2L-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：136)；

137 NB2L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：137)；

138 NB2L-Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ IDNO：138)；

139 NB2L-2Aca-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：139)；

140 NB2L-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：140)；

141 NB2L-PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：141)；

142 NB2L-PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol(SEQ ID NO：142)；

in one embodiment of the invention, the polypeptide of formula I, its derivatives, its stereoisomers, or its physiologically non-toxic salts, is selected from the above compounds.

Another aspect of the present invention relates to a pharmaceutical composition comprising at least one polypeptide of formula I, a derivative, a stereoisomer, or a physiologically nontoxic salt thereof, as described above, and a pharmaceutically acceptable carrier or excipient.

Yet another aspect of the present invention relates to an HIV fusion inhibitor comprising at least one polypeptide of formula I, a derivative thereof, a stereoisomer thereof, or a physiologically nontoxic salt thereof.

Yet another aspect of the present invention relates to the use of a polypeptide of formula I as described above, a derivative thereof, a stereoisomer thereof, or a physiologically nontoxic salt thereof, for the preparation of an HIV fusion inhibitor.

In a further aspect, the present invention relates to the use of the polypeptide of formula I, its derivatives, its stereoisomers, or its physiologically non-toxic salts for the preparation of a medicament for the treatment or prevention of diseases associated with HIV infection, in particular aids.

Yet another aspect of the present invention relates to a method for the treatment or prevention of a disease associated with HIV infection, in particular aids, said method comprising administering to a subject to be treated or prevented an effective amount of a polypeptide of formula I, a derivative thereof, a stereoisomer thereof, or a physiologically non-toxic salt thereof.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The abbreviations used in the present invention have the following meanings:

the solid-phase synthesis carrier Rink amide resin used in the examples is a product of Tianjin Nankai synthesis responsibility Co., Ltd; natural amino acids protected by HBTU, HOBT, DIEA and Fmoc or unnatural amino acids of D-type are products of shanghai gill biochemical company and cheng nuo new technology, llc. N-methylpyrrolidone (NMP) is a product of ACROS company; trifluoroacetic acid (TFA) is a product of Beijing Bomeijie science and technology Limited; 3, 4-dihydroxyphenylacetic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, 2, 5-hexanedione, tert-butanol and ethyl bromoacetate are the products of ALFA company; DMF and DCM are products of Samsung Corp.Korea; the chromatographic pure acetonitrile is a product of Fisher company. Other reagents are domestic analytical pure products unless specified.

Example 1: preparation of Compound 1

1.1 preparation of HT and its attachment to linker arms

Synthesis of intermediate 2:

0.2g (1.19mmol) of Compound 1 is dissolved in 18.5ml of methanol under nitrogen, and concentrated H is added dropwise₂SO₄3 drops, and reflux for 2 hours in dark. After the reaction was complete, the reaction mixture was evaporated to dryness, redissolved with ethyl acetate and then saturated NaHCO₃Washing for three times. The aqueous phases were combined, extracted three times with ethyl acetate and the ester phases were combined and washed to neutrality with saturated NaCl solution. Anhydrous Na for ester phase₂SO₄Drying overnight, evaporating the solvent to dryness to obtain intermediate 2. The yield thereof was found to be 96%.

Synthesis of intermediate 3:

0.21g (1.19mmol) of intermediate 2 was dissolved in 15ml of anhydrous chloroform under nitrogen protection, and 1.6ml of 2, 2-Dimethoxypropane (DMP) and 0.06g (0.24mmol) of camphorsulfonic acid were added. Reflux for 4h in the dark, TLC monitor reaction (petroleum ether: ether ═ 7: 1) and saturated NaHCO₃Washing for three times, and using anhydrous Na for chloroform phase₂SO₄Drying overnight, evaporating the solvent to dryness to obtain the crude product. Column chromatography separation and purification (petroleum ether: 80: 1) gave intermediate 3 in 71% yield.

