Intermediates and methods for their preparation, catenulobactin b natural products and methods and uses thereof

Abstract

The application discloses an intermediate and a preparation method thereof, a Catenulobactin natural compound and a preparation method and application thereof. The intermediate I has a structural formula shown in formula I. The intermediate I is used for synthesizing Catenulobactin B, and the intermediate I is used as raw material, so that a total synthesis route is short, and a yield is high.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to an intermediate and its preparation method, the natural compound Catenobactin and its preparation method and application. Background Technology

[0002] In 2018, Catenulobactins A and B were discovered by the Abe team using co-culture technology from mycobacteria. T. pulmonis TP-B0596 and Streptomyces Catenuloplanes A novel heterocyclic peptide was isolated from a coculture of sp. RD067331 (J. Nat. Prod. 2018, 81, 9, 2106-2110). The team analyzed its structure using nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), and chiral-phase gas chromatography-mass spectrometry (chiral-phase GC-MS), determining the planar structure and absolute configuration of Catenulobactins A and B (structural formulas shown below). Catenulobactin A possesses both oxazoline and isoxazoline ketone structures, while Catenulobactin B has two identical oxazoline structures and an N-hydroxyornithine structure. The study showed that Catenulobactin B exhibited Fe(III) chelating activity and moderate cytotoxicity (IC50) against mouse leukemia P388 cells. 50 = 22.4 μM).

[0003]

[0004] Currently, there are no reports on the total synthesis and bioactivity studies of Catenuolobactin family natural products. Therefore, there is an urgent need to develop a new intermediate for the total synthesis of Catenuolobactin family natural products. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides an intermediate I that can be used for the synthesis of Catenobactin B with a high yield.

[0006] A second aspect of the present invention also provides a method for preparing intermediate I.

[0007] A third aspect of the present invention also provides an intermediate II.

[0008] The fourth aspect of the present invention also provides a method for preparing intermediate II.

[0009] The fifth aspect of this invention also provides a method for preparing the natural product Catenobactin B.

[0010] The sixth aspect of the present invention also provides an application of the natural product Catenobactin B.

[0011] According to a first aspect of the present invention, an intermediate I is provided, having the structural formula shown in Formula I: ; R1 and R2 are each independently selected from H, benzyl, trimethylsilylethyl, and trimethylsilylethoxymethylene; R3 is selected from -NH2 and / or -NBoc2.

[0012] According to a preferred embodiment of the present invention, the intermediate I is selected from the following structural formula: , , .

[0013] Intermediate I according to embodiments of the present invention has at least the following beneficial effects: This invention provides intermediate I for the synthesis of Catenobactin B, which provides a short total synthetic route with high yield using intermediate I as a starting material.

[0014] According to a second aspect of the present invention, a method for preparing intermediate I is provided, comprising the following steps: The reaction is carried out under condensing reagent, solvent, and alkaline conditions, and compound A and compound B undergo an amide condensation reaction to obtain the product. The structural formulas of compounds A and B are as follows: .

[0015] According to a preferred embodiment of the present invention, the reaction temperature of the amide condensation reaction is 0~40°C.

[0016] According to a preferred embodiment of the present invention, the molar ratio of compound A to compound B is 1:(1~3).

[0017] According to a preferred embodiment of the present invention, the condensing agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI). N, N -Dicyclohexylcarbodiimide (DCC) NAt least one of the following: hydroxy-7-azabenzotriazole (HOAt), (7-azabenzotriazole-1-oxo)tripyrrolephosphonium hexafluorophosphate (PyAOP), 3-(diethoxy-o-acyloxy)-1,2,3-benzotriazine-4-one (DEPBT), and bis(2-oxo-3-oxazolyl)phosphine chloride (BOPCl).

[0018] According to a preferred embodiment of the present invention, an alkali is added to achieve the alkaline conditions, for example, the alkali includes triethylamine, diisopropylethylamine, pyridine, etc. N,N -Dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N-methylmorpholine.

[0019] According to a preferred embodiment of the present invention, the solvent includes at least one of haloalkanes, ethers, and polar aprotic solvents.

[0020] According to a preferred embodiment of the present invention, the halogenated hydrocarbon solvent includes dichloromethane, chloroform, 1,2-dichloroethane, and tetrachloroethane.

[0021] According to a preferred embodiment of the present invention, the ether solvent includes tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, and 1,4-dioxane.

[0022] According to a preferred embodiment of the present invention, the polar aprotic solvent includes N,N -Dimethylformamide, N,N - Dimethylacetamide, acetonitrile.

[0023] According to a third aspect of the present invention, an intermediate II is provided, having the structural formula shown in Formula II: ; Among them, R4 is selected from OH and C. 1~6 Alkyl and benzyl groups.

[0024] According to a preferred embodiment of the present invention, the intermediate II is selected from the following structural formula: , , .

[0025] Intermediate II according to embodiments of the present invention has at least the following beneficial effects: This invention provides intermediate II for the synthesis of Catenobactin B, which yields high production rates. Furthermore, intermediate II of this invention exhibits neuroprotective effects and can be used in the field of drugs for neurodegenerative diseases.

