CN110627727A

CN110627727A - Pseudomonas aeruginosa metabolite and derivative, synthesis method and application thereof

Info

Publication number: CN110627727A
Application number: CN201810651018.6A
Authority: CN
Inventors: 雷晓光; 张健; 杨荣文; 赵天湖
Original assignee: Peking University
Current assignee: Haipaitec Beijing Biomedical Technology Co ltd
Priority date: 2018-06-22
Filing date: 2018-06-22
Publication date: 2019-12-31
Anticipated expiration: 2038-06-22
Also published as: CN110627727B

Abstract

The invention relates to a Pseudomonas aeruginosa metabolite and a derivative thereof, and the structural general formula is shown as I. The pseudomonas aeruginosa metabolite and the derivative thereof provided by the invention have specific spatial configuration, are novel metal carriers secreted by gram-negative bacteria pseudomonas aeruginosa, and can help the pseudomonas aeruginosa to obtain metal ions in an environment with metal deficiency. Based on the characteristic that the metabolite and the derivative of the pseudomonas aeruginosa can help the pseudomonas aeruginosa to obtain metal ions, the metabolite and the derivative of the pseudomonas aeruginosa can be connected with known antibiotics to generate a novel high-ferritin antibiotic. The invention also relates to a synthesis method of the pseudomonas aeruginosa metabolite and the derivative thereof, and the method provides a complete synthesis route of the pseudomonas aeruginosa metabolite and the derivative thereof with specific spatial configuration, and the modularized synthesis route can be applied to the chemical synthesis of compounds with similar structures and related derivatives, thereby opening up a wide development space for novel antibiotic drugs.

Description

Pseudomonas aeruginosa metabolite and derivative, synthesis method and application thereof

Technical Field

The invention relates to the field of innovative drug research and development, in particular to a pseudomonas aeruginosa metabolite with a specific spatial configuration and a derivative thereof, and also relates to a chemical synthesis method of the pseudomonas aeruginosa metabolite and the derivative thereof and application of the pseudomonas aeruginosa metabolite and the derivative thereof in preparation of drugs.

Background

In modern society, as antibiotics are used more and more generally, the problem of antibiotic resistance has become one of the main factors affecting global health, and clinically available antibiotics are decreasing. Compared with gram-positive bacteria, gram-negative bacteria have the outer membrane protection, are easier to generate drug resistance, and are more difficult to treat. The outer membrane is the specific structure of gram-negative bacteria cell wall, and can prevent a plurality of high-activity gram-positive bacteria resistant medicaments from entering cells to play an antibacterial activity. The current strategy of "trojan horses" to solve the problem of resistance caused by gram-outer membranes is a feasible approach. The research strategy of "Trojan horse" refers to the synthesis of antibiotics of the ferricin class by linking a metal vector secreted by bacteria to an antibiotic, which can penetrate the outer membrane of gram-negative bacteria by virtue of the active transport of the metal vector by the bacteria. Therefore, the discovery and utilization of metal carriers secreted by bacteria themselves become a hotspot for research on overcoming the drug resistance of gram-negative bacteria.

Ghssein et al reported in Science 2016 a novel multifunctional metal complexing agent secreted by Staphylococcus aureus, staphynopine, which complexes with a variety of extracellular metal ions when they are taken up by bacteria and is recognized by the bacteria's solute binding proteins, thereby allowing the extracellular metal ions to enter the bacteria. In the next year, a multifunctional metal complexing agent pseudocaline secreted by pseudomonas aeruginosa was reported, and researchers discovered a biosynthetic pathway of pseudocaline and proposed the structure of pseudocaline through transcriptomics studies on gram-negative bacteria pseudomonas aeruginosa. Unfortunately, in their proposed structures, the spatial configuration of glutamate is not confirmed, and the other two stereocenters are also determined only by biological isotope feeding experiments, without any chemical nuclear magnetic or single crystal characterization confirming the stereostructures of pseudocaline, especially the stereocenters of glutamate fragments. Therefore, the development of an asymmetric synthetic route of pseudoephedrine and the determination of the stereochemistry of three chiral centers of pseudoephedrine are of great significance to the research of pseudoephedrine biology and the subsequent development of innovative antibiotic drugs.

Disclosure of Invention

A first object of the present invention is to provide a metabolite of pseudomonas aeruginosa and a derivative thereof having a specific steric configuration. The Pseudomonas aeruginosa metabolite provided by the invention can be connected with known antibiotics to obtain the high-ferritin antibiotics, and has wide application prospect.

Specifically, the Pseudomonas aeruginosa metabolite and the derivative thereof provided by the invention contain a structure shown as a general formula I:

in each chemical formula, the (S) or (R) represents the stereo configuration of the chiral carbon at the corresponding position and is S or R.

In the general formula I, R₁、R₂、R₃Independently of one another represent-H or- (CH)₂)m-NH₂(ii) a Wherein m represents an integer of 1-10; x represents C or N, preferably C.

The- (CH)₂)m-NH₂The group-may be a straight-chain structure or a structure having a branched chain. This structure reacts with an antibiotic containing an active ester group to form an amide bond (CH)₂) m-NHCO, coupling the Pseudomonas aeruginosa metabolite and the derivative thereof with antibiotics.

In a preferred embodiment of the present invention, in the general formula I, R is₁、R₂、R₃Any two groups in (A) each independently represent- (CH)₂)m-NH₂And the remaining one group represents H.

In a preferred embodiment of the present invention, in the general formula I, R is₁、R₂、R₃Any one of the groups represents- (CH)₂)m-NH₂And the remaining two groups each represent H; more preferably, X is C, and R₁Represent-(CH₂)m-NH₂And the remaining two groups both represent H.

In a preferred embodiment of the present invention, in the general formula I, R is₁、R₂、R₃All represent H; more preferably, X is C, and R₁、R₂、R₃All represent H.

The- (CH)₂)m-NH₂In the group, m further preferably represents an integer of 1 to 5.

As a particularly preferred embodiment of the present invention, the pseudomonas aeruginosa metabolite and derivative thereof are selected from compounds of the following structure:

wherein, the compound represented by I-1 is pseudomonas aeruginosa metabolite pseudoephedrine of the invention, wherein the three-dimensional configurations of three chiral carbons are S, S, S respectively, and pseudoephedrine is adopted to represent the specific compound of the specific configuration in the invention.

The second purpose of the invention is to provide a synthesis method of the pseudomonas aeruginosa metabolite and the derivative thereof.

Specifically, in the general formula I, when R is₁、R₂、R₃When all represent H, the synthesis method comprises the following steps:

in the general formula I, when R₁Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10, and R₂、R₃When all represent H, the synthesis method comprises the following steps:

in the general formula I, when R₂Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10, and R₁、R₃All represent HThe synthesis method comprises the following steps:

in the general formula I, R₃Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10, and R₁、R₂When all represent H, the synthesis method comprises the following steps:

as specific embodiments of the present invention:

the compound I-1, namely pseudoephedrine, can be synthesized by adopting a method comprising the following specific steps:

(1) dissolving the compound 12 in dichloromethane, adding imidazole and tert-butyldimethylsilyl chloride in sequence under the protection of argon in an ice water bath, reacting for 12 hours at room temperature, cooling the mixture in the ice water bath, adding water to quench the reaction, washing an organic phase with water, performing rotary evaporation, and performing column chromatography to obtain a compound 13;

