Oligopeptide compound with anti-influenza virus activity and related application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to an oligopeptide compound with anti-influenza virus activity and related application thereof. The oligopeptide compound provided by the invention has a structure as shown in a general formula I. The oligopeptide compound provided by the invention plays a role in inhibiting influenza virus by inhibiting the activity of TMPRSS2, and further plays a role in resisting influenza virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceuticals, and particularly relates to an oligopeptide compound with anti-influenza virus activity and related applications thereof. BACKGROUND

[0002] Influenza viruses (such as influenza A H1N1, H3N2 and influenza B) cause about 3-5 million severe cases and 290,000-650,000 deaths worldwide each year, with high-risk groups (such as children, the elderly and immunodeficient patients) facing higher risks. Existing anti-influenza drugs (such as the neuraminidase inhibitor oseltamivir and the RNA polymerase inhibitor baloxavir) face increasing drug resistance, limited efficacy (which requires early use), and insufficient spectrum (which is only directed against specific subtypes). In addition, the high mutation rate of influenza viruses leads to unstable vaccine protection. Therefore, the development of new anti-influenza drugs, especially broad-spectrum drugs targeting conserved viral targets or host factors, has important clinical significance.

[0003] TMPRSS2 (transmembrane serine protease 2) is a type II transmembrane protease on the surface of host cells, which plays a key role in influenza virus activation and infection spread. The hemagglutinin (HA) protein of influenza virus needs to be cleaved into HA1 and HA2 by host proteases before it can mediate fusion of the viral envelope with the host cell membrane. TMPRSS2 can directly cleave HA to promote viral entry into host cells. In addition, TMPRSS2 can also activate other proteases (such as plasmin) in respiratory tract cells, enhancing the spread of viruses in host tissues. TMPRSS2 knockout mice show significant resistance to multiple influenza viruses, with significantly reduced viral load and lung damage after infection. TMPRSS2 is a common host-dependent factor for infection by multiple influenza viruses, and targeting this protein may overcome the limitations of viral subtypes and develop broad-spectrum drugs. Targeting host proteins rather than viral targets can avoid the problem of drug resistance caused by viral gene mutations.

[0004] Inhibiting TMPRSS2 can block infection at the viral entry stage, which is earlier than neuraminidase inhibitors (which act on the viral release stage). Existing inhibitors have been verified, and clinically approved TMPRSS2 inhibitors (such as camostat and nafamostat) have shown anti-influenza activity in cell and animal models, suggesting the possibility of repurposing old drugs. Therefore, it is of great significance to develop inhibitors that target TMPRSS2 for new broad-spectrum anti-influenza drugs, which have both prophylactic and therapeutic efficacy, are less likely to cause drug resistance, and have broad-spectrum anti-influenza activity. SUMMARY

[0005] Therefore, the application provides application of an oligopeptide compound in preparation of an anti-influenza virus drug.

[0006] In the actual operation of the application, halogen refers to fluorine, chlorine, bromine and iodine. C1-C6 alkyl refers to a straight chain and branched chain saturated hydrocarbon group containing 1-6 carbon atoms, which is usually, for example, methyl, ethyl, isopropyl, n-butyl, pentyl or hexyl, etc. The term "alkyl" also includes cycloalkyl, i.e. cyclic C3-C6 hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0007] C1-C6 alkoxy refers to a straight chain and branched chain saturated hydrocarbon group containing 1-6 carbon atoms, which is usually, for example, methoxy, ethoxy, isopropoxy, n-butoxy, pentoxy or hexyloxy, etc. The term "alkoxy" also includes cycloalkoxy, i.e. cyclic C3-C6 hydrocarbon groups, such as cyclopropoxy, cyclobutoxy, cyclopentoxy and cyclohexoxy.

[0008] C1-C6 acyl refers to a straight chain and branched chain saturated acyl group containing 1-6 carbon atoms, which is usually, for example, acetyl, isopropyl acetyl, n-butyl acetyl, pentyl acetyl or hexyl acetyl, etc. The term "acyl" also includes cycloacyl, i.e. cyclic C3-C6 acyl groups, such as cyclopropyl acyl, cyclobutyl acyl, cyclopentyl acyl.

[0009] The oligopeptide compound includes any one of the following compounds or a pharmaceutically acceptable salt thereof: INT1, INT2, INT3, INT4, INT5, INT6, INT7, INT10, INT1-LXY, I1, I2, I3, I4, C1-1, C2-2, C3-1, C4-2, C5-2, C7-2, MC25-06, ST-24-8, ST-24-9, ST-24-11, ST-23-12 and ST-23-18.

[0010] Particularly preferred compounds of general formula I are shown in Table 1.

[0011] Table 1 Preferred compounds of general formula I In the application, the dosage form of the pharmaceutical composition includes oral preparations, and further, the oral preparations are preferably tablets.

[0012] By adopting the above technical scheme, the application has the following beneficial effects: The chemical name of the oligopeptide compound is "substituted N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17-diaimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadecan-12-yl)-amide". The oligopeptide compound can inhibit the activity of TMPRSS2 cutting oligopeptide substrate in vitro, and has inhibitory activity of influenza virus H3N2. Toxicity detection shows that the oligopeptide compound does not show growth inhibition activity on Vero, Huh-7 and HEK-293T cells at a concentration of 50 μg / mL, and can be used for preparing anti-influenza virus drugs and / or preparations. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Synthetic route of the peptide compound with anti-influenza virus activity; Figure 2 Principle diagram of TMPRSS2 cell screening model based on fluorescent substrate; Figure 3 Construction and verification diagram of TMPRSS2 cell screening model based on fluorescent substrate; Figure 4 Result diagram of the inhibitory activity of the oligopeptide compound on TMPRSS2 in the cell model; Figure 5 Result diagram of the binding activity of the oligopeptide compound and TMPRSS2 detected by SPR technology. DETAILED DESCRIPTION

[0014] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0015] Synthesis of the oligopeptide compound Synthesis of a peptide compound with anti-influenza virus activity: Taking compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17-diaimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadecan-12-yl)-3-methylisoxazole-5-carboxamide <INT7(M2)> as a synthesis example, the preparation synthesis route is as shown in Figure 1 The specific synthesis steps are as follows: 1. Preparation synthesis of compound 3 Compound 1 and compound 2 were added to acetonitrile in a molar ratio of 1:1, TCFH molar ratio was 1.5, NMI molar ratio was 3.0, nitrogen protection was 2 h, LCMS detection was carried out, direct sample mixing Flash purification, and vacuum drying was carried out to obtain compound 3.

[0016] 2. Preparation of compound 5 Compound 3 was dissolved in DEA / DCM (1 / 1), stirred at room temperature for 1 h, and LCMS detection was performed. After the reaction was completed, rotary evaporation was performed under reduced pressure, column chromatography purification was performed, and freeze drying was performed to obtain compound 5.

[0017] 3. Preparation of compound 7 Compound 5 and compound 6 were added to acetonitrile at a molar ratio of 1:1, a TCFH molar ratio of 1.5, and an NMI molar ratio of 3.0, and nitrogen protection was performed for 2 h. LCMS detection was performed, direct sample mixing Flash purification was performed, and rotary evaporation was performed under reduced pressure to obtain compound 7.

[0018] 4. Preparation of compound 9 Compound 7 was dissolved in DEA / DCM (1 / 1), stirred at room temperature for 1 h, and LCMS detection was performed. After the reaction was completed, rotary evaporation was performed under reduced pressure, column chromatography purification was performed, and freeze drying was performed to obtain compound 9.

[0019] 5. Preparation of compound 11 Compound 9 and compound 10 were added to acetonitrile at a molar ratio of 1:1, a TCFH molar ratio of 1.5, and an NMI molar ratio of 3.0, and nitrogen protection was performed for 2 h. LCMS detection was performed, direct sample mixing Flash purification was performed, and rotary evaporation was performed under reduced pressure to obtain compound 11.

