A benzo[nitrogen-containing heterocyclic compound, its preparation method and application

Abstract

This invention belongs to the field of microbiology and novel pharmaceuticals, specifically relating to a benzo[a]-nitrogenous heterocyclic compound, particularly a novel multi-target active benzo[a]-nitrogenous heterocyclic compound derived from marine micromonospora, and further disclosing its preparation method and applications. This invention isolated and screened two novel nitrogen-containing small molecule compounds, FWYZ52-D and FWYZ52-E, from the preserved marine actinomycete micromonospora FIMYZ52. Compound FWYZ52-D shows promise as a multi-target neuromodulator, while compound FWYZ52-E shows promise as a multi-target antitumor / immunomodulatory active substance.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and novel medicines, specifically relating to a benzo[a] nitrogen-containing heterocyclic compound, particularly a novel multi-target active benzo[a] nitrogen-containing heterocyclic compound derived from marine micromonospora, and further disclosing its preparation method and application. Background Technology

[0002] Studies show that Micromonospora can produce a wide variety of bioactive substances. For example, macrolides produced by Micromonospora mainly include Levantilide A and Levantilide C, which exhibit moderate anti-tumor cell proliferation activity against various tumor cell lines such as human leukemia tumor (HL-60), human breast tumor (MDA-MB-231), human colon cancer (SW620), and human liver cancer (SMMC7721); bafilomycin, which can be used as an insecticide to inhibit the growth of malignant cells; Rosamicin, which has broad-spectrum antibacterial activity; and juvenimicin C, which can enhance the activity of QR1 enzyme and the folding level of glutathione.

[0003] Therefore, the field looks forward to discovering new types of active compounds, especially nitrogen-containing active substances, from micromonospora, which will help us to continue to expand our current drug reserves and provide protection for human health. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a benzo[a] nitrogen-containing heterocyclic compound derived from marine micromonospora, wherein the benzo[a] nitrogen-containing heterocyclic compound has multi-target neuromodulatory activity; The second technical problem to be solved by the present invention is to provide a method for preparing and applying the above-mentioned benzo[a] nitrogen-containing heterocyclic compounds derived from marine micromonospora.

[0005] To address the aforementioned technical problems, the present invention provides a benzo[a] nitrogen-containing heterocyclic compound, including compound FWYZ52-D having the structure shown in formula (I) below, and compound FWYZ52-E having the structure shown in formula (II) below: .

[0006] The present invention also discloses a method for preparing the benzo[a] nitrogen-containing heterocyclic compound as described above, comprising the step of inoculating the micromonospora strain FIMYZ52 into a suitable fermentation medium for fermentation culture; The micromonosporal strain FIMYZ52 is classified as Micromonospora. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24479.

[0007] Specifically, the preparation method of the benzo[a] nitrogen-containing heterocyclic compound includes the following steps: (1) Seed culture: The preserved micromonosporal strain FIMYZ52 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; (2) Fermentation broth culture: The seed liquid is transferred to the fermentation medium for fermentation culture to obtain the fermentation product containing the desired benzo[a] nitrogen-containing heterocyclic compound.

[0008] Specifically, in the preparation method of the benzo[a] nitrogen-containing heterocyclic compound, in step (1), the seed culture medium comprises: 1.0-3.0 wt% maltodextrin, 0.3-0.8 wt% corn steep liquor powder, 0.3-0.8 wt% glucose, 0.3-0.8 wt% soybean meal, and 0.03-0.08 wt% potassium chloride. 2HPO4, 0.03-0.08wt% MgSO4 • 7H2O, 0.03-0.08wt% NaCl, 0.03-0.08wt% (NH4)2SO4, pH6.5-7.5; Preferably, the temperature for the seed culture step is 25-35℃, and the culture time is 2-3 days.

[0009] Preferably, in the method for preparing the polyhydroxy macrocyclic lactone compound, in step (1), the seed culture medium comprises: 2.0 wt% maltodextrin, 0.5 wt% corn steep liquor powder, 0.5 wt% glucose, 0.5 wt% soybean meal, 0.05 wt% K2HPO4, and 0.05 wt% MgSO4. • 7H2O, 0.05wt% NaCl, 0.05wt% (NH4)2SO4, pH6.5-7.5.