[0026] According to a fourth aspect of the present invention, a method for preparing intermediate II is provided, comprising the following steps: The intermediate I described in the first aspect of the present invention is mixed with a protic acid and subjected to a tert-butoxycarbonyl group protection removal reaction of the amino functional group; then, under condensation reagent and alkaline conditions, it is subjected to an amide condensation reaction with compound A to obtain the product. Alternatively, under condensing reagent and alkaline conditions, compound A and compound C can be obtained by amide condensation reaction; The structural formulas of compounds A and C are as follows: , .

[0027] According to a preferred embodiment of the present invention, the protic acid includes at least one selected from hydrochloric acid, sulfuric acid, trifluoroacetic acid, and acetic acid.

[0028] According to a preferred embodiment of the present invention, the tert-butyloxycarbonyl protection removal reaction is carried out in a solvent; the solvent is selected from: halogenated hydrocarbons, such as dichloromethane, chloroform, 1,2-dichloroethane; ethers, such as tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butylmethyl ether, 1,4-dioxane; polar aprotic solvents, such as N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile; or any mixture of solvents thereof in any proportion.

[0029] According to a preferred embodiment of the present invention, the condensing agent comprises at least one of: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-dicyclohexylcarbodiimide (DCC), N-hydroxy-7-azabenzotriazole (HOAt), (7-azabenzotriazole-1-oxo)tripyrrolephosphonohexafluorophosphate (PyAOP), 3-(diethoxy-o-acyloxy)-1,2,3-benzotriazine-4-one (DEPBT), and bis(2-oxo-3-oxazolyl)phosphine chloride (BOPCl).

[0030] According to a fifth aspect of the present invention, a method for preparing a natural product of Catenulobactin B is provided, comprising the following steps: The intermediate II described in the third aspect of the present invention and the solvent are subjected to a benzyl removal reaction under catalytic hydrogenation conditions to obtain the product. The structural formula of the natural product Catenulobactin B is as follows: .

[0031] According to a preferred embodiment of the present invention, the reaction temperature for the benzyl removal reaction is 20~40°C.

[0032] According to a preferred embodiment of the present invention, the catalyst in the catalytic hydrogenation conditions is selected from Pd / C and / or Pd(OH)2 / C.

[0033] According to a preferred embodiment of the present invention, the hydrogenation in the catalytic hydrogenation conditions refers to the reaction being carried out under hydrogen conditions.

[0034] According to a preferred embodiment of the present invention, the solvent includes at least one of alcohol solvents, nitrile solvents, ether solvents, ester solvents, and amide solvents.

[0035] According to a preferred embodiment of the present invention, the alcohol solvent includes methanol, ethanol, and ethylene glycol.

[0036] According to a preferred embodiment of the present invention, the nitrile solvent includes acetonitrile and propionitrile.

[0037] According to a preferred embodiment of the present invention, the ether solvent includes diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane.

[0038] According to a preferred embodiment of the present invention, the ester solvent includes methyl formate, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, and ethyl propionate.

[0039] According to a preferred embodiment of the present invention, the amide solvent includes N,N -Dimethylformamide, N, N -Dimethylacetamide.

[0040] The sixth aspect of this invention provides the use of intermediate I as described in the first aspect of this invention, intermediate II as described in the third aspect of this invention, or the natural product of Catenobactin B prepared by the preparation method described in the fifth aspect of this invention in the preparation of drugs for the treatment and / or prevention of neuroprotective effects, drugs for neurodegenerative diseases, or drugs for Parkinson's disease.

[0041] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0042] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1(A) Untreated NL5901 nematode α-Syn fluorescence images treated with L-DOPA (2 mM, 30 μM) and Catenulobactin B (10, 20, 30 μM); (B) Quantitative values ​​of NL5901 nematode α-Syn fluorescence intensity.

[0043] Figure 2 (A) Untreated NL5901 nematode α-Syn fluorescence images treated with Catenobactin B, intermediate I-1, intermediate I-3, intermediate I-2, intermediate II-1, intermediate II-2 and intermediate II-3. (B) Quantitative values ​​of NL5901 nematode α-Syn fluorescence intensity, a, b, c, d, e, f correspond to intermediates I-1, I-3, I-2, II-1, II-2 and II-3 respectively. Data are the mean of three independent experiments (n = 3), mean ± SEM. Compared with the control group NL5901 nematodes, *** p < 0.001, ** p < 0.01, *p < 0.05, ns indicate no significant difference, #p < 0.050, (L-DOPA (2mM) versus Catenulobactin B (30 μM)), p = 0.9049 (Catenulobactin B versus 2-11). Figure 3 This is a graph showing the effect of Catenobactin B on 6-OHDA-induced degeneration of DA neurons in BZ555 nematodes. (A) represents the dat-1p::GFP fluorescence image of BZ555 nematodes; (B) represents the quantitative value of fluorescence intensity of DA neurons in BZ555 nematodes. Data are the mean of three independent experiments (n = 3), mean ± SEM. Compared with the control group BZ555 nematodes, ###p<0.001; compared with the positive control group 6-OHDA, ***p<0.001, p = 0.0450 (L-DOPA (2mM) versus Catenobactin B (30 μM)). Figure 4 This is a bar chart showing the effect of Catenobactin B on the expression of iron(III) metabolism-related genes in NL5901 nematodes. Data are the mean of three independent experiments (n = 3), mean ± SEM. Compared with the control group NL5901 nematodes, ***p<0.001, **p<0.01, ns indicates no significant difference. Detailed Implementation

[0044] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0045] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0046] Experimental methods: Caenorhabditis elegans strains and culture The Caenorhabditis elegans strains used in this study included NL5901 (pkIs2386 [unc-54p::α-synuclein::YFP + unc-119(+)]) and BZ555 (egIs1 [dat-1p::GFP]). All strains were purchased from the Nematode Genetic Center (CGC) and cultured at 20 °C on standard nematode growth medium (NGM) plates coated with Escherichia coli OP50.