(2) dissolving the compound 13 in dichloromethane, adding 2, 6-dimethylpyridine, cooling in an ice water bath, adding trimethylsilyl trifluoromethanesulfonate, reacting at room temperature for 1.5 hours, cooling in an ice water bath, adding methanol, stirring at room temperature for 3 hours, then carrying out rotary evaporation to obtain a crude product, adding ethyl acetate to dissolve the crude product, adjusting the pH value of saturated sodium bicarbonate to be more than 9, adding o-nitrobenzenesulfonyl chloride, stirring at room temperature overnight, washing an organic phase with water, drying, concentrating, and carrying out column chromatography to obtain a compound 10;

(3) adding the compound 10 into a tetrahydrofuran solution in which tris (dibenzylideneacetone) dipalladium and (1R,2R) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand are dissolved in an ice-water bath, adding the compound 11, stirring for 5 hours in the ice-water bath, adding a pyridine hydrofluoric acid solution, and reacting at room temperature overnight. Diluting with ethyl acetate, adding saturated sodium bicarbonate water solution for quenching, washing with organic phase sodium bicarbonate, drying, concentrating, and purifying with column to obtain compound 15 a;

(4) dissolving the compound 8a in tetrahydrofuran, adding the solution into a tetrahydrofuran solution containing azodicarboxylic acid ethyl ester and pyridine diphenyl phosphine under an ice-water bath, and adding a tetrahydrofuran solution of the compound 15 a. Reacting at room temperature for 2 hours, concentrating, dissolving crude ethyl acetate, washing with 2 moles of hydrochloric acid, drying an organic phase, concentrating, and passing through a column to obtain a compound 7 a;

(5) dissolving the compound 7a in dioxane and water, adding sodium periodate and osmium tetroxide, and carrying out oxidation cutting reaction at room temperature; reacting for 2 hours, adding sodium thiosulfate for quenching, diluting with ethyl acetate, washing with water, washing with 5% sodium dihydrogen phosphate, concentrating, drying, dissolving the obtained product in acetone, performing aldehyde group oxidation reaction with Jones reagent in ice water bath for 2 hours, adding isopropanol for quenching, diluting with ethyl acetate, washing with sodium thiosulfate, and purifying to obtain a compound 16 a;

(6) dissolving the compound 16a in dichloromethane, adding anisole and trifluoromethanesulfonic acid, hydrolyzing to remove a protecting group, dissolving a crude product in dimethylformamide, adding thiophenol, and removing an o-nitrobenzenesulfonyl protecting group under an alkaline condition to obtain a compound 2a, namely pseudoplane;

(7) dissolving the compound 17a in a mixture solvent of acetone and water, adding sodium bicarbonate to adjust the pH to about 10, slowly adding benzyl chloroformate, and reacting at room temperature to obtain the imidazole benzyl chloroformate protected compound. Then dissolving the obtained product in dimethylformamide, adding potassium carbonate and benzyl bromide, and stirring at room temperature to obtain a compound 14 a;

(8) and dissolving the compound 14a in dichloromethane and trifluoroacetic acid, performing deprotection to obtain a crude product, dissolving the crude product in ethyl acetate and water, and adding sodium bicarbonate and o-nitrobenzenesulfonyl chloride to react to obtain a compound 8 a.

The compounds I-2, I-3 and I-4 according to the invention can be synthesized according to the above-mentioned methods starting from specific compounds containing the corresponding groups.

The derivative I-2 of the invention can be synthesized according to the method of the compound I-1, except that the synthesis block 8a is replaced by a corresponding synthesis block 8 b. The synthetic block 8b may be synthesized by the following method:

the specific steps of the compound 8b are as follows:

(1) under the alkaline condition, benzyl chloroformate protects imidazole, and benzyl carboxylate protects to obtain a compound 18 b;

(2) performing a Sonogashira coupling reaction on the compound 18b and the compound 19b to obtain a compound 20 b;

(3) hydrogenating and reducing the compound 20b to obtain a compound 21 b;

(4) the compound 21b is protected by benzyl chloroformate again to obtain a compound 22 b;

(5) the compound 22b is subjected to acid deprotection and is subjected to deprotection again to obtain a compound 8 b; compound 2b was synthesized using synthesis block 8b according to steps (4) to (6) of Synthesis I-1 (i.e., Compound 2 a).

The derivative I-3 of the invention can be synthesized according to the method of the compound I-1, except that the synthesis block 8a is replaced by a corresponding synthesis block 8 c. The synthetic block 8c may be synthesized by the following method:

the specific steps for compound 8c are as follows:

(1) the compound 17c is protected by o-nitrobenzenesulfonyl chloride at amino under alkaline condition, and then protected by carboxyl benzyl to obtain a compound 18 c;

(2) carrying out click reaction on the compound 18c and the compound 19c under the catalysis of copper to obtain a compound 8 c; compound 2c was synthesized using synthesis block 8c according to steps (4) to (6) of Synthesis I-1 (i.e., Compound 2 a).

The derivative I-4 of the invention can be synthesized according to the method of the compound I-1, except that the synthesis block 8a is replaced by a corresponding synthesis block 8 d. The synthetic block 8d may be synthesized by the following method:

the synthesis method of the compound 8d comprises the following specific steps:

(1) protecting the compound 17d with benzyl chloroformate under alkaline conditions to obtain a compound 18 d;

(2) performing Sonogashira coupling reaction on the compound 18d and the compound 19b to obtain a compound 20 d;

(3) hydrogenating reduction of the compound 20d to give a compound 21 d;

(4) the compound 21d is protected by benzyl chloroformate again to obtain a compound 22 d;

(5) deprotecting the compound 22d under an acidic condition, and re-protecting the o-nitrobenzenesulfonyl chloride to obtain a compound 8 d; compound 2d was synthesized using synthesis block 8d according to steps (4) to (6) of Synthesis I-1 (i.e., Compound 2 a).

The method provides a synthetic route of pseudopaline and derivatives thereof with specific spatial configuration, and the modularized synthetic route can be applied to the chemical synthesis of compounds with similar structures and related derivatives, so that a wide development space is opened up for novel antibiotic drugs.

The third purpose of the invention is to protect the application of the pseudomonas aeruginosa metabolite and the derivative thereof in preparing the high-iron-mycin antibiotics. Specifically, the Pseudomonas aeruginosa metabolite and the derivative thereof may be coupled to an antibiotic by reacting the Pseudomonas aeruginosa metabolite and the derivative thereof with an antibiotic having an ester group to form an amide bond. The pseudomonas aeruginosa metabolite and the derivative thereof can be connected with known antibiotics to generate novel high-ferritin antibiotics, and the capability of the connected antibiotics on gram-negative bacteria to permeate outer membranes can be obviously improved, so that the antibacterial activity of the antibiotics on the gram-negative bacteria is enhanced. In particular, the invention provides that primary amines in metabolites of pseudomonas aeruginosa and derivatives thereof can be linked as a linking site to a β -lactam antibiotic. The beta-lactam antibiotics comprise carbapenem antibiotics, penicillin antibiotics, cephalosporin antibiotics and the like; preferably, the beta-lactam antibiotic is linked by an amide bond.