Synthesis of intermediate 4:

2.54g (11.43mmol) of intermediate 3 are dissolved in 100ml of anhydrous tetrahydrofuran under nitrogen protection, LiAlH is added₄0.22g (5.72mmol) was reacted for 6 hours with exclusion of light. After the reaction, 5ml of diethyl ether moistened with water was added, and then 0.5ml of water was added, and a precipitate was formed. Filtering to obtain precipitate, and adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness to obtain the crude product. The intermediate 4 was isolated and purified by column chromatography (15: 1 petroleum ether: diethyl ether) in 70% yield.

Synthesis of intermediate 5:

0.2g (1.03mmol) of intermediate 4 was dissolved in 5ml of dichloromethane, and 0.3ml of triethylamine and 0.01g of DMAP were added thereto, followed by stirring in an ice bath. 0.24g (2.4mmol) of succinic anhydride was weighed and dissolved in 3ml of dichloromethane to give a suspension, which was refluxed in an oil bath. After 10 minutes, the dichloromethane solution of the intermediate 4 was dropped into the flask through a constant pressure funnel, and the reaction was completed 10 minutes after the dropping. Washing the organic phase with 10% citric acid aqueous solution for 1 time, washing with saturated NaCl solution to neutrality, and washing with anhydrous Na₂SO₄Dry overnight. The solvent is evaporated to dryness to obtain a crude product. The compound 5 was isolated and purified by column chromatography (petroleum ether: ethyl acetate: 8: 1) with 80% yield.

1.2 preparation of HT and peptide conjugates (Compound 1)

Polypeptide synthesis was performed using standard Fmoc solid phase methods. Rink Amide resin is selected, and a peptide chain is extended from a C end to an N end. The condensing agent is HBTU/HOBt/DIEA. The deprotection agent is piperidine/DMF solution. The cracking agent is trifluoroacetic acid (TFA), and the crude peptide is dissolved in water and then is freeze-dried and stored. Separating and purifying by medium pressure liquid chromatography or High Pressure Liquid Chromatography (HPLC), wherein the content of pure peptide is more than 90%. And determining the molecular weight of the peptide sequence by matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS).

A peptide sequence is synthesized by using a CEM microwave polypeptide synthesizer, a small molecular compound can be used as a final residue to be condensed with an amino group at the N end of the polypeptide, and then a hydroxyl protecting group is removed under the condition of polypeptide cracking, so that a small molecule-polypeptide conjugate is finally obtained.

The synthesis conditions were as follows:

protecting amino acids or small molecule compounds: 0.2M of a solution in DMF,

activating agent: 0.45M HBTU/HOBt in DMF,

activating alkali: a 2M solution of DIEA in NMP,

deprotection agent: 20% v/v piperidine in DMF,

blocking reagent: 20% v/v acetic anhydride in DMF.

Weighing 0.5g (0.25mmol) of Rink Amide resin, placing the Rink Amide resin into a reactor of a CEM microwave polypeptide synthesizer, preparing amino acid, micromolecule, activating agent, activated base, deprotection reagent and blocking reagent according to the concentration, and synthesizing by using the CEM microwave full-automatic polypeptide synthesizer. After completion, the peptide resin was washed with DMF for 3 times, then shrunk with anhydrous methanol, and vacuum-dried at room temperature to obtain 2.05g of peptide resin.

Lysis buffer (volume percent): trifluoroacetic acid, ethanedithiol, m-cresol and water (82.5: 10: 5: 2.5).

Cleavage of peptide resin (linking small molecule HT): 2.05g of peptide resin synthesized by a microwave synthesizer is weighed and put into a 250ml eggplant-shaped bottle, and the mixture is subjected to ice bath and electromagnetic stirring. The lysate was prepared by adding 10ml of 1g of peptide resin. The TFA needs to be cooled for 30min in an ice bath in advance or stored in a refrigerator for use in advance; and adding the prepared lysate into peptide resin under the ice bath condition, electromagnetically stirring, enabling the resin to turn orange red, reacting for 30min under the ice bath condition, then removing the ice bath, and continuing stirring and reacting for 90min at room temperature to finish the reaction. Adding 200ml of cold ether into the reactor under vigorous stirring to separate out a white precipitate, and continuing stirring for 30 min; the precipitate was filtered off using a sand-core filter funnel of G4, washed repeatedly with cold diethyl ether 3 times and dried. Adding 50ml of double distilled water and 5ml of acetonitrile to fully dissolve the solid, filtering, and freeze-drying the filtrate to obtain 1.03g of crude peptide with small molecules connected to the N end.