[0047] Synuclein aggregation analysis To investigate whether catenobactin B affects α-synuclein aggregation, we conducted experiments using the transgenic nematode strain NL5901. Synchronized nematodes were cultured at 20 °C until the eggs hatched and developed into L1 larvae. The L1 larvae were then transferred to NGM plates supplemented with catenobactin B (10, 20, 30 μM), its six analogues (30 μM each), or levodopa (L-DOPA, 2 mM, 30 μM), with the untreated group serving as a control. Nematodes were further cultured at 20 °C until the L4 larval stage, then collected, washed with M9 buffer, anesthetized with 20 mM sodium azide, and mounted on slides. Fluorescence images were acquired using an Olympus BX63 microscope, and fluorescence intensity was quantified using ImageJ software.

[0048] Dopaminergic neuron analysis To investigate whether catenobactin B can improve 6-OHDA-induced dopaminergic neurodegeneration, this study used the transgenic nematode strain BZ555. Synchronized BZ555 nematodes were cultured at 20 °C until the eggs hatched and developed into L1 larvae. The L1 larvae were then transferred to NGM plates containing catenobactin B (30 μM), NGM plates containing L-DOPA (2 mM, 30 μM), and an untreated control plate, and cultured at 20 °C until the L4 larval stage. L4 nematodes were collected and incubated in 50 mM 6-OHDA solution for 1 hour, gently inverted and mixed every 10 minutes. After treatment, the nematodes were transferred to fresh NGM plates for 24 hours to recover. Subsequently, the nematodes were collected, washed with M9 buffer, anesthetized with 20 mM sodium azide, and mounted on slides. Fluorescence images were acquired using an Olympus BX63 microscope, and fluorescence intensity was quantified using ImageJ software.

[0049] RT-qPCR L4-stage NL5901 nematodes, treated with or without 30 μM Catenulobactin B, were collected. Total RNA was extracted from the nematodes using Trizol and reverse transcribed into complementary DNA (cDNA) using a PrimeScript RT kit (Takara). RT-qPCR reactions were performed using TB Green Premix Ex TaqII (Takara). Primer sequences are shown in the table below.

[0050] Table 1 Primer sequences for RT-qPCR.

[0051] The raw material synthesis methods used in the embodiments of the present invention are as follows: Synthesis of compound 3:

[0052] Compound 1 (1.381 g, 10 mmol), compound 2 (1.696 g, 10 mmol), and HOBt (135 mg, 1.0 mmol) were dissolved in anhydrous dichloromethane (30 mL). Triethylamine (1.39 mL, 10 mmol) and EDCI·HCl (2.109 g, 11 mmol) in dichloromethane solutions (30 mL) were added sequentially at 0 °C. The mixture was stirred at room temperature for 12 h. After removing the dichloromethane under reduced pressure, the mixture was dissolved in ethyl acetate. The organic phase was washed sequentially with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give a colorless oily compound 3 (2.278 g, 90%). 1 H NMR (500 MHz, CDCl3) δ 7.58 (dd, J = 1.5, 7.9 Hz, 1H), 7.43 (ddd, J = 1.5, 7.2, 8.4 Hz, 1H), 6.96 (dd, J = 1.2, 8.7 Hz, 1H), 6.84(ddd, J = 1.2, 7.2, 7.9 Hz, 1H), 4.79 (td, J = 7.8, 6.9, 2.6 Hz, 1H), 4.47 (dq, J = 2.1, 6.4 Hz, 1H), 3.76 (s, 3H), 1.26 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ171.5, 170.4, 161.3, 134.7, 126.3, 119.0, 118.4, 113.9, 68.0, 57.2, 52.9,20.1. Synthesis of compound 4:

[0053] Compound 3 (1.266 g, 5.0 mmol), (NH4)2MoO4 (98 mg, 0.5 mmol), and C6F5COOH (212 mg, 1.0 mmol) were dissolved in toluene (50 mL). The mixture was heated to azeotropic reflux using a Dean-Stark apparatus to remove water. After 12 h, the reaction mixture was cooled to room temperature, concentrated under reduced pressure, dissolved in ethyl acetate, and washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15 / 1) to give compound 4 (658 mg, 56%) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ7.65 (dd, J = 7.9, 1.8 Hz, 1H), 7.39 (ddd, J = 1.5, 7.2, 8.4 Hz, 1H), 7.01 (dd, J = 8.4, 1.1 Hz, 1H), 6.87 (ddd, J = 8.1, 7.3, 1.1 Hz, 1H), 4.97 (qd, J = 6.5, 6.9 Hz, 1H), 4.50 (d, J = 7.1 Hz, 1H), 3.79 (s, 3H), 1.54 (d, J = 6.4 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 171.0, 167.0, 160.1, 134.0, 128.4, 118.8, 117.0,110.3, 78.5, 73.6, 52.8, 20.8. Synthesis of compound 5:

[0054] Compound 4 (235 mg, 1.0 mmol) was dissolved in methanol (5 mL), and an aqueous solution of lithium hydroxide (96 mg, 4 mmol) (5 mL) was added dropwise at room temperature with stirring for 2 h. After the reaction was complete, the mixture was acidified with 1 M hydrochloric acid (pH = 2), the methanol was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a white solid acid 5 (215 mg, 97%). 1 H NMR (500 MHz, Chloroform-d) δ 7.62 (d, J= 7.9 Hz, 1H), 7.37 (dd, J = 7.5, 7.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.84 (dd, J = 7.5, 8.1Hz, 1H), 4.95 (qd, J = 6.3, 7.6 Hz, 1H), 4.48 (d, J = 7.4 Hz, 1H), 1.45 (d, J =6.3 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 174.0, 167.6, 160.4, 135.0, 128.9, 119.1, 117.2, 109.5, 79.9, 72.3, 21.0. Synthesis of compound 7:

[0055] Compound 6 (261 mg, 1.0 mmol) was dissolved in dichloromethane (10 mL), and DMAP (24 mg, 0.2 mmol), BnOH (0.09 mL, 0.9 mmol), and DCC (309 mg, 1.5 mmol) were added. The reaction system was stirred at room temperature for 5 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1) to obtain a colorless oily liquid of the compound (344 mg, 98%). 1 H NMR (500 MHz, CDCl3) δ7.34 (m, 5H), 5.15 (d, J = 4.9 Hz, 2H), 4.37 (td, J = 8.4, 4.9 Hz, 1H), 3.64(s, 3H), 2.44 – 2.30 (m, 2H), 2.19 (dt, J = 13.9, 7.2 Hz, 1H), 1.95 (td, J =14.8, 8.3 Hz, 1H), 1.42 (s, 9H). 13C NMR(126 MHz, CDCl3) δ 173.2, 172.1, 155.4,135.3, 128.6, 128.5, 128.3, 80.0, 67.2, 53.0, 51.8, 30.0, 28.3, 27.7. A colorless oily liquid (351 mg, 1 mmol) was dissolved in acetonitrile (10 mL), and DMAP (146 mg, 1.2 mmol) and Boc anhydride (0.46 mL, 2 mmol) were added. The reaction system was stirred at room temperature for 3 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum water pump, and the residue was dissolved in ethyl acetate and washed with water. The resulting aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure using a vacuum water pump, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1) to give compound 7 as a colorless oily liquid (420 mg, 93%). 1 H NMR (500 MHz, CDCl3) δ 7.36 – 7.27 (m, 5H), 5.14 (d, J = 2.7Hz, 2H), 4.96 (dd, J = 9.7, 4.9 Hz, 1H), 3.65 (s, 3H), 2.49 (m, 1H), 2.44 –2.36 (m, 2H), 2.25 – 2.16 (m, 1H), 1.44 (d, J = 2.9 Hz, 18H). 13 C NMR (126 MHz, CDCl3) δ 173.1, 170.2, 152.0, 135.6, 128.5, 128.2, 128.0, 83.3, 66.8, 57.5, 51.7, 30.6, 27.9, 24.8. Synthesis of compound 10:

[0056] Compound 7 (2.0 g, 4.43 mmol) was dissolved in diethyl ether (50 mL), cooled to -78 °C, and DIBAL-H (7.1 mL, 1.0 M in hexanes) was slowly added dropwise. The reaction mixture was stirred at -78 °C for 30 min, then water (0.5 mL) was added to quench the reaction, followed by the addition of saturated sodium potassium tartrate aqueous solution (25 mL), and the mixture was stirred at room temperature for 1 h. The mixture was extracted with ethyl acetate, and the combined organic phases were washed with water and saturated brine, dried over colorless sodium sulfate solid, filtered, and the filtrate was concentrated under reduced pressure using a vacuum pump. The resulting crude product 8 was used directly in the next step without further purification.

[0057] The compound 8 obtained in the previous step was directly dissolved in anhydrous methanol (50 mL), cooled to 0 °C, and pyridine (3.8 mL, 44.3 mmol) was added. O -Benzylhydroxylamine (0.67 mL, 5.76 mmol). The mixture was brought to room temperature and stirred continuously for 16 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum water pump. The residue was dissolved in ethyl acetate, washed with water, and the resulting aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure using a vacuum water pump. The resulting crude product 9 was used directly in the next step without further purification.

[0058] Compound 9 obtained in the previous step was directly dissolved in methanol (50 mL), cooled to 0 °C, and the pH was adjusted to 5.0 with acetic acid / methanol. Sodium cyanoborohydride (557 mg, 8.86 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, a saturated sodium bicarbonate solution was added to quench the reaction. The reaction solution was concentrated under reduced pressure using a vacuum water pump, and the residue was dissolved in ethyl acetate, washed with water, and the resulting aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure using a vacuum water pump, and the crude product was subjected to silica gel column chromatography to obtain compound 10 (1.709 g, 73%), a colorless oily liquid. 1 H NMR(500 MHz, CDCl3) δ 7.36 – 7.27(m, 10H), 5.19 – 5.11 (m, 2H), 4.91 (dd, J = 9.7, 5.1 Hz, 1H), 4.70 (s, 2H), 3.02 – 2.90 (m, 2H), 2.19 (dtd, J = 13.7, 8.3, 7.9, 5.2 Hz, 1H), 1.96 (m, 1H), 1.60 (td,J = 14.5, 13.6, 6.5 Hz, 2H), 1.45 (s, 18H). 13 C NMR(126 MHz, CDCl3) δ170.7, 152.3, 137.9, 135.7, 128.5, 128.4, 128.4, 128.1, 128.0, 127.8, 83.1,66.8, 58.1, 51.6, 27.9, 27.1, 24.2. Synthesis of compound 11:

[0059] Compound 10 (529 mg, 1.0 mmol) was dissolved in dichloromethane (10 mL), cooled to 0 °C, and TFA (0.46 mL, 6 mmol) was added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum water pump, and the resulting crude product 11 was used directly in the next step without further purification.