Drawings

Fig. 1 and 2 are nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 13, respectively;

fig. 3 and 4 are nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of the compound 10 respectively;

fig. 5 and 6 are nmr hydrogen spectra and nmr carbon spectra of compound 15a, respectively;

fig. 7 and 8 are nmr hydrogen spectra and nmr carbon spectra of compound 7a, respectively;

fig. 9 and 10 are nmr hydrogen spectra and nmr carbon spectra of compound 16a, respectively;

fig. 11 and 12 are nmr hydrogen spectra and nmr carbon spectra of compound 2a, respectively;

fig. 13 and 14 are nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 8, respectively;

fig. 15 and 16 are nmr hydrogen spectra and nmr carbon spectra of compound 15d, respectively;

fig. 17 and 18 are nmr hydrogen spectra and nmr carbon spectra of compound 7d, respectively;

fig. 19 and 20 are nmr hydrogen spectra and nmr carbon spectra of compound 16d, respectively;

fig. 21 and 22 are nmr hydrogen spectra and nmr carbon spectra of compound 2d, respectively;

FIG. 23 is a schematic representation of the effect of pseudoperaline on the growth of Pseudomonas aeruginosa; wherein, FIG. 23A illustrates that Pseudomonas aeruginosa promotes the growth of Pseudomonas aeruginosa but that (R, S, S) -Pseudomonas aeruginosa does not promote the growth of Pseudomonas aeruginosa in M9 medium lacking metal ions; FIG. 23B illustrates the concentration dependence of pseudocaline on the growth-promoting effect of Pseudomonas aeruginosa in M9 medium lacking metal ions; FIG. 23C illustrates that pseudopropanine can help Pseudomonas aeruginosa transport zinc ions in VBMM medium supplemented with 10 micromolar zinc sulfate and 50 micromolar EDTA; FIG. 23D illustrates that zinc ions and pseudocaline are able to inhibit the expression of the pseudocaline synthon in VBMM medium.

FIG. 24 is a schematic diagram showing the effect of pseudopalaine on the growth of Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii in a metal ion deficient environment;

FIG. 25 is a graph showing the effect of pseudoplaine on the growth of E.coli, S.aureus and A.baumannii in a metal-free environment.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example (b): synthesis of Pseudomonas aeruginosa metabolites and derivatives thereof

Pseudocaline was synthesized according to the following steps:

synthesis of compound 13:

compound 12(7.8g, 25.2mmol, 1eq) was dissolved in DCM (100mL) and imidazole (2.58g, 37.8mmol, 1.5eq) and tert-butyldimethylsilyl chloride (4.76g, 31.6mmol, 1.25eq) were added at 0 ℃. The mixture was stirred at room temperature for 1 hour, then quenched at 0 ℃ with water (50ml) and the aqueous phase extracted with dichloromethane (100 ml. times.3). The organic phase was dried and concentrated in vacuo. Further purification by silica gel column chromatography (petroleum ether/ethyl acetate ═ 20: 1) gave the desired product 13(91g, 23.4 mmol, 93%) as a colorless oil.¹H NMR(400MHz,Chloroform-d)δ7.43–7.28(m,5H),5.76(d,J＝7.5Hz, 1H),5.16(dd,J＝40.0,12.4Hz,2H),4.42(dd,J＝11.6,7.0Hz,1H),3.76–3.57(m,2H),2.11–1.98(m, 1H),1.94(dd,J＝12.9,7.4Hz,1H),1.42(s,9H),0.88(s,9H),0.02(d,J＝1.5Hz,6H).¹³C NMR(101 MHz,CDCl₃)δ172.39,155.56,135.66,128.52,128.26,128.20,79.45,66.82,60.18,52.57,33.67,28.33, 25.85,25.69,18.12,-5.63,-5.67；IR(neat)νmax 2916,1723,1496,1460,1378,1159,836,730cm-1； HRMS(ESI):[M+H]+ calculated value C₂₂H₃₈NO₅Si 424.2514, found 424.2528. [ alpha ] to]¹⁹D-25.4(c ═ 1, chloroform); rf is 0.80 (petroleum ether/ethyl acetate 3/1). The above results of the hydrogen spectrum and the carbon spectrum are shown in detail in FIGS. 1 and 2.

Synthesis of compound 10:

trimethylsilyl triflate (TMSOTf) (18.8mL, 104mmol, 8.0eq) was added to a solution of 2, 6-lutidine (15mL, 130mmol, 10eq) and compound 13(5.23mL, 13 mmol,1.0 eq) in dichloromethane (200mL) at 0 ℃. After stirring at room temperature for 1.5h, the reaction was cooled to 0 deg.C, methanol (20mL) was added and stirring continued at room temperature for 3 h. The reaction mixture was concentrated in vacuo to give a colorless oil, which was used immediately in the next reaction without further purification. O-nitrobenzenesulfonyl chloride (3.17g, 1.5mmol) was added to a solution of the crude amine in ethyl acetate and 100mL of an aqueous solution containing sodium bicarbonate solution (5.46g, 65mmol, 5.0eq) was added to the mixture. After 8h, the mixture was diluted with ethyl acetate (100ml), the organic phase washed with water, rotary evaporated and concentrated to give the crude product which was purified by column chromatography (petroleum ether/ethyl acetate 30:1) to give compound 10(4.95g, 75%) as a colourless oil.¹H NMR(400MHz,Chloroform–d)δ7.98(d,J＝7.6Hz,1H), 7.78(d,J＝7.6Hz,1H),7.62–7.57(m,2H),7.32–7.30(m,3H),7.22–7.13(m,2H),6.48(d,J＝8.8Hz, 1H),4.91(q,J＝12.0Hz,2H),4.47–4.42(m,1H),3.79–3.67(m,2H),2.14–1.99(m,2H),0.89(s,9H), 0.06(s,6H)；¹³C NMR(101MHz,CDCl₃)δ170.88,147.50,134.89,134.39,133.28,132.61,130.26, 128.52,128.44,128.16,125.43,67.11,59.05,54.66,35.19,25.88,18.31,–5.56.IR(neat)νmax 3329,2957, 2857,1745,1542,1368,1172,1122,837cm–1；HRMS(ESI):[M+H]+ calculated value C₂₃H₃₂N₂O₇SSi: 509.1772, found 509.1768; [ alpha ] to]²⁴ _D-110 (c 1, chloroform). The above results of the hydrogen spectrum and the carbon spectrum are shown in detail in FIGS. 3 and 4.

Synthesis of compound 15 a:

at 0 deg.C, tris (dibenzylideneacetone) dipalladium (63mg,0.061mmol,0.05eq) and ligand (1R,2R)) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand (144mg,0.16mmol,0.15eq) was dissolved in tetrahydrofuran (2mL) under argon, a solution of compound 10(620mg,1.22mmol,1eq) in tetrahydrofuran (3mL) and compound 11 were added, stirred at 0 ℃ for 5 hours, a solution of pyridine hydrofluoric acid (0.67mL,4.88mmol,4eq) was added, stirred at room temperature overnight, diluted with ethyl acetate (20mL), quenched with saturated sodium bicarbonate solution (20mL), extracted with ethyl acetate in the aqueous phase (20mL x 2), combined with the organic phase, dried, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) compound 15a (553mg, 94% yield) was obtained as an oil.¹H NMR(400MHz,Chloroform–d)δ8.04(dd,J＝8.0,4.0Hz,1H),7.59–7.55(m,1H),7.48(t,J＝ 7.4Hz,2H),7.34–7.28(m,3H),7.24–7.18(m,2H),5.96(dq,J＝8.0,4.0Hz,1H),5.52(dq,J＝8.0,4.0 Hz,1H),5.05(q,J＝12Hz,1H),4.82(dd,J＝9.2,5.2Hz,1H),4.64–4.58(m,1H),3.85–3.75(m,2H), 2.67–2.58(m,1H),2.54–2.44(m,1H),2.36(brs,1H),2.33–2.27(m,1H),2.25–2.23(m,1H),2.20–2.15 (m,1H),2.13–2.06(m,1H),1.99–1.95(m,1H).¹³C NMR(101MHz,CDCl₃)δ171.27,148.12,136.45, 134.82,134.41,133.30,131.24,128.96,128.49,128.39,123.77,67.48,66.37,58.30,57.57,34.02,31.57,29.75；IR(neat)νmax 3557,2949,1737,1543,1372,1160,1061cm–1；HRMS(ESI):[M+NH4]+ calculated value C₂₂H₂₈N₃O₇478.1642, found value 478.1636; [ alpha ] to]²⁴ _D1.5(c 1, chloroform). The above results of the hydrogen spectrum and the carbon spectrum are shown in FIGS. 5 and 6.