The resulting crude small molecule-linked peptide is purified by medium or high pressure chromatography. The chromatographic column is C8 column, and the eluent is acetonitrile, water and small amount of acetic acid. The method comprises the following specific operation steps: 1.00g of crude peptide was weighed, 20ml of water and 5ml of acetonitrile were added to dissolve the solid, and the mixture was centrifuged for 10min (3000 rpm) to obtain a supernatant. The column was equilibrated beforehand with 200ml of a 15% acetonitrile/water/0.1% glacial acetic acid solution. After the sample is loaded, the sample is continuously washed by 200ml of 15 percent acetonitrile/water/0.1 percent glacial acetic acid solution, and the eluent components are detected by a high performance liquid phase. And gradually increasing the acetonitrile content according to the liquid phase detection result until the main peak of the purified polypeptide conjugate is eluted. Combining the eluates, rotary evaporating to remove most solvent, and lyophilizing to obtain pure small molecule-polypeptide conjugate (compound 1) with HPLC content higher than 90%.

EXAMPLES 2-7 preparation of Compounds 2-7

The method is the same as example 1, except that the linker connecting the small molecule and the polypeptide is replaced by β -alanine (β Ala), 6-aminocaproic acid (Aca), PEG₁，PEG₂，PEG₃The compounds serving as the connecting arms are connected with the polypeptide and then connected with the small molecule respectively to obtain the compounds 2-7.

EXAMPLE 8 preparation of Compound 8

Synthesis of Cholesterol Bromoacetate (Chol-Br)

6.95g of bromoacetic acid, 7.73g of cholesterol, 13.43g of EDC & HCl and 122mg of DMAP were sequentially weighed out and put into a 500ml eggplant-shaped bottle, 500ml of DCM was added, and the solution was stirred in an ice bath to be yellow. After 30min, the ice bath was removed and the reaction was carried out at room temperature for 24h, giving a reddish brown solution. Sequentially with saturated NaHCO₃Saturated NaCl wash, anhydrous MgSO₄And (5) drying. After appropriate concentration, the mixture is loaded on a silica gel column by a wet method and purified. Petroleum ether and ethyl acetate in the ratio of 10 to 1 are used as the washing liquidAfter 500ml of eluent is removed, the eluent is changed into petroleum ether and ethyl acetate which are 9: 1, the product is eluted, the solvent is evaporated and dried in vacuum, 6.3g of white solid is obtained, and the yield is 62%.

Firstly, the polypeptide is synthesized according to the method of example 1, then 20mg of the purified polypeptide (> 90%) is dissolved in 0.3ml DMSO, 0.2ml of Chol-Br 3.0mg THF solution is respectively added, 2 drops of DIEA are added by a dropper after uniform mixing, the mixture reacts for 3h at room temperature, and is separated and purified by a C4 semi-preparative reversed phase chromatographic column, and the molecular weight is confirmed by MALDI-TOF-MS.

EXAMPLES 9-15 preparation of Compounds 9-15

First, a conjugate of small molecule HT and polypeptide was synthesized according to the method of example 1, and then, it was linked to cholesterol according to the method of example 8 to obtain the target compound, and molecular weight was confirmed by MALDI-TOF-MS.

EXAMPLES 16-30 preparation of Compounds 16-30

Polypeptide and small molecule HT conjugates were synthesized as described in example 1, example 8 and example 9, respectively, except that the polypeptide sequence was varied.

EXAMPLE 31 preparation of Compound 31

31.1 Synthesis of Small molecule Compound MNB 2: NB₂The carboxyl of the benzene ring is protected by tert-butyl ester, so that the phenolic hydroxyl of the benzene ring reacts with ethyl bromoacetate, and then the benzene ring is saponified and hydrolyzed into ester bond to be carboxyl so as to react with a connecting arm or the N end of polypeptide. The synthetic route is as follows:

synthesis of intermediate 2:

weighing 4-nitro group0.2g of salicylic acid was put in a 50ml eggplant-shaped bottle, 0.006g of DMAP was added thereto, and 3ml of t-butanol was added thereto and dissolved to obtain a suspension. 0.24g of DCC was weighed and dissolved in 3ml of anhydrous THF. The solution was dropped into a bottle of eggplant shape using a constant pressure funnel and refluxed for 4 hours. Evaporating the solvent after the reaction is completed, dissolving the solvent by ethyl acetate again, and then using saturated NaHCO₃Washing for three times, washing for three times with saturated NaCl solution, anhydrous Na₂SO₄Dry overnight. Purifying with 20: 1 petroleum ether and ethyl acetate column.