[0020] 6. Synthesis of target compound INT7 (M2) Compound 11 was dissolved in TFA, stirred at room temperature overnight, and LCMS detection was performed. Rotary evaporation was performed under reduced pressure, column chromatography purification was performed, and finally the final product INT7 (M2) was obtained.

[0021] According to the above-mentioned route and method, all the compounds described in the present application can be stably and reproducibly synthesized.

[0022] Example 1 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17-diaza-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadec-12-yl)-3-iodo-5-bromobenzamide (INT1) Compound INT1 was synthesized by the above-mentioned method, with a yield of 41% and an M.W of 854.12. The structure of INT1 (1-INT) is shown in Table 1.

[0023] 1H NMR (400 MHz, MeOD) δ 8.17-8.21 (m, 2H), 8.10-8.14 (m, 2H), 8.01-8.02 (t, J = 16 Hz, 1H), 7.61-7.66 (m, 2H), 5.66-5.69 (m, 1H), 4.53-4.57 (m, 1H), 4.26 (d, J = 64 Hz, 1H), 3.20-3.27 (m, 4H), 2.09-2.18 (m, 3H), 1.79-1.92 (m, 5H), 1.66-1.70 (m, 2H), 0.95-1.00 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.32 (s, 1C), 172.57 (s, 2C), 166.72 (s, 1C), 164.26 (s, 1C), 157.34 (s, 1C), 153.40 (s, 1C), 137.05 (s, 1C), 134.62 (s, 1C), 134.37 (s, 1C), 133.83 (s, 2C), 129.14 (d, J = 17.6 Hz, 1C), 128.02 (s, 1C), 127.18 (s, 1C), 126.97 (s, 1C), 125.70 (s, 1C), 125.09 (s, 1C), 122.43 (s, 1C), 58.87 (s, 1C), 54.77 (s, 1C), 53.77 (s, 1C), 40.50 (d, J = 20 Hz, 2C), 30.56 (s, 1C), 28.49 (s, 1C), 28.07 (s, 1C), 25.04 (d, J = 31.6 Hz, 2C), 18.36 (d, J = 40.4 Hz, 1C), 17.39 (s, 1C).

[0024] Example 2 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)picolinamide Compound INT2 (MPV1) was synthesized by the above method, yield 36%, M.W: 651.31; The structure of INT2 (MPV1) is shown in Table 1.

[0025] 1 H NMR (400 MHz, MeOD) δ 8.74 (d, J = 4.8 Hz, 1H), 8.35 (d, J = 8 Hz, 1H), 8.20-8.26 (m, 2H), 8.12-8.14 (m, 1H), 7.78-7.81 (m, 1H), 7.60-7.67 (m, 1H), 5.67-5.71 (m, 1H), 4.69-4.73 (m, 1H), 4.27 (d, J = 32 Hz, 1H), 3.23-3.29 (m, 4H), 2.09-2.13 (m, 2H), 1.74-2.01 (m, 5H), 1.70-1.79 (m, 2H), 0.97-1.00 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.33 (s, 1C), 172.55 (s, 1C), 172.29 (s, 1C), 164.26 (s, 1C), 163.14 (s, 1C), 157.24 (s, 1C), 153.40 (s, 1C), 146.97 (s, 1C), 146.68 (s, 1C), 140.75 (s, 1C), 137.05 (s, 1C), 128.05 (s, 1C), 127.62 (s, 2C), 127.21 (s, 1C), 125.09 (s, 1C), 123.00 (s, 1C), 122.45 (s, 1C), 59.06 (s, 1C), 54.81 (s, 1C), 53.12 (s, 1C), 40.58 (d, J = 14.4 Hz, 2C), 30.44 (s, 1C), 19.194 (s, 1C), 28.04 (s, 1C), 25.17 (s, 1C), 24.83 (s, 1C), 18.30 (s, 1C), 17.46 (s, 1C).

[0026] Example 3 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-3-iodo-4-methylbenzamide Compound INT3 (MPV2) was synthesized by the above method, yield 28%, M.W: 790.22; The structure of INT3 (MPV2) is shown in Table 1.

[0027] 1 H NMR (400 MHz, MeOD) δ 8.53 (s, 2H), 8.30-8.32 (m, 1H), 8.11-8.21 (m, 1H), 7.76-7.78 (m, 1H), 7.59-7.67 (m, 1H), 7.36-7.38 (m, 1H), 5.67-5.70 (m, 1H), 4.56-4.60 (m, 1H), 4.26 (d, J = 72 Hz, 1H), 3.18-3.27 (m, 4H), 2.46 (s, 3H), 2.07-2.19 (m, 2H), 1.71-1.94 (m, 4H), 1.66-1.69 (m, 3H), 0.94-0.994 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.32 (s, 0.3C), 172.89 (s, 1C), 172.56 (s, 1C), 169.04 (s, 1C), 167.29 (s, 1C), 164.27 (s, 1C), 157.33 (s, 1C), 153.40 (s, 1C), 145.53 (s, 1C), 137.76 (s, 2C), 137.05 (s, 1C), 132.90 (s, 1C), 129.32 (s, 1C), 128.02 (s, 1C), 127.16 (d, J = 14.8 Hz, 2C), 125.10 (s, 1C), 122.43 (s, 1C), 99.82 (s, 1C), 58.84 (s, 1C), 54.76 (s, 1C), 53.61 (s, 1C), 40.50 (d, J = 20 Hz, 2C), 30.57 (s, 1C), 28.49 (s, 1C), 28.07 (s, 1C), 26.88 (s, 1C), 25.05 (d, J = 20 Hz, 1C), 18.33 (d, J = 20.4 Hz, 1C), 17.36 (s, 1C).

[0028] Example 4 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-4-bromo-3-chloro- benzamide Compound INT4 (4-4-M2) was synthesized by the above method, yield 45%, M.W: 762.18; The structure of INT4 (4-4-M2) is shown in Table 1.

[0029] 1 H NMR (400 MHz, MeOD) δ 8.53 (s, 2H), 8.11-8.21 (m, 1H), 7.99-8.03 (m, 1H), 7.77-7.81 (m, 1H), 7.61-7.70 (m, 2H), 5.67-5.69 (m, 1H), 4.56-4.60 (m, 1H), 4.26 (d, J = 72 Hz, 1H), 3.18-3.27 (m, 4H), 2.09-2.19 (m, 2H), 1.81-1.94 (m, 4H), 1.68-1.79 (m, 3H), 0.94-0.99 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.32 (s, 1C), 172.66 (d, J = 14.8 Hz, 1C), 166.72 (s, 1C), 164.26 (s, 1C), 157.34 (s, 1C), 153.40 (s, 1C), 137.05 (s, 1C), 134.62 (s, 1C), 134.37 (s, 1C), 133.83 (s, 2C), 129.16 (s, 2C), 128.02 (s, 1C), 127.18 (s, 1C), 126.97 (s, 1C), 125.70 (s, 1C), 125.09 (s, 1C), 122.43 (s, 1C), 58.87 (s, 1C), 54.77 (s, 1C), 53.77 (s, 1C), 40.50 (d, J = 20 Hz, 2C), 30.56 (s, 1C), 28.49 (s, 1C), 28.07 (s, 1C), 25.08 (s, 1C), 25.00 (s, 1C), 18.36 (d, J = 40.4 Hz, 1C), 17.39 (s, 1C).

[0030] Example 5 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2- carbonyl)-1,17-diazenyl-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadecan-12-yl)-2,5- difluoro-4-methylbenzamide Compound INT5 (MPV3) was obtained by the above method with a yield of 39%, M.W: 700.31; The structure of INT5 (MPV3) is shown in Table 1.