[0010] Specifically, in the preparation method of the benzo[a] nitrogen-containing heterocyclic compound, in step (2), the fermentation culture medium comprises: 0.5-2.0 wt% glucose, 1.0-3.0 wt% soluble starch, 0.3-0.8 wt% yeast extract, 0.3-0.8 wt% acid-hydrolyzed casein, 1.0-2.0 wt% sea salt, 0.1-0.3 wt% CaCO3, pH 6.5-7.5; Preferably, the fermentation culture step is carried out at a temperature of 25-35℃ for 3-5 days.

[0011] Preferably, the fermentation medium comprises: 1.0 wt% glucose, 2.0 wt% soluble starch, 0.5 wt% yeast extract, 0.5 wt% acid-hydrolyzed casein, 1.5 wt% sea salt, 0.2 wt% CaCO3, and pH 6.5-7.5.

[0012] Specifically, the method for preparing the benzo[a] nitrogen-containing heterocyclic compound further includes a step of extracting the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (3) Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is extracted with an extractant, the extract is collected and evaporated to dryness to obtain crude extract A of fermentation broth; the mycelium is extracted with an alcohol solvent, the extract is collected and evaporated to dryness to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired benzo[a] nitrogen-containing heterocyclic compounds; Preferably, the extractant comprises ethyl acetate; Preferably, the alcohol solvent includes ethanol; or, (3') Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and then desorbed with desorption solution to obtain crude extract A of fermentation broth; the mycelium is added to alcohol solvent for extraction, and the extract is collected and concentrated to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired benzo[a] nitrogen-containing heterocyclic compounds; Preferably, the macroporous resin includes at least one of D101, AB-8, HZ816, Amberlite XAD-4, XAD-8, XAD-16 or HP20 resin; Preferably, the desorption solution includes ethanol; Preferably, the alcohol solvent includes ethanol or methanol.

[0013] Specifically, the method for preparing the benzo[a] nitrogen-containing heterocyclic compound further includes a step of separating the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (4) Separation: The crude extract obtained in step (3) and / or (3') is subjected to normal-phase silica gel column chromatography, eluted with a dichloromethane-methanol solvent with a volume ratio of 99:1-90:10, and the dichloromethane-methanol solvent fractions with a volume ratio of 95:5-94:6 are collected to obtain the eluent containing compound FWYZ52-D; then subjected to C 18Reversed-phase column chromatography was performed, with methanol-water solvent at a volume ratio of 10%-100% for elution. The eluent containing FWYZ52-D was collected in the methanol content range of 45%-50% by high-performance liquid chromatography. or, (4') Separation: The crude extract obtained in step (3) and / or (3') is separated using C 18 Reversed-phase column chromatography was performed, with elution using a methanol-water solvent at a volume ratio of 10%-100%. High-performance liquid chromatography (HPLC) was used to detect the elution, and the eluent containing FWYZ52-E was collected in the methanol content range of 40%-60%. Then, normal-phase silica gel column chromatography was performed, with elution using a dichloromethane-methanol solvent at a volume ratio of 99:1-90:10. Fractions in the dichloromethane-methanol solvent range of 95:5-94:6 were collected, yielding the eluent containing compound FWYZ52-E.

[0014] Specifically, the method for preparing the benzo[a] nitrogen-containing heterocyclic compound further includes a purification step of the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (5) Purification: Take the eluent containing FWYZ52-D collected in step (4) to prepare type C 18 The mixture was processed by reversed-phase high-performance liquid chromatography and isocratic elution with a 45% (v / v) methanol-water solution to obtain the pure compound FWYZ52-D. or, (5') Purification: Take the eluent containing FWYZ52-E collected in step (4'), perform LH-20 gel column chromatography, and then use preparative C 18 The compound was processed by reversed-phase high-performance liquid chromatography and isocratic elution with acetonitrile-water solution at a volume ratio of 25% to obtain the pure compound FWYZ52-E.

[0015] The present invention also discloses the use of the benzo[a] nitrogen-containing heterocyclic compounds in the preparation of multi-target neuromodulators, multi-target antitumor drugs, or multi-target immunomodulators.

[0016] This invention also discloses the use of a micromonosporal strain FIMYZ52 for the fermentation preparation of the aforementioned benzo[a]-nitrogenous heterocyclic compound, wherein the micromonosporal strain FIMYZ52 is classified as Micromonospora. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24479.

[0017] This invention utilizes preserved marine actinomycetes Micromonospora (Micromonospora spp.) MicromonosporaTwo novel nitrogen-containing small molecule compounds, FWYZ52-D and FWYZ52-E, were isolated and screened from FIMYZ52 sp. These compounds are nitrogen-containing compounds and are analogs of indole and indole-like compounds. In particular, the nitrogen-containing compound FWYZ52-D exhibits inhibitory activity against potential tumor cells, providing a lead compound for the research and development of new nitrogen-containing indole and indole-like active drugs, and is of significant value for the development and utilization of China's marine drug resources.

