Synthesis method of axially chiral cyclopeptide-containing compound

Abstract

The invention discloses a synthesis method of axially chiral cyclopeptide compounds, which comprises the following steps of: synthesizing a series of axially chiral cyclopeptide compounds with diastereoisomers in a ratio of 1: 1 by catalyzing Ullmann coupling reaction of tryptophan residues through copper; a new thought and a new way are provided for synthesis of the cyclic peptide. The preparation method disclosed by the invention is simple, the reaction conditions are milder than those of the prior art, the selectivity on tryptophan residues is excellent, a more effective and direct method is provided for synthesis of cyclopeptide, and meanwhile, a powerful synthesis means is provided for modern organic synthesis, natural products and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and in particular relates to a method for synthesizing cyclic peptide compounds containing axial chirality. Background Art

[0002] Cyclic peptides and other polypeptides are ubiquitous in nature and possess a wide range of biological activities, such as antitumor, antibacterial, immunosuppressive, and antiviral properties. Peptide cyclization can occur at the C-terminus (head), N-terminus (tail, NH2, or OH), or R group (side chain) of an amino acid residue. Therefore, four cyclization directions are possible: head-to-tail, head-to-side chain, tail-to-side chain, and side chain-to-side chain. Head-to-tail cyclization is the most common, resulting in a complete ring closure between the N-terminal amine and the C-terminal carboxylic acid. Head-to-side chain cyclization involves the bonding of a nucleophilic side chain to the C-terminus. Tail-to-side chain cyclization connects the N-terminus to an electrophilic side chain. Finally, side chain-to-side chain cyclization connects the R groups of adjacent amino acid residues, for example, through the oxidation of Cys (cysteine) to form a disulfide bond. These cyclization strategies are selected based on the target structure, synthetic route, and reaction conditions. In addition to utilizing functional moieties present at the peptide termini and side chains for peptide cyclization, peptide side chains may also be linked using external linkers to form cyclic peptides.

[0003] At present, the synthesis of cyclic peptides mainly focuses on the side chain, such as Wang and colleagues through palladium-catalyzed C(sp 3 )-H activation and synthesis of cyclic peptides with unique hydrocarbon bonds between the β-carbon of amino acids and the aromatic side chains of Phe and Trp (Chem. Sci., 2017, 8, 4565-4570).

[0004] Pentelute and co-workers reported macrocyclization between lysine residues via nitrogen arylation of a perfluoroaryl linker (J. Am. Chem. Soc., 2016, 138, 8340-8343).

[0005] The Chen group developed a method to reshape the structural landscape of homologous peptides by cross-linking two natural aromatic side chains with aryl linkers connected to various heteroatoms (X). The aryl linkers can be easily installed through copper-catalyzed heteroatom arylation reaction with aryl diiodides (J.Am.Chem.Soc., 2023, 145, 13968-13978).

[0006] Due to the unique properties of amino acid units, precise modification of side chains can directly alter the backbone structure, effectively regulating the structure and function of polypeptides. The synthesis of cyclic peptides based on polypeptide side chains has great potential for development. Therefore, exploring a simple, mild, and efficient method for preparing cyclic peptides remains an important and challenging task. Summary of the Invention

[0007] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a cyclic peptide compound containing axial chirality, wherein the cyclic peptide compound containing axial chirality has a general structural formula as shown in Formula III.

[0010]

[0011] In formula III, AA represents a natural amino acid or a non-natural amino acid, and R is selected from any one of methyl ester, tert-butyl ester, tert-amyl ester, and benzophenone.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for synthesizing cyclic peptide compounds containing axial chirality.

[0013] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0014] A polypeptide substrate having a structural formula as shown in Formula I and containing tryptophan residues at both ends and an aryl iodide having a structural formula as shown in Formula II are reacted with a catalyst, a ligand, an additive, an inorganic base, and an organic solvent under a nitrogen atmosphere with heating and stirring. After the reaction is completed, the reaction product is separated and purified to obtain a cyclic peptide compound containing axial chirality as shown in Formula III;

[0015]

[0016] AA in Formula I and Formula III represents a natural amino acid or a non-natural amino acid, the protecting group on the nitrogen atom at the nitrogen terminal is a tert-butyloxycarbonyl group, and the protecting group on the carbon atom at the carbon terminal is a methyl ester group;

[0017] R in formula II is selected from any one of methyl ester, tert-butyl ester, tert-amyl ester and benzophenone.

[0018] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the molar ratio of the polypeptide substrate with tryptophan residues at both ends, as shown in the structural formula of Formula I, to the aryl iodide, as shown in the structural formula of Formula II, is 1:(1.0-1.2), the molar ratio to the catalyst is 1:(0.3-1.2), the molar ratio to the ligand is 1:(0.4-0.8), the molar ratio to the additive is 1:(0.2-0.5), and the molar ratio to the inorganic base is 1:(6-12).