[0031] 1 H NMR (400 MHz, MeOD) δ 8.19-8.22 (m, 1H), 8.11-8.13 (m, 1H), 7.61-7.68 (m, 2H), 7.41-7.45 (m, 1H), 7.13-7.17 (m, 1H), 5.67-5.70 (m, 1H), 4.64-4.64 (m, 1H), 4.27 (d, J = 64 Hz, 1H), 3.18-3.27 (m, 4H), 2.31 (s, 3H), 2.09-2.11 (m, 2H), 1.77-1.93 (m, 5H), 1.66-1.70 (m, 2H), 0.96-1.00 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.34 (s, 1C), 172.512 (s, 1C), 172.31 (s, 1C), 164.27 (s, 1C), 157.27 (d, J = 16 Hz, 2C), 153.40 (s, 1C), 137.05 (s, 1C), 128.40 (s, 2C), 127.18 (s, 2C), 125.08 (s, 2C), 122.43 (s, 2C), 118.52 (s, 2C), 115.43 (s, 1C), 58.79 (s, 1C), 54.79 (s, 1C), 53.28 (s, 1C), 40.51 (d, J = 14.4 Hz, 2C), 30.57 (s, 1C), 28.93 (s, 1C), 28.03 (s, 1C), 25.09 (s, 1C), 24.74 (s, 1C), 18.27 (s, 1C), 17.24 (s, 1C), 13.17 (d, J = 8.8 Hz, 1C).

[0032] Example 6 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosan-12-yl)-7-fluoroimidazo[1,2- a]pyridine-3-carboxamide Compound INT6 was synthesized by the above method, yield 53%, M.W: 708.31; The structure of INT6 is shown in Table 1.

[0033] 1 H NMR (400 MHz, MeOD) δ 9.54-9.58 (m, 1H), 8.46 (s, 1H), 8.19-8.21 (m, 1H), 8.11-8.13 (m, 1H), 7.59-7.63 (m, 2H), 7.50-7.53 (m, 1H), 7.18-7.21 (m, 1H) 5.66-5.69 (m, 1H), 4.61-4.64 (m, 1H), 4.27-4.36 (m, 1H), 3.18-3.29 (m, 4H), 2.09-2.19 (m, 2H), 1.70-1.97 (m, 7H), 0.95-1.00 (m, 6H). 13C NMR (400 MHz, MeOD) δ 192.23 (s, 1C), 172.73 (d, J = 136.8 Hz, 2C), 164.26 (s, 1C), 161.76 (s, 1C), 160.27 (s, 1C), 157.28 (s, 1C), 153.38 (s, 1C), 137.03 (s, 1C), 134.22 (s, 2C), 130.65 (d, J = 17.6 Hz, 2C), 128.06 (d, J = 17.6 Hz, 1C), 127.21 (d, J = 14.4 Hz, 1C), 125.09 (d, J = 23.2 Hz, 1C), 122.43 (s, 1C), 58.71 (s, 1C), 54.80 (s, 1C), 53.00 (s, 1C), 40.49 (d, J = 33.6 Hz, 2C), 30.66 (s, 1C), 28.46 (d, J = 29.2 Hz, 1C), 28.01 (s, 1C), 24.93-25.28 (m, 2C), 18.36 (d, J = 40.8 Hz, 1C), 17.25 (d, J = 40.8 Hz, 1C).

[0034] Example 7 Synthesis of compound N-((9S,12S)-l,17-diamino-6-(benzo[d]thiazole-2-carbonyl)- 1,17-diazenido-9-isopropyl-8,l l-dioxo-2,7,10,16-tetraazheptadecyl)-3-methylisoxazole- 5-carboxamide Compound INT7 (M2) was synthesized by the above method, yield 22%, M.W: 655.30; The structure of INT7 (M2) is shown in Table 1.

[0035] 1H NMR (400 MHz, MeOD) δ 8.19-8.22 (m, 1H), 8.13-8.13 (m, 1H), 7.59-7.67 (m, 2H), 6.86-6.88 (m, 1H) 5.67-5.70 (m, 1H), 4.57-4.60 (m, 1H), 4.24-4.26 (m, 1H), 3.19-3.29 (m, 4H), 2.34 (s, 3H), 2.07-2.20 (m, 2H), 1.72-1.96 (m, 5H), 1.65-1.69 (m, 2H), 0.94-0.99 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.23 (s, 1C), 172.52 (s, 1C), 172.11 (s, 1C), 164.24 (s, 1C), 162.48 (s, 1C), 160.95 (s, 1C), 157.28 (s, 1C), 157.06 (s, 1C), 153.39 (s, 1C), 137.04 (s, 1C), 128.03 (s, 1C), 127.19 (s, 1C), 125.09 (s, 1C), 122.43 (s, 1C), 107.392 (s, 2C), 58.91 (s, 1C), 54.77 (s, 1C), 53.10 (s, 1C), 40.49 (d, J = 17.2 Hz, 2C), 30.50 (s, 1C), 28.53 (s, 1C), 28.02 (s, 1C), 25.09 (s, 1C), 24.92 (s, 1C), 18.27 (s, 1C), 17.36 (s, 1C).

[0036] Example 8 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-5-bromo-6-methylpicolinamide Compound INT10 (10) was synthesized by the above method, yield 46%, M.W: 743.23; The structure of INT10 (10) is shown in Table 1.

[0037] 1H NMR (400 MHz, MeOD) δ 8.20-8.23 (m, 1H), 8.11-8.15 (m, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.60-7.67 (m, 2H), 5.67-5.70 (m, 1H), 4.69-4.73 (m, 1H), 4.27 (d, J = 7.2 Hz, 1H), 3.22-3.27 (m, 4H), 2.71 (s, 3H), 2.09-2.13 (m, 2H), 1.18-1.91 (m, 5H), 1.66-1.70 (m, 2H), 0.95-1.00 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.33 (s, 1C), 172.53 (s, 1C), 172.30 (s, 1C), 164.25 (d, J = 11.6 Hz, 1C), 157.23 (d, J = 14.4 Hz, 1C), 156.55 (s, 1C), 153.39 (s, 1C), 147.15 (s, 1C), 141.55 (s, 2C), 137.04 (s, 1C), 128.04 (s, 1C), 127.21 (s, 1C), 125.11 (s, 1C), 124.83 (s, 1C), 122.44 (s, 1C), 121.04 (s, 2C), 59.04 (s, 1C), 54.85 (s, 1C), 52.58 (s, 1C), 40.57 (s, 2C), 30.43 (s, 1C), 29.53 (s, 1C), 28.04 (s, 1C), 25.19 (s, 1C), 24.73 (s, 1C), 23.55 (s, 1C), 18.30 (s, 1C), 17.44 (s, 1C).

[0038] Example 9 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-1-naphthalenecarboxamide Compound INT1-LXY was synthesized by the above method, with a yield of 47%, M.W: 700.33; The structure of INT1-LXY is shown in Table 1.

[0039] 1 H NMR (400 MHz, MeOD) δ 8.47 (m, 1H), 8.19-8.21 (m, 1H), 8.10-8.13 (m, 1H), 7.93-8.00 (m, 1H), 7.90-7.94 (m, 3H), 7.54-7.66 (m, 4H), 5.68-5.71 (m, 1H), 4.68-4.72 (m, 1H), 4.31 (d, J = 7.2 Hz, 1H), 3.25-3.28 (m, 4H), 2.11-2.18 (m, 2H), 1.82-2.02 (m, 3H), 1.73-1.80 (m, 4H), 0.98-1.01 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.32 (s, 1C), 173.00 (s, 1C), 172.59 (s, 1C), 169.19 (s, 1C), 164.25 (s, 1C), 157.23 (s, 1C), 153.39 (s, 1C), 137.04 (s, 1C), 135.03 (s, 1C), 132.62 (s, 1C), 130.84 (s, 1C), 128.70 (s, 1C), 128.02 (s, 1C), 127.85 (s, 1C), 127.65 (s, 1C), 127.42 (s, 1C), 127.19 (s, 1C), 126.56 (s, 1C), 125.16 (s, 1C), 123.63 (s, 1C), 122.44 (s, 1C), 58.90 (s, 1C), 54.78 (s, 1C), 53.71 (s, 1C), 40.59 (d, J = 43.2 Hz, 2C), 30.60 (s, 1C), 28.61 (s, 1C), 28.07 (s, 1C), 25.16 (s, 2C), 18.35 (s, 1C), 17.41 (s, 1C).