[0018] This invention further validates the activities of compounds FWYZ52-D and FWYZ52-E. Compound FWYZ52-D shows promise as a multi-target neuromodulator, while compound FWYZ52-E shows promise as a multi-target antitumor / immunomodulator.

[0019] This invention provides small monosporous bacteria ( Micromonospora sp.) This invention discloses the preparation method and potential applications of novel nitrogen-containing small molecule compounds FWYZ52-D and FWYZ52-E, extracted and isolated from the fermentation broth of FIMYZ52. The advantages of this invention are: it provides lead compounds for the research and development of new drugs and has significant value for the development and utilization of China's marine drug resources. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Picture 1 This is the high-resolution mass spectrum of the nitrogen-containing compound FWYZ52-D in this invention; Picture 2 This is the proton nuclear magnetic resonance image of the nitrogen-containing compound FWYZ52-D in this invention. 1 H spectrum; Picture 3 This is the carbon NMR spectrum of the nitrogen-containing compound FWYZ52-D in this invention. 13 C spectrum; Picture 4 It is the nitrogen-containing compound FWYZ52-D in this invention. 1 H- 1 HCOSY related graphs; Picture 5 This is the HSQC spectrum of the nitrogen-containing compound FWYZ52-D in this invention; Picture 6 This is the HMBC spectrum of the nitrogen-containing compound FWYZ52-D in this invention; Picture 7 This is the high-resolution mass spectrum of the nitrogen-containing compound FWYZ52-E in this invention; Picture 8 This is the proton nuclear magnetic resonance image of the nitrogen-containing compound FWYZ52-E in this invention. 1 H spectrum; Picture 9 This is the carbon NMR spectrum of the nitrogen-containing compound FWYZ52-E in this invention. 13 C spectrum; Picture 10 It is the nitrogen-containing compound FWYZ52-E in this invention. 1 H- 1 HCOSY related graphs; Picture 11 This is the HSQC spectrum of the nitrogen-containing compound FWYZ52-E in this invention; Picture 12 This is the HMBC spectrum of the nitrogen-containing compound FWYZ52-E in this invention. Detailed Implementation

[0021] In the following embodiments of the present invention, a nitrogen-containing heterocyclic compound derived from marine micromonads is provided, including compound FWYZ52-D having the structure shown in formula (I) and compound FWYZ52-E having the structure shown in formula (II): .

[0022] In the following embodiments of the present invention, a method for preparing the nitrogen-containing heterocyclic compound as described above is also disclosed, comprising the step of inoculating the micromonospora strain FIMYZ52 into a suitable fermentation medium for fermentation culture; The micromonosporal strain FIMYZ52 is classified as Micromonospora. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24479.

[0023] In the following embodiments of the present invention, a method using Micromonospora is also disclosed. Micromonospora sp The method for preparing the nitrogen-containing heterocyclic compound by FIMYZ52 includes the following steps: (1) Seed culture: The preserved micromonosporal strain FIMYZ52 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; The seed culture medium comprises: 1.0-3.0 wt% maltodextrin, 0.3-0.8 wt% corn steep liquor powder, 0.3-0.8 wt% glucose, 0.3-0.8 wt% soybean meal, 0.03-0.08 wt% K₂HPO₄, and 0.03-0.08 wt% MgSO₄. •7H2O, 0.03-0.08wt% NaCl, 0.03-0.08wt% (NH4)2SO4, pH6.5-7.5; The temperature for the seed culture step is 25-35℃, and the culture time is 2-3 days.