Synthesis of compound 7 a:

pyridine diphenylphosphine (264mg, 1.01mmol, 1.5eq) was dissolved in THF (10mL) at 0 deg.C, and diethyl azodicarboxylate (158uL, 1.01mmol, 1.5eq) was added. The mixture was stirred for 10 min, then compound 8(265mg, 0.47mmol,0.7eq) was added, stirred for 10 min, and a solution of 15a (310mg, 0.67mmol, 1eq) in tetrahydrofuran (2mL) was added. After 2h, rotary evaporation, concentration to give crude product, dilution with ethyl acetate, 2N hydrochloric acid (5X 10ml),washed with saturated brine. The organic phase was rotary evaporated, concentrated and the residue was further purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 7: 1) to give the desired product (410mg, 0.407mmol, 85%) as a colorless oil;¹H NMR(400MHz,CDCl₃)δ8.06–7.99(m,1H),7.97(d,J ＝0.9Hz,1H),7.87(dd,J＝7.9,1.1Hz,1H),7.62–7.54(m,1H),7.53–7.26(m,18H),7.22–7.12(m, 2H),5.96(dd,J＝5.5,2.1Hz,1H),5.55(dd,J＝5.5,2.1Hz,1H),5.38(s,2H),5.16–4.86(m,5H),4.68 (d,J＝6.8Hz,1H),4.36(t,J＝6.5Hz,1H),3.79–3.58(m,1H),3.53–3.39(m,1H),3.34(dd,J＝15.6, 5.1Hz,1H),3.12(dd,J＝15.6,9.6Hz,1H),2.58(dd,J＝13.0,5.8Hz,2H),2.31–2.16(m,3H),2.00– 1.90(m,1H)；¹³C NMR(101MHz,CDCl₃)δ170.67,169.86,148.45,148.40,147.95,138.87,136.79, 136.67,134.96,134.84,134.43,134.12,133.45,133.32,132.22,131.45,131.37,131.19,130.95,129.39,129.10,128.84,128.81,128.62,128.55,128.52,128.50,128.39,123.90,123.71,114.99,77.38,77.06, 76.74,69.76,67.71,67.51,66.25,60.28,57.79,45.30,33.60,31.56,29.56,29.24.IR(neat)νmax 2955, 1739,1542,1213,1162,970,730,580cm–1；HRMS(ESI):[M+H]+ calculated value C₄₉H₄₇N₆O₁₄S₂1007.2586, found 1007.2585; [ alpha ] to]¹⁹ _D+3.70(c ═ 1, chloroform). The above results of the hydrogen spectrum and the carbon spectrum are shown in FIGS. 7 and 8.

Synthesis of compound 16 a:

compound 7a (190mg, 0.189mmol, 1eq) was dissolved in dioxane (4mL) and water (2mL) at room temperature, and 2, 6-lutidine (87uL, 0.756mmol, 4eq), osmium tetroxide (3.8mL, 0.038mmol, 0.2eq) and sodium periodate (404mg,1.89mmol,10eq) were added. After stirring at room temperature for 2 hours, sodium thiosulfate solution (6mL) was added for quenching, ethyl acetate extraction (8 mL. times.3) was performed, and the combined organic phases were washed with sodium thiosulfate solution (10 mL. times.2), 5% sodium dihydrogen phosphate solution (10 mL. times.2), saturated brine, dried over sodium sulfate, and the filtrate was concentrated to give a crude product. Dissolving the crude product inAcetone, cooled to 0 ℃, added jones' reagent (210uL, 0.416mmol, 2.2eq), stirred at room temperature for 2h, quenched by addition of isopropanol (1mL), diluted the reaction solution with ethyl acetate (20mL), sodium thiosulfate solution (10mL × 2), washed with saturated brine (10mL), dried over sodium sulfate, and the filtrate concentrated in vacuo. The residue was purified by gel column chromatography to give pure product 16a as a white solid (107mg, 0.100mmol, 53%).¹H NMR(400MHz,CDCl₃)δ10.35(br,2H),8.42(s,1H),7.97(d,J＝7.2Hz,1H),7.86(d,J＝ 7.2Hz,1H),7.65–7.01(m,23H),5.47–5.28(m,2H),5.13–4.63(m,6H),4.47(s,1H),3.89–3.65(m, 1H),3.43(dd,J＝36.5,8.4Hz,2H),3.14(dd,J＝16.0,9.6Hz,1H),2.67–2.25(m,4H),2.13(s,1H), 1.93(d,J＝6.1Hz,1H)；¹³C NMR(101MHz,CDCl₃)δ176.96,172.99,170.87,169.12,147.88,147.77, 147.26,136.69,135.43,134.52,134.43,133.83,133.40,132.70,131.89,131.82,131.75,131.62,131.14, 129.23,128.95,128.92,128.80,128.59,128.54,123.94,123.82,116.18,77.42,77.30,77.10,76.78,70.92, 68.12,67.86,60.05,58.24,56.80,45.22,31.30,28.05,16.26；IR(neat)ν_max3148,2961,1739,1544,1372, 1165,753,591cm^–1；HRMS(ESI):[M+H]⁺Calculated value C₄₉H₄₇N₆O₁₈S₂1071.2384, found 1071.2585; [ alpha ] to]¹⁹ _D+76.26(c 1, chloroform); the above results of the hydrogen spectrum and the carbon spectrum are shown in FIGS. 9 and 10.

Synthesis of compound 2 a:

compound 16a (92mg,0.086mmol,1eq) was dissolved in dichloromethane (15mL), anisole (76ul, 0.945mmol, 11eq) was added, the temperature was reduced to 0 deg.C, and trifluoromethanesulfonic acid (76ul, 0.859mmol, 10eq) was added. The reaction mixture was stirred at 0 ℃ for 0.5 h, then warmed to room temperature, stirred for 2h, cooled to 0 ℃ and added with an aqueous solution (10mL) of sodium bicarbonate (109mg, 1.29mmol, 15eq), stirred for an additional 0.5 h and washed with dichloromethane (10 mL. times.3). The aqueous phase was concentrated to give the crude product. The crude mixture and potassium carbonate (95mg, 0.687mmol) were dissolved in dryDimethylformamide (9mL), thiophenol (176ul, 1.718mmol, 20eq) was added, stirred at room temperature for 10 hours, then washed with water (10mL), dichloromethane (10 mL. times.3). The aqueous phase was then concentrated in vacuo and the resulting solid was purified on a gel column to give 2a (23.6mg, 0.042 mmol, 68%) as a white solid.¹H NMR(400MHz,D₂O)δ7.72(d,J＝0.9Hz,1H),6.92(s,1H),3.61(t,J＝ 6.3Hz,1H),3.32–3.19(m,2H),3.10(dt,J＝11.2,5.4Hz,1H),3.03(d,J＝6.4Hz,2H),2.90(ddd,J＝ 12.4,8.9,5.8Hz,1H),2.20(t,J＝7.9Hz,2H),2.03–1.78(m,4H)；¹³C NMR(101MHz,D₂O)δ181.79, 176.93,176.13,175.46,135.60,131.29,117.41,62.89,62.79,62.15,45.65,33.83,28.59,28.36,28.12；IR； 3359 1570 1395 1103 995 831cm-1；IR(neat)ν_max 2923,1737,1543,1372,1259,1017cm^–1；HRMS(ESI): [M-H]^-Calculated value C₁₅H₂₁N₄O₈385.1365, found 385.1372; [ alpha ] to]¹⁹ _D+5.873(c ═ 1, water); m.p.>330 ℃ is adopted. The above results of the hydrogen spectrum and the carbon spectrum are shown in detail in FIGS. 11 and 12.