[0040] Example 10 Synthesis of compound N-((9S,12R)-l,17-diamino-6-(benzo[d]thiazole-2- carbonyl)-l,17-diazenido-9-isopropyl-8,l l-dioxo-2,7,10,16-tetraazaheneicosan- 12-yl)-5-methylfuran-2-carboxamide Compound I1 was synthesized by the above method, yield 33%, M.W: 654.31; The structure of I1 is shown in Table 1.

[0041] 1 H NMR (400 MHz, MeOD) δ 8.08-8.21 (m, 2H), 7.61-7.65 (m, 2H), 7.06 (s, 1H), 6.21 (s, 1H) 5.64-5.69 (m, 1H), 4.56-4.58 (m, 1H), 4.25-4.45 (m, 1H), 3.19-3.30 (m, 4H), 2.36 (s, 3H), 2.09-2.17 (m, 2H), 1.79-2.08 (m, 4H), 1.64-1.62 (m, 3H), 0.82-0.95 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.23 (s, 1C), 172.52 (s, 1C), 172.11 (s, 1C), 164.24 (s, 1C), 162.48 (s, 1C), 160.95 (s, 1C), 157.17 (d, J = 49.6 Hz, 3C), 153.39 (s, 1C), 137.04 (s, 1C), 128.033 (s, 1C), 127.19 (s, 1C), 125.09 (s, 1C), 122.43 (s, 1C), 107.39 (s, 2C), 58.91 (s, 1C), 54.77 (s, 1C), 53.10 (s, 1C), 40.49 (d, J = 17.2 Hz, 2C), 30.50 (s, 1C), 28.53 (s, 1C), 28.02 (s, 1C), 25.09 (s, 1C), 24.92 (s, 1C), 18.27 (s, 1C), 17.36 (s, 1C), 9.79 (s, 1C).

[0042] Example 11 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-2-fluoro-5-methylbenzamide Compound I2 was synthesized by the above method, yield 56%, M.W: 682.32; The structure of I2 is shown in Table 1.

[0043] 1 H NMR (400 MHz, MeOD) δ 8.199-8.22 (m, 1H), 8.11-8.13 (m, 1H), 7.61-7.65 (m, 2H), 7.54-7.56 (m, 1H), 733-7.36 (m, 1H), 7.07-7.12 (m, 1H), 5.68-5.71 (m, 1H), 4.63-4.67 (m, 1H), 4.28 (d, J = 7.2 Hz, 1H), 3.20-3.25 (m, 4H), 2.34 (s, 3H), 2.08-2.12 (m, 2H), 1.78-1.94 (m, 5H), 1.68-1.71 (m, 2H), 0.96-1.01 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.34 (s, 1C), 172.46 (d, J = 32 Hz, 2C), 165.52 (s, 1C), 164.26 (s, 1C), 157.30 (d, J = 20.4 Hz, 1C), 153.40 (s, 1C), 137.05 (s, 1C), 134.33 (d, J = 12 Hz, 1C), 133.55 (d, J = 31.6 Hz, 1C), 130.28 (d, J = 8.8 Hz, 2C), 128.03 (s, 1C), 127.19 (s, 1C), 125.09 (s, 1C), 122.43 (s, 1C), 121.65 (s, 1C), 115.74 (s, 1C), 115.51 (s, 1C), 58.77 (s, 1C), 54.78 (s, 1C), 53.28 (s, 1C), 40.50 (d, J = 26 Hz, 2C), 30.60 (s, 1C), 28.86 (s, 1C), 28.03 (s, 1C), 25.09 (s, 1C), 24.80 (s, 1C), 19.11 (s, 1C), 18.28 (s, 1C), 17.24 (s, 1C).

[0044] Example 12 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadec-12-yl)-4-cyano-2- fluorobenzamide Compound I3 was synthesized by the above method, yield 65%, M.W: 693.30; The structure of I3 is shown in Table 1.

[0045] 1 H NMR (400 MHz, MeOD) δ 8.20-8.22 (m, 1H), 8.11-8.14 (m, 1H), 7.8-7.867 (m, 1H), 7.61-7.71 (m, 4H), 5.66-5.70 (m, 1H), 4.61-4.65 (m, 1H), 4.29 (d, J = 6.8 Hz, 1H), 3.21-3.28 (m, 4H), 2.10-2.18 (m, 2H), 1.80-1.93 (m, 4H), 1.68-1.72 (m, 3H), 0.96-1.01 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.35 (s, 1C), 172.32 (d, J = 141.6 Hz, 2C), 164.28 (s, 1C), 157.28 (s, 2C), 153.40 (s, 1C), 137.05 (s, 1C), 131.25 (d, J = 14.4 Hz, 2C), 128.35 (d, J = 14.4 Hz, 2C), 128.04 (s, 1C), 127.55 (s, 1C), 127.20 (s, 1C), 125.08 (s, 1C), 122.44 (s, 1C), 120.09 (s, 1C), 119.83 (s, 1C), 116.45 (s, 1C), 115.58 (d, J = 40.8 Hz, 1C), 58.71 (s, 1C), 54.80 (s, 1C), 53.35 (s, 1C), 40.50 (d, J = 14.4 Hz, 2C), 30.66 (s, 1C), 28.69 (s, 1C), 28.02 (s, 1C), 25.11 (s, 1C), 24.82 (s, 1C), 18.30 (s, 1C), 17.21 (s, 1C).

[0046] Example 13 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-2-(4-isobutylphenyl)- propionamide Compound I4 was synthesized by the above method with a yield of 29% and M.W: 734.41. The structure of I4 is shown in Table 1.

[0047] 1 H NMR (400 MHz, MeOD) δ 8.19-8.22 (m, 1H), 8.11-8.13 (m, 1H), 7.61-7.66 (m, 2H), 7.24 (d, J = 8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 5.64-5.67 (m, 1H), 4.38-4.42 (m, 1H), 4.16 (d, J = 6.8 Hz, 1H), 3.70-3.72 (m, 1H), 3.16-3.25 (m, 4H), 2.418 (t, J = 3.2 Hz, 2H), 2.13 (m, 1H), 1.96-1.97 (m, 1H), 1.58-1.87 (m, 7H), 1.41-1.45 (m, 3H), 0.84-0.89 (m, 9H), 0.78 (d, J = 6.8 Hz, 3H). 13C NMR (400 MHz, MeOD) δ 192.31 (s, 1C), 176.27 (s, 1C), 172.35 (d, J = 20.4 Hz, 2C), 164.27 (s, 1C), 157.23 (d, J = 11.6 Hz, 1C), 153.40 (s, 1C), 140.22 (s, 1C), 138.41 (s, 1C), 137.05 (s, 1C), 129.03 (s, 2C), 128.03 (s, 1C), 127.18 (s, 1C), 126.85 (s, 2C), 125.07 (s, 2C), 122.44 (s, 1C), 58.46 (s, 1C), 54.65 (s, 1C), 52.63 (s, 1C), 45.38 (s, 1C), 44.62 (s, 1C), 40.52 (s, 1C), 40.43 (s, 1C), 30.56 (s, 1C), 30.04 (s, 1C), 28.37 (s, 1C), 28.05 (s, 1C), 25.02 (s, 1C), 24.87 (s, 1C), 21.32 (s, 2C), 18.23 (s, 1C), 17.50 (s, 1C), 17.08 (s, 1C).

[0048] Example 14 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazheptadecyl)-3-iodobenzamide Compound C1-1 was synthesized by the above method, with a yield of 55%, M.W: 776.21; The structure of C1-1 is shown in Table 1.