[0024] (2) Fermentation broth culture: The seed liquid is transferred to the fermentation medium for fermentation culture to obtain the fermentation product containing the desired polyhydroxy macrocyclic lactone compound; The inoculation volume ratio of the seed liquid is 1:5-1:15, preferably 1:10; The fermentation medium comprises: 0.5-2.0 wt% glucose, 1.0-3.0 wt% soluble starch, 0.3-0.8 wt% yeast extract, 0.3-0.8 wt% acid-hydrolyzed casein, 1.0-2.0 wt% sea salt, 0.1-0.3 wt% CaCO3, pH 6.5-7.5; The fermentation culture step is carried out at a temperature of 25-35℃ for 3-5 days. (3) Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is extracted with an extractant, the extract is collected and evaporated to dryness to obtain crude extract A of fermentation broth; the mycelium is extracted with an alcohol solvent, the extract is collected and evaporated to dryness to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired polyhydroxy macrocyclic lactone compound; And / or, (3') Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and then desorbed with desorption solution to obtain crude extract A of fermentation broth; the mycelium is added to alcohol solvent for extraction, and the extract is collected and concentrated to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired polyhydroxy macrocyclic lactone compound; (4) Separation: The crude extract obtained in step (3) and / or (3') is subjected to normal-phase silica gel column chromatography with gradient elution in a dichloromethane-methanol solvent with a volume ratio of 99:1-90:10 (elution at a gradient concentration of 1% v / v, the elution volume is based on the complete elution of the separated substances). The dichloromethane-methanol solvent fractions with a volume ratio of 95:5-94:6 (i.e., the 95%-96% v / v dichloromethane content fractions) are collected separately, and the eluent containing compound FWYZ52-D is obtained; then subjected to C 18Reversed-phase column chromatography was performed using a gradient elution of methanol-water solvent with a volume ratio of 10%-100% (elution was performed with a gradient concentration of 1% v / v, and the elution volume was based on the complete separation of the substances). The eluent containing FWYZ52-D was collected in the methanol content range of 45%-50% by high performance liquid chromatography. or, (4') Separation: The crude extract obtained in step (3) and / or (3') is separated using C 18 Reversed-phase column chromatography was performed using a gradient elution with methanol-water solvent at a volume ratio of 10%-100% (elution at a gradient concentration of 1% v / v, with the elution volume determined by complete elution of the separated substances). High-performance liquid chromatography (HPLC) was used to detect the elution fraction containing FWYZ52-E in the methanol content range of 40%-60%. Normal-phase silica gel column chromatography was then performed using a gradient elution with dichloromethane-methanol solvent at a volume ratio of 99:1-90:10 (elution at a gradient concentration of 1% v / v, with the elution volume determined by complete elution of the separated substances). Fractions in the dichloromethane-methanol solvent range of 95:5-94:6 (i.e., the 95%-96% v / v dichloromethane content range) were collected, yielding the eluent containing compound FWYZ52-E. (5) Purification: Take the eluent containing FWYZ52-D collected in step (4) to prepare type C 18 The mixture was processed by reversed-phase high-performance liquid chromatography and isocratic elution with a 45% (v / v) methanol-water solution to obtain the pure compound FWYZ52-D. or, (5') Purification: Take the eluent containing FWYZ52-E collected in step (4'), perform LH-20 gel column chromatography, and then use preparative C 18 The compound was processed by reversed-phase high-performance liquid chromatography and isocratic elution with acetonitrile-water solution at a volume ratio of 25% to obtain the pure compound FWYZ52-E.

[0025] In the following embodiments of the present invention, the high-performance liquid chromatography detection step employs a Welch reverse C-phase flow. 18 (4.6* 250mm) analytical column, gradient elution with 10-100% v / v methanol-water solution, elution time 60 min, flow rate 1 ml / min, column temperature 42℃.

[0026] In the following embodiments of the present invention, the preparative C 18 The reversed-phase high-performance liquid chromatography (RP-HPLC) detection procedure was performed using a Welch Materials XB-C18 column (20 * 250 mm) with isocratic elution using a methanol-water solution of a selected concentration.

[0027] The present invention also discloses the active use of compound FWYZ52-D as a multi-target neuromodulator and the active use of compound FWYZ52-E as a multi-target antitumor / immunomodulator in the following embodiments.

[0028] The marine micromonospora used in the following embodiments of the present invention Micromonospora sp FIMYZ52, identified by 16S rRNA gene analysis, belongs to the genus Micromonospora within the actinomycetes, and is classified as Micromonospora actinomycetes. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24479, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] In the following embodiments of the present invention, micromonospora strains ( Micromonospora sp. Two novel nitrogen-containing heterocyclic compounds, FWYZ52-D and FWYZ52-E, were extracted and isolated from the FIMYZ52 fermentation broth. Example 1

[0030] This embodiment is based on a preserved Micromonospora strain ( Micromonospora sp. A novel macrocyclic lactone compound, FWYZ52-D, was prepared by fermentation of FIMYZ52.