Synthesis of compound 8:

compound 14(21.2g,44.2mmol,1eq) was dissolved in dichloromethane (120mL), trifluoroacetic acid (30mL) was added while cooling to 0 deg.C, the mixture was stirred at room temperature for 1 hour, and rotary evaporation gave a crude product which was dissolved in ethyl acetate (100mL), o-nitrobenzenesulfonyl chloride (9.9g,44.6mmol,1.01eq) and saturated sodium bicarbonate solution (200 mL). The reaction mixture was stirred overnight at room temperature, the organic phase was washed with saturated sodium bicarbonate (100mL x 2), saturated brine (100mL x 1), dried over sodium sulfate, concentrated to give crude product, which was recrystallized from dichloromethane and n-hexane to give pure product (20.1g, 87% yield)¹H NMR(400MHz, Chloroform–d)δ8.00–7.96(m,2H),7.75–7.66(m,1H),7.60–7.52(m,2H),7.46–7.38(m,5H),7.25 –7.19(m,2H),7.12–7.09(m,3H),7.05(s,1H),5.38(s,2H),4.87(dd,J＝44,12Hz,2H),4.64(m,1H), 3.12–3.09(m,2H)；¹³C NMR(101MHz,CDCl₃)δ170.13,148.17,147.41,138.06,137.01,134.89, 133.89,133.11,132.62,130.25,129.29,128.92,128.86,128.43,128.30,125.33,114.86,69.92,67.24, 56.18,31.04；IR(neat)νmax1683,1515,1387,1362,1319,1148,928cm–1；HRMS(ESI):[M+H]+ calcd for C27H25N4O8S:565.1388, found 565.1381; [ alpha ] to]²⁴ _D88.6(c 0.5, chloroform); m.p. 130 ℃. The above results of the hydrogen spectrum and the carbon spectrum are shown in fig. 13 and 14.

In this example, (R, S) -pseudocaline was also synthesized, and the specific steps and reaction conditions were the same as those of the pseudocaline synthesis described above, except that the (1R,2R) - (-) -N, N '-bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand of the synthetic compound 15a was replaced with (1S,2S) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand. The reaction process and the related product characterization information of each step are as follows:

synthesis of compound 15 d:

tris (dibenzylideneacetone) dipalladium (63mg,0.061mmol,0.05eq) and (1S,2S) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand (144mg,0.16mmol,0.15eq) were dissolved in tetrahydrofuran (2mL) under argon at 0 ℃, a tetrahydrofuran solution (3mL) of compound 10(620mg,1.22mmol,1eq) and compound 11 were added, stirred at 0 ℃ for 5 hours, a pyridine hydrofluoric acid solution (0.67mL,4.88mmol,4eq) was added, stirred at room temperature overnight, diluted with ethyl acetate (20mL), quenched with saturated sodium bicarbonate solution (20mL), extracted with aqueous ethyl acetate (20mL x 2), combined organically, dried, concentrated, passed through a column (petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 15d (553mg, yield 87%) oil.¹H NMR (400MHz,Chloroform–d)δ8.05(d,J＝8.0Hz,1H),7.58–7.53(m,1H),7.48(d,J＝8.2Hz,2H),7.33(m, 3H),7.25–7.23(m,2H),5.99(dd,J＝5.6,2.4Hz,1H),5.56(dd,J＝5.6,2.4Hz,1H),5.13–4.97(m,3H), 4.50(t,J＝6.8Hz,1H),3.80–3.72(m,2H),2.54–2.48(m,2H),2.32–2.27(m,2H),2.00–1.97(m,3H).¹³C NMR(101MHz,CDCl₃)δ171.34,148.35,137.54,134.73,133.68,133.39,131.37,130.69,128.60, 128.57,128.50,128.45,123.94,67.68,65.46,59.24,55.60,34.43,31.13,29.54；IR(neat)νmax 3559, 2961,2924,1738,1542,1373,1256,1158cm–1；HRMS(ESI):[M+NH4]+ calculated value C₂₂H₂₈N₃O₇478.1642, found value 478.1648; [ alpha ] to]²² _D31.7(c 1, chloroform); the above results of the hydrogen spectrum and the carbon spectrum are shown in detail in FIGS. 15 and 16.

Synthesis of compound 7 d:

¹H NMR(400MHz,CDCl₃)δ8.06–7.99(m,1H),7.97(d,J＝0.9Hz,1H),7.87(dd,J＝7.9,1.1Hz,1H), 7.62–7.54(m,1H),7.53–7.26(m,18H),7.22–7.12(m,2H),5.96(dd,J＝5.5,2.1Hz,1H),5.55(dd,J ＝5.5,2.1Hz,1H),5.38(s,2H),5.16–5.03(m,3H),4.97–4.90(m,3H),4.10(d,J＝8.0Hz,1H),3.79– 3.58(m,1H),3.44–3.37(m,1H),3.34(dd,J＝15.6,5.1Hz,1H),3.12(dd,J＝15.6,9.6Hz,1H),2.52– 2.38(m,2H),2.31–2.16(m,3H),2.00–1.90(m,1H)；¹³C NMR(101MHz,CDCl₃)δ170.74,169.82, 148.43,148.32,147.84,138.89,138.06,136.66,134.82,134.78,134.03,133.77,133.39,133.36,132.07, 131.62,131.26,131.01,130.71,129.02,128.76,128.75,128.54,128.51,128.43,128.41,128.36,128.32, 128.23,123.98,123.65,114.85,69.69,67.71,67.33,65.28,60.22,55.85,45.10,33.69,31.19,29.36,28.63； IR(neat)ν_max 2958,1742,1543,1405,1372,1244,1163cm^–1；HRMS(ESI):[M+H]⁺calculated value C₄₉H₄₇N₆O₁₄S₂1007.2586, found 1007.2587; [ alpha ] to]¹⁹ _D-23.7 (c ═ 1, chloroform); rf 0.29 (petroleum ether/ethyl acetate 1/1); the above results of the hydrogen spectrum and the carbon spectrum are shown in FIGS. 17 and 18.

Synthesis of compound 16 d:

¹H NMR(400MHz,CDCl₃)δ8.42(s,2H contain COOH),7.97(d,J＝7.2Hz,1H),7.86(d,J＝7.2Hz, 1H),7.65–7.01(m,23H),5.38(s,2H),5.18–5.08(m,2H),4.94(s,2H),4.89(t,J＝6Hz),4.47(m,1H), 4.27(m,1H),3.65–3.51(m,2H),3.44(dd,J＝8.4,4.2Hz,1H),3.11(dd,J＝8.4,4.2Hz,1H),2.67– 2.62(m,1H),2.51–2.40(m,2H),2.35–2.28(m,2H),2.13(m,1H).¹³C NMR(126MHz,CDCl₃)δ 176.74,172.62,170.01,169.60,148.70,147.80,147.49,137.04,136.51,134.79,134.69,134.02,133.57, 133.50,132.65,132.24,131.72,131.16,130.92,129.35,128.95,128.89,128.79,128.68,128.64,128.61,128.58,128.56,128.52,128.48,128.44,128.38,128.31,127.66,127.00,123.84,123.77,115.83,70.62, 69.72,68.05,67.59,60.53,59.42,57.76,32.27,30.42,28.28,25.64；IR(neat)ν_max 2923,1737,1543, 1372,1259,1017cm^–1；HRMS(ESI):[M+H]⁺calculated value C₄₉H₄₇N₆O₁₈S₂1071.2383, found 1071.2396; [ alpha ] to]²¹ _D-2.5 (c 0.8, chloroform); m.p. ═ 89-91 ℃; the above results of the hydrogen spectrum and the carbon spectrum are shown in detail in FIGS. 19 and 20.