Synthesis of intermediate 3:

intermediate 2 was dissolved in 3ml 2-butanone and solid K was added₂CO₃0.15g, heated to reflux for 30 minutes. To the above solution was added 0.22g of t-butyl bromoacetate, and refluxing was continued for 3 hours. After the reaction was completed, 15ml of water was added, and the mixture was extracted once with chloroform, and the chloroform layer was washed twice with 5% NaOH and then with saturated NaCl solution three times, anhydrous Na₂SO₄Dry overnight. Evaporating to dryness to obtain brown oil. The mixture was purified by column chromatography (petroleum ether: ethyl acetate: 8: 1) to give 0.18g of a colorless oil. The yield thereof was found to be 80%.

Synthesis of intermediate 4:

0.2g of intermediate 3 was weighed out and dissolved in 5ml of anhydrous methanol. Catalytic hydrogenation was carried out at 50psi with the addition of 0.02g of 10% Pd/C. After 1 hour of reaction, 5% Pd/C was filtered off, and the solution was evaporated to dryness to obtain a colorless oil which was used directly in the next reaction. The yield thereof was found to be 98%.

Synthesis of intermediate 5:

intermediate 40.3 g was weighed and dissolved in 5ml of toluene. 0.1ml of NMM, 0.23g of 2, 5-hexanedione and 0.01g of p-toluenesulfonic acid were added thereto, and the mixture was refluxed for 5 hours. After the reaction, the mixture is placed at room temperature, and the solvent is removed by evaporation under reduced pressure to obtain a brownish red oily substance. Ethyl acetate (15 ml) was added to dissolve the product and washed three times with saturated NaCl solution, anhydrous Na₂SO₄Dry overnight. Separating and purifying by column chromatography (petroleum ether: ethyl acetate: 30: 1) to obtain colorless oily substance. The yield was 70%.

Synthesis of intermediate 6:

the oily substance obtained above was dissolved in 3ml of methanol, and 1ml of 1N NaOH was added dropwise to the solution in ice bath. After 15 minutes of reaction, the reaction mixture was neutralized with 1N hydrochloric acid. After a part of the solvent was distilled off under reduced pressure, the pH was adjusted to 2-3 with 1N hydrochloric acid. White solid is precipitated to obtain the compound 7. The yield was 95%.

31.2 Small molecule MNB₂Attachment to a polypeptide

MNB was synthesized according to the method of example 1.2₂Conjugates with polypeptides.

EXAMPLES 32-58 preparation of Compounds 32-58

Compounds 32-58 were synthesized according to the methods of example 31, example 8 and example 9, respectively, and molecular weights were confirmed by MALDI-TOF-MS.

EXAMPLE 59 preparation of Compound 59

59.1 Small molecule Compound A₁₂L synthesis:

synthesis of carboxy protected A according to the synthetic route₁₂And the phenolic hydroxyl group is etherified, the influence on the electrical property caused by modification is reduced, and the carboxyl group of the carboxymethyl is utilized to be combined with the N end of the polypeptide.

Synthesis of intermediate 2:

0.2g of 5-nitro salicylic acid is weighed into a 50ml eggplant-shaped bottle, 0.006g of DMAP is added, and 3ml of tert-butyl alcohol is added for dissolution to obtain suspension. 0.24g of DCC was weighed and dissolved in 3ml of anhydrous THF. The solution was dropped into a bottle of eggplant shape with a constant pressure funnel and refluxed for 2 hours. After the reaction is completed, the solvent is evaporated to dryness, and is dissolved again by ethyl acetate, and then saturated NaHCO is used₃Washing for three times, washing for three times with saturated NaCl solution, anhydrous Na₂SO₄Dry overnight. Stone (stone)Oil ether ethyl acetate 20: 1.