[0031] Preserved marine micromonospora Micromonospora sp FIMYZ52 was inoculated onto ISP2 agar slant culture and then inoculated into liquid seed culture medium. The temperature was controlled at 28℃. After 2 days of culture, the resulting seed culture was inoculated into fermentation medium at a volume ratio of 1:10 and cultured with shaking at 28℃ for 5 days. The fermentation broth was then collected.

[0032] The seed culture medium components (wt%) are: 2.0% maltodextrin, 0.5% corn steep liquor powder, 0.5% glucose, 0.5% soybean meal, 0.05% K₂HPO₄, and 0.05% MgSO₄. • 7H2O, 0.05% NaCl, 0.05% (NH4)2SO4, pH7.0.

[0033] The fermentation medium components (wt%) were: 1.0% glucose, 2.0% soluble starch, 0.5% yeast extract, 0.5% acid-hydrolyzed casein, 1.5% sea salt, 0.2% CaCO3, pH 7.0.

[0034] The fermentation broth was collected and the fermentation broth and mycelium were collected separately through solid-liquid separation. The fermentation broth was extracted three times with ethyl acetate. The ethyl acetate extract was collected and evaporated to dryness using a rotary evaporator to obtain crude fermentation broth extract A. The separated mycelium was extracted three times with alcohol. The alcohol extract was collected and evaporated to dryness using a rotary evaporator to obtain crude mycelium extract B. The crude fermentation broth extract A and crude mycelium extract B were combined to obtain the crude extract.

[0035] The crude extract was subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane-methanol solvent at a volume ratio (v / v) of 99:1-90:10, using a 1% v / v gradient concentration. The elution volume was determined based on the complete elution of the separated substances. Fractions in the dichloromethane-methanol solvent section (i.e., the 95%-94% v / v dichloromethane content section) at a volume ratio of 95:5-94:6 were collected, yielding the eluent containing the nitrogen-containing compound FWYZ52-D. This eluent was then subjected to C... 18 Reversed-phase column chromatography was performed, eluting with a methanol-water solvent at a volume ratio of 10%-100% using a 1% v / v gradient concentration. Detection was performed by high-performance liquid chromatography (HPLC). The eluent containing FWYZ52-D was collected in the methanol concentration range of 45%-50%. This was followed by preparative C... 18 The mixture was processed by reversed-phase high-performance liquid chromatography and eluted isocratically with a 45% (v / v) methanol-water solution to obtain pure nitrogen-containing compound FWYZ52-D with the new structure. Example 2

[0036] This embodiment is based on a preserved Micromonospora strain ( Micromonospora sp. A novel macrocyclic lactone compound, FWYZ52-E, was prepared by fermentation of FIMYZ52.

[0037] Preserved marine micromonospora Micromonospora sp FIMYZ52 was inoculated onto ISP2 agar slant culture and then inoculated into liquid seed culture medium. After culturing at 28°C for 2 days, the seed culture was inoculated into fermentation medium at a volume ratio of 1:10 and cultured with shaking at 28°C for 4 days to obtain fermentation broth.

[0038] The seed culture medium components (wt%) are: 2.0% maltodextrin, 0.5% corn steep liquor powder, 0.5% glucose, 0.5% soybean meal, 0.05% K₂HPO₄, and 0.05% MgSO₄. • 7H2O, 0.05% NaCl, 0.05% (NH4)2SO4, pH7.0.

[0039] The fermentation medium components (wt%) were: 1.0% glucose, 2.0% soluble starch, 0.5% yeast extract, 0.5% acid-hydrolyzed casein, 1.5% sea salt, 0.2% CaCO3, pH 7.0.

[0040] The fermentation broth was collected and separated into fermentation broth and mycelium through solid-liquid separation. The fermentation broth was added to HP20 macroporous resin for adsorption, with the resin-to-fermentation broth mass ratio controlled at 1:10-1:30; in this embodiment, a mass ratio of 1:20 was selected. The mixture was then loaded onto a resin column for adsorption. After adsorption, the mixture was washed with distilled water, and 10-20% ethanol was added to remove impurities. Then, it was desorbed with 100% ethanol, and the ethanol solvent was recovered to obtain crude extract A from the fermentation broth. The separated mycelium was extracted three times with ethanol, and the soaking liquid was collected and concentrated under reduced pressure to obtain crude extract B from the mycelium. The crude extracts A and B were combined to obtain the crude extract.

