Bicyclic peptide compound oxintamycin A and application thereof

Abstract

The invention relates to a bicyclopeptide compound, namely oxins A and an application thereof. The oxins A has a structure shown in the specification. The invention also relates to an application of the oxins A in treatment of coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a bicyclic peptide compound, styromycin A, and its applications. Background Technology

[0002] Cinnamoyl-containing nonribosomal peptides (CCNPs) [1] These are natural products formed by the condensation of ortho-substituted cinnamoyl lipid (CL) groups with cyclic or linear peptides via amide bonds, and possess antibacterial, antifungal, antitumor, immunomodulatory, anti-inflammatory, and antiviral activities. [1] CCNPs (Cellular Polypeptides) exhibit diverse structures, with their structural differences primarily manifested in the following aspects: peptide chain length (5–11 amino acids), amino acid types (including d-amino acids, hydroxylated amino acids, and diaminoalanine, etc.), CL group modifications (epoxidation, glycosylation, etc.), and the different positions where cyclic peptides or cyclic ester peptides are formed. [1] .

[0003] Most reported CCNPs are monocyclic peptides, with only four bicyclic peptides, including cihanmycin. [2] epoxinnamide [3] and nyuzenamide [4,5] It forms a bicyclic peptide backbone through aromatic ether bonds, while the grisgenomycin previously discovered by our group is the first CCNP-type natural product with a bicyclic structure formed by carbon-carbon bond bridging. [6] It is also the first cyclic peptide with anti-coronavirus activity reported among CCNPs to date, and has potential value in the development of antiviral lead drugs. [6] .

[0004] Based on a gray synergist-based genome-directed mining and activation strategy, this study found Streptomycesscopuliridis The target gene cluster and gray lignin ggm The gene clusters are highly homologous, all containing tandem LuxR regulatory genes, large nonribosomal peptide synthetase (NRPS) and type II polyketone synthase (PKS) genes, but they differ in A domain substrate specificity and modified gene composition, suggesting that they may encode novel natural products containing a bicyclic CCNP backbone, which has certain development potential.

[0005] Based on this, the present invention is proposed.

[0006] [References] 1. Li Yihong, Fu Jie, Li Xingxing, Hong Bin. Research progress on cinnamoyl-modified nonribosomal peptides. Chinese Journal of Antibiotics, 2025, 50(09):1044-1058. 2. Fang C, Zhang L, Wang Y, et al. Discovery and biosynthesis ofcihanmycins reveal cytochrome P450-catalyzed intramolecular CO phenolcoupling reactions. J Am Chem Soc, 2024; 146(24): 16478-89. 3. Kang S, Han J, Jang S, et al. Epoxinnamide: an epoxy cinnamoyl-containing nonribosomal peptide from an intertidal mudflat-derivedS treptomyces sp. Mar Drugs, 2022, 20(7): 455. 4. Karim MRU, In Y, Zhou T, et al. Nyuzenamides A and B: Bicyclicpeptides with antifungal and cytotoxic activity from a marine-derived Streptomyces sp. Org Lett, 2021, 23(6): 2109-2113. 5. An JS, Kim MS, Han J, et al. Nyuzenamide C, an antiangiogenicepoxy cinnamic acid-containing bicyclic peptide from ariverine Streptomyces sp. J Nat Prod, 2022, 85(4): 804-814. 6. Li Y, Ding X, Du Y, et al. Genome-directed discovery of bicycliccinnamoyl-containing nonribosomal peptides with anticoronaviral activity from Streptomycesgriseus . Org Lett, 2023, 25(26): 4874-4879. Summary of the Invention

[0007] This invention first relates to a bicyclic peptide compound: Cintyromycin A, which has the structure shown in formula (1). (1) In the structure of the bicyclic peptide compound of Cintyromycin A, The ortho-substituted cinnamic yl analog group (Cm) is linked to Dap (diaminoalanine) 1 via a peptide bond; Dap1 and Phe are linked by peptide bonds; Phe and Leu1 are linked by peptide bonds; Leu1 and β- OH-Asp ( β- Hydroxylated aspartic acid is linked by peptide bonds; β- OH-Asp is linked to Tyr via peptide bonds; Tyr and Dap2 β The amino groups are linked by peptide bonds; Dap2 and Dap3 are linked by peptide bonds; Dap3 and Leu2 are linked by peptide bonds; Leu2 is linked to 4'-MeO-Trp (4'-methoxytryptophan) via a peptide bond; 4'-MeO-Trp is linked to Leu3 via a peptide bond; Leu3 and Dap1 β The amino groups are linked by peptide bonds; The 3' position of Tyr is connected to C-11 on the Cm group via a C-C bond.