Synthesis of intermediate 3:

Synthesis of intermediate 4:

0.2g of intermediate 3 was weighed out and dissolved in 5ml of anhydrous methanol. Catalytic hydrogenation was carried out at 50psi with the addition of 10% Pd/C0.02g. After 1 hour of reaction, 5% Pd/C was filtered off, and the solution was evaporated to dryness to obtain a colorless oil which was used directly in the next reaction. The yield thereof was found to be 98%.

Synthesis of intermediate 5:

Synthesis of intermediate 6:

59.2 Small molecule Compound A₁₂Preparation of L and polypeptide conjugates

Synthesis of A as in example 1.2₁₂A conjugate of L and a polypeptide.

EXAMPLES 60-86 preparation of Compounds 60-86

Compounds 60 to 86 were synthesized according to the methods of example 59, example 8 and example 9, respectively, and molecular weights were confirmed by MALDI-TOF-MS.

EXAMPLE 87 preparation of Compound 87

87.1 Synthesis of Small molecule Compound MA 12:

the synthesis of the small molecular compound MA12, which uses benzyl ester to protect benzene ring carboxyl and introduces carboxyl at the 3-position of pyrrole ring to connect with peptide chain, is carried out according to the following synthetic route.

Synthesis of intermediate 2:

to an eggplant flask were added t-butyl acetoacetate (5.0ml, 0.03mol) and 25ml of absolute ethanol, and the mixture was cooled to 0 ℃ in an ice bath. Sodium ethoxide (12.3ml, 0.03mol) was added dropwise thereto, and the mixture was reacted for 15 minutes under ice-bath conditions. The mixture was added to a stirred mixture of compound 6(2.2ml, 0.03mol) in absolute ethanol/toluene [30ml, V (absolute ethanol): V (toluene) ═ 2: 1]The solution was stirred at room temperature for 4 hours. Acidifying to neutrality with 2N HCl, evaporating to remove ethanol, adding EtOAc, washing organic phase with water for three times, and removing anhydrous MgSO₄And (5) drying. The crude compound 7 was purified by silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) ═ 7: 2: -]3.79g of a pale yellow liquid are obtained. The yield thereof was found to be 57.80%.

Synthesis of intermediate 4:

5-Aminosalicylic acid (1.80g, 6.44mmol) was added to an eggplant flask and dissolved in 30ml of toluene. N-methylmorpholine (0.71ml, 6) was added44mmol), Compound 2(1.52g, 6.44mmol) and p-toluenesulfonic acid 0.08 g. The reaction was heated under reflux for 3 hours and then cooled to room temperature. Evaporating under reduced pressure to remove solvent, dissolving in ethyl acetate, washing organic phase with water for 3 times, and collecting anhydrous MgSO₄And (5) drying. The crude compound 4 was purified by silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) ═ 9: 1%]1.02g of a pale yellow liquid are obtained. The yield thereof was found to be 65.12%.

Synthesis of intermediate 5:

compound 4(10.0g, 0.04mol) was treated with 30ml CH₂Cl₂And 5ml DMF, and after dissolution, benzyl alcohol (12.85g, 0.12mol), DCC (9.0g, 0.04mol) and 4-pyrrolidinopyridine (1.0 g) were added, and the mixture was stirred at room temperature for 12 hours, and the progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was distilled off, 200ml of ethyl acetate was added to the reaction mixture, and DCU was filtered off. The organic phase is successively treated with saturated NaHCO₃5% citric acid, H₂O washing, anhydrous MgSO₄And (5) drying. The crude compound 5 was purified by silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) ═ 7: 2: -]5.8g of a colorless oil was obtained in 42.12% yield.

Synthesis of intermediate 6:

the intermediate 5 was added with 6N HCl/EtOAc solution, stirred at room temperature until the starting material disappeared, evaporated to remove the solvent, added with petroleum ether, and placed in a refrigerator to obtain 1.3g of light brown powder. The yield thereof was found to be 83%.