[0041] Take the crude extract and use C 18 Reversed-phase column chromatography was performed using a gradient elution with a methanol-water solution of 10%–100% (v / v) at a concentration gradient of 1% v / v. The elution volume was determined to ensure complete elution of the separated substances. High-performance liquid chromatography (HPLC) was used to detect the FWYZ52-E-containing eluent in the 40%–60% methanol concentration range. Next, normal-phase silica gel column chromatography was performed using a dichloromethane-methane solvent gradient elution of 99:1–90:10 (v / v) at a concentration gradient of 1% v / v. Fractional collection was conducted, and the eluent containing the nitrogen-containing compound FWYZ52-E was collected in the 95:5–94:6 (v / v) dichloromethane concentration range (i.e., the 95%–94% v / v dichloromethane concentration range). Then, fractional collection was performed using LH-20 gel column chromatography, with one tube collecting 2-3 ml at a time. High-performance liquid chromatography (HPLC) was used to detect the nitrogen-containing compound FWYZ52-E in the collection tube. Finally, preparative C... 18 The nitrogen-containing compound FWYZ52-E with the new structure was obtained by reversed-phase high-performance liquid chromatography followed by isocratic elution with 25% (v / v) acetonitrile-water solution. Example 3

[0042] This embodiment focuses on the structural analysis of the previously isolated compounds FWYZ52-D and FWYZ52-E.

[0043] In this embodiment, the structural characteristics of macrocyclic lactone compounds FWYZ52-D and FWYZ52-E were identified by MS and NMR techniques as follows.

[0044] The physicochemical properties of compound FWYZ52-D are as follows: pale yellow amorphous solid, molecular formula: C 12 H 11ClN2O2, High-resolution mass spectrometry: Measured values: m / z 251.0578[M+H] + Theoretical value: m / z 251.0582 [M+H] + Its degree of unsaturation is 8. It is soluble in organic solvents such as methanol, ethanol, ethyl acetate, and dimethyl sulfoxide.

[0045] Compound FWYZ52-D 1 1H NMR spectrum (DMSO-d6, 600MHz): δ 12.13 (s, 1H), 8.45 (s, 1H), 8.17 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.22 (dd, J = 8.5, 2.0 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 1.91 (s, 3H).

[0046] Compound FWYZ52-D 13 C NMR spectrum (DMSO-d6, 150MHz): δ 190.50, 169.51, 136.91, 127.42, 124.18, 122.61, 122.43, 122.15, 114.01, 111.94, 45.71, 22.47.

[0047] The physicochemical properties of compound FWYZ52-E are as follows: pale yellow solid, molecular formula: C 14 H 18 N₂O₃, High-resolution mass spectrometry: Measured value: m / z 263.1399 [M+H] + Theoretical value: m / z 263.1391 [M+H] + Its degree of unsaturation is 7. It is soluble in organic solvents such as methanol, ethanol, ethyl acetate, and dimethyl sulfoxide.

[0048] Compound FWYZ52-E 11H NMR spectrum (DMSO-d6, 600MHz): δ 8.19 (s, 1H), 7.40 (s, 1H), 7.32 (s, 1H), 5.72 (d, J = 5.4 Hz, 1H), 5.45 (s, 1H), 5.18 (s, 1H), 4.36 (s, 1H), 3.94 (m, 1H), 3.81 (d, J = 5.1 Hz, 1H), 3.30 (s, 6H), 2.29 (s, 3H), 2.26 (s, 3H), 1.29 (d, J = 6.4 Hz, 3H).

[0049] Compound FWYZ52-E 13 C NMR spectrum (DMSO-d6, 150MHz): δ 141.11, 135.94, 131.37, 130.40, 119.64, 111.06, 88.95, 79.70, 74.50, 73.08, 20.12, 19.84, 19.15.

[0050] In addition, this invention also determined various nuclear magnetic resonance spectra of compounds FWYZ52-D and FWYZ52-E, as shown in the appendix. Picture 1-12 The relevant test data are shown in Tables 1-2 below.

[0051]

[0052]

[0053] This determined the assignment of all carbon and hydrogen atoms in compounds FWYZ52-D and FWYZ52-E, as well as their chemical structures, confirming them as novel nitrogen-containing compounds with the following structural formula: FWYZ52-D: ; FWYZ52-E: ; Example 4

[0054] This embodiment uses virtual screening and activity prediction based on the isolated nitrogen-containing compounds FWYZ52-D and FWYZ52-E.