[0060] Example 1 This example provides an intermediate I-1, and the preparation steps are as follows:

[0061] Compound 5 (78 mg, 0.35 mmol), compound 10 (125 mg, 0.24 mmol), and HOAt (49 mg, 0.35 mmol) were dissolved in anhydrous dichloromethane (30 mL). DIPEA (0.13 mL, 0.72 mmol) and HATU (183 mg, 0.48 mmol) were added at 0 °C. The mixture was stirred at room temperature for 2 h. After removing the dichloromethane under reduced pressure, the mixture was dissolved in ethyl acetate, quenched with saturated ammonium chloride solution, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give a colorless oily intermediate I-1 (143 mg, 82%).

[0062] The NMR data are as follows: 1 H NMR (500 MHz, CDCl3) δ 7.65 (d, J = 6.1 Hz, 1H), 7.44 – 7.36 (m, 6H), 7.31 (d, J = 4.0 Hz, 5H), 7.05 (d, J = 8.4 Hz, 1H), 6.86 (t,J = 7.5 Hz, 1H), 5.13 (q, J = 12.4 Hz, 3H), 5.00 (q, J = 10.1 Hz, 2H), 4.92 (dd, J = 9.6, 5.1 Hz, 2H), 3.96 (dt, J = 14.1, 7.0 Hz, 1H), 3.56 (dt, J = 14.4, 6.9 Hz, 1H), 2.21 –2.11 (m, 1H), 1.95 (dtd, J = 14.7, 9.6, 5.5 Hz, 1H), 1.77 (dt, J = 15.6, 7.5Hz, 2H), 1.43 (s, 18H), 1.37 (d, J = 6.4 Hz, 3H). 13 C NMR(126 MHz, CDCl3) δ170.76, 170.50, 167.27, 160.45, 152.64, 135.95, 134.49, 134.27, 129.79,129.54, 129.18, 128.94, 128.80, 128.48, 128.31, 119.13, 117.25, 83.59, 70.97,67.17, 58.12, 45.59, 28.25, 27.03, 24.01, 20.66. Example 2 This example provides an intermediate II-1, the preparation method of which is as follows:

[0063] Intermediate I (732 mg, 1.0 mmol) was dissolved in dichloromethane (10 mL), cooled to 0 °C, and TFA (0.76 mL, 10 mmol) was added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum water pump. The crude product obtained was used directly in the next step without further purification.

[0064] The crude product obtained was dissolved in anhydrous dichloromethane (20 mL) along with compound 5 (254 mg, 1.15 mmol) and HOAt (136 mg, 1 mmol). DIPEA (0.52 mL, 1 mmol) and HATU (760 mg, 2 mmol) were added at 0 °C. The mixture was stirred at room temperature for 5 h. After removing the dichloromethane under reduced pressure, the mixture was dissolved in ethyl acetate, quenched with saturated ammonium chloride solution, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane / acetone = 3 / 1) to give a colorless oily intermediate II-1 (566 mg, 77%).

[0065] Alternatively, it can be prepared using the following method:

[0066] Under a nitrogen atmosphere, the crude product 11, compound 5 (664 mg, 3.0 mmol), and HOBt (135 mg, 1.0 mmol) obtained in the previous step were dissolved in dichloromethane (20 mL) and cooled to 0 °C. DIPEA (1.05 mL, 6.0 mmol) and EDCI·HCl (767 mg, 4.0 mmol) were added. After stirring for 3 hours, the dichloromethane was removed under reduced pressure, and the product was dissolved in ethyl acetate. The organic phase was washed successively with 1M hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / acetone = 3 / 1) to obtain a white solid powder, intermediate II (468 mg, 64%).

[0067] The NMR data are as follows: 1 H NMR (500 MHz, CDCl3) δ 7.70 (t, J = 7.9 Hz, 2H), 7.48 – 7.36 (m, 7H), 7.30 (s, 5H), 7.03 (dd, J = 16.9, 8.3 Hz, 3H), 6.91 (dt, J = 22.0, 7.5 Hz, 2H),5.17 – 5.06 (m, 3H), 5.05 – 4.95 (m, 2H), 4.86 (p, J = 6.6 Hz, 2H), 4.70 (q, J = 7.5 Hz, 1H), 4.41 (d, J = 7.9 Hz, 1H), 3.95 (dt,J = 14.1, 6.6 Hz, 1H), 3.63(dt, J = 14.0, 6.1 Hz, 1H), 2.00 (q, J = 9.8, 8.9 Hz, 1H), 1.86 – 1.70 (m, 3H), 1.60 (d, J = 6.3 Hz, 3H), 1.36 (d, J = 6.4 Hz, 3H). 13 C NMR(126 MHz, CDCl3) δ171.2, 170.5, 167.4, 166.7, 160.0, 135.1, 134.2, 134.0, 133.8, 129.5, 129.3,128.9, 128.7, 128.6, 128.5, 128.5, 128.3, 119.0, 118.8, 117.0, 116.8, 110.6,110.3, 79.3, 77.9, 74.2, 71.2, 67.4, 51.8, 44.7, 29.1, 22.8, 21.6, 20.3. Example 3 This example provides a method for preparing the natural product Catenobactin B, with the following steps:

[0068] Under a nitrogen atmosphere, intermediate II (50 mg, 0.07 mmol) was dissolved in anhydrous methanol (2 mL), and Pd / C (6 mg, 10 wt%) was added. The mixture was then bubbled with a hydrogen balloon for 5 min, and the reaction mixture was stirred overnight at room temperature under a hydrogen atmosphere (1 atm). After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure using a vacuum water pump. The crude product was then subjected to silica gel column chromatography to obtain the compound, a white solid called Catenulobactin B (30 mg, 78%).

[0069] The NMR data are as follows: 1 H NMR (500 MHz, DMSO-) d 6) δ 12.14(s, 1H), 11.92(s, 1H), 8.08(d, J = 7.3Hz, 1H), 7.61(t, J= 6.7 Hz, 2H), 7.48-7.41 (m, 2H), 7.05 – 6.87 (m, 4H), 5.03(d, J = 5.6 Hz, 1H), 4.88 (m, 2H), 4.55 (d, J = 7.1 Hz, 1H), 4.25 – 4.15 (m,1H), 3.64 – 3.44 (m, 2H), 1.82 – 1.71 (m, 1H), 1.63-1.57 (m, 3H), 1.45 (d, J =5.2, Hz, 6H). 13 C NMR (126 MHz, DMSO-) d 6) δ 173.9, 169.9, 169.5, 165.8, 165.8,159.7, 159.6, 134.5, 134.4, 128.5, 128.4, 119.4, 119.4, 117.0, 117.0, 110.6,110.5, 79.4, 79.3, 73.9, 70.8, 52.5, 47.7, 28.7, 23.2, 20.9, 20.9. Example 4 This example provides an intermediate I-2, which is prepared as follows:

[0070] Under a nitrogen atmosphere, intermediate I-1 (45 mg, 0.06 mmol) was dissolved in anhydrous methanol (1 mL), and Pd / C (5 mg, 10 wt%) was added. The mixture was then bubbled with a hydrogen balloon for 5 min, and the reaction mixture was stirred overnight at room temperature under a hydrogen atmosphere (1 atm). After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure using a vacuum water pump. The crude product was then subjected to silica gel column chromatography to obtain intermediate I-2 (31 mg, 89%), a white solid. 1 H NMR (500 MHz, DMSO-) d 6)δ 7.60 (d, J = 7.5 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 5.04 (d, J = 5.6 Hz, 1H), 4.90 (p,J = 6.0 Hz, 1H), 4.57 (dd, J = 9.8, 5.0 Hz, 1H), 3.59 - 3.50 (m, 2H), 2.06 - 1.94 (m, 1H), 1.83-1.73 (m, 1H), 1.59 - 1.52 (m, 2H), 1.41 (s, 21H). 13 C NMR (126 MHz, DMSO-) d 6) δ 169.2, 165.5, 159.5, 152.4, 134.1, 128.1, 119.1, 116.7, 110.3, 81.8,79.0, 70.52, 47.7, 27.8, 27.3, 23.7, 20.6. Example 5 This example provides an intermediate I-3, which is prepared as follows:

[0071] Intermediate I-1 (147 mg, 0.2 mmol) was dissolved in dichloromethane (2 mL), cooled to 0 °C, and trifluoroacetic acid (0.15 mL, 2 mmol) was added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum water pump, quenched with saturated sodium bicarbonate, washed with saturated brine, and the combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was then subjected to silica gel column chromatography to obtain intermediate I-3 (98 mg, 93%). 1 H NMR (500MHz, Methanol-) d 4) δ 7.62 (d, J = 6.4 Hz, 1H), 7.51 – 7.36 (m, 6H), 7.36 – 7.24(m, 5H), 6.94 (d, J = 8.3 Hz, 1H), 6.88 (t, J = 7.6 Hz, 1H), 5.12 (d, J = 2.9Hz, 2H), 5.04 – 4.94 (m, 2H), 4.87 – 4.82 (m, 2H), 3.86 (dt, J = 13.1, 5.6 Hz, 1H), 3.70 (dd, J = 14.4, 6.5 Hz, 1H), 3.57 (t, J= 5.5 Hz, 1H), 1.85 – 1.62 (m,4H), 1.30 (d, J = 5.0 Hz, 3H). 13 C NMR (126 MHz, Methanol-) d 4) δ 175.9, 173.1,168.3, 161.4, 137.6, 135.9, 135.3, 131.3, 130.7, 130.2, 129.9, 129.7, 129.7,120.3, 117.9, 80.2, 77.8, 72.9, 68.2, 55.1, 46.0, 32.5, 24.2, 21.1. Example 6 This example provides an intermediate II-2, which is prepared as follows:

[0072] Intermediate II-1 (45 mg, 0.06 mmol) was dissolved in methanol (1 mL), and an aqueous solution of lithium hydroxide (6 mg, 0.24 mmol) (0.5 mL) was added dropwise at room temperature with stirring for 10 min. After the reaction was complete, the mixture was acidified with 1 M hydrochloric acid (pH = 2), the methanol was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a white solid acid intermediate II-2 (31 mg, 90%). 1 H NMR (500 MHz, DMSO-) d 6) δ 8.03 (s, 1H), 7.60 (t, J = 7.3 Hz, 2H), 7.45 (d, J = 7.4 Hz, 4H), 7.39 (s, 3H), 7.02 –6.90 (m, 4H), 5.04 – 4.89 (m, 3H), 4.84 (p, J = 6.1 Hz, 1H), 4.74 (p, J = 6.6Hz, 1H), 4.53 (d, J = 7.3 Hz, 1H), 3.99 (d, J = 19.2 Hz, 1H), 3.78 (dt, J =13.9, 6.6 Hz, 2H), 1.77 (s, 1H), 1.67 – 1.47 (m, 3H), 1.42 (d, J= 6.3 Hz, 3H), 1.26 (d, J = 6.2 Hz, 3H). 13 C NMR (126 MHz, DMSO-) d 6) δ 170.1, 169.1, 166.0,165.9, 159.6, 159.6, 134.6, 134.5, 130.1, 129.3, 128.9, 128.4, 128.3, 119.4,119.4, 117.0, 116.9, 110.3, 79.71, 79.0, 75.9, 73.9, 70.8, 53.5, 44.7, 29.4,21.0, 20.6. Example 7 This example provides an intermediate II-3, which is prepared as follows:

[0073] Intermediate II-1 (45 mg, 0.06 mmol) was dissolved in methanol (2 mL), and excess sodium bicarbonate was added at room temperature and stirred for 3 h. After the reaction was complete, the methanol was removed under reduced pressure, and the solution was dissolved in ethyl acetate. The solution was then washed with water and saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to give intermediate II-3 (33 mg, 84%) as a white solid. 1 HNMR(500 MHz, CDCl3) δ 7.66 (ddd, J = 18.6, 7.9, 1.7 Hz, 2H), 7.34-7.42 (m,8H), 6.99 (dd, J = 20.7, 8.3 Hz, 3H), 6.87 (dt, J = 15.9, 7.5 Hz, 2H), 5.08(q, J = 6.6 Hz, 1H), 4.95 (d, J = 10.6 Hz, 1H), 4.92 – 4.84 (m, 2H), 4.76 (d,J = 6.3 Hz, 1H), 4.64 – 4.57 (m, 1H), 4.43 (d, J = 7.8 Hz, 1H), 3.83 (q, J =7.8, 6.2 Hz, 1H), 3.68 (s, 3H), 3.60 (d, J = 11.7 Hz, 1H), 1.88 (d, J = 10.9Hz, 1H), 1.73 – 1.63 (m, 3H), 1.57 (d, J = 6.2 Hz, 3H), 1.32 (d, J = 6.4 Hz,3H).13 C NMR(126 MHz, CDCl3) δ 172.0, 170.7, 170.6, 167.3, 166.7, 160.0, 159.9,134.4, 134.1, 133.8, 129.5, 129.3, 128.9, 128.7, 128.6, 119.1, 118.9, 117.1,116.9, 110.7, 110.3, 79.5, 74.4, 71.3, 52.7, 51.8, 44.7, 29.2, 21.6, 20.3. Performance testing 1. The bioactivity of intermediates I and II and the natural product Catenobactin B prepared in the embodiments of the present invention was studied. Using the Caenorhabditis elegans Parkinson's disease (PD) model, the neuroprotective activity of Catenobactin B was systematically investigated, as follows: (1) Catenulobactin B reduces the aggregation of α-Syn in NL5901 nematodes. First, the NL5901 nematode model was used to evaluate the effect of catenobactin B on protein aggregation. The results are as follows: Figure 1 As shown, in the NL5901 nematode model, Catenuolobactin B significantly reduced the concentration-dependent aggregation of α-Syn compared to the untreated group. Notably, at a concentration of 30 μM, Catenuolobactin B significantly reduced the fluorescence intensity of α-Syn, and its inhibitory effect was comparable to that of the classic anti-Parkinson's drug L-DOPA, indicating its promising anti-PD potential.

[0074] Secondly, to further evaluate the effect of structural modification on activity, the effects of intermediate I, intermediate II, and the natural product Catenuolobactin B at a concentration of 30 μM on α-Syn aggregation were also investigated. The results are as follows: Figure 2 As shown, the results indicate that intermediates II-1, II-2, and II-3 all reduced the fluorescence intensity of α-Syn to varying degrees, with intermediate II-1 exhibiting the most significant inhibitory effect, comparable to that of Catenobactin B.

[0075] 2. Catenulobactin B restores 6-OHDA-induced dopaminergic neuronal degeneration in BZ555 nematodes. In the pathophysiological study of Parkinson's disease (PD), damage and loss of dopaminergic (DA) neurons are another core pathological feature besides motor dysfunction. The transgenic nematode strain BZ555 is an important model tool for in vivo tracing of DA neurons. It achieves precise targeting and visualization of DA neurons in nematodes by specifically inserting green fluorescent protein (GFP) into the regulatory region of the dopamine transporter gene dat-1. Based on this model, selective damage, degeneration, and apoptosis of DA neurons can be induced by administering the neurotoxin 6-hydroxydopamine (6-OHDA), allowing for the monitoring of specific degeneration of DA neurons.