[0008] Furthermore, in the structure of the bicyclic peptide compound of Cintyromycin A, The three-dimensional configuration of Dap1 is L-shaped; Phenylalanine (Phe) has a d-type stereoconfiguration; Leucine (Leu1, 2, 3) all have the d-type stereoconfiguration; β- The stereoconfiguration of OH-Asp is l- erythro - β -OH-Asp; Tyrosine (Tyr) has a d-type stereoconfiguration; The three-dimensional configuration of Dap2 and 3 is d-type; The stereo configuration of 4'-MeO-Trp is of the l-type. The relative configurations of the C-10 and C-11 are 10. S* ,11 R* .

[0009] This invention also relates to a drug or drug composition comprising, (1) A therapeutically effective amount of the bicyclic peptide compound Cintyromycin A; (2) Necessary pharmaceutical excipients.

[0010] Furthermore, the drug or drug composition is an antiviral drug or drug composition, preferably, the virus is a coronavirus; more preferably, the virus is a human coronavirus.

[0011] This invention also relates to the use of the bicyclic peptide compound Cintyromycin A in the preparation of antiviral drugs; preferably, the antiviral drug is an anti-coronavirus drug.

[0012] The beneficial effects of this invention are as follows: (1) Introducing LuxR family regulatory genes tandemly into the grisgenomycin A gene cluster into Streptomyces. S.scopuliridis In this study, efficient heterologous activation of the target gene cluster was achieved. Various chemical experimental methods were used to isolate and confirm the differentially expressed components in the overexpressing strain, and finally a new bicyclic CCNP product, Cintyromycin A, with inhibitory activity against human coronavirus was obtained. Attached Figure Description

[0013] Figure 1 , S.scopuliridis / pSET152 and S.scopuliridis Differential analysis of metabolites of / pL-3284-3285 in ISP2 solid medium.

[0014] Figure 2 (+)-HRESI-MS spectrum of Cintyromycin A (compound 1).

[0015] Figure 3The planar structure of Cintyromycin A (compound 1), 1 H- 1 H COSY (Bold) and HMBC (Arrow) related signals.

[0016] Figure 4 Marfey's analysis of the acid hydrolysis products of compound 1. m / z The extractive ion chromatogram (EIC) of 487 corresponds to the FDAA (1-fluoro-2,4-dinitrophenyl-5-propanediamide) derivative of 4'-MeO-l-Trp.

[0017] Figure 5 Marfey's analysis of the acid hydrolysis products of compound 1. m / z The extractable ion chromatogram (EIC) of 402 corresponds to dl- threo - β FDAA derivatives of -OH-Asp.

[0018] Figure 6 Marfey's analysis of the acid hydrolysis products of compound 1. m / z The extractive ion chromatogram (EIC) of 384 corresponds to the FDAA derivative of Leu.

[0019] Figure 7 Marfey's analysis of the acid hydrolysis products of compound 1. m / z The extractive ion chromatogram (EIC) of 418 corresponds to the FDAA derivative of Phe.

[0020] Figure 8 Marfey's analysis of the acid hydrolysis products of compound 1. m / z The extractive ion chromatogram (EIC) of 609 corresponds to the FDAA derivative of Dap.

[0021] Figure 9 8 possible configurations of compound 1.

[0022] Figure 10 Experimental ECD spectra of compound 1 in methanol solution and theoretically calculated ECD spectra of 8 configurations 1a−1h.