[0049] 1H NMR (400 MHz, MeOD) δ 8.201-8.220 (m, 2H), 8.11-8.13 (m, 1H), 7.83-7.91 (m, 2H), 7.59-7.67 (m, 2H), 7.22-7.26 (m, 1H), 5.65-5.68 (m, 1H), 4.54-4.58 (m, 1H), 4.34 (d, J = 7.2 Hz, 1H), 3.17-3.27 (m, 4H), 2.09-2.18 (m, 2H), 1.77-1.93 (m, 5H), 1.64-1.70 (m, 2H), 0.94-0.99 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.32 (s, 1C), 172.55 (d, J = 43.2 Hz, 1C), 167.46 (s, 1C), 157.29 (s, 1C), 153.40 (s, 1C), 140.57 (s, 2C), 137.03 (s, 1C), 136.25 (s, 2C), 135.69 (s, 1C), 130.02 (s, 1C), 128.05 (s, 1C), 127.21 (s, 1C), 126.481 (s, 2C), 125.13 (s, 1C), 122.41 (s, 1C), 93.32 (s, 1C), 58.64 (s, 1C), 54.84 (s, 1C), 53.73 (s, 1C), 40.50 (d, J = 64.8 Hz, 2C), 30.59 (s, 1C), 28.39 (s, 1C), 27.99 (s, 1C), 25.30 (s, 1C), 24.88 (s, 1C), 18.40 (s, 1C), 17.13 (s, 1C).

[0050] Example 15 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2- carboxamido)-1,17-diaza-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazahexadec-12-yl)-1,2,3,4- tetrahydroisoquinoline-6-carboxamide

[0051] Compound C2-2 was synthesized by the above method, with a yield of 60%, M.W: 705.35; The structure of C2-2 is shown in Table 1.

[0052] 1 H NMR (400 MHz, MeOD) δ8.19-8.21 (m, 1H), 8.05-8.13 (m, 1H), 7.89-7.94 (m, 1H), 7.74-7.76 (m, 1H), 7.49-7.56 (m, 2H), 7.31-7.33 (m, 1H), 5.66-5.69 (m, 1H), 4.60-4.64 (m, 1H), 4.41 (s, 2H), 4.23-4.30 (m, 1H), 3.50-3.55 (m, 2H), 3.12-3.21 (m, 6H), 2.04-2.19 (m, 2H), 1.73-1.96 (m, 5H), 1.66-1.79 (m, 2H), 0.89-1.00 (m, 6H).

[0053] Example 16 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazo-2-carbonyl)-1,17-diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheptadecane-12-yl)-4-bromo-2,3,6-trifluorophenylacetamide Compound C3-1 was synthesized by the above method with a yield of 51% and a MW of 796.21. The structure of C3-1 is shown in Table 1.

[0054] 1 H NMR (400 MHz, MeOD) δ8.19-8.21 (m, 1H), 8.10-8.12 (m, 1H), 7.59-7.65 (m, 1H), 7.45-7.49 (m, 1H), 7.27-7.36 (m, 2H), 5.62-5.64 (m, 1H), 4.23-4.41 (m, 2H), 3.72-3.74 (m, 2H), 3.01-3.26 (m, 4H), 1.70-2.15 (m, 7H), 1.62-1.68 (m, 2H), 0.91-0.97 (m,6H). 13C NMR (400 MHz, MeOD) δ 192.21 (s, 1C), 172.40 (d, J = 55.2 Hz, 2C), 169.53 (s, 1C), 157.25 (d, J = 23.2 Hz, 1C), 153.38 (s, 1C), 137.01 (s, 1C), 129.66 (s, 1C), 128.04 (s, 1C), 127.19 (s, 3C), 125.12 (s, 1C), 124.86 (s, 2C), 122.40 (s, 2C), 114.45 (s, 1C), 58.60 (s, 1C), 54.88 (s, 1C), 53.32 (s, 1C), 40.54 (s, 2C), 40.40 (s, 1C), 30.47 (s, 1C), 28.58 (s, 2C), 27.88 (s, 1C), 25.31 (s, 1C), 24.65 (s, 1C), 18.37 (s, 1C), 17.02 (s, 1C).

[0055] Example 17 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)pyrazine-2-carboxamide Compound C4-2 was synthesized by the above method, with a yield of 45%, M.W: 652.30; The structure of C4-2 is shown in Table 1.

[0056] 1 H NMR (400 MHz, MeOD) δ 9.24 (s, 1H), 8.81 (d, J = 2.4 Hz, 1H), 8.70 (t, J = 1.8 Hz, 1H), 8.19-8.21 (m, 1H), 8.11-8.13 (m, 1H), 7.61-7.65 (m, 2H), 5.66-5.70 (m, 1H), 4.69-4.72 (m, 1H), 42.25 (d, J = 7.2 Hz, 1H), 3.19-3.27 (m, 4H), 2.07-2.18 (m, 4H), 1.80-1.90 (m, 3H), 1.66-1.71 (m, 2H), 0.94-1.02 (m, 6H). 13C NMR (400 MHz, MeOD) δ 192.33 (s, 1C), 172.52 (s, 1C), 172.23 (s, 1C), 164.27 (s, 1C), 157.28 (s, 1C), 153.40 (s, 1C), 147.55 (s, 1C), 144.34 (s, 1C), 143.46 (s, 4C), 137.05 (s, 1C), 58.95 (s, 1C), 54.78 (s, 1C), 52.66 (s, 1C), 40.52 (d, J = 31.6 Hz, 2C), 30.44 (s, 1C), 29.32 (s, 1C), 28.02 (s, 1C), 25.09 (s, 1C), 24.72 (s, 1C), 18.25 (s, 1C), 17.33 (s, 1C).

[0057] Example 18 Synthesis of compound N-((9S,12S)-l,17-diamino-6-(benzo[d]thiazole-2-carbonyl)- 1,17-diazenido-9-isopropyl-8,l l-dioxo-2,7,10,16-tetraazaheneicosan-12-yl)-3-methyl-2,6- difluorophenylacetamide Compound C5-2 was synthesized by the above method, yield 40%, M.W: 714.32; The structure of C5-2 is shown in Table 1.

[0058] 1 H NMR (400 MHz, MeOD) δ 8.119-8.22 (m, 2H), 7.61-7.67 (m, 2H), 7.12-7.16 (m, 1H), 6.82-6.87 (m, 1H), 5.66-5.70 (m, 1H), 4.39-4.42 (m, 1H), 4.24 (d, J = 7.2 Hz, 1H), 3.69 (s, 2H), 3.21-3.24 (m, 4H), 2.22 (s, 3H), 2.05-2.17 (m, 2H), 1.65-1.87 (m, 7H), 0.91-0.97 (m, 6H). 13C NMR (400 MHz, MeOD) δ 192.31 (s, 1C), 172.53 (s, 1C), 172.45 (s, 1C), 170.91 (s, 1C), 164.25 (s, 1C), 157.26 (s, 1C), 153.41 (s, 1C), 137.06 (s, 1C), 129.82 (d, J = 115.6 Hz, 2C), 128.04 (s, 2C), 127.19 (s, 2C), 125.08 (s, 2C), 122.44 (s, 1C), 110.21 (s, 1C), 109.98 (s, 1C), 58.68 (s, 1C), 54.66 (s, 1C), 53.06 (s, 1C), 40.46 (d, J = 32 Hz, 2C), 30.51 (s, 1C), 28.60 (d, J = 34.8 Hz, 1C), 28.04 (s, 1C), 25.01 (s, 1C), 24.82 (s, 1C), 18.25 (s, 1C), 17.20 (s, 1C), 12.73 (d, J = 14.4 Hz, 2C).

[0059] Example 19 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosan-12-yl)-2,3-dihydrobenzo[b][1,4] dioxepine-5-carboxamide Compound C7-2 was synthesized by the above method with a yield of 53% and M.W: 708.32. The structure of C7-2 is shown in Table 1.