Synthesis of compound 2 d:

¹H NMR(400MHz,Deuterium Oxide)δ7.70(s,1H),6.96(s,1H),3.68(t,J＝7.2Hz,1H),3.43(t,J＝ 7.2Hz,1H),3.32(t,J＝7.2Hz,1H),3.07(m,2H),2.99(m,2H),2.25(t,J＝7.2Hz,2H),2.00(m,,2H), 1.89(q,J＝7.2Hz,2H).¹³C NMR(101MHz,D₂O)δ181.77,175.73,174.87,174.77,135.89,131.66, 117.08,62.32,61.54,59.88,44.30,33.69,27.69,26.96,26.28；IR(neat)ν_max 3395,1628,1574,1399, 1318,1118,835,758cm^–1；HRMS(ESI):[M+H]+ calculated value C₁₇H₁₉N₂O₇565.1388, found value 565.1381; [ alpha ] to]²¹ _D+9.5(c 0.9, water); m.p. ═>At 330 ℃; the above results of the hydrogen spectrum and the carbon spectrum are shown in FIGS. 21 and 22.

The specific steps and reaction conditions for the synthesis of derivatives 2b,2c and 2d are the same as for the synthesis of compound 2a, except that the synthesis block 8a is replaced by 8b, 8c and 8d, respectively; compounds 8b and 8d have the same synthetic procedures and conditions except that the positions of the iodides of 8b and 8d are at the 2-and 4-positions of imidazole, respectively. The specific steps and conditions for the synthesis of 8b and 8c are as follows:

synthesis of Compound 8b

Compound 22b (60mg,0.090mmol,1eq) was dissolved in dichloromethane (5mL), trifluoroacetic acid (1mL) was added while cooling to 0 deg.C, the mixture was warmed to room temperature and stirred for 1 hour, rotary evaporation gave a crude product, which was dissolved in ethyl acetate (5mL), o-nitrobenzenesulfonyl chloride (21mg,0.094mmol,1.01eq) and saturated sodium bicarbonate solution (2mL) were added. The reaction mixture was stirred overnight at rt, the organic phase washed with saturated sodium bicarbonate (10mL x 2), saturated brine (10mL x 1), dried over sodium sulfate and concentrated to give the crude product which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 10: 1) to give the pure product (22mg, 78% yield)¹H NMR(400 MHz,Chloroform-d)δ8.01–7.93(m,1H),7.73–7.66(m,1H),7.60–7.48(m,3H),7.41(s,5H),7.38– 7.28(m,5H),7.20(dd,J＝5.1,2.0Hz,3H),7.04(dd,J＝6.8,2.7Hz,2H),6.99(s,1H),5.33(s,2H),5.18 (t,J＝6.0Hz,1H),5.09(s,2H),4.90(d,J＝12.1Hz,1H),4.74(d,J＝12.2Hz,1H),4.62(dt,J＝9.2,4.8 Hz,1H),3.19(q,J＝6.6Hz,2H),3.11–2.85(m,4H),2.05–1.89(m,2H).

Synthesis of Compound 8c

Compound 18c (38.8mg, 0.1mmol, 1eq) was dissolved in DMSO, compound 19c (27.3mg, 0.11 mmol, 1.1eq), copper sulfate (5mg,0.02mmol,0.2eq) and sodium ascorbate (10mg,0.05mmol,0.5eq) were added, reacted overnight at room temperature, diluted with ethyl acetate, washed with 10% brine, dried, concentrated, and column chromatographed (dichloromethane/methanol ═ 20: 1) to give pure compound8c (51mg, 80% yield)¹H NMR(400MHz,Chloroform-d)δ8.04–7.98(m,1H),7.80– 7.75(m,1H),7.67–7.55(m,2H),7.37–7.27(m,4H),7.22–7.15(m,2H),6.59(d,J＝8.5Hz,1H),5.01 –4.87(m,2H),4.58(ddd,J＝8.7,6.0,5.0Hz,1H),4.30(t,J＝7.0Hz,2H),3.38–3.23(m,2H),3.12(q, J＝6.6Hz,2H),1.88(p,J＝7.1Hz,2H),1.68–1.55(m,2H),1.43(s,9H)。

Experimental example 1

This experimental example further examined the activity of the compound pseudomonas aeruginosa metabolite and derivative thereof obtained in the above example. The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

LB solid medium (1L): 5g of yeast extract, 10g of tryptone, 10g of NaCl and 20g of agarose.

LB liquid medium (1L): 5g of yeast extract, 10g of tryptone and 10g of NaCl.

M9 liquid medium (1L): na (Na)₂HPO₄·12H₂O 17.1g、KH₂PO₄ 3g、NaCl 0.5g、NH₄Cl 1g, glucose 4g, MgSO₄ 0.24g、CaCl₂ 0.011g。

M9 metal-deficient medium (1L): na (Na)₂HPO₄·12H₂O 17.1g、KH₂PO₄ 3g、NaCl 0.5g、NH₄Cl 1g and glucose 4 g.

VBMM medium (1L): MgSO (MgSO)₄0.04g, 2g of citric acid and KH₂PO₄ 10g、NaNH₄HPO₄·4H₂O 3.5g。

CDM metal-deficient medium (1L): 4g of glucose, 0.48g of L-cysteine, 2.4g of L-aspartic acid, 2.4g of L-glutamic acid, 2.4g of proline, 0.36g of arginine, 2.4g of glycine, 0.48g of histidine, 0.6g of lysine, 2.4g of serine, 0.48g of valine, 2.4g of threonine, and 2.4g of alanine2.4g, isoleucine 0.6g, leucine 0.6g, tryptophan 0.06g, methionine 0.18g, tyrosine 0.18g, phenylalanine 0.2g, Na₂HPO₄ 5.7g、NH₄SO₄ 6.84g、KH₂PO₄1.34g, 0.01194g nicotinic acid, 0.0006g thiamine nicotinate, 0.002g calcium pantothenate and 0.0001g biotin.

CDM medium (1L): adding MgSO (MgSO) into CDM metal-deficient culture medium₄·7H₂O 0.247g。

1. (S, S, S) -pseudoplaline and its stereochemical isomer (R, S, S) -pseudoplaline influence on growth of pseudomonas aeruginosa in metal-deficient environment

The specific method comprises the following steps: pseudomonas aeruginosa was inoculated on LB solid medium plate and cultured overnight at 37 ℃. Single colonies were picked from the plate, inoculated into LB liquid medium, and cultured overnight at 37 ℃ with shaking at 200 rpm. 1mL of overnight culture was centrifuged, and the cells were washed 2 times with PBS buffer and then resuspended in 1mL of PBS buffer. Taking 1 mu L of the resuspended bacterial liquid, inoculating the bacterial liquid into 100 mu L M9 of metal-deficient culture medium, respectively adding (S, S, S) -pseudocaline or a stereochemical isomer (R, S, S) -pseudocaline with the final concentration of 100 mu M, taking any pseudocaline not provided as a control group, adding the pseudocaline into a 96-well cell culture plate, and placing the cell culture plate in a TECAN F200PRO microplate reader for culture at 37 ℃ and 200rpm in a shaking manner. Reading the light absorption value (OD) of the bacterial liquid at 600nm every other hour₆₀₀) OD in terms of turbidity of bacterial liquid₆₀₀Reflecting the growth of the bacteria, the results are shown in Table 1. The results show that the (S, S, S) -pseudoephedrine can obviously promote the growth of the pseudomonas aeruginosa in the metal-deficient culture medium, and the isomer (R, S, S) -pseudoephedrine has no promotion effect on the growth of the pseudomonas aeruginosa and is not different from a control group, thereby indicating that the (S, S, S) -pseudoephedrine is a bioactive configuration.