[0023] Figure 11 The stereostructure of Cintyromycin A (compound 1). Detailed Implementation

[0024] Example 1: Fermentation of the strain and purification of the product 1. Fermentation by bacterial strain The recombinant plasmid pL-3284-3285 (sequence shown in reference 6) was introduced into Streptomyces via conjugation transfer. S.scopuliridis In this study, positive conjugates were obtained through alpramycin resistance screening and PCR verification, leading to the overexpression of the strain. S.scopuliridis / pL-3284-3285. The overexpressing strain was inoculated into ISP2 solid medium and fermented at 28 °C for 7 days. High-resolution UPLC-HRMS analysis showed that compound 1 m / z 1497.7083 [M+H] + Compound 2 m / z 1497.7083 [M+H] + and compound 3 m / z 277.1181 [M+H] + Stable generation ( Figure 1 , Figure 2 High-resolution UPLC-HRMS detection conditions: Instrument: Waters UPLC-Xevo G2-S Qtof Column: ACQUITY UPLC BEH C 18 1.7 μm 2.1×50 mm Mobile phase: A 0.1% FA water, B 0.1% FA acetonitrile Flow rate: 0.3 mL / min Column temperature: 30 ℃ Liquid phase program: 0-10 min 5% ~ 100% B; 10-12 min 100% B Detection wavelength: 280 nm and full wavelength scanning Mass spectrometry conditions: Analysis was performed using positive ion data-dependent (DDA) acquisition. Optimized source parameters were: Source Voltage, 3 kV; Cone voltage, 15 V; Source Offset Voltage, 30 V; Source temperature, 125 ℃; Desolvation temperature, 400 ℃; Cone gas flow, 100 L / h; Desolvation gas flow, 800 L / h; MS primary scan range. m / z 100 ~ 1600 Da.

[0025] 2. Separation and purification of metabolites Fermentation was expanded using the above method, with a total fermentation volume of 10 L. The solid culture was extracted twice with 10 L of ethyl acetate–methanol (4:1, v / v), and lyophilized to obtain 2.5 g of crude extract. Separation and purification were performed using Sephadex LH-20 gel column chromatography and reversed-phase semi-preparative HPLC, with the specific steps as follows: (1) Preliminary separation of crude dextran using LH-20 gel column The crude product was dissolved in an appropriate amount of methanol and centrifuged. The supernatant was directly loaded onto a self-packed dextran gel LH-20 open column. Using pure methanol as the mobile phase, elution was performed at a flow rate of approximately 1 mL / min under normal pressure, with fractions collected approximately every 30 min. HPLC analysis showed that the differentially expressed components were mainly enriched in the Fr.2 and Fr.3 fractions: Fr.2 was the methanol elution fraction for approximately 31–60 min; while Fr.3 was the elution fraction for the subsequent 61–90 min.

[0026] (2) Preparation of compound 1 after purification Fractions Fr.2 and Fr.3 were purified by semi-preparative HPLC under the conditions shown below, yielding a total of 41 mg of pure compound 1.

[0027] Instrument: Waters Alliance e2695 Column: Waters Xbridge C 18 (5 μm, 10 × 150 mm) semi-preparative column Mobile phase: A 0.5% TFA-water B Acetonitrile Flow rate: 3 mL / min Column temperature: 40 ℃ Elution program: 0-5 min 10% B; 5-15 min 10-40% B; 15-55 min 40% B Detection wavelength: 280 nm and full wavelength scanning Retention time: Approximately 28 minutes Example 2: Structural Identification of Compound 1 Compound 1, named Cintyromycin A, was determined using a combination of modern spectroscopic techniques, the Marfey reaction, and ECD calculations.

[0028] Cintyromycin A (Compound 1) It is a white amorphous powder, readily soluble in methanol and DMSO; [α] 20 D +86 c 0.10, MeOH); UV (MeOH) λmax (log) ε 209 (4.78), 281 (4.31). HRESI-MS analysis showed [M+2H] 2+ Quasi-molecular ions m / z The value is 749.3708, [M+H] + Quasi-molecular ions m / z The value was 1497.7083, and its molecular formula was determined to be C based on NMR data. 75 H 96 N 14 O 19 The degree of unsaturation is 35. High-performance liquid chromatography (HPLC) analysis showed that compound 1 exhibited a single and sharp chromatographic peak under different chromatographic conditions. Simultaneously, high-resolution mass spectrometry (HPLC) analysis revealed that compound 1 was a single molecular weight ion peak, suggesting that this compound is a single-component pure compound.