[0076] To further explore the neuroprotective effect of Catenobactin B, this invention employed 6-OHDA to induce a PD model of DA neuron damage in *C. elegans* BZ555. After induction treatment with the neurotoxin 6-hydroxydopamine (6-OHDA), fluorescence imaging and analysis were performed on dopaminergic neurons in the head region of *C. elegans*. The results are as follows: Figure 3 As shown, compared to the blank control group, the mean fluorescence intensity in the dopaminergic neuronal region of the head of BZ555 nematodes treated with the neurotoxin 6-OHDA was significantly reduced. This result confirms that the treatment induced severe neurodegenerative lesions or neuronal loss. In contrast, treatment with Catenulobactin B significantly enhanced this fluorescence intensity, with an effect comparable to that of 2 mM L-DOPA, indicating its protective effect on dopaminergic neurons. These results suggest that Catenulobactin B can effectively alleviate 6-OHDA-induced dopaminergic neurodegenerative diseases in BZ555 nematodes and has a significant neuroprotective effect.

[0077] (3) Catenulobactin B exerts its neuroprotective effect by regulating iron metabolism. To investigate whether catenobactin B exerts neuroprotective effects by regulating iron(III) metabolism, qRT-PCR was used to analyze the expression changes of key iron metabolism genes in NL5901 nematodes. The results are as follows: Figure 4As shown, the results indicated that treatment with Catenulobactin B significantly upregulated the expression of the ferritin homolog FTN-1, while other iron metabolism-related genes tested were downregulated compared to the untreated control group. Among these, the divalent metal ion transporter genes SMF-1 and SMF-3 are primarily responsible for mediating the uptake of exogenous non-transferrin-bound iron and possess broad transmembrane transport activity for divalent metal ions such as iron and manganese. The synergistic downregulation of these two genes after Catenulobactin B treatment suggests that the drug may reduce the intracellular influx of exogenous iron, thereby limiting the excessive accumulation of intracellular free iron and helping to alleviate iron-mediated oxidative stress damage. Simultaneously, the expression of the ferrotransferrin homologs FPN-1.1 and FPN-1.2, responsible for intracellular iron efflux, also decreased after drug treatment. This further suggests that Catenulobactin B may normalize intracellular iron(III) levels by regulating iron homeostasis, thereby reducing the cell's need for iron efflux. Catenulobactin B can coordinate the expression of key genes involved in iron uptake, efflux, and storage. By inhibiting exogenous iron uptake while increasing intracellular iron storage levels, it may exert a neuroprotective effect by regulating iron metabolism and alleviating iron-mediated oxidative stress.

[0078] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An intermediate I, characterized in that, It has the structural formula shown in Equation I: ； R1 and R2 are each independently selected from H, benzyl, and C. 1~6 Alkyl, trimethylsilylethyl, trimethylsilylethoxymethylene; R3 is selected from -NH2 and / or -NBoc2.

2. The intermediate I according to claim 1, characterized in that, Selected from the following structural formula: 、 、 。 3. A method for preparing intermediate I as described in claim 1 or 2, characterized in that, Includes the following steps: The reaction is carried out under condensing reagent, solvent, and alkaline conditions, and compound A and compound B undergo an amide condensation reaction to obtain the product. The structural formulas of compounds A and B are as follows: 。 4. The preparation method according to claim 3, characterized in that, The condensing agent comprises at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, N-hydroxy-7-azabenzotriazole, (7-azabenzotriazole-1-oxo)tripyrrolephosphonohexafluorophosphate, 3-(diethoxy-o-acyloxy)-1,2,3-benzotriazine-4-one, and bis(2-oxo-3-oxazolyl)phosphine chloride.

5. An intermediate II, characterized in that, It has the structural formula shown in Formula II: ； Among them, R4 is selected from OH and C. 1~6 Alkyl and benzyl groups.

6. The intermediate II according to claim 5, characterized in that, Choose the following structural formula: 、 、 。 7. A method for preparing intermediate II as described in claim 5 or 6, characterized in that, Includes the following steps: The intermediate I described in claim 1 is mixed with a protic acid and subjected to a tert-butoxycarbonyl group protection removal reaction of the amino functional group; then, under condensation reagent and alkaline conditions, it is subjected to an amide condensation reaction with compound A to obtain the final product. Alternatively, under condensing reagent and alkaline conditions, compound A and compound C can be obtained by undergoing an amide condensation reaction. The structural formulas of compounds A and C are as follows: 、 。 8. A method for preparing the natural product Catenobactin B, characterized in that, Includes the following steps: The intermediate II described in claim 5 or 6 is obtained by removing the benzyl group from the solvent under catalytic hydrogenation conditions; The structural formula of the natural product Catenulobactin B is as follows: 。 9. The preparation method according to claim 8, characterized in that, The catalyst used in the catalytic hydrogenation conditions is selected from Pd / C and / or Pd(OH)2 / C.

10. The use of intermediate I as described in claim 1 or 2, intermediate II as described in claim 5 or 6, or the natural product of Catenobactin B prepared by the preparation method described in any one of claims 8 to 9 in the preparation of drugs for the treatment and / or prevention of neuroprotective effects, drugs for neurodegenerative diseases, or drugs for Parkinson's disease.

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