[0019] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the organic solvent is a mixed solution of 1,2-dichloroethane and acetonitrile, and the volume ratio of the two is 1:(0.8-1.2).

[0020] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the concentration of the polypeptide substrate with tryptophan residues at both ends, as shown in the structural formula of Formula I, in the reaction system is 0.05M.

[0021] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the catalyst includes one of CuI, Cu(OAc)2, CuOAc, and Cu powder.

[0022] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the inorganic base includes one of cesium carbonate and potassium carbonate.

[0023] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the ligand includes one of 2,2'-bipyridine, 2,2'-bipiperazine, benzoylacetone, and 4,4'-di-tert-butyl-2,2'-bipyridine.

[0024] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the additive includes one of pyridine and Ni(py)4Cl2.

[0025] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the reaction time of the heating and stirring reaction is 8-10h, and the reaction temperature is 95-105℃.

[0026] As a preferred scheme of the synthetic method of the axially chiral cyclic peptide compound, the compound synthesized by the method has antibacterial effect.

[0027] The present application has the following beneficial effects:

[0028] The application reacts the polypeptide compound with tryptophan residues at both nitrogen end and carbon end and aryl iodide, synthesizes a cyclic peptide through copper-catalyzed Ullmann coupling reaction of the tryptophan residues, realizes the construction of C-N bond in the polypeptide, and through a series of substrate expansion experiments, it is found that the reaction can be smoothly carried out with the increase of the carbon chain and the change of the amino acid sequence in the carbon chain, which embodies the universality of the method.

[0029] The method only acts on the N-H bond on the indole ring of the tryptophan residue, compared with the prior art, the application has excellent selectivity for the tryptophan residue, and the cyclic peptide product synthesized by the application is an axially chiral cyclic peptide compound, has diastereoisomers, and the ratio is about 1:1, the application synthesizes a series of isomer cyclic peptide compounds, and provides a new idea and a new way for the synthesis of cyclic peptides. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor. Among them:

[0031] Figure 1 The nuclear magnetic hydrogen spectrum of the 1c-1 compound prepared in the embodiment 1 of the application.

[0032] Figure 2 The nuclear magnetic carbon spectrum of the 1c-1 compound prepared in the embodiment 1 of the application.

[0033] Figure 3 The nuclear magnetic hydrogen spectrum of the 1c-2 compound prepared in the embodiment 1 of the application.

[0034] Figure 4 The nuclear magnetic carbon spectrum of the 1c-2 compound prepared in the embodiment 1 of the application.

[0035] Figure 5 The nuclear magnetic hydrogen spectrum of the 2c-1 compound prepared in the embodiment 2 of the application.

[0036] Figure 6 The nuclear magnetic hydrogen spectrum of the 2c-2 compound prepared in the embodiment 2 of the application.

[0037] Figure 7 The nuclear magnetic carbon spectrum of the 2c-2 compound prepared in the embodiment 2 of the application.

[0038] Figure 8 The nuclear magnetic hydrogen spectrum of the 3c-1 compound prepared in the embodiment 3 of the application.

[0039] Figure 9 The nuclear magnetic hydrogen spectrum of the compound 3c-2 prepared in Example 3 of the present application is shown in the following figure.

[0040] Figure 10 The nuclear magnetic hydrogen spectrum of the compound 3c-2 prepared in Example 3 of the present application is shown in the following figure.

[0041] Figure 11 The nuclear magnetic hydrogen spectrum of the compound 3c-2 prepared in Example 3 of the present application is shown in the following figure.

[0042] Figure 12 The nuclear magnetic hydrogen spectrum of the mixture of compounds 4c-1, 4c-2 prepared in Example 4 of the present application is shown in the following figure.

[0043] Figure 13 The nuclear magnetic hydrogen spectrum of the mixture of compounds 4c-1, 4c-2 prepared in Example 4 of the present application is shown in the following figure.

[0044] Figure 14 The comparison chart of antibacterial effect of the mixture of isomer cyclic peptide products prepared in Example 4 of the present application at different concentrations is shown in the following figure.

[0045] Figure 15 The comparison chart of antibacterial effect of the mixture of isomer cyclic peptide products prepared in Example 4 of the present application at different concentrations is shown in the following figure. 5α The activity inhibition curve of E. coli. DETAILED DESCRIPTION

[0046] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application will be described in detail below with reference to the embodiments of the present application.

[0047] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, therefore, the present application is not limited by the specific embodiments disclosed below.

[0048] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is independent or alternative to other embodiments.