[0029] Detailed analysis of its 1D (¹H, ¹³C, DEPT) and 2D (HSQC, ¹H–¹H COSY, HMBC) NMR data (Table 1) revealed that the compound consists of 10 amino acids: 3 leucines (Leu), 3 diaminoalanines (Dap), 1 phenylalanine (Phe), 1 4'-methoxytryptophan (4'-MeO-Trp), and 1... β- Hydroxylated aspartic acid ( β -OH-Asp) and 1 tyrosine (Tyr). For β -OH-Asp residues, β Hydroxylation modification can be achieved by... β Carbon signal ( δ C The deshielding chemical shift of 72.4) was clearly deduced. Furthermore, the methoxy proton (H3, ) of 4'-MeO δ H 3.83) and C-4' ( δ C The HMBC correlation signal between 154.1) indicates that the methoxy substitution is located at the C-4' position of Trp, while the HMBC correlation signal between NH-2' / C-7' and H3-4'-OMe / C-5' further confirms the presence of the 4'-MeO-Trp residue.

[0030] In addition to the amino acid residues mentioned above, the 14 additional ¹³C signals appearing in the spectrum were attributed to a structural unit resembling a cinnamon yl group (Cm). The COSY spectrum showed three spin-coupled segments (H-2 / H-3, H-5 / H-6 / H-7 / H-8 and H-10 / H-11 / H-12 / H-13), and the remaining four ¹³C signals included two aprotonated aromatic carbons (…). δ C-4133.3; δ C-9 142.6) and two carbonyl carbons ( δ C-1 168.1; δ C-14 167.8). HMBC spectroscopy shows that H-2 is associated with C-1 and C-4, H-3 with C-4, C-5, and C-9, H-10 with C-8 and C-9, and H-12 and H-13 with C-14, further supporting the assembly mode of this structural unit. Furthermore, the C-10 hydroxylation modification can be determined by its chemical shift (…). δ C The demasking characteristics of 75.9) are clearly inferred. The coupling constant of H-2 / H-3 ( J 2,3 = 15.9 Hz) and the coupling constants of H-12 / H-13 ( J 12 , 13 = 16.0 Hz) confirmed C-2 and C-3, and C-12 and C- The double bond configurations between 1 and 3 are all E type .

[0031] Based on the HMBC correlation signal between the amide proton (NH) and the adjacent amide carbon, and the relationship between the amide proton (NH) and amino acids... α - The ROESY correlation signal between protons determined the amino acid linkage sequence of the compound and confirmed that its peptide backbone is a cyclic decapeptide. In the HMBC spectrum, H-11 in Cm and C-2′ in Tyr ( δ C 127.6), C-3′ ( δ C 125.8) and C-4′ ( δ C 152.9) is related to H-2′ in Tyr and C-11 in Cm ( δ C 55.7) is related, while H-2′ and H-6′ in Tyr are related to C-3 ( δ C 32.5) Correlation, these correlation signals collectively indicate that, C-11 in Cm is linked to C-3′ in Tyr via carbon-carbon bonds. Furthermore, the NH₂ of Dap-1 and the C₁ of Cm ( δ C 168.1) shows an HMBC correlation, suggesting that the NH-2 group of Dap-1 forms an amide bond with the acyl group of the Cm group, thus constructing the bicyclic peptide structure of the compound. Combined with molecular formula analysis, C-14 was confirmed as a carboxyl group. In summary, the planar structure of compound 1 is thus clarified. Figure 3 ).

[0032] Table 1. Cintyromycin A (compound 1) in DMSO-d ¹H (800 MHz) and ¹³C (201 MHz) NMR data in 6 Example 3: Identification of the stereoconfiguration of compound 1 The structure of Cintyromycin A contains 13 chiral centers, which are activated via the Marfey reaction (…). Figures 4-8 The 10 chiral centers contained in the 9 amino acids in the structure were identified as follows: 2 d-Dap, 1 l-Dap, 1 d-Phe, 1 l- erythro -β-OH-Asp, 1 4'-MeO-l-Trp, 3 d-Leu. Marfey reaction method: Acid hydrolysis and derivatization of the target compound: Weigh 0.5 mg of sample, add 100 μL of 6 N hydrochloric acid, seal, acid hydrolyze at 110 ℃ for 1 h and dry. Add 1 M NaHCO3 (30 μL), add 1% l / d-FDAA acetone solution (40 μL), and incubate in a water bath at 40 ℃ for 1 h. Neutralize the reaction solution with 1 M HCl (30 μL), dissolve with 10% acetonitrile-water (500 μL), centrifuge at 14,500 rpm / min, and analyze by LC-ESI-MS. Derivatization of standard amino acids: Add 50 μL of amino acid stock solution, add 1 M NaHCO3 (30 μL), add 1% l / d-FDAA acetone solution (40 μL), and incubate in a water bath at 40 ℃ for 1 h. The reaction solution was neutralized with 1M HCl (30 μL), dissolved with 10% acetonitrile-water (500 μL), centrifuged at 14,500 rpm, and analyzed by LC-ESI-MS.