87.2 Small molecule MA₁₂Preparation of conjugates with polypeptides

Conjugates of MA12 with polypeptides were synthesized according to the method of example 1.2.

EXAMPLES 88-114 preparation of Compounds 88-114

Compounds 88 to 114 were synthesized according to the methods of example 87, example 8 and example 9, respectively, and molecular weights were confirmed by MALDI-TOF-MS.

EXAMPLE 115 preparation of Compound 115

115.1 NB₂Protection of carboxyl group and introduction of linker arm: NB₂The carboxyl of the benzene ring is protected by benzyl ester, and then a carboxyl is introduced into the 3-position of the pyrrole ring so as to react with the connecting arm or the N-terminal of the polypeptide. The synthetic route is as follows:

2, synthesis of intermediate:

compound 1(10.0g, 0.04mol) was treated with 30ml CH₂Cl₂And 5ml DMF, and after dissolution, benzyl alcohol (12.85g, 0.12mol), DCC (9.0g, 0.04mol) and 4-pyrrolidinopyridine (1.0 g) were added, and the mixture was stirred at room temperature for 12 hours, and the progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was distilled off, 200ml of ethyl acetate was added to the reaction mixture, and DCU was filtered off. The organic phase is successively treated with saturated NaHCO₃5% citric acid, H₂O washing, anhydrous MgSO₄And (5) drying. The crude compound 2 was purified by silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) ═ 7: 2%]6.2g of a colorless oil were obtained in a yield of 45.76%.

Synthesis of intermediate 3:

to compound 2(6.2g, 0.02mol) was added 4N HCl/EtOAc solution, stirred at room temperature for 1 hour, and the reaction solution was evaporated to dryness to give compound 2 (white solid) which was used directly in the next reaction.

Synthesis of intermediate 6:

acetone (508ml, 6.90mol), H was added to a three-necked flask₂O 1000ml，KClO₃(120g, 1.10 mol). Dropping Br under mechanical stirring₂(206ml, 4.00mol), and the reaction solution was dropped over about 1.5 hours. Stirring is continued until the reaction solution is colorless, and KClO₃And (4) completely dissolving and completing the reaction. Extracting and separating, adding MgO into organic phase, shaking, washing with water for 3 times, and adding anhydrous CaCl₂And (5) drying. Vacuum distilling, and collecting distillate with boiling point of 50-51 deg.C to obtain 140.9g colorless irritant liquid. The yield thereof was found to be 14.93%.

Synthesis of intermediate 7:

Synthesis of intermediate 8:

compound 3(1.80g, 6.44mmol) was added to an eggplant bottle and dissolved in 30ml of toluene. N-methylmorpholine (0.71ml, 6.44mmol), compound 7(1.52g, 6.44mmol) and p-toluenesulfonic acid 0.08g were added. The reaction was heated under reflux for 3 hours and then cooled to room temperature. Evaporating under reduced pressure to remove solvent, dissolving in ethyl acetate, washing organic phase with water for 3 times, and collecting anhydrous MgSO₄And (5) drying. The crude compound 8 was purified by silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) ═ 9: 1%]1.91g of a pale yellow liquid are obtained. The yield thereof was found to be 70.48%.

Synthesis of target compound (compound 9):

the intermediate 8 was added to 6N HCl/EtOAc solution, stirred at room temperature until the starting material disappeared, evaporated to remove the solvent, added with petroleum ether and placed in a refrigerator to obtain 1.3g of light brown powder. The yield thereof was found to be 83%.

115.2 Small molecule Compound NB₂L ligation to a polypeptide

NB was synthesized according to the procedure of example 1.2₂A conjugate of L and a polypeptide.

Example 116 preparation of the 142 Compounds 116-142

The molecular weight was confirmed by MALDI-TOF-MS for the synthesis of compound 116-142 in accordance with the methods of example 115, example 8 and example 9, respectively.