[0049] The raw materials used in the present application are commercially available in the art without special instructions.

[0050] Example 1

[0051] Referring to Reaction Scheme A, the present embodiment provides a preparation method of isomer cyclic peptide, specifically:

[0052]

[0053] 1) In a clean and oven-dried reaction tube of 8 mL, sequentially add the tripeptide substrate (0.05 mmol, 1.0 eq.) with structural formula as shown in 1a, aryl iodide (0.06 mmol, 1.2 eq.) with structural formula as shown in 1b, catalyst CuI (60 mol%), additive pyridine (20 mmol%), ligand 2,2'-bipyridine (0.4 eq.), inorganic base Cs2CO3 (8 eq.), to obtain a reaction system;

[0054] 2) Then add anhydrous DCE and MeCN (1:1 in volume, a total of 1 mL) to the reaction system in the glove box, seal with a plastic cap with a polytetrafluoroethylene gasket, maintain the N2 atmosphere, and react at 100°C for 10 h;

[0055] 3) After the reaction is completed, add saturated NaCl solution to the reaction tube to stop the reaction, pour the reaction system into a separatory funnel, extract with ethyl acetate multiple times (10 mL x 3), combine the organic phase, dry with Na2SO4, and concentrate under vacuum to obtain a reaction liquid, separate and purify by column chromatography (PE:EA:DCM=2:1:1 as eluent) to obtain isomeric cyclic peptide products with structural formulas as shown in 1c-1 and 1c-2, respectively, at a ratio of about 1:1, with a total separation yield of 70%.

[0056] Figure 1 、 2 The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the compound with structural formula as shown in 1c-1 (yellow solid) are shown in Figures 1 and 2, respectively, and the characterization data are as follows:

[0057] 1H NMR (400 MHz, CDC13) δ 8.04 (s, 1H), 7.90 (s, 1H), 7.66 (dd, J = 8.2, 4.6 Hz, 3H), 7.51 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.28 (d, J = 7.4 Hz, 1H), 7.26 - 7.20 (m, 1H), 6.65 (d, J = 7.0 Hz, 1H), 6.41 (s, 1H), 6.28 (s, 1H), 5.66 (d, J = 9.6 Hz, 1H), 5.18 (t, J = 10.2 Hz, 1H), 5.04 (d, J = 9.2 Hz, 1H), 4.08 (d, J = 7.0 Hz, 1H), 3.97 (t, J = 9.4 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.67 (s, 3H), 3.44 - 3.22 (m, 2H), 2.99 - 2.83 (m, 2H), 1.41 (s, 9H), 1.29 (d, J = 7.0 Hz, 3H).

[0058] 13 C NMR (100 MHz, CDC13) δ 172.55, 171.39, 170.87, 165.21, 164.68, 154.81, 140.07, 137.49, 137.31, 132.60, 131.37, 130.40, 130.30, 128.43, 128.20, 128.04, 127.84, 123.70, 123.59, 121.34, 119.33, 118.93, 115.70, 114.57, 112.48, 112.39, 79.98, 56.03, 52.84, 52.76, 49.82, 48.63, 29.66, 28.20, 27.74, 25.28, 19.30.

[0059] Figure 3 、 4 H NMR and C NMR spectra of the compound (yellow solid) represented by structural formula lc-2, respectively, are shown below:

[0060] 1H NMR (400 MHz, CDC13) δ 7.93 (s, 1H), 7.62-7.54 (m, 4H), 7.53 (d, J = 3.2 Hz, 1H), 7.32 (q, J = 8.0 Hz, 2H), 7.22 (dd, J = 14.6, 7.4 Hz, 2H), 6.89 (d, J = 7.0 Hz, 1H), 6.41 (s, 1H), 6.30 (s, 1H), 5.88 (d, J = 9.6 Hz, 1H), 5.02 (d, J = 9.2 Hz, 1H), 4.75 (t, J = 9.6 Hz, 1H), 4.50-4.38 (m, 1H), 3.99 (p, J = 7.0 Hz, 1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.47 (s, 3H), 3.44-3.21 (m, 2H), 2.92-2.74 (m, 2H), 1.42 (s, 9H), 1.29 (d, J = 7.0 Hz, 3H).

[0061] 13 C NMR (100 MHz, CDC13) δ 172.39, 171.55, 170.11, 165.15, 164.71, 155.17, 140.92, 139.44, 138.53, 138.26, 133.19, 131.03, 130.64, 130.47, 130.11, 129.43, 128.75, 127.95, 123.65, 123.51, 121.29, 121.23, 119.49, 118.93, 116.20, 114.65, 112.32, 112.08, 80.23, 53.60, 52.90, 52.76, 52.62, 52.45, 48.94, 28.21, 27.02, 26.16, 19.20.