[0060] 1H NMR (400 MHz, MeOD) δ 8.19-8.21 (m, 1H), 8.11-8.14 (m, 1H), 7.60-7.67 (m, 2H), 7.38-7.40 (m, 1H), 7.00-7.03 (m, 1H), 6.89-6.93 (m, 1H), 5.67-5.70 (m, 1H), 4.67-4.70 (m, 1H), 4.42-4.44 (m, 2H), 4.28-4.31 (m, 3H), 3.18-3.26 (m, 4H), 2.08-2.19 (m, 2H), 1.64-1.96 (m, 7H), 0.95-1.00 (m, 6H). 13 C NMR (400 MHz, MeOD) δ 192.35 (s, 1C), 172.54 (s, 1C), 172.41 (s, 1C), 166.33 (s, 1C), 164.29 (s, 1C), 157.28 (s, 1C), 153.40 (s, 1C), 144.08 (s, 1C), 142.26 (s, 1C), 137.05 (s, 1C), 128.04 (s, 1C), 127.20 (s, 2C), 125.08 (s, 1C), 122.44 (s, 1C), 122.27 (s, 1C), 121.95 (s, 1C), 120.80 (s, 1C), 120.55 (s, 1C), 65.00 (s, 1C), 63.69 (s, 1C), 58.71 (s, 1C), 54.77 (s, 1C), 53.00 (s, 1C), 40.51 (d, J = 20.4 Hz, 2C), 30.67 (s, 1C), 29.32 (s, 1C), 28.03 (s, 1C), 25.08 (s, 1C), 24.74 (s, 1C), 18.27 (s, 1C), 17.23 (s, 1C).

[0061] Example 20 Synthesis of compound N-((6S, 12S)-1, 17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1, 17-diamino-9-isopropyl-8, 11-dioxo-2, 7, 10, 16-tetraazahexadec-12-yl)cyclopropanecarboxamide Compound MC25-06 was synthesized by the above method, yield 29%, M.W: 614.31; The structure of MC25-06 is shown in Table 1.

[0062] 1 HNMR (MeOD-d4), δ 8.14-8.24 (m, 2H), 7.64-7.70 (m, 2H), 5.70-5.73 (m, 1H), 4.26-4.40 (m, 2H), 3.60-3.65 (m, 1H), 3.28-3.29 (m, 2H), 3.18-3.23 (m, 2H), 2.01-2.22 (m, 7H), 1.81-1.92 (m, 2H), 1.64-1.73 (m, 2H), 1.19-1.22 (m, 2H), 0.96-1.01 (m, 6H). 13 CNMR (MeOD-d4), δ 192.33 (s, 1C), 172.82 (s, 1C), 172.54 (s, 1C), 172.20 (s, 1C), 164.26 (s, 1C), 157.28 (s, 1C), 153.41 (s, 1C), 137.05 (s, 1C), 128.05 (s, 2C), 127.20 (s, 1C), 125.08 (s, 1C), 122.44 (s, 1C), 58.67 (s, 1C), 56.93 (s, 1C), 54.72 (s, 1C), 52.98 (s, 1C), 40.47-40.53 (m, 2C), 30.58 (s, 1C), 28.58 (s, 1C), 28.05 (s, 1C), 25.06 (s, 1C), 24.85 (s, 1C), 21.02 (s, 1C), 18.28 (s, 1C), 17.25 (s, 1C), 16.96 (s, 1C).

[0063] Example 21 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-2-chloro-5- trifluoromethylbenzamide Compound ST-24-8 was synthesized by the above method, with a yield of 34%, M.W: 752.19; The structure of ST-24-8 is shown in Table 1.

[0064] 1H NMR (500 MHz, MeOD) δ 8.31-7.93 (m, 3H), 7.65-7.34 (m, 1H), 7.18-6.91 (m, 3H), 4.79-4.63 (m, 1H), 4.35-4.09 (m, 1H), 3.66-3.02 (m, 5H), 2.25-1.46 (m, 8H), 1.22-0.74 (m, 7H). ESI-MS: 687.3 (M+H).

[0065] Example 22 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)- 1,17-diazenido-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazheptadec-12-yl)-2,4- difluorobenzamide Compound ST-24-9 was synthesized by the above method with a yield of 22% and M.W: 686.29; The structure of ST-24-9 is shown in Table 1.

[0066] 1 H NMR (500 MHz, MeOD) δ 8.31-7.93 (m, 3H), 7.65-7.34 (m, 1H), 7.18-6.91 (m, 3H), 4.79-4.63 (m, 1H), 4.35-4.09 (m, 1H), 3.66-3.02 (m, 5H), 2.25-1.46 (m, 8H), 1.22-0.74 (m, 7H). ESI-MS: 687.3 (M+H).

[0067] Example 23 Synthesis of compound N-((9S,12S)-1-((4-methoxy-2,3,6-trimethylphenyl)sulfonamido)- 17-amino-6-(benzo[d]thiazole-2-carbonyl)-1,17-diazenido-9-isopropyl-8,11-dioxo-2,7,10,16- tetraazheptadec-12-yl)-2,4,6-trichlorobenzamide Compound ST-24-11 was synthesized by the above method with a yield of 11% and M.W: 964.25; The structure of ST-24-11 is shown in Table 1.

[0068] 1H NMR (500 MHz, MeOD) δ 8.38-7.96 (m, 3H), 7.90-7.38 (m, 5H), 6.70-6.39 (m, 1H), 4.74-4.18 (m, 2H), 3.86-3.70 (m, 3H), 3.30-3.04 (m, 5H), 2.64-2.32 (m, 7H), 2.25-1.96 (m, 5H), 1.89-1.65 (m, 5H), 1.06-0.79 (m, 7H). 13 CNMR (151 MHz, MeOD) δ 192.42, 172.41, 167.05, 164.11, 161.36, 158.46, 157.30, 156.83, 153.37, 142.57, 137.57, 136.97, 136.59, 134.80, 132.19, 130.57, 127.92, 127.05, 126.31, 124.32, 122.30, 118.31, 117.77, 115.71, 111.40, 54.61, 53.54, 42.49, 40.60, 30.64, 28.66, 27.25, 25.00, 22.92, 22.24, 18.37, 17.44, 12.04, 10.69. ESI-MS: 965.3 (M+H).

[0069] Example 24 Synthesis of compound N-((9S,12S)-1,17-diamino-6-(benzo[d]thiazole-2-carbonyl)-1,17- diimino-9-isopropyl-8,11-dioxo-2,7,10,16-tetraazaheneicosyl)-3,4-dichlorobenzamide Compound ST-23-12 was synthesized by the above method, yield 32%, M.W: 718.23; The structure of ST-23-12 is shown in Table 1.

[0070] 1 H NMR (500 MHz, MeOD) δ 8.38-7.96 (m, 3H), 7.90-7.38 (m, 5H), 6.70-6.39 (m, 1H), 4.74-4.18 (m, 2H), 3.86-3.70 (m, 3H), 3.30-3.04 (m, 5H), 2.64-2.32 (m, 7H), 2.25-1.96 (m, 5H), 1.89-1.65 (m, 5H), 1.06-0.79 (m, 7H). J = 8.0 Hz, 1H), 8.19-8.07 (m, 1H), 7.73-7.32 (m, 5H), 5.75-5.68 (m, 1H), 4.64 (ddd, J= 14.5, 8.6, 5.5 Hz, 1H), 4.46-4.23 (m, 1H), 3.34-3.18 (m, 4H), 2.25-1.68 (m, 9H), 1.10-0.92 (m, 6H). 13 C NMR (126 MHz, MeOD) δ 192.37, 172.55, 167.75, 164.22, 160.96, 157.38, 153.41, 137.05, 136.18, 131.80, 130.10, 129.39, 128.07, 127.18, 125.19, 122.46, 117.48, 115.17, 58.84, 55.06, 53.99, 40.61, 30.77, 28.60, 27.93, 27.32, 25.09, 23.95, 18.54, 17.27. ESI-MS: 719.2 (M+H).

[0071] Example 25 Synthesis of compound N-((9S,)-l-amino-6-(benzo[d]thiazole-2-carbonyl)-l-imino-9- isopropyl-8-oxo-2,7-diazanona-9-yl)-3,4-dichlorobenzamide Compound ST-23-18 was synthesized by the above method, yield 26%, M.W: 562.13; The structure of ST-23-18 is shown in Table 1.