Table 1: three groups of experimental results OD₆₀₀Percentage of (2)

2. Effect of Pseudoplaine on the growth of Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus in a Metal deficient Environment

The specific method is substantially the same as described above. Taking 1 mu L of the resuspended escherichia coli liquid to inoculate into 100 mu L M9 of the metal-deficient culture medium, inoculating 1 mu L of the resuspended acinetobacter baumannii liquid or staphylococcus aureus liquid into 100 mu L of the CDM metal-deficient culture medium, respectively adding pseudotropine with the final concentration of 100 mu M, and taking any pseudotropine not provided as a control group, wherein the detection method is the same as the above. The results show that pseudotropine can also significantly promote the growth of escherichia coli, acinetobacter baumannii and staphylococcus aureus in a metal-deficient culture medium.

3. Effect of Pseudoplaine on the growth of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus in a metal ion-rich environment

Compared with study 1, the only difference was that the resuspended pellet was inoculated into VBMM or LB medium without metal ions. The results show that pseudopaline has no effect on the growth of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus in a metal ion-rich medium. Indicating that pseudoplaline can assist in transporting trace metal elements in environments of pseudomonas aeruginosa, escherichia coli and acinetobacter baumannii in environments lacking metal ions.

4. Effect of pseudoplaine on zinc ion transport by pseudomonas aeruginosa

The specific method comprises the following steps: refer to the technology of Mastropasuqua. The pseudomonas aeruginosa liquid cultured overnight in 1mL of LB was centrifuged, washed twice with PBS, and then resuspended in 1mL of PBS. 100 μ L of the resuspended suspension was inoculated into 10mL of a suspension containing 10 μ M ZnSO₄And 50. mu.M EDTA in VBMM medium, 100. mu.M pseudoephedrine was added to the experimental group, and no pseudoephedrine was added to the control group. After shaking culture at 37 ℃ and 200rpm for 18 hours, the two groups were centrifuged at 8000rpm for 5min to collect the bacterial liquid. The cells were washed 2 times with 10mL of PBS buffer containing 1. mu.M EDTA and once with 10mL of PBS buffer. Enzyme-linked immunosorbent assay (ELISA) for detecting bacterial liquid OD (origin-destination) of bacteria₆₀₀After that, the supernatant was discarded by centrifugation, and the cells were dried at 95 ℃ overnight. The dried cells were digested by heating with 5mL of concentrated nitric acid at 140 ℃ for 3 hours, and then cooled to room temperature and diluted with 15mL of water.Determination of the content, OD, of Zinc ions by inductively coupled plasma Mass Spectrometry₆₀₀For Zn in cells²⁺And carrying out normalization treatment on the concentration. The experimental result shows that compared with the control group without pseudocaline, the pseudomonas aeruginosa intracellular Zn in the pseudocaline added experimental group is added²⁺The concentration content is improved by 43 percent, which shows that pseudopaline can help pseudomonas aeruginosa to transport zinc ions in the environment of zinc ion deficiency.

5. Effect of Pseudoplaine on the transcriptional level of the Pseudoplaine Synthesis operon in Pseudomonas aeruginosa

The specific method comprises the following steps: first, the promoter region of the pseudocaline operon was amplified using primers pPA4837F (5'-GGAATTCGCGCCAGGGTGCGGCTGA-3') and pPA4837R (5'-CGCAAGCTTGGGAAATCGCACCAGAAAAGAA-3'), and the resulting fragment was digested with EcoRI and HindIII, inserted into plasmid pPROBE-AT ' containing the GFP reporter gene, to construct recombinant plasmid pPA4837:: GFP.^[8]The recombinant plasmid is chemically transformed into pseudomonas aeruginosa PAO1 to construct a strain PAO1/pPA4837: gfp. The overnight cultured PAO1/pPA4837 in 1mL LB was centrifuged and washed twice with PBS and resuspended in 1mL PBS. Inoculating 1 μ L of the resuspended bacterial liquid into 100 μ L of VBMM culture medium, adding pseudoephedrine with final concentration of 100 μ M into experimental group, and adding pseudoephedrine or 5 μ M ZnSO into control group₄And the cells were added to a 96-well cell culture plate and cultured in a TECAN F200PRO microplate reader at 37 ℃ and 200rpm with shaking. Reading GFP green fluorescence (485nm excitation, 535nm emission) and absorbance (OD) of bacterial liquid at 600nm every hour₆₀₀) Using OD₆₀₀Normalization of GFP fluorescence results to GFP/OD₆₀₀In response to the transcriptional level of the pseudocaline operon. The experimental result shows that pseudocaline or zinc ions can inhibit the transcription level of a pseudocaline operon in pseudomonas aeruginosa, and the pseudocaline can play a feedback inhibition role in the synthesis of the pseudocaline.

In this experimental example, a schematic diagram of the effect of pseudoperanine on the growth of Pseudomonas aeruginosa is shown in FIG. 23. Specifically, FIG. 23A illustrates that Pseudomonas aeruginosa promotes the growth of Pseudomonas aeruginosa but that (R, S, S) -Pseudomonas aeruginosa does not promote the growth of Pseudomonas aeruginosa in M9 medium lacking metal ions; FIG. 23B illustrates the concentration dependence of pseudocaline on the growth-promoting effect of Pseudomonas aeruginosa in M9 medium lacking metal ions; FIG. 23C illustrates that pseudopropanine can help Pseudomonas aeruginosa transport zinc ions in VBMM medium supplemented with 10 micromolar zinc sulfate and 50 micromolar EDTA; FIG. 23D illustrates that zinc ions and pseudocaline are able to inhibit the expression of the pseudocaline synthon in VBMM medium.

In this experimental example, a schematic diagram of the influence of pseudopinane on the growth of escherichia coli, staphylococcus aureus and acinetobacter baumannii in the environment lacking metal ions is shown in fig. 24; a schematic diagram of the effect of pseudoplaine on the growth of E.coli, S.aureus and A.baumannii in a metal-free environment is shown in FIG. 25.

Experimental example 2

1. The influence of the pseudoplasine derivatives 2b,2c and 2d on the growth of the pseudomonas aeruginosa in the metal-deficient environment is tested in the embodiment, and the specific test method is consistent with that of the pseudoplasine, but the pseudoplasine is replaced by the pseudoplasine derivatives 2b,2c and 2 d. The results of the experiment are shown in table 2.

Table 2: experimental results OD₆₀₀Percentage of (2)

2. The influence of pseudoplaline derivatives 2b,2c and 2d on the growth of escherichia coli, acinetobacter baumannii and staphylococcus aureus in a metal-deficient environment is tested in the embodiment, and the specific test method is consistent with that of pseudoplaline, except that the pseudoplaline is replaced by the pseudoplaline derivatives 2b,2c and 2 d. The results show that the pseudoplaline derivatives 2b,2c and 2d can also significantly promote the growth of escherichia coli, acinetobacter baumannii and staphylococcus aureus in a metal-deficient culture medium, wherein 2b is basically consistent with the pseudoplaline activity.

3. This example tests the effect of Pseudopaline derivatives 2b,2c and 2d on the growth of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus in a metal ion rich environment. The method is identical to the steps described above. The results show that pseudoplaline derivatives 2b,2c and 2d have no effect on the growth of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus in metal ion rich media. Indicating that pseudoplaline derivatives 2b,2c and 2d can assist in transporting trace metal elements in environments of pseudomonas aeruginosa, escherichia coli and acinetobacter baumannii in environments lacking metal ions.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A pseudomonas aeruginosa metabolite and derivatives thereof, characterized by comprising a structure shown in general formula I:

in the general formula I, R₁、R₂、R₃Independently of one another represent-H or- (CH)₂)m-NH₂(ii) a Wherein m represents an integer of 1-10;

x represents C or N, preferably C.