[0062] The above characterization results show that the structure of the product prepared in this example is correct, which is the target product.

[0063] Example 2

[0064] With reference to Reaction Formula B, the present example provides a method for preparing an isomeric cyclic peptide, in particular:

[0065]

[0066] 1) In a clean and oven-dried reaction tube of 8 mL, sequentially add tetrapeptide substrate (0.05 mmol, 1.0 eq.) with structural formula as shown in 2a, aryl iodide (0.06 mmol, 1.2 eq.) with structural formula as shown in 1b, catalyst CuI (60 mol%), additive Ni(py)4Cl2(20 mmol%), ligand 2,2'-bipyridine (0.4 eq.), inorganic base Cs2CO3(8 eq.), to obtain a reaction system;

[0067] 2) Subsequently, add anhydrous DCE and MeCN (volume ratio of 1:1, a total of 2 mL) to the reaction system in the glove box, seal with a plastic cap with a polytetrafluoroethylene gasket, maintain its N2 atmosphere, and react at 100 °C for 10 h;

[0068] 3) After the reaction is completed, add saturated NaCl solution to the reaction tube to stop the reaction, pour the reaction system into a separatory funnel, extract with ethyl acetate multiple times (10 mL x 3), combine the organic phase, dry with Na2SO4, and concentrate under vacuum to obtain a reaction liquid, separate and purify by column chromatography (PE:EA:DCM = 1:4:1 as eluent) to obtain isomeric cyclic peptide products with structural formulas as shown in 2c-1 and 2c-2, respectively, at a ratio of about 1:1, with a total separation yield of 67%.

[0069] Figure 5 The nuclear magnetic resonance spectrum of the compound with structural formula as shown in 2c-1 (yellow solid) is shown in the following table:

[0070] 1 H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 7.87 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.35-7.28 (m, 2H), 7.23 (d, J = 6.0 Hz, 2H), 7.15 (t, J = 7.8 Hz, 2H), 6.50 (s, 1H), 6.45 (s, 1H), 6.34-6.27 (m, 1H), 6.25 (d, J = 8.8 Hz, 1H), 5.23 (d, J = 7.0 Hz, 1H), 4.76 (s, 1H), 4.33-4.20 (m, 3H), 3.80 (s, 3H), 3.54 (s, 3H), 3.51-3.42 (m, 3H), 3.37 (s, 3H), 3.13-3.01 (m, 1H), 1.44 (s, 9H), 1.22 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H).

[0071] Figure 6 、 7The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the compound (yellow solid) represented by structural formula 2c-2 are shown in the following table, and the characterization data are as follows:

[0072] 1 H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.2 Hz, 2H), 7.31-7.26 (m, 3H), 7.25-7.22 (m, 1H), 7.17 (t, J = 7.4 Hz, 1H), 6.88 (s, 1H), 6.66 (d, J = 6.0 Hz, 1H), 6.47 (s, 1H), 6.38 (d, J = 9.2 Hz, 1H), 6.11 (d, J = 7.0 Hz, 1H), 5.45 (d, J = 9.2 Hz, 1H), 4.81 (q, J = 5.6 Hz, 1H), 4.56-4.44 (m, 1H), 4.34-4.21 (m, 3H), 3.85 (s, 3H), 3.63 (s, 3H), 3.44 (s, 3H), 3.38 (dd, J = 15.2, 5.0 Hz, 1H), 3.16 (dd, J = 14.4, 7.2 Hz, 1H), 1.36 (s, 9H), 1.26 (d, J = 7.4 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H).

[0073] 13 C NMR (100 MHz, CDCl3) δ 172.05, 171.72, 171.63, 170.54, 164.68, 164.38, 155.34, 139.63, 138.92, 138.33, 137.50, 133.08, 132.38, 132.01, 131.73, 131.01, 129.47, 129.42, 128.02, 123.15, 123.01, 121.08, 120.28, 119.36, 118.70, 113.24, 110.85, 110.68, 110.57, 79.29, 56.02, 52.98, 52.92, 52.82, 52.74, 52.29, 49.61, 48.13, 28.22, 26.94, 26.10, 19.49, 18.88.

[0074] The above characterization results show that the structure of the product prepared in this embodiment is correct, which is the target product.