[0055] This embodiment first performs virtual target screening and bioactivity prediction based on the isolated nitrogen-containing compounds FWYZ52-D and FWYZ52-E. Specifically, the PPB3 multi-target prediction tool based on ligand similarity and deep neural network (DNN) is used, with seven fingerprint models for prediction: ECFP4 (common extended connection fingerprint with radius 2); ECFP6 (extended connection fingerprint with radius 3); MHFP6 (molecular hash-based fingerprint); RDKit (fingerprint generated by RDKit software); Layered (layered structure fingerprint); AtomPair (atom pair fingerprint); and Fused (ECFP4+MHFP6) (optimal fusion fingerprint with the highest accuracy). The high-probability target types are obtained, and then the bioactivity of the corresponding targets is predicted. The final output is a result with a threshold greater than 0.1, where a larger threshold (0-1) indicates a higher probability.

[0056] The results showed that the compound FWYZ52-D, through virtual screening, identified potential targets including (threshold range 0.11-0.56): Melatonin receptor, GABA-A receptor; anion channel, Serotonin 6 (5-HT6) receptor, Group X secretory phospholipase A2, Regulator of G-protein signaling 12, Serotonin transporter, Ras-related protein Rab-9A, Beta-lactamase AmpC, Solute carrier organic anion transporter family member 1B3, Solute carrier organic anion transporter family member 1B1, Nonstructural protein 1, and LIM domain kinase 1.

[0057] The results showed that the compound FWYZ52-E, through virtual screening, identified potential targets including (threshold range 0.18-0.73): L1210, Prelamin-A / C, Hypoxia-inducible factor 1 alpha, Nuclear receptor ROR-gamma, Thrombopoietin, Tyrosine-protein kinase SYK, KB, Nuclear factor NF-kappa-B p105 subunit, Microtubule-associated protein tau, Ras-related protein Rab-9A, Solute carrier organic anion transporter family member 1B1, Solute carrier organic anion transporter family member 1B3, Geminin, Peripheral myelin protein 22, Survival motor neuron protein, microRNA21, Aldehyde dehydrogenase 1A1, Heat shock protein HSP 90-alpha, Serine / threonine-protein kinase mTOR, and Cytochrome. P450 2C19.

[0058] Biological activity results from the novel nitrogen-chloroindole compound FWYZ52-D indicate that this compound is a potential multi-target neuromodulator, with its core activity being the synergistic improvement of sleep through melatonin and GABA-A receptors; and potential neuroprotective effects through anti-anxiety / sedative, antioxidant, and anti-inflammatory mechanisms via GABA-A and 5-HT6 receptors.

[0059] Biological activity results of the novel glyconitrogen compound FWYZ52-E indicate that it is a potential multi-target antitumor / immunomodulator with the following key characteristics: potent antitumor activity (L1210 high confidence 0.73), immunomodulatory effects (multiple inhibition of RORγt, SYK, and NF-κB), and potential hematologic and neuroprotective effects (TPO, HIF-1α, Tau).

[0060] In summary, this embodiment conducted virtual target screening for nitrogen-containing compounds FWYZ52-D and FWYZ52-E. The virtual screening results showed that compounds FWYZ52-D and FWYZ52-E exhibit different bioactivities against different targets, as well as shared common applications. Therefore, nitrogen-containing indole compound FWYZ52-D shows promise as a multi-target neuromodulator. Sugar-containing nitrogen compound FWYZ52-E shows promise as a multi-target antitumor / immunomodulator.

[0061] It should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described in this application.

Claims

1. A benzo[a] nitrogen-containing heterocyclic compound, characterized in that, This includes compound FWYZ52-D having the structure shown in formula (Ⅰ), and compound FWYZ52-E having the structure shown in formula (Ⅱ): 。 2. A method for preparing the benzo[a] nitrogen-containing heterocyclic compound as described in claim 1, characterized in that, This includes the step of inoculating the micromonosporal strain FIMYZ52 into a suitable fermentation medium for fermentation culture; The micromonosporal strain FIMYZ52 is classified as Micromonospora. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24479.

3. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to claim 2, characterized in that, Includes the following steps: (1) Seed culture: The preserved micromonosporal strain FIMYZ52 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; (2) Fermentation broth culture: The seed liquid is transferred to the fermentation medium for fermentation culture to obtain the fermentation product containing the desired benzo[a] nitrogen-containing heterocyclic compound.

4. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to claim 3, characterized in that, In step (1), the seed culture medium comprises: 1.0-3.0 wt% maltodextrin, 0.3-0.8 wt% corn steep liquor powder, 0.3-0.8 wt% glucose, 0.3-0.8 wt% soybean meal, 0.03-0.08 wt% K₂HPO₄, and 0.03-0.08 wt% MgSO₄. • 7H2O, 0.03-0.08wt% NaCl, 0.03-0.08wt% (NH4)2SO4, pH6.5-7.5; Preferably, the temperature for the seed culture step is 25-35℃, and the culture time is 2-3 days.

5. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to claim 3 or 4, characterized in that, In step (2), the fermentation medium comprises: 0.5-2.0 wt% glucose, 1.0-3.0 wt% soluble starch, 0.3-0.8 wt% yeast extract, 0.3-0.8 wt% acid-hydrolyzed casein, 1.0-2.0 wt% sea salt, 0.1-0.3 wt% CaCO3, pH 6.5-7.5; Preferably, the fermentation culture step is carried out at a temperature of 25-35℃ for 3-5 days.

6. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to any one of claims 2-5, characterized in that, The method further includes a step of extracting the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (3) Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is extracted with an extractant, the extract is collected and evaporated to dryness to obtain crude extract A of fermentation broth; the mycelium is extracted with an alcohol solvent, the extract is collected and evaporated to dryness to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired benzo[a] nitrogen-containing heterocyclic compounds; Preferably, the extractant comprises ethyl acetate; Preferably, the alcohol solvent includes ethanol; or, (3') Extraction: The collected fermentation products are separated into solid and liquid components to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and then desorbed with desorption solution to obtain crude extract A of fermentation broth; the mycelium is added to alcohol solvent for extraction, and the extract is collected and concentrated to obtain crude extract B of mycelium; the crude extract A of fermentation broth and crude extract B of mycelium are combined to obtain a crude extract containing the desired benzo[a] nitrogen-containing heterocyclic compounds; Preferably, the macroporous resin includes at least one of D101, AB-8, HZ816, Amberlite XAD-4, XAD-8, XAD-16 or HP20 resin; Preferably, the desorption solution includes ethanol; Preferably, the alcohol solvent includes ethanol or methanol.

7. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to claim 6, characterized in that, The method further includes a step of separating the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (4) Separation: The crude extract obtained in step (3) and / or (3') is subjected to normal-phase silica gel column chromatography, eluted with a dichloromethane-methanol solvent with a volume ratio of 99:1-90:10, and the dichloromethane-methanol solvent fractions with a volume ratio of 95:5-94:6 are collected to obtain the eluent containing compound FWYZ52-D; then subjected to C 18 Reversed-phase column chromatography was performed, with methanol-water solvent at a volume ratio of 10%-100% for elution. The eluent containing FWYZ52-D was collected in the methanol content range of 45%-50% by high-performance liquid chromatography. or, (4') Separation: The crude extract obtained in step (3) and / or (3') is separated using C 18 Reversed-phase column chromatography was performed, with elution using a methanol-water solvent at a volume ratio of 10%-100%. High-performance liquid chromatography (HPLC) was used to detect the elution, and the eluent containing FWYZ52-E was collected in the methanol content range of 40%-60%. Then, normal-phase silica gel column chromatography was performed, with elution using a dichloromethane-methanol solvent at a volume ratio of 99:1-90:

10. Fractions in the dichloromethane-methanol solvent range of 95:5-94:6 were collected, yielding the eluent containing compound FWYZ52-E.

8. The method for preparing the benzo[a] nitrogen-containing heterocyclic compound according to claim 7, characterized in that, The method further includes a purification step of the benzo[a] nitrogen-containing heterocyclic compound, specifically including: (5) Purification: Take the eluent containing FWYZ52-D collected in step (4) to prepare type C 18 The mixture was processed by reversed-phase high-performance liquid chromatography and isocratic elution with a 45% (v / v) methanol-water solution to obtain the pure compound FWYZ52-D. or, (5') Purification: Take the eluent containing FWYZ52-E collected in step (4'), perform LH-20 gel column chromatography, and then use preparative C 18 The compound was processed by reversed-phase high-performance liquid chromatography and isocratic elution with acetonitrile-water solution at a volume ratio of 25% to obtain the pure compound FWYZ52-E.

9. The use of the benzo[a] nitrogen-containing heterocyclic compound of claim 1 in the preparation of multi-target neuromodulators, multi-target antitumor drugs or multi-target immunomodulators.

10. The use of a micromonosporal strain FIMYZ52 for fermentation preparation of the benzo[a]-nitrogen-containing heterocyclic compound of claim 1, characterized in that, The micromonosporal strain FIMYZ52 is classified as Micromonospora. Micromonospora sp. was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24479.

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