[0072] 1 H NMR (500 MHz, CD3OD) δ 8.20 (d, J = 6.8 Hz, 1H), 8.09 (t, J = 7.7 Hz, 1H), 7.71-7.22 (m, 5H), 5.82-5.56 (m, 1H), 4.53-4.32 (m, 1H), 3.36-3.25 (m, 2H), 2.24-2.18 (m, 2H), 1.93-1.81 (m, 2H), 1.15-0.81 (m, 6H). 13C NMR (126 MHz, MeOD) δ 192.34, 171.61, 167.61, 164.13, 164.08, 157.34, 153.39, 137.04, 136.02, 130.00, 129.35, 128.05, 127.21, 125.20, 122.41, 59.64, 54.59, 40.47, 30.21, 28.00, 25.36, 17.82, 10.81. ESI-MS: 563.2 (M+H).

[0073] Application Example 1 Construction of TMPRSS2 inhibitor cell screening model based on fluorescently labeled substrate (1) Model construction strategy The construction strategy is shown in Figure 2 TMPRSS2 can hydrolyze Boc-Gln-Ala-Arg-AMC (Boc-QAR-AMC) to produce free 7-amino-4-methylcoumarin (AMC), which can be detected by an enzyme label under the condition of excitation wavelength (Ex) 340 nm and emission wavelength (Em) 440 nm. The change in fluorescence intensity in a 96-well plate can be used to judge the activity of TMPRSS2, thereby achieving the purpose of screening TMPRSS2 inhibitors.

[0074] (2) Model construction African green monkey kidney cells (Vero E6) with high expression of TMPRSS2 (Vero E6 / TMPRSS2) 100 μL were uniformly plated in a 96-well plate with a white bottom and a transparent cover at 1~2×10 4 / well, and cultured at 37 ℃, 5% CO2 for 24 h; after washing with PBS for 3 times, 100 μL of fluorescent substrate Boc-QAR-AMC with a final concentration of 400, 200, 150, 100, 50, 25, 12.5, 6.25 μmol·L -1 were added to each well in 3 groups of duplicate wells; the multifunctional enzyme label was set at Ex 340 nm and Em 440 nm, and the relative fluorescence unit (RFU) was read once every 30 s; the Michaelis equation was fitted using GraphPad Prism 9.5.1 software to calculate the Michaelis constant (Km) and maximum reaction speed (Vmax) to determine the usability and optimal substrate concentration of the model.

[0075] Figure 3 The results showed that Vero E6 / TMPRSS2 cells endogenous TMPRSS2 can cleave the substrate Boc-Gln-Ala-Arg-AMC, causing changes in RFU Figure 3 , by the hydrolysis trend of different concentrations of substrate within 50 min, fitting its Michaelis constant reaction curve Figure 3 , calculated K m 37.36 μmol·L -1 , V max 15.60 RFU·min -1 .

[0076] (3) Inhibitory activity verification of Camostat Mesilate, a TMPRSS2 inhibitor, on cell model Camostat Mesilate is a known TMPRSS2 inhibitor, 100 μL of Vero E6 / TMPRSS2 cells were uniformly plated in a 96-well plate with a white background and transparent cover at 1~2×10 4 per well, and cultured at 37 ℃, 5% CO2 for 24 h; after washing with PBS for 3 times, 50 μL of Camostat Mesilate diluted by PBS in a ratio of 1:2 was added to each well, with concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.20 nmol·L -1 , and incubated at RT for 30 min in a microplate incubator shaker. DMSO was added to Vero E6 / TMPRSS2 cells as a positive control, and the group containing only DMEM was the negative control. The final concentration of the fluorescent substrate Boc-Gln-Ala-Arg-AMC added to each well was 50 μmol·L -1 , and incubated at RT in the dark for 1 h. The RFU was read in a multifunctional enzyme label instrument, and the inhibition rate of each well was calculated according to the formula. The halfmaximal inhibitory concentration (IC 50 ) was calculated using GraphPad Prism 9.5.1 software.

[0077]

[0078] As shown in Figure 3 , the IC 50 of Camostat Mesilate on the cell model was 13.47 ± 6.60 nmol·L -1 , which was comparable to the literature reported, proving that the cell model can be used for the activity measurement of TMPRSS2 inhibitors.

[0079] (4) Reproducibility analysis of cell screening model The 96-well plate was divided into two parts, one was positive control Vero E6 / TMPRSS2 cells, and the other group contained only DMEM as negative control, and was cultured at 37°C, 5% CO2 for 24 h, washed with PBS for 3 times, and 100 μL of fluorescent substrate Boc-Gln-Ala-Arg-AMC with a final concentration of 50 μmol·L -1 was added to each well, and incubated at RT in the dark for 1 h. The multifunctional enzyme label instrument was used to read RFU. According to the formula, the Z factor was calculated, SD was the standard deviation of the control group, and μ was the average value of the control group.

[0080]

[0081] The Z factor can reflect the stability and reliability of the drug screening model. The Z factor of the established cell screening model was 0.73 ( Figure 3 ), which met the requirements of high-throughput screening model.

[0082] Application Example 2 Inhibition activity detection of oligopeptide compounds on cell model The final concentration of oligopeptide compounds was 200-1.56 μmol·L -1 , PBS was used to dilute the compounds by multiple times, 3 groups of duplicate wells were set, and the inhibition rate of each concentration of the compounds was calculated to obtain IC 50 .

[0083] As shown in Figure 4 and Table 2, oligopeptide compounds can inhibit TMPRSS2 cleavage of the substrate oligopeptide at the nM level.

[0084] Table 2 IC 50 of oligopeptide compounds on TMPRSS2 cleavage of oligopeptide substrate (nM)

[0085] Application Example 3 Cytotoxicity detection of oligopeptide compounds CCK-8 kit (Cell Counting Kit-8) was used to detect the cytotoxicity of IMB-9C. Vero, Huh-7 and HEK-293T cells were taken out from liquid nitrogen, recovered in a 37 ℃ water bath, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with 1 mL of 1640 culture medium containing 10% fetal bovine serum, transferred to a culture dish, and 9 mL of culture medium was added. The cells were cultured at 37 ℃ and 5% CO2. When the cells grew to more than 80% of the culture dish area, they were trypsinized and subcultured. The cells in the logarithmic phase were plated at a density of 8000 cells per well, and the outermost circle was sealed with sterile water. The active oligopeptide compounds obtained by screening were added to each well to a final concentration of 50 μg / mL, and the culture was continued for 48 h. 10 μL of CCK-8 detection reagent was added to each well, and the culture was incubated in the dark for 2-4 h. The absorbance at 450 nm was read by a microplate reader. Compared with the control group without oligopeptide compounds, the relative survival rate of cells was calculated. The same method was used to detect the toxicity of oligopeptide compound IMB-9C on Vero cells and Huh-7 cells.

[0086] The results showed that the oligopeptide compounds did not show growth inhibition activity on Vero, Huh-7 and HEK-293T cells at a concentration of 50 μg / mL.

[0087] Application Example 4 Detection of the inhibitory activity of oligopeptide compounds on influenza virus H3N2 Influenza virus A / Hangyong / 359 / 95 (H3N2) was cultured and passaged in the allantoic cavity of chicken embryos and stored at -80 ℃. When detecting oligopeptide compounds, the culture medium was diluted to a certain concentration and then diluted by 3 times, with 8 dilutions for each. The positive control drugs were ribavirin (RBV) and oseltamivir phosphate. MDCK cells were inoculated into a 96-well culture plate and cultured at 37 ℃ in 5% CO2. After 24 h, the cells were infected with influenza virus at a multiplicity of infection (MOI) of 1 / 30 -5 , adsorbed for 2 h, and the virus solution was discarded. The maintenance solution containing different dilutions of samples and positive control drugs was added, and cell control wells and virus control wells were set up. The culture was incubated at 37 ℃ in 5% CO2. The TC 50 and IC 50 of the samples were calculated based on the virus control.