[0075] Example 3

[0076] With reference to Reaction Formula C, the embodiment provides a preparation method of an isomer cyclic peptide, in particular:

[0077]

[0078] 1) In a clean and oven-dried reaction tube of 8 mL, sequentially added a tripeptide substrate (0.05 mmol, 1.0 eq.) with structural formula as shown in 1a, an aryl iodide (0.06 mmol, 1.2 eq.) with structural formula as shown in 2b, a catalyst Cu(OAc)2(60 mol%), an additive Ni(py)4Cl2(20 mmol%), a ligand 2,2'-bipiperazine (0.4 eq.), an inorganic base Cs2CO3(8 eq.), to obtain a reaction system;

[0079] 2) Subsequently, in the glove box, anhydrous DCE and MeCN (1:1 by volume, a total of 1 mL) were added to the reaction system, and the reaction system was sealed with a plastic cap with a polytetrafluoroethylene gasket, and kept in an N2atmosphere, and reacted at 100°C for 10 h;

[0080] 3) After the reaction was completed, saturated NaCl solution was added to the reaction tube to stop the reaction, and the reaction system was poured into a separatory funnel, extracted with ethyl acetate several times (10 mL x 3), the organic phase was combined, dried with Na2SO4, and concentrated under vacuum to obtain a reaction liquid, which was separated and purified by column chromatography (PE:EA:DCM = 2:1:1 as eluent) to obtain isomeric cyclic peptide products with structural formulas as shown in 3c-1 and 3c-2, respectively, at a ratio of about 1:1, with a total separation yield of 60%.

[0081] Figure 8 9 The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the compound with structural formula as shown in 3c-1 (yellow solid) are shown in Figures 1 and 2, respectively, and the characterization data are as follows:

[0082] 1 ​H NMR (400MHz, CDCl3) δ7.92(d,J=7.8Hz,2H),7.83(d,J=7.8Hz,2H),7.80(s,1H),7.79(s,1H),7.61(d,J=8.2Hz,1H),7.56(d,J=8. 2Hz,1H),7.49(d,J=8.2Hz,1H),7.43(d,J=7.4Hz,1H),7.41–7.31(m,5H),7.29(d,J=7.4Hz,2H),7.25–7.20(m,1H),7.14(q,J=8. 0Hz,2H),6.78(d,J=7.4Hz,1H),6.43(s,1H),6.26(s,1H),5.87(d,J=10.2Hz,1H),5.11(t,J=10.2Hz,1H),5.04(d,J=9.2Hz,1H), 4.28–4.15(m,1H),3.95(t,J=10.8Hz,1H),3.76(s,3H),3.25–3.07(m,2H),2.90–2.70(m,2H),1.40(s,9H),1.36(d,J=7.0Hz,3H).

[0083] 13 C NMR (100MHz, CDCl3) δ194.23,193.65,172.60,171.46,171.02,154.77,140.21,140.11,137.68 ,137.00,136.48,135.95,135.16,133.59,133.54,132.54,131.68,130.64,130.57,129.48,129 .21,128.43,128.30,126.58,126.03,123.59,123.54,121.52,121.46,119.30,118.86,116.08,115.05,112.09,111.99,79.94,55.92,52.73,49.87,48.83,29.67,28.21,27.42,25.22,19.39.

[0084] Figure 10 、 11 The H-NMR spectrum and C-NMR spectrum of the compound (yellow solid) shown in the structural formula 3c-2 are respectively, and the characterization data are as follows:

[0085] 1H NMR (400 MHz, CDC13) δ 7.76 (d, J = 7.8 Hz, 2H), 7.61 (dd, J = 13.8, 7.8 Hz, 4H), 7.53 - 7.44 (m, 4H), 7.43 - 7.36 (m, 4H), 7.35 - 7.29 (m, 2H), 7.21 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.4 Hz, 2H), 7.10 (d, J = 7.8 Hz, 1H), 7.08 - 7.02 (m, 1H), 6.54 (s, 1H), 6.34 (s, 1H), 6.25 (d, J = 9.6 Hz, 1H), 5.02 (d, J = 9.2 Hz, 1H), 4.72 (t, J = 10.8 Hz, 1H), 4.36 - 4.24 (m, 1H), 4.17 (t, J = 12.4 Hz, 1H), 3.78 (s, 3H), 3.35 - 3.09 (m, 2H), 2.72 - 2.59 (m, 2H), 1.42 (s, 9H), 1.39 (d, J = 6.4 Hz, 3H).

[0086] 13 C NMR (100 MHz, CDC13) δ 194.39, 194.26, 172.33, 171.96, 170.50, 155.08, 139.60, 139.56, 137.67, 137.39, 136.51, 135.85, 133.86, 133.76, 133.33, 130.91, 130.24, 129.35, 129.26, 128.45, 128.37, 126.62, 125.72, 123.44, 123.35, 121.48, 121.04, 119.21, 118.91, 115.91, 115.48, 112.29, 111.98, 80.10, 53.71, 52.73, 49.05, 29.67, 28.22, 26.63, 25.75, 19.47.