2. Pseudomonas aeruginosa metabolite and derivatives thereof according to claim 1, characterized in that R is₁、R₂、R₃Any two groups in (A) each independently represent- (CH)₂)m-NH₂And the remaining one group represents H;

or, said R₁、R₂、R₃Any one of the groups represents- (CH)₂)m-NH₂And the remaining two groups each represent H;

or, said R₁、R₂、R₃All represent H.

3. Pseudomonas aeruginosa metabolite and derivatives thereof according to claim 1 or 2, characterized in that X is C, R₁Represents- (CH)₂)m-NH₂，R₂、R₃All represent H.

4. A pseudomonas aeruginosa metabolite and derivatives thereof according to claim 1 characterised by a compound selected from the following structures:

5. the method for synthesizing Pseudomonas aeruginosa metabolites and derivatives thereof according to any one of claims 1 to 4, wherein in the general formula I, R is₁、R₂、R₃All represent H; the synthesis method comprises the following steps:

the synthesis method comprises the following specific steps:

(1) under the alkaline condition, benzyl chloroformate protection of imidazole is firstly carried out on the compound 17a, and then benzyl protection is carried out on carboxyl to obtain a compound 14 a;

(2) removing a protecting group of the compound 14a under an acidic condition, and then protecting with in-situ o-nitrobenzenesulfonyl chloride to obtain a compound 8 a;

(3) protecting hydroxyl of the compound 12 and tert-butyldimethylsilyl chloride under the action of imidazole, removing tert-butyloxycarbonyl protecting groups under the action of trimethylsilyl trifluoromethanesulfonate and 2, 6-dimethylpyridine to obtain free amino, and reacting the amino with o-nitrobenzenesulfonyl chloride by a one-pot method to generate a compound 10;

(4) the compound 10 reacts with a compound 11 under the action of tris (dibenzylideneacetone) dipalladium and (1R,2R) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand to introduce cyclopentenyl on amino, and then hydrofluoric acid pyridine is added in the reaction to remove silicon protecting group to obtain a compound 15 a;

(5) the compound 15a and the compound 8a generate a compound 7a under the action of ethyl azodicarboxylate and pyridine diphenylphosphine;

(6) after the compound 7a is subjected to oxidation cutting reaction, performing aldehyde group oxidation reaction with a Jones reagent to obtain a compound 16 a;

(7) and (3) hydrolyzing the compound 16a under an acidic condition to completely remove the protecting group, and then removing the o-nitrobenzenesulfonyl protecting group under an alkaline condition to obtain a compound 2 a.

6. The method for synthesizing Pseudomonas aeruginosa metabolites and derivatives thereof according to any one of claims 1 to 4, wherein in the general formula I, R is₁Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10; r₂、R₃All represent H; the synthesis method comprises the following steps:

the synthesis method comprises the following specific steps:

(1) protecting the compound 17b by benzyl chloroformate under an alkaline condition, and protecting benzyl carboxylate to obtain a compound 18 b;

(2) performing Sonogashira coupling reaction on the compound 18b and the compound 19b to obtain a compound 20 b;

(3) hydrogenating and reducing the compound 20b to obtain a compound 21 b;

(5) the compound 22b is subjected to acid deprotection and is subjected to deprotection again to obtain a compound 8 b;

(6) protecting hydroxyl of the compound 12 and tert-butyldimethylsilyl chloride under the action of imidazole, removing tert-butyloxycarbonyl protecting groups under the action of trimethylsilyl trifluoromethanesulfonate and 2, 6-dimethylpyridine to obtain free amino, and reacting the amino with o-nitrobenzenesulfonyl chloride by a one-pot method to generate a compound 10;

(7) the compound 10 reacts with a compound 11 under the action of tris (dibenzylideneacetone) dipalladium and (1R,2R) - (-) -N, N' -bis (2-diphenylphosphino-1-naphthoyl) -1, 2-cyclohexanediamine ligand to introduce cyclopentenyl on amino, and then hydrofluoric acid pyridine is added in the reaction to remove silicon protecting group to obtain a compound 15 a;

(8) the compound 15a and a compound 8b generate a compound 7b under the action of ethyl azodicarboxylate and pyridine diphenylphosphine;

(9) after the compound 7b is subjected to oxidation cutting reaction, performing aldehyde group oxidation reaction with a Jones reagent to obtain a compound 16 b;

(10) and removing the protecting group of the compound 16b under an acidic condition, and then removing the o-nitrobenzenesulfonyl protecting group under an alkaline condition to obtain a compound 2 b.

7. The method for synthesizing Pseudomonas aeruginosa metabolites and derivatives thereof according to any one of claims 1 to 4, wherein in the general formula I, R is₂Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10, R₁、R₃All represent H; the synthesis method comprises the following steps:

the synthesis method comprises the following specific steps:

(1) the compound 17c is protected by o-nitrobenzenesulfonyl chloride and carboxyl benzyl to obtain a compound 18 c;

(2) carrying out click reaction on the compound 18c and the compound 19c under the catalysis of copper to obtain a compound 8 c;

(3) protecting hydroxyl of a compound 12 and tert-butyldimethylchlorosilane under the action of imidazole, removing a tert-butylcarboxycarbonyl protecting group under the action of trimethylsilyl trifluoromethanesulfonate and 2, 6-dimethylpyridine to obtain free amino, and reacting the amino with o-nitrobenzenesulfonyl chloride by a one-pot method to generate a compound 10;

(5) the compound 15a and the compound 8c generate a compound 7c under the action of diethyl azodicarboxylate and pyridine diphenylphosphine;

(6) after the compound 7c is subjected to oxidation cutting reaction, aldehyde group oxidation reaction is carried out on the compound and Jones reagent to obtain a compound 16 c;

(7) and (3) hydrolyzing the compound 16c under an acidic condition to remove a protecting group, and then removing o-nitrobenzenesulfonyl under an alkaline condition to obtain a compound 2 c.

8. The method for synthesizing Pseudomonas aeruginosa metabolites and derivatives thereof according to any one of claims 1 to 4, wherein in the general formula I, R is₃Represents- (CH)₂)m-NH₂Wherein m represents an integer of 1 to 10, R₁、R₂All represent H; the synthesis method comprises the following steps:

the synthesis method comprises the following specific steps:

(3) hydrogenating reduction of the compound 20d to give a compound 21 d;

(5) deprotecting the compound 22d under an acidic condition, and re-protecting the o-nitrobenzenesulfonyl chloride to obtain a compound 8 d;

(6) the compound 8d and the compound 15a generate a compound 7d under the action of diethyl azodicarboxylate and pyridine diphenylphosphine;

(2) after the compound 7d is subjected to oxidation cutting reaction, aldehyde group oxidation reaction is carried out on the compound and a Jones reagent to obtain a compound 16 d;

(3) and (3) hydrolyzing the compound 16d under an acidic condition to remove a protecting group, and then removing o-nitrobenzenesulfonyl under an alkaline condition to obtain a compound 2 d.

9. Use of the Pseudomonas aeruginosa metabolite and the derivative thereof according to any one of claims 1 to 4 in the preparation of a methemomycin antibiotic.

10. The use according to claim 9, characterized in that the metabolites of pseudomonas aeruginosa and their derivatives are reacted with an antibiotic containing an ester group to form an amide bond; the antibiotic containing an ester group is preferably a beta-lactam antibiotic.