[0088] As shown in Table 3, all oligopeptide compounds had inhibitory activity on influenza virus H3N2, and the SI was above 90, which was better than the positive control drugs RBV and oseltamivir phosphate.

[0089] Table 3 IC 50 (μg / ml) and TC of oligopeptide compounds on influenza virus H3N250 (µg / ml)

[0090] TC 50 : Drug half-maximal toxic concentration; IC50 50 : Half-maximal inhibitory concentration (IC50) of the drug against the virus; SI: Selectivity index, SI = TC 50 / IC 50 .

[0091] Application Example 5 SPR method for detecting the affinity of oligopeptides for TMPRSS2 Clean the instrument tubing in the following order: 20% ethanol, H2O, and PBST. Install the dextran chip before PBST cleaning and check for SPR angle deviation after installation. Mix EDC and NHS in a 1:1 ratio into the sample tube at a flow rate of 10 μL·min⁻¹ for 7 min to activate the chip. Dilute TMPRSS2 to 80 μg·mL⁻¹ using sodium acetate solution at pH 4.5. -1 The sample was loaded into a sample tube, and protein immobilization was performed in the left channel at a flow rate of 10 μL / min. -1 Inject for 7 minutes, with the right channel serving as a blank control. Once the protein has fixed to the ideal signal, inject 1 mol·L⁻¹. -1 Ethanolamine (pH 8.5) was used to block unbound protein sites on the chip surface. The tubing was then rinsed with PBSTD. The compound was diluted with PBSTD buffer to final concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μmol·L⁻¹. -1 PBSTD was used as a blank control. Sample loading was performed for 0.5 min, followed by natural dissociation for 2.5 min. The sample flow rate was set to 25 μL / min. -1 The equilibrium binding constant (KD) was calculated using Trace Drawer software. After the detection, the instrument tubing was cleaned sequentially with H2O, SDS-glycine, and 20% ethanol.

[0092] like Figure 5 As shown in Table 4, oligopeptide compounds can bind to TMPRSS2 with moderate strength, which is basically a fast binding and fast dissociation mechanism.

[0093] Table 4. Affinity test results of oligopeptides with TMPRSS2

[0094] Application Example 6 ADMET prediction of oligopeptide compounds The ADME of the oligopeptide compound is predicted by using Chemdraw software. The operation process is as follows: the structure of the active oligopeptide compound is converted into SMILES format and input into the search box, and the in vivo ADME of the oligopeptide compound is predicted by clicking run.

[0095] The ADME prediction results are shown in Table 5. The oligopeptide compounds do not comply with the five-fold rule due to the large molecular weight of the peptide structure, have low intestinal absorption, cannot pass through the blood-brain barrier, are slightly soluble in water, and except for ST-23-18, do not inhibit liver enzymes.

[0096] In terms of toxicity, as shown in Table 6, none of them cause eye irritation, but most of them have certain liver toxicity and respiratory toxicity, and most of them do not have acute oral toxicity in rats.

[0097] Table 5 ADME prediction of oligopeptide compounds

[0098] Table 6 Toxicity prediction of oligopeptide compounds

[0099] +++ / ++ represents high risk, and --- / -- represents low risk.

[0100] As can be seen from the above examples, the application provides the use of the oligopeptide compound in the preparation of an anti-influenza virus drug. The oligopeptide compound of the application has an inhibitory effect on influenza virus and its mutant strains, and can be used to prepare an anti-influenza virus drug and / or preparation.

[0101] Although the specific embodiments of the application have been described in detail, those skilled in the art can further modify and replace the details of the technical solutions of the application according to all the teachings disclosed herein. These changes are within the scope of the protection of the application. The entire scope of the application is given by the appended claims and any equivalents thereof.

Claims

1. An oligopeptide compound having anti-influenza virus activity, characterized in that, The structure of the oligopeptide compound is shown in general formula I: Ⅰ R1 is independently hydrogen atom, C1-C6 alkyl, phenyl, mono- or poly-substituted phenyl, pyridyl, mono- or poly-substituted pyridyl, naphthyl, mono- or poly-substituted naphthyl, furanyl, mono- or poly-substituted furanyl, thienyl, mono- or poly-substituted thienyl, thiazolyl, mono- or poly-substituted thiazolyl, oxazolyl, mono- or poly-substituted oxazolyl, isoxazolyl, mono- or poly-substituted isoxazolyl, imidazolyl, mono- or poly-substituted imidazolyl, pyrazinyl, mono- or poly-substituted pyrazinyl, pyrimidinyl, mono- or poly-substituted pyrimidinyl, pyridazinyl, mono- or poly-substituted pyridazinyl, indolyl, mono- or poly-substituted indolyl, purinyl, mono- or poly-substituted purinyl, quinolinyl, mono- or poly-substituted quinolinyl, isoquinolinyl, mono- or poly-substituted isoquinolinyl, imidazopyridinyl, mono- or poly-substituted imidazopyridinyl; R2 is independently hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy, nitro, hydroxyl, amine, amine methyl, C1-C6 acyl or halogen substitution; R3 is independently hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy, nitro, hydroxyl, amine, amine methyl, C1-C6 acyl or halogen substitution; R4 is independently hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy, nitro, hydroxyl, amine, amine methyl, C1-C6 acyl or halogen substitution; R5 is independently hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy, nitro, hydroxyl, amine, amine methyl, C1-C6 acyl or halogen substitution; R6 is independently hydrogen atom or 4-methoxy-2,3,6-trimethylbenzenesulfonyl; X is independently NH, S or O; M is independently absent or C1-C6 alkyl; Y is independently absent or L-arginine fragment.

2. The oligopeptide compound according to claim 1, wherein R1 is independently hydrogen atom, C1-C6 alkyl, phenyl, mono- or poly-substituted phenyl, pyridyl, mono- or poly-substituted pyridyl, oxazolyl, mono- or poly-substituted oxazolyl, naphthyl, mono- or poly-substituted naphthyl, furanyl, mono- or poly-substituted furanyl, imidazolyl, mono- or poly-substituted imidazolyl, pyrazinyl, mono- or poly-substituted pyrazinyl; R2 is independently halogen, hydrogen atom, methyl or acetyl; R3 is independently halogen, hydrogen atom, methyl or acetyl; R4 is independently halogen, hydrogen atom, methyl or acetyl; R5 is independently halogen, hydrogen atom, methyl or acetyl; R6 is independently hydrogen atom or 4-methoxy-2,3,6-trimethylbenzenesulfonyl; X is independently NH, S or O; M is independently absent or C1-C3 alkyl; Y is independently absent or L-arginine fragment. ​ 3. The oligopeptidic compound according to claim 1, characterized in that, The oligopeptide compound includes any one of the following compounds or a pharmaceutically acceptable salt thereof: INT1, INT2, INT3, INT4, INT5, INT6, INT7, INT10, INT1-LXY, I1, I2, I3, I4, C1-1, C2-2, C3-1, C4-2, C5-2, C7-2, MC25-06, ST-24-8, ST-24-9, ST-24-11, ST-23-12, and ST-23-18.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition includes a therapeutically effective amount of the oligopeptide compound of any one of claims 1-3 and one or more pharmaceutically acceptable carriers.

5. The pharmaceutical composition of claim 4, wherein, The dosage form of the pharmaceutical composition includes oral preparations.

6. Use of the oligopeptide compound of any one of claims 1-3 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicament and / or preparation for resisting influenza virus.

7. Use according to claim 6, characterized in that, The influenza virus is of H3N2 subtype.

8. Use of the oligopeptide compound of any one of claims 1-3 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicament and / or preparation for inhibiting TMPRSS2 protease activity.

9. Use of the oligopeptide compound of any one of claims 1-3 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicament and / or preparation for binding to TMPRSS2 protein.

10. A method for preventing or treating an infection with an influenza virus, characterized in that, The method includes administering a therapeutically effective amount of the oligopeptide compound of any one of claims 1-3 or the pharmaceutical composition of any one of claims 4-5 to an infected individual.

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