[0087] Example 4

[0088] With reference to Reaction Scheme D, this example provides a method for preparing an isomeric cyclic peptide, specifically:

[0089]

[0090] 1) In a clean and oven-dried reaction tube of 8 mL, add the pentapeptide substrate (0.05 mmol, 1.0 eq.) with the structure as shown in 4a, aryl iodide (0.06 mmol, 1.2 eq.) with the structure as shown in 1b, catalyst Cu powder (60 mol%), additive pyridine (20 mmol%), ligand 4,4'-di-tert-butyl-2,2'-bipyridine (0.4 eq.), inorganic base K2CO3 (8 eq.) successively to obtain the reaction system;

[0091] 2) Then add anhydrous DCE and MeCN (volume ratio 1:1, total 2 mL) to the reaction system in the glove box, seal with a plastic cap with a polytetrafluoroethylene gasket, maintain the N2 atmosphere, and react at 100°C for 10 h;

[0092] 3) After the reaction is completed, add saturated NaCl solution to the reaction tube to stop the reaction, pour the reaction system into a separatory funnel, extract with ethyl acetate for several times (10 mL x 3), combine the organic phase, dry with Na2SO4 and concentrate under vacuum to obtain the reaction liquid, separate and purify by column chromatography (PE:EA:DCM = 1:4:1 as eluent) to obtain isomeric cyclic peptide products with the structures as shown in 4c-1 and 4c-2 respectively, with a total separation yield of 48%, and the ratio of about 1:1.

[0093] Figure 12 、 13 The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the compound mixture (yellow solid) with the structures as shown in 4c-1 and 4c-2 respectively are shown in the following:

[0094] 1H NMR (400 MHz, CDC13) δ 8.06 (s, 2 / 3 x 1 H), 8.05 (s, 1 / 3 x 1 H), 8.02 (s, 2 / 3 x 1 H), 7.91 (s, 1 / 3 x 1 H), 7.88-7.81 (m, 1 / 3 x 1 H, 2 / 3 x 1 H), 7.71-7.57 (m, 1 / 3 x 2 H, 2 / 3 x 2 H), 7.50-7.44 (m, 1 / 3 x 1 H, 2 / 3 x 1 H), 7.40-7.27 (m, 1 / 3 x 3 H, 2 / 3 x 3 H), 7.25-7.14 (m, 1 / 3 x 4 H, 2 / 3 x 4 H), 7.05-7.01 (m, 1 / 3 x 1 H, 2 / 3 x 1 H), 6.95 (s, 1 / 3 x 1 H), 6.87 (s, 1 / 3 x 1 H), 6.73 (s, 2 / 3 x 1 H), 6.68 (s, 2 / 3 x 1 H), 5.96 (d, J = 8.2 Hz, 2 / 3 x 1 H), 5.75 (d, J = 7.8 Hz, 1 / 3 x 1 H), 5.02-4.95 (m, 2 / 3 x 1 H), 4.94-4.83 (m, 1 / 3 x 1 H, 2 / 3 x 1 H), 4.82-4.77 (m, 1 / 3 x 1 H), 4.66-4.59 (m, 2 / 3 x 2 H), 4.57-4.49 (m, 1 / 3 x 2 H), 4.46-4.40 (m, 1 / 3 x 1 H), 4.18-4.06 (m, 2 / 3 x 1 H), 3.87 (s, 2 / 3 x 3 H), 3.79 (s, 1 / 3 x 3 H), 3.63-3.60 (m, 1 / 3 x 2 H), 3.57 (s, 1 / 3 x 3 H), 3.56 (s, 2 / 3 x 3 H), 3.48 (s, 1 / 3 x 3 H), 3.44 (s, 2 / 3 x 3 H), 3.41-3.37 (m, 1 / 3 x 2 H), 3.36-3.25 (m, 2 / 3 x 2 H), 3.23-3.09 (m, 2 / 3 x 2 H), 1.54 (s, 2 / 3 x 9 H), 1.53 (s, 1 / 3 x 9 H), 1.38 (d, J = 4.4 Hz, 1 / 3 x 3 H), 1.32 (d, J = 7.8 Hz, 1 / 3 x 3 H), 1.28 (d, J = 6.6 Hz, 2 / 3 x 3 H), 1.23 (d, J = 7.2 Hz, 2 / 3 x 3 H), 0.82 (d, J = 7.2 Hz, 1 / 3 x 3 H), 0.75 (d, J = 7.2 Hz, 2 / 3 x 3 H).

[0095] 13C NMR (150 MHz, CDC13) δ 172.56, 172.34, 172.15, 172.01, 171.98, 171.52, 171.47, 171.25, 169.89, 169.84, 166.35, 166.01, 165.85, 164.53, 155.36, 155.26, 137.77, 137.44, 137.33, 137.30, 137.21, 136.90, 136.70, 136.53, 132.39, 131.98, 131.39, 130.85, 130.12, 129.08, 128.65, 128.43, 128.14, 128.03, 127.52, 127.16, 123.10, 123.02, 122.86, 122.79, 120.69, 120.64, 120.46, 120.35, 119.71, 119.64, 118.91, 118.65, 113.31, 112.60, 112.55, 112.06, 110.25, 110.22, 110.18, 110.15, 109.91, 109.88, 109.63, 79.48, 79.42, 55.20, 54.86, 53.16, 53.08, 52.83, 52.77, 52.73, 52.59, 52.55, 51.97, 49.40, 49.05, 48.92, 48.80, 48.37, 48.24, 28.48, 28.46, 28.21, 20.32, 19.46, 17.45.

[0096] Antibacterial experiment

[0097] Prepare solid culture medium according to the ratio in Table 1:

[0098] Table 1

[0099] LB medium Tryptone Yeast extract NaCl Agar 100 mL 1g 0.5g 1g 1.5g

[0100] Prepare liquid culture medium according to the ratio in Table 2:

[0101] Table 2

[0102] LB medium Tryptone Yeast extract NaCl 100 mL 1g 0.5g 1g

[0103] 1) Take DH from -20 °C freezer 5α E. coli, single colony culture on solid medium by plate streaking, inverted in a 37 °C incubator for 12 h, observe whether single colony grows, use tweezers to pick out single colony and put into liquid culture medium, shake on a shaker for 12 h for inoculation;

[0104] 2) The OD600 value of the bacterial solution was 1.384, and according to 1 OD600 = 10 8 CFU / mL, it was inferred that the concentration of the bacterial solution was 1.384 x 10 8 CFU / mL. The bacterial stock solution was diluted with liquid medium to 10 7 , 10 6 , 10 5 , 10 4 , 10 3 CFU / mL, respectively, 1 mL each.

[0105] 2) The 5 x 10 -3 mol / L sample (DMSO as solvent, isomer cyclopeptide product mixture as solute, the structural formula is shown in 4c-1 and 4c-2, respectively) was diluted with sterilized primary water to 4 x 10 -3 mol / L, 3 x 10 -3 mol / L, 2 x 10 - 3 mol / L, 1 x 10 -3 mol / L, 2 x 10 -4 mol / L, 1 x 10 -4 mol / L, 2 x 10 -5 mol / L, respectively.

[0106] 3) 100 μL of 10 3 CFU / mL bacterial solution was taken in 9 1.5 mL centrifuge tubes, and the same volume of diluted and undiluted samples and primary water blank sample were mixed with the bacterial solution, and then the mixture was placed for 30 min, and then the mixture was coated on the solid medium by plate coating method, and then inverted in a 37°C constant temperature incubator for 12 h, and then the growth of bacterial colonies was observed, and the results are shown in Table 1. Figure 14 、 15 .

[0107] Figure 14 a ~ Figure 14 i are blank group, 5 x 10 -3 mol / L undiluted sample group, and sample groups diluted to 4 x 10 -3 mol / L, 3 x 10 -3 mol / L, 2 x 10 -3 mol / L, 1 x 10 -3 mol / L, 2 x 10 -4 mol / L, 1 x 10 -4 mol / L, 2 x 10 -5 mol / L, respectively.

[0108] After repeated experiments, it was found that the number of colonies in the blank control was less than that in the concentration of 10 -5 Therefore, it is believed that samples with too low concentrations are beneficial to bacterial growth.

[0109] Figure 15 This is the activity inhibition curve of the isomeric cyclic peptide product mixture prepared in Example 4 of the present invention against DH5α Escherichia coli. It can be seen that the IC50 value of the mixture is 251 μM.

[0110] In summary, the isomeric cyclic peptide product synthesized by the method of the present application has good antibacterial properties.

[0111] Comparative Example 1

[0112] The difference between this comparative example and Examples 1 to 4 is that the inorganic base Cs2CO3 in step 1) is adjusted to the inorganic base Na2CO3, and the remaining steps and processes are all referred to Examples 1 to 4. After the reaction is completed, a saturated NaCl solution is added to the reaction tube to stop the reaction, and the reaction system is poured into a separatory funnel and extracted with ethyl acetate multiple times (10 mL×3). The organic phases are combined, dried with Na2SO4 and concentrated in vacuo to obtain a reaction solution. No product point is found in the reaction system through TLC monitoring.

[0113] Comparative Example 2

[0114] The difference between this comparative example and Examples 1 to 4 is that the inorganic base Cs2CO3 in step 1) is adjusted to an organic base 4-dimethylaminopyridine, and the remaining steps and processes are all referred to Examples 1 to 4. After the reaction is completed, a saturated NaCl solution is added to the reaction tube to stop the reaction, and the reaction system is poured into a separatory funnel and extracted with ethyl acetate multiple times (10 mL×3). The organic phases are combined, dried with Na2SO4 and concentrated in vacuo to obtain a reaction solution. No product point is found in the reaction system by TLC monitoring.

[0115] It can be seen from Comparative Examples 1 and 2 that when the inorganic base in the present application is replaced by sodium carbonate or the organic base is 4-dimethylaminopyridine, no reaction occurs under the present application system.

[0116] In summary, the present invention reacts a polypeptide compound containing tryptophan residues at both ends with protected nitrogen and carbon ends with an aryl iodide, and synthesizes a cyclic peptide through a copper-catalyzed Ullmann coupling reaction of the tryptophan residues, thereby achieving the construction of a C-N bond in the polypeptide. Through a series of substrate extension experiments, it was found that the reaction can proceed smoothly with the growth of the carbon chain and the change of the amino acid sequence in the carbon chain, reflecting the universality of this method.

[0117] The method only acts on the N-H bond on the indole ring of the tryptophan residue, and the present application has excellent selectivity for the tryptophan residue compared with the prior art, and the cyclic peptide product synthesized by the present application is an axially chiral cyclic peptide compound having diastereoisomers with a ratio of about 1:1, and the present application synthesizes a series of cyclic peptide compounds which are isomers of each other, thereby providing a new idea and a new way for the synthesis of cyclic peptides.

[0118] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A cyclic peptide compound containing axial chirality, characterized in that: Having the general formula shown in formula III, In formula III, AA represents a natural amino acid or a non-natural amino acid, and R is selected from any one of methyl ester, tert-butyl ester, tert-amyl ester, and benzophenone.

2. The method for synthesizing axial chiral cyclic peptide compounds according to claim 1, wherein: include, A polypeptide substrate having a structural formula as shown in Formula I and containing tryptophan residues at both ends and an aryl iodide having a structural formula as shown in Formula II are reacted with a catalyst, a ligand, an additive, an inorganic base, and an organic solvent as a reaction system under a nitrogen atmosphere with heating and stirring. The reaction time is 8 to 10 hours and the reaction temperature is 95 to 105° C. After the reaction, the reaction product is separated and purified to obtain a cyclic peptide compound containing axial chirality as shown in Formula III. AA in Formula I and Formula III represents a natural amino acid or a non-natural amino acid, the protecting group on the nitrogen atom at the nitrogen terminal is a tert-butyloxycarbonyl group, and the protecting group on the carbon atom at the carbon terminal is a methyl ester group; R in Formula II and Formula III is selected from any one of methyl ester, tert-butyl ester, tert-amyl ester, and phenone.

3. The method for synthesizing axial chiral cyclic peptide compounds according to claim 2, wherein: The molar ratio of the polypeptide substrate having a structural formula as shown in Formula I and containing tryptophan residues at both ends to the aryl iodide having a structural formula as shown in Formula II is 1:(1.0-1.2), the molar ratio to the catalyst is 1:(0.3-1.2), the molar ratio to the ligand is 1:(0.4-0.8), the molar ratio to the additive is 1:(0.2-0.5), and the molar ratio to the inorganic base is 1:(6-12).

4. The method for synthesizing axial chiral cyclic peptide compounds according to claim 2, wherein: The organic solvent is a mixed solution of 1,2-dichloroethane and acetonitrile, and the volume ratio of the two is 1:(0.8-1.2).

5. The method for synthesizing a cyclic peptide compound containing axial chirality according to any one of claims 2 or 3, wherein: The concentration of the polypeptide substrate having a structural formula as shown in Formula I and containing tryptophan residues at both ends in the reaction system is 0.05M.

6. The method for synthesizing a cyclic peptide compound containing axial chirality according to any one of claims 2 or 3, wherein: The catalyst includes one of CuI, Cu(OAc)2, CuOAc, and Cu powder.

7. The method for synthesizing axial chiral cyclic peptide compounds according to claim 3, wherein: The inorganic base includes one of cesium carbonate and potassium carbonate.

8. The method for synthesizing axial chiral cyclic peptide compounds according to claim 3, wherein: The ligand includes one of 2,2'-bipyridine, 2,2'-bipiperazine, benzoyl acetone, and 4,4'-di-tert-butyl-2,2'-bipyridine.

9. The method for synthesizing axial chiral cyclic peptide compounds according to claim 3, wherein: The additive includes one of pyridine and Ni(py)4Cl2.

10. The cyclic peptide compound containing axial chirality synthesized by the synthesis method of claim 1, characterized in that: The compounds have antibacterial effects.

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