[0033] The chirality of Tyr was determined using ECD calculations. The relative configurations of the two chiral centers (C-10 and C-11) on the cinnamic yl group side chain were determined using ECD calculations: Comparison of the calculated and experimental ECD spectra showed that the calculated ECD spectra for configurations 1a and 1d were consistent with the experimental spectra trends. Figure 9-10 Therefore, it can be determined that C in compound 1... α -Tyr's absolute configuration is R The relative configurations of the C-10 and C-11 are as follows: S *and R *

[0034] Thus, we have determined the complete planar structure of Cintyromycin A and the chiral centers of all amino acids. Figure 11 ).

[0035] Example 4: Test of antiviral activity of the compound The antiviral activity of the compound was determined using the cytopathic effect method. Shintamycin A showed antiviral activity against human coronavirus HCoV-229E in the Huh7 cell line, EC50. 50 The concentration was 6.42 μM (Table 1), and its activity was basically equivalent to that of grisgenomycin A and ribavirin.

[0036] Table 1. Results of the anti-human coronavirus HCoV-229E activity of compound cintyromycin A in Huh7 cell line. a CC was measured by a cytopathic effect inhibition assay. 50 (Hydrotoxic concentration) and EC 50 (Drug half-maximal effective concentration) value.

[0037] b Select the index (SI) value by CC. 50 / EC 50 Calculation of the ratio.

[0038] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A bicyclic peptide compound, cintyromycin A, having the structure shown below, ； In the aforementioned styrycin A structure: The ortho-substituted cinnamic yl analog group (Cm) is linked to Dap (diaminoalanine) 1 via a peptide bond; Dap1 and Phe are linked by peptide bonds; Phe and Leu1 are linked by peptide bonds; Leu1 and β- OH-Asp ( β- Hydroxylated aspartic acid is linked by peptide bonds; β- OH-Asp is linked to Tyr via peptide bonds; Tyr and Dap2 β The amino groups are linked by peptide bonds; Dap2 and Dap3 are linked by peptide bonds; Dap3 and Leu2 are linked by peptide bonds; Leu2 is linked to 4'-MeO-Trp (4'-methoxytryptophan) via a peptide bond; 4'-MeO-Trp is linked to Leu3 via a peptide bond; Leu3 and Dap1 β The amino groups are linked by peptide bonds; The 3' position of Tyr is connected to C-11 on the Cm group via a C-C bond.

2. The styromycin A according to claim 1, characterized in that, In the structure of the aforementioned styromycin A, The three-dimensional configuration of Dap1 is L-shaped; Phenylalanine (Phe) has a d-type stereoconfiguration; Leucine (Leu1, 2, 3) all have the d-type stereoconfiguration; β The stereoconfiguration of -OH-Asp is l- erythro - β -OH-Asp; Tyrosine (Tyr) has a d-type stereoconfiguration; The three-dimensional configuration of Dap2 and 3 is d-type; The stereo configuration of 4'-MeO-Trp is of the l-type. The relative configurations of the C-10 and C-11 are 10. S* ,11 R* .

3. A drug or drug composition comprising, (1) A therapeutically effective amount of styrycin A as described in claim 1 or 2; (2) Necessary pharmaceutical excipients.

4. The drug or drug composition according to claim 3, characterized in that, The drug or drug composition is an antiviral drug or drug composition, preferably, the virus is a coronavirus; more preferably, the virus is a human coronavirus.

5. The use of sinetetracycline A as described in claim 1 or 2 in the preparation of an antiviral drug; preferably, the antiviral drug is an anticoronavirus drug.

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