EXAMPLE 143 evaluation of the Activity of Compounds to inhibit HIV-1 mediated cell-cell fusion (IC)₅₀)

Staining transfer method for detection of HIV-1 mediated cell-cell fusion: HIV-1_IIIBInfected H9 cells (H9/HIV-1)_IIIB) Labeled with a fluorescent reagent, Calcein-AM (Molecular Probes, Inc., Eugene, OR), and co-cultured with OR without test compound and MT-2 cells (ratio 1: 10) in 96-well plates at 37 ℃ for 2 h. Test compounds were diluted from a two-fold gradient at a concentration of 250 μ g/ml. Fused and unfused Calcein-labeled HIV-1 infected cells were counted using an inverse fluorescence microscope (Zeiss, Germany). Computing IC₅₀The value is obtained.

The results of the activity measurements according to the above method are shown in Table 1 below.

Table 1: inhibition of HIV-1 mediated cell fusion activity (IC)₅₀)

As can be seen from the activity results in Table 1, all the small molecule-polypeptide conjugates showed HIV-1 cell fusion inhibitory activity, wherein the compounds 8-15, 23-30, 38-44, 52-58, 66-72, 80-86, 94-100, 108-114, 122-128, 136-142 inhibited HIV fusion to a low nM level, comparable to the positive controls T20 (compound 144) and C34 (compound 143).

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula I or a physiologically non-toxic salt thereof,

Sm-L₁-XNNYTSLIHSLIEESQNQQEKNEQELL-L₂-Chol formula I

Wherein,

sm is a small molecule compound capable of being specifically combined with HIV-1gp 41N-trimer surface hydrophobic pocket region;

x is L-isoleucine (L-Ile), or X is deleted;

L₂is a linking arm for linking the polypeptide and the cholesterol molecule;

chol is cholesterol;

wherein Sm is selected from the following small molecule compounds:

2. A compound according to claim 1, or a physiologically non-toxic salt thereof, wherein L₁Selected from:

natural or unnatural amino acids; a diacid; a diamine; a diol; polyethylene glycol with one end being amino and the other end being carboxyl.

3. A compound according to claim 1, or a physiologically non-toxic salt thereof, wherein L₁Selected from:

glycine (Gly) and alanine (Ala), leucine (Leu), isoleucine (Ile), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), valine (Val), lysine (Lys), serine (Ser), threonine (Thr), arginine (Arg), histidine (His), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), cysteine (Cys) or methionine (Met) in L-or D-form;

beta-alanine (beta-Ala);

gamma-aminobutyric acid (GABA);

6-aminocaproic acid (Aca);

oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid;

ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine;

ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol;

NH₂-CH₂CH₂-O-CH₂CH₂-COOH(PEG₁)；

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₂)；

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₃)。

4. a compound according to claim 1, or a physiologically non-toxic salt thereof, wherein L₂Selected from:

chloroacetic acid;

bromoacetic acid;

beta-alanine (beta-Ala);

gamma-aminobutyric acid (GABA);

6-aminocaproic acid (Aca);

oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid;

NH₂-CH₂CH₂-O-CH₂CH₂-COOH(PEG₁)；

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₂)；

NH₂-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-O-CH₂CH₂-COOH(PEG₃)。

5. a compound according to claim 1, wherein the compound is selected from:

HT-NNYTSIIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₁--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₂--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

HT--PEG₃--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MNB2--PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-PEG₃--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L--Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-2Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L--PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

A12L--PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12--Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12--PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12--PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12--PEG₃-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-2Aca-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

MA12-PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-βAla-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-2Aca--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₁--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₂--NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₃-NNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-βAla-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-Aca--INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-2Aca-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₁-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₂-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol；

NB2L-PEG₃-INNYTSLIHSLIEESQNQQEKNEQELL-βAla-C-Chol。

6. a pharmaceutical composition comprising at least one compound according to any one of claims 1 to 5 or a physiologically nontoxic salt thereof, and a pharmaceutically acceptable carrier or adjuvant.

7. An HIV fusion inhibitor comprising at least one compound according to any one of claims 1 to 5, or a physiologically nontoxic salt thereof.

8. Use of a compound according to any one of claims 1 to 5, or a physiologically non-toxic salt thereof, for the manufacture of an HIV fusion inhibitor.

9. Use of a compound according to any one of claims 1 to 5, or a physiologically non-toxic salt thereof, for the manufacture of a medicament for the treatment or prophylaxis of a disease associated with HIV infection, wherein the disease associated with HIV